EP4689666A1

EP4689666A1 - Methods for determining the likelihood of occurrence of bronchiolitis obliterans syndrome in a lung transplant recipient

Info

Publication number: EP4689666A1
Application number: EP24716735.6A
Authority: EP
Inventors: Joël Lemaoult; Nathalie Rouas-Freiss; Edgardo CAROSELLA; Olivier BRUGIERE; Ronan GUILET; Dora DREYFUSS
Original assignee: Commissariat a lEnergie Atomique CEA; Assistance Publique Hopitaux de Paris APHP; HOPITAL FOCH; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Assistance Publique Hopitaux de Paris APHP; HOPITAL FOCH; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2023-03-31
Filing date: 2024-03-29
Publication date: 2026-02-11
Also published as: WO2024200804A1

Abstract

The present invention relates to in vitro method for determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual who is a lung transplant recipient comprising at least the steps of (a) measuring the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from said individual, (b) comparing the level of (EVs-HLA-G+) obtained at step (a) with a reference value; and (c) determining the likelihood of occurrence of BOS in said individual based on the comparison of step (c).

Description

[TITLE]

METHODS FOR DETERMINING THE LIKELIHOOD OF OCCURRENCE OF

BRONCHIOLITIS OBLITERANS SYNDROME IN A LUNG TRANSPLANT RECIPIENT

[TECHNICAL FIELD]

The present disclosure relates to the field of human lung transplantation (Tx). It relates to the description of a new blood biomarker, used as a predictive test of bronchiolitis obliterans syndrome (BOS) considered as a chronic rejection, and which remains one of the first causes limiting the long-term survival of lung transplant patients.

In particular, the present invention relates to a method for determining the likelihood of occurrence of BOS in a lung transplant recipient.

[TECHNICAL BACKGROUND]

Long-term survival after lung transplantation (LTx) still remains limited because of chronic lung allograft dysfunction (CLAD), thought to represent a form of chronic rejection (Kotloff et al., Lung transplantation. Am J Respir Crit Care Med 2011; 184: 159-171). The most prevalent morphologic feature in CLAD is bronchiolitis obliterans syndrome (BOS) representing about 80 % of CLAD cases and affecting over 50 % of the patients after 5 years (Verleden et al., Chronic lung allograft dysfunction: Definition, diagnostic criteria, and approaches to treatment-A consensus report from the Pulmonary Council of the ISHLT. J Heart Lung Transplant 2019; 38: 493-503 and Glanville et al., Chronic lung allograft dysfunction: Definition and update of restrictive allograft syndrome-A consensus report from the Pulmonary Council of the ISHLT. J Heart Lung Transplant 2019; 38: 483-492). It is defined as a persistent decline in forced expiratory volume in 1 second (FEV1) of more than 20% from baseline post-transplant FEV 1 , after ruling out other obvious causes of FEV 1 decline (such as bronchial stenosis, pneumonia acute, acute rejection, ...) (Verleden et al., (2019). Chronic lung allograft dysfunction: Definition, diagnostic criteria, and approaches to treatment-A consensus report from the Pulmonary Council of the ISHLT. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation 38, 493-503)

The current immunosuppressive therapies cannot prevent the occurrence of CLAD in many LTx patients (Chambers et al., International Society for H, Lung T. The Registry of the International Society for Heart and Lung Transplantation: Thirty-fourth Adult Lung And Heart-Lung Transplantation Report-2017; Focus Theme: Allograft ischemic time. J Heart Lung Transplant 2017; 36: 1047-1059), whereas other recipients, under the same standard immunosuppression therapy, remain free of CLAD for a prolonged time, as also reported in other solid organ Tx (SOT) (Girlanda R, Kirk AD. Frontiers in nephrology: immune tolerance to allografts in humans. J Am Soc Nephrol 2007; 18: 2242-2251). In particular, pathogenesis of BOS includes a series of attacks on the bronchial epithelium, alloimmune or not, such as viral infections or acute rejection, allowing activation of the recipient's T lymphocytes by the presenting cells of the graft antigen. This specific alloimmune response to graft antigens ultimately leads to aberrant bronchiolar repair leading to fibro-proliferation with progressive reduction in the caliber of the airways (Neuringer et al., (2005). Obliterative bronchiolitis or chronic lung allograft rejection: a basic science review. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation 24, 3-19; and Verleden et al., 2019). Among the validated treatments for BOS, macrolides are currently the only treatment that has shown a reduction in its incidence and is used as a preventive or curative treatment depending on the centre. The other treatments have an uncertain level of proof of efficacy and include: increasing the intensity of immunosuppressive treatment, IV bolus of corticosteroids, anti -lymphocyte serum, extracorporeal photopheresis, and more recently treatment aimed at deimmunization during rejection, antibody-mediated (Plasmapheresis, Immunoglobulin IV, Rituximab) (Verleden et al., 2019). However, these treatments are often ineffective in slowing down or stabilizing respiratory decline because they are administered at too late a stage in the chronic rejection process. Thus, the only current treatment remains re -transplantation and concerns about 5% of lung transplants, reserved for hyper-selected candidates (Verleden et al., 2019). Hence, there remains a need for a better understanding of immune checkpoints leading to a tolerogenic state in some patients and conversely, an identification of predictive markers of graft acceptance breakdown in the medium- and long-term.

In the last decade, attention has been paid to the immune checkpoint human leukocyte antigen G (HLA-G) molecule (Carosella et al. , HLA-G: An Immune Checkpoint Molecule. Adv Immunol 2015; 127: 33-144), also commonly neo-expressed in some pathologic situations, including allograft in SOT and cancer. HLA-G expression in the graft has been associated with acceptance of various allografts, as reflected by fewer episodes of acute rejection (AR) and reduced incidence of chronic rejection (Lila et al., Implication of HLA- G molecule in heart-graft acceptance. Lancet 2000; 355: 2138, Creput et al., Detection of HLA-G in serum and graft biopsy associated with fewer acute rejections following combined liver-kidney transplantation: possible implications for monitoring patients. Hum Immunol 2003; 64: 1033-1038, Qiu et al., Soluble HLA-G expression and renal graft acceptance. Am J Transplant 2006; 6: 2152-2156 and Brugiere et al., Role of HLA-G as a predictive marker of low risk of chronic rejection in lung transplant recipients: a clinical prospective study. Am J Transplant 2015; 15: 461-471). In the context of LTx, HLA-G expression in bronchial epithelial cells, the primary targets of rejection, is associated with allograft acceptance (Brugiere et al. , Immunohistochemical study of HLA-G expression in lung transplant recipients. Am J Transplant 2009; 9: 1427-1438). HLA-G exerts its immune modulatory functions via 2 main inhibitory receptors: leukocyte immunoglobulin (Ig)-like receptor Bl(LILRBl) and LILRB2 (also known as Ig-like transcript 2[ILT2]/CD85j and ILT4/CD85d), which are differentially expressed by immune cells. ILT2 is expressed by some T and natural killer (NK) cells, B cells and antigen-presenting cells (APCs), whereas ILT4 is myeloid-specific.

In order to use a non-invasive tool of HLA-G expression as a predictor of graft tolerance, the potential of the soluble form of HLA-G (sHLA-G) in the blood and bronchoalveolar lavage (BAL) in LTx recipient has been assessed. In opposite to expected results, an increase in sHLA-G blood levels (Brugiere et al., 2015) or sHLA-G BAL levels (White et al., Association of soluble HLA-G with acute rejection episodes and early development of bronchiolitis obliterans in lung transplantation. PLoS One 2014; 9: el03643) was observed in patients with subsequent CLAD development (Brugiere et al., 2015) or those undergoing acute rejection (White et al., 2014), respectively. Further, no association of HLA-G graft expression and peripheral sHLA-G plasma levels was observed in previous studies (Brugiere et al., 2015), supporting an opposite correlation between, in one hand, graft HLA- G expression and functional stability, and in the other hand increased sHLA-G plasma levels. Conflicting results of sHLA-G plasma levels were also observed in the context of cancer, which can be considered as a negative mirror of SOT, where HLA-G has been evidenced as an immune checkpoint associated with invasive tumors. Indeed, the prognostic values of sHLA-G plasma dosage, and of these different sub-components, including the free soluble HLA-G form (sHLA-G free) and the form of extracellular vesicles (HLA-GEV), have been shown to be associated to cancer progression in different ways (Li et al., HLA- G/sHLA-G and HLA-G-Bearing Extracellular Vesicles in Cancers: Potential Role as Biomarkers. Front Immunol 2021; 12: 791535 and Konig et al., The prognostic impact of soluble and vesicular HLA-G and its relationship to circulating tumor cells in neoadjuvant treated breast cancer patients. Hum Immunol 2016; 77: 791-799). However, no association between total sHLA-G (HLA-G TOT) plasma dosage and progression or recurrence of cancer have been shown. Nevertheless, and of note, HLA-GEV subpart was recently shown as a strong independent predictor for progression in ovarian cancer (Schwich et al., Vesicular-Bound HLA-G as a Predictive Marker for Disease Progression in Epithelial Ovarian Cancer. Cancers (Basel) 2019; 11) or breast cancer whereas additional studies showed in an opposite way that sHLA-G free plasma levels were associated with an improved prognosis in cancers (Konig et al., 2016 and Nardi et al., Soluble monomers, dimers and HLA-G-expressing extracellular vesicles: the three dimensions of structural complexity to use HLA-G as a clinical biomarker. HLA 2016; 88: 77-86).

Taken together, these data may suggest that blood sHLA-G levels associated with the occurrence of acute and chronic rejection (Brugiere et al., 2015 and White et al., 2014) do not reflect the possible link between increased SHLA-GEV plasma levels and acceptance of the lung graft. One of the hypotheses for these opposite effects of vesicular and non- vesicular forms in LTx could be linked to their respective origins: from the cells of the donor graft for the vesicular form, or secreted by the immune cells of the recipient for sHLA-G free during a negative feedback situation in a situation of alloreactivity, with the aim of inhibiting this immune reaction.

There is currently no validated early marker predictive of the evolution of a lung graft at term, either towards tolerance (acceptance of the graft), or towards rejection of the CLAD type. The diagnosis of CLAD is functional, and is based on the occurrence of a persistent decline in the maximum volume exhaled in 1 second (FEV 1) < 80% of the basal value posttransplantation on the respiratory function explorations (Verleden et al., 2019) . This diagnostic test is often late in the process of development of the intra -pulmonary histological lesions of CLAD because the fibre -proliferative lesions of the terminal bronchioles are already irreversible at an early stage of the diagnosis. Moreover, systematic posttransplantation monitoring programs using transbronchial biopsies have not shown any interest in early diagnosis of CLAD because of frequent false-negative histology results (due to the heterogeneous distribution of histological lesions within graft parenchyma) and the overly invasive nature of this method.

Among the candidate markers predictive of the subsequent pulmonary function in CLAD, several types of lymphocyte populations have already been studied in lung transplantation. First, the CD4+CD25hiCD1271o regulatory T cells (TREG), producing immunoregulatory cytokines such as TGFbeta and IL-10, were studied in organ transplantation, with conflicting results in lung transplantation (Salman et al., 2017; Meloni et al., 2006; Durand et al., 2018). Second, we previously reported increased expression of human leukocyte antigen G (HLA-G) in the bronchial epithelium of some lung transplantation recipients, which was associated with a stable condition at the date of biopsies (Brugiere et al., 2009), and we assessed the role of HLA-G expression as a predictor of graft acceptance (Brugiere et al., 2015).

In renal transplantation, cytotoxic CD4+CD57+ T cells have been reported to be associated with the risk of rejection resistant to Belatacept (recombinant CTLA4 immunoglobulin, blocking CD28-mediated costimulatory lymphocytes (Espinosa et al, 2016).

However, there is still a need to identify non-invasive optimized biomarkers, predictive of the occurrence of a CLAD, in particular of a BOS, in order to identify early at-risk patients, and adapt the immunosuppressive treatments as early as possible, and avoid the development of low-level rejection lesions.

Therefore, there remains a need for a method of determining the likeliness of occurrence of a BOS, in particular a method that is non-invasive and may be implemented early enough to identify and treat patients at risk.

The present disclosure has for purpose to satisfy all or part of those needs.

[SUMMARY]

The present invention relates to an in vitro method for determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual who is a lung transplant recipient, comprising at least the steps of:

(a) measuring the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from said individual;

(b) comparing the level of EVs-HLA-G+ obtained at step (a) with a reference value; and

(c) determining the likelihood of occurrence of BOS in said individual based on the comparison of step (b).

The inventors were able to show that high plasma levels of HLA-G+ extracellular vesicles (EVs-HLA-G+) are significantly associated with graft tolerance at 3 years posttransplantation, discriminating patients who would therefore be less at risk of developing chronic rejection, in particular BOS, from those likely to experience such rejection.

In a particular embodiment, the individual’s sample at step (a) is selected in the group consisting of whole blood, blood plasma and blood serum, in particular is blood serum.

In a particular embodiment, the level of EVs-HLA-G+ is measured in step (a) in a sample previously collected from the individual at least 12 months, in particular about 12 months, after said individual received the lung transplant. In a particular embodiment, at step (b), the reference value is the level of EVs-HLA-G+ measured in a sample previously collected from an individual who received and did not reject a lung transplant.

In a particular embodiment, at step (b) the reference value is a level of EVs-HLA-G+ from about 5ng/mL to about 30ng/mL, in particular from about lOng/mL to about 25ng/mL.

In a particular embodiment, in step (a) the level of EVs-HLA-G+ is measured by (i) isolating the EVs-HLA-G+ from the sample and then (ii) quantifying the level of EVs-HLA-G+.

In particular, the EVs-HLA-G+ may be isolated from the sample via a size exclusion method, in particular by size exclusion chromatography.

In particular, the EVs-HLA-G+ level may be quantified by an enzyme immunoassay. In a particular embodiment, the enzyme immunoassay is selected from the group consisting of Enzyme-linked immunosorbent assay (ELISA), Enzyme multiplied immunoassay technique (EMIT), Fluorescent enzyme immunoassays (FEIAs), Chemiluminescent immunoassays (CLIAs), Radioimmunoassays and in particular is an ELISA.

In a particular embodiment, when the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from an individual who is a lung transplant recipient is lower than the reference value, then said individual is more likely to develop bronchiolitis obliterans syndrome (BOS), in particular within 3 years after the lung transplant.

In a particular embodiment, the in vitro method comprises at least the steps of:

(a) measuring the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from said individual wherein the individual’s sample is selected in the group consisting of whole blood, blood plasma and blood serum, in particular is blood serum, wherein the level of EVs-HLA-G+ is measured in a sample previously collected from the individual at least 12 months, in particular about 12 months, after said individual received the lung transplant,

(b) comparing the level of EVs-HLA-G+ obtained at step (a) with a reference value, wherein the reference value is the level of EVs-HLA-G+ measured in a sample previously collected from an individual who is a stable lung transplant recipient at least three years after receiving said transplant.; and

(c) determining the likelihood of occurrence of BOS in said individual based on the comparison of step (b), wherein when the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from an individual who is a lung transplant recipient is lower than the reference value, then said individual is more likely to develop bronchiolitis obliterans syndrome (BOS), in particular within 3 years after the lung transplant, wherein the individual is stable with regards to the lung transplant at least twelve months after receiving the transplant.

The invention further relates to a kit for determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual who is a lung transplant recipient, comprising means to detect and/or quantify the level of extracellular- vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from the individual.

In a particular embodiment, the level of extracellular-vesicular (EVs)-bound HLA-G (EVs- HLA-G+) is detected and/or quantified on the outside surface of the extracellular vesicles. In a particular embodiment, the means to detect and/or quantify the level of extracellular- vesicular (EVs)-bound HLA-G (EVs-HLA-G+)+) allow to maintain the structural integrity of the extracellular vesicles.

[BRIEF DESCRIPTION OF THE FIGURES]

Figure 1 shows the study flow chart. LTx, lung transplantation; COLT, cohort for Lung Transplantation; PFTs, pulmonary function tests; CLAD, chronic lung allograft dysfunction; BOS: bronchiolitis obliterans syndrome; RAS: restrictive allograft syndrome. Figure 2 shows the plasma concentrations of vesicular EVs-HLA-G+ (in ng/mL) at 1 year post-transplant (Ml 2) determined by ELISA method in lung transplant patients and in healthy control donors. STA M12: stable patients (n=41); BOS M12: patients with bronchiolitis obliterans syndrome 3 years post-transplant (n=32); RAS M12: patients with restrictive syndrome post-lung transplantation at 3 years post-transplant (n=5); and HD: healthy donors (n=20) (ANOVA, p=0.039).

Figure 3 provides the survival curves without BOS (freedom from BOS) and without CLAD according to the Kaplan-Meier method as a function of the plasma concentration threshold of EVs-HLA-G+ at M12 (probability of survival (%) as a function of time (years)). (A) Comparison of BOS-free survival at 3 years post-Tx according to the 25% IQR threshold (21.3 ng/ml) and threshold 15ng/ml at M12 post-TxP in the whole cohort. Freedom from BOS was higher in patients with a EVs-HLA-G+ value at M12 > 21.3 ng/ml (top curve) and > 15 ng/ml (bottom curve) as compared to those with value <21.3 ng/ml and <15 ng/ml, respectively (top curve p=0.019; and bottom curve p=0.0353, respectively). (B) Comparison of CLAD-free survival at 3 years post Tx according to the 25% IQR threshold and threshold 15 ng/ml in all transplanted patients. Freedom from CLAD was higher in patients with a EVs-HLA-G+ value at Ml 2^21 .3 ng/ml (top curve) and >15 ng/ml (bottom curve) as compared to those with value <21.3 ng/ml and <_15 ng/ml, respectively (top curve, p=0.017; and bottom curve, p=0.0045, respectively).

Figure 4 shows the graft survival curves according to the 25% IQR threshold (21.3 ng/ml) at M12 post-TxP in the whole cohort. Patients with EVs-HLA-G+ plasma levels > 21.3 ng/ml at M12 had a better graft survival as compared with those with plasma levels <21.3 ng/ml (p=0.037).

[DETAILED DESCRIPTION]

Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance in describing the methods and combinations of biomarkers of the disclosure and how to identify, quantify and use them. The following definitions are provided for the present specification, including the claims.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within three or more than three standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Also, particularly with respect to systems or processes, the term can mean within an order of magnitude, preferably within five-fold, and more preferably within two-fold, of a value. Within the meaning of the invention, “biomarker” intends to refer to a biologically derived indicator (such as a metabolite) of a process, event, or condition (such as aging, disease). A biomarker is a quantifiable characteristic that is objectively measured and evaluated as an indicator of normal or pathogenic biological processes, or of pharmacologic responses to a therapeutic intervention. It can be any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease, the effect of a treatment, or an intervention. A biomarker can be a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like. In the present disclosure, extracellular vesicular (EVs)-bound HLA-Gs (EVs-HLA-G+) are biomarkers. The term "comprise" is to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. Also, it may specify strictly the stated features, integers, steps or components, and therefore in such case it may be replaced with “consist”.

The terms “determine”, “measure”, and “compare” or any equivalent thereof used within the invention in relation with the biomarkers intend to mean the quantification or qualification of the biomarkers.

The quantification is a measure of a quantity of a biomarker which may be expressed in volume, in mole, in weight, in weight by weight or by volume of the matrix containing the biomarker, such as a concentration, in particular a molar concentration. For example, a quantification of a biomarker may be expressed in ng/ml or pg/ml. Also, the quantification of a biomarker may be expressed relatively to the quantification of another biomarker or to a reference (or standard). In such case, the quantification of the concerned biomarkers may be expressed as a ratio, such as a weight:weight ratio or a molar ratio.

The qualification of a biomarker is the determination of the presence or absence of the concerned biomarker. It may also be a non-numerical value of the status of a patient, such as sex (male/female) or menopause status (pre/post-menopause).

The determination, such as quantification or qualification, of a biomarker may be carried out by any known techniques in the art applicable to the concerned biomarker.

In one exemplary embodiment, determination of a biomarker intends to mean quantification of the given biomarker.

Within the scope of the present disclosure, the expression “determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual” is synonym of determining or predicting the risk of occurrence of BOS.

As used herein, an “individual” or a “patient” considered within the present invention is a mammal, and more preferably an animal of economic importance which encompasses primarily human individuals as well as farms, laboratories or food industries animals, such as sheep, swine, cattle, goats, dogs, cats, horses, poultry, mice, rats. Most preferably, an individual is a human.

As used herein, an “individual who is a lung transplant recipient” is an individual who has undergone a surgical procedure to replace a diseased or failing lung with a healthy lung from a donor.

It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

All lists of items are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of ... list of items ... “and combinations and mixtures thereof.”

Referenced herein may be trade names for components utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g., those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.

In the description of the various embodiments of the present disclosure, various embodiments or individual features are disclosed. As will be apparent to the ordinarily skilled practitioner, all combinations of such embodiments and features are possible and can result in preferred executions of the present disclosure. While various embodiments and individual features of the present invention have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the invention. As will also be apparent, all combinations of the embodiments and features taught in the present disclosure are possible and can result in preferred executions of the invention.

(a) measuring the level of extracellular- vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from said individual;

Extracellular vesicles (EVs) are membrane structures presenting a protein, lipid and genetic cargo. In the majority of studies, extracellular vesicles are classified into three groups according to their size and mode of biogenesis: exosomes (30-100 nm) produced in intracellular multivesicular bodies, microvesicles (or ectosomes) (100-1000 nm ) produced by active membrane budding, and apoptosomes (>lpm) produced in late stages of apoptosis.

At the pulmonary level, extracellular vesicles have demonstrated an important involvement in many pathological mechanisms, with the interesting parallel between the characteristics of plasma EVs and the EVs of the alveolar and bronchial compartment, contained in the broncholaveolar fluid (McVey et al., Journal or extracellular vesicles, 2018, vol. 7, 1454776, https://doi.org/10.1080/20013078.2018.1454776). In the field of lung transplantation specifically, extracellular vesicles have shown an important role in the presentation of HLA and non-HLA antigens to the immune system of the recipient, initiating the acute or chronic rejection reaction (Hwang et al., J Thorac Cardiovasc Surg. 2021 February; 161(2): el29-el34. doi:10.1016/j.jtcvs.2020.04.183).

Extracellular- vesicular (EVs)-bound HLA-G (EVs-HLA-G+) as used herein are extracellular vesicles expressing HLA-G. They are present in a soluble form, as opposed to a membrane form. Throughout the text, the EVs-HLA-G+ may also be called “SHLA-GEV” indifferently.

Methods for measuring the level of extracellular-vesicular (EVs)-bound HLA-G (EVs- HLA-G+) in a biological sample are known in the art.

In a particular embodiment of the method, the level of EVs-HLA-G+ is measured in step (a) by (i) isolating the EVs-HLA-G+ from the sample and then (ii) quantifying the level of EVs-HLA-G+.

Isolation of the EVs-HLA-G+ may be performed by any means known to one skilled in the art, such as isolation by size, by affinity to other compounds, by electrical charge, etc.

In a particular embodiment, the EVs-HLA-G+ are isolated from the sample via a size exclusion method. Size exclusion methods for proteins are well known in the art and allow to separate proteins according to their size and molecule weight.

In a particular embodiment, the EVs-HLA-G+ are isolated from the sample via a size exclusion method, in particular is isolated by size exclusion chromatography (SEC).

Methods for quantifying the level of EVs-HLA-G+, and more generally for quantifying the level of proteins in a sample are known in the art.

In a particular embodiment, the level of EVs-HLA-G+ is quantified by an enzyme immunoassay.

In a particular embodiment, the enzyme immunoassay performed in (ii) is selected from the group consisting of Enzyme-linked immunosorbent assay (ELISA), Enzyme multiplied immunoassay technique (EMIT), Fluorescent enzyme immunoassays (FEIAs), Chemiluminescent immunoassays (CEIAs), Radioimmunoassays and in particular is an EEISA.

ELISA is an immunological test that allows the detection or assay of molecules in a biological sample and is well known in the art.

The enzyme immunoassay may allow to control HLA-G expression of the extracellular vesicles.

In a particular embodiment, the method may further comprise, between step (i) and step (ii), an additional control step consisting in controlling that the compounds obtained after step (i) are in fact extracellular vesicles. In a particular embodiment, this control step may be completed by nanoparticle tracking analysis (NTA).

Nanoparticle tracking analysis may allow to determine the size and the amount of extracellular vesicles in the sample.

The level of EVs-HLA-G+ is measured in a sample previously collected from the individual subject of the method. As such, obtaining the sample from said individual is not part of the method of the invention. Indeed, the present method is exclusively an in vitro method.

In a particular embodiment, the individual’s previously collected sample is selected in the group consisting of whole blood, blood plasma and blood serum, in particular is blood plasma.

Blood plasma is a liquid component of blood in which blood cells are absent, but which contains proteins and other constituents of whole blood in suspension.

In a particular embodiment, the level of EVs-HLA-G+ is measured in step (a) in a sample previously collected from the individual at least 12 months after said individual received the lung transplant.

The expression “at least 12 months after said individual received the lung transplant” should be considered as meaning at least 12 months from the day when said individual underwent the surgery which provided them with a lung transplant. In a particular embodiment, the level of EVs-HLA-G+ is measured in step (a) in a sample previously collected from the individual about 12 months after said individual received the lung transplant.

In a particular embodiment, the individual is stable with regards to the lung transplant at least twelve months after receiving the transplant. Definitions of stable transplants, and in particular of stable lung transplants are provided in the art, for example in Verleden et al. (Chronic lung allograft dysfunction: Definition, diagnostic criteria, and approaches to treatment-A consensus report from the Pulmonary Council of the ISHLT. J Heart Lung Transplant 2019; 38: 493-503) and in Glanville et al. (Chronic lung allograft dysfunction: Definition and update of restrictive allograft syndrome - A consensus report from the Pulmonary Council of the ISHLT. J Heart Lung Transplant 2019; 38: 483-492).

By “a stable individual with regards to the lung transplant at least twelve months after receiving the transplant” it is understood herein an individual who has a stable respiratory function at least twelve months after having received the lung transplant.

In a particular embodiment, the reference value used in step (b) is the level of EVs-HLA- G+ measured in a sample previously collected from an individual who is a stable lung transplant recipient at least three years after receiving said transplant.

By “an individual who is a stable lung transplant recipient at least three years after receiving said transplant” it is understood an individual who has a stable respiratory function at least three years after receiving the transplant. In other words, the individual did not reject the transplant. In particular, it is understood that the individual did not develop a CLAD within three years after the lung transplant.

In a particular embodiment, the reference value used in step (b) is a level of EVs-HLA-G+ from about 5ng/mL to about 30ng/mL, in particular from about lOng/mL to about 25ng/mL. In a particular embodiment, the reference value is a level of EVs-HLA-G+ of about 15ng/mL. In another particular embodiment, the reference value is a level of EVs-HLA-G+ of about 21,3ng/mL.

In a particular embodiment, the reference value used in step (b) is a level of EVs-HLA-G+ from about 20ng/mL to about 24ng/mL, in particular from about 21ng/mL to about 23ng/mL.

When the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from an individual who is a lung transplant recipient is lower than the reference value, then said individual is more likely to develop bronchiolitis obliterans syndrome (BOS) , in particular within 3 years after the lung transplant.

When the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from an individual who is a lung transplant recipient is higher than the reference value, then said individual is less likely to develop bronchiolitis obliterans syndrome (BOS), in particular within 3 years after the lung transplant. In a particular embodiment, the reference value used in step (b) is the level of EVs-HLA- G+ measured in a sample previously collected from an individual who is a lung transplant recipient who developed BOS within three years from said transplant.

The invention further relates to a kit for determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual who is a lung transplant recipient, comprising means to detect and/or quantify the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from the individual.

Means to detect and/or quantify the level of EVs-HLA-G+ are known in the art, and are described in further detail above. In particular, means to quantify the level of EVs-HLA-G+ in a sample previously collected from an individual may include (i) means to isolate EVs- HLA-G+ from the sample and (ii) means to quantify the level of EVs-HLA-G+ after isolation, in particular as described above.

In a particular embodiment, the level of extracellular-vesicular (EVs)-bound HLA-G (EVs- HLA-G+) is detected and/or quantified on the outside surface of the extracellular vesicles. In other words, the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) is not detected and/or quantified inside the extracellular vesicles. Only the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) on the exterior of the extracellular vesicles is detected and/or quantified.

According to a particular embodiment, the means to detect and/or quantify the level of extracellular- vesicular (EVs)-bound HLA-G (EVs-HLA-G+) allow to maintain the structural integrity of the extracellular vesicles. In particular said means allow to detect and/or quantify the levels of EVs-HLA-G+ without breaking or harming the extracellular vesicles. As such, any HLA-G on the inside of the extracellular vesicles should not be detected and/or quantified with said means.

[EXAMPLES]

Example 1:

Materials and Methods

Inclusion and patient follow-up.

The COLT (COhort in Lung Transplantation) study, started in 2009, allowed the constitution of a large European cohort of lung transplant patients, associated with a bio- collection. This is a national, multicentre, prospective cohort, in which the majority of lung transplant centres in France participate (including Foch Hospital). More than 1,500 transplanted patients were included, among whom more than 400 patients were already classified according to their respiratory status during the 3 -year follow-up: identified as stable patients, or patients with chronic graft dysfunction (BOS or RAS) (Tissot et al., COLT : 10 ans de recherche en transplantation pulmonaire, resultats et perspectives. Revue des Maladies Respiratoires. sept 2018;35(7):699 705). The COLT study has received an agreement from the CPP n°2009-R4 n°ID RB 2009-A00035-5.

As part of this study, the inventors selected 79 patients in chronological order of inclusion in COLT (from the date of 19/11/2009) with the selection criterion being the availability of plasma samples at 6 and 12 months post-lung transplantation (TxP), knowledge of their functional status at 3 years post-TxP (stable or carrying a CLAD), with a time to onset of CLAD > 12 months for the patients concerned. The follow-up of these patients corresponds to that planned by the COLT study, including a visit every 6 months with prospective collection of clinical data, EFR, blood sampling and bronchial fibroscopy with bronchoalveolar lavage.

At the beginning of the study, this monitoring and the taking of biological samples had already been carried out. The collection of pre-, per- and post-Tx data of interest up to 3 years was directly extracted from the already existing COLT database. These data included in particular the known or suspected predisposing factors for CLAD such as: donor/recipient age, donor/recipient sex, duration of graft ischemia, donor/recipient CMV mismatch, cell rejection score the 1st year post-TxP (sum histological grades of histologically proven cellular acute rejection episodes during the 1st year post-TxP), number of probable or certain humoral rejection episodes according to ISHLT criteria (Levine DJ. Antibody- mediated rejection of the lung: A consensus report of the International Society for Heart and Lung Transplantation. The Journal of Heart and Lung Transplantation. 2016;35(4): 10.), number of post-transplant viral and bacterial infections, detection post-transplant HLA alloimmunization (Luminex technique), HLA mismatch in class 1 and 2 donor/recipient, type of immunosuppressive treatment, immunosuppressive induction treatment.

The diagnosis of acute rejection (AR) was retained after histological confirmation and graded according to the ISHLT criteria (Verleden GM. A new classification system for chronic lung allograft dysfunction. The Journal of Heart and Lung Transplantation. 2014;33(2):7). Diagnosis of BOS-type CLAD was made according to the ISHLT International Functional Definition (Meyer et al. An international ISHLT/ATS/ERS clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome. Eur Respir J. dec 2014;44(6): 1479-503) and graded as BOS grade 1, grade 2, and grade 3. Diagnosis of RAS-type CLAD was made according to recommended diagnostic criteria of the ISHLT (Glanville et al. Chronic lung allograft dysfunction: Definition and update of restrictive allograft syndrome — A consensus report from the Pulmonary Council of the ISHLT. The Journal of Heart and Lung Transplantation, mai 2019;38(5):483 92).

Patient classification.

Seventy-nine patients were able to be selected for this study and were classified into two equivalent groups according to their functional status at 3 years post-TxP: a first group of 41 patients in stable condition at 3 years post-TxP ( STABLE group); and a second of 38 patients with CLAD (either BOS (n=32) or RAS (n=5)) at 3 years post-TxP (CLAD group). For each patient, the plasmas at visits 3 and 4, noted respectively V3 (6 months posttransplant) and V4 (12 months post-transplant) were recovered and stored in the laboratory of Saint Louis Hospital (Paris, France) for analysis.

Among the 78 patients, 41 remained with stable condition throughout the study (ST A group), and in 37, CLAD developed within 3 years post-LTx, with BOS (n=32) or RAS (n=5) phenotype (Figure 1).

Biological samples.

At each visit of the COLT study, a blood sample was to be taken for the biocollection, in parallel and according to the local monitoring protocol of each transplant center. For the blood sample, 60 mL were taken during the first visit, then 50 mL during subsequent visits. The anticoagulant used for all the samples in this study was heparin. The plasmas of the 79 selected patients were transferred at -80°C from the Nantes, France, CRB (in charge of managing the COLT study) to our laboratory at Saint-Louis Hospital in Paris, France. Upon receipt, the samples were stored in a freezer at -80°C awaiting analysis. In addition, plasma samples (n=20, volume=500 pL) were collected from blood samples taken from healthy adult individuals, voluntary donors at the EFS of Saint-Louis Hospital, in order to constitute a control group (EFS-CEA agreement).

Isolation and analysis of extracellular vesicles.

The extracellular vesicles (EVs) of interest (exosomes and micro vesicles) were isolated by the Size Exclusion Chromatography (SEC) method using qEVl 35nm columns (Izon Science), a method of choice for a rapid and relatively pure isolation of EVs with a size between 35 and 350 nm from plasma samples (Thery et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 1 dec 2018;7(l): 1535750). The particles were separated in decreasing order of size as they passed through the column, which was made up of porous resin beads. The largest particles did not penetrate the beads, and flow quickly (obtaining a fraction of interest). Conversely, small particles (< 35 nm) were retained in the pores of the beads and flowed more slowly. For each plasma sample, 1 mL of sample was deposited on a chromatography column then a buffer solution (Phosphate Buffered Saline, PBS IX) was added to cause the sample to migrate through the column. After the flow of a dead volume of 4.5 mL (1 mL from the sample + 3.5 mL of PBS for migration), the inventors collected the fraction of interest (Purified Collection Volume, PCV) from a volume of 3 mL of purified vesicles contained in the buffer. For each PCV, 1 mL was stored at +4°C for the analysis of this study, and 2 mL were aliquoted into 2 x 1 mL tubes and frozen without preservative at -80°C for subsequent use.

Subsequently, the EVs contained in each PCV were analyzed quantitatively and qualitatively by the NTA Nanoparticle Tracking Analysis technique (NanoSight NS300, Malvern Panalytical, MSCMed antenna, Saints Peres university site, Paris) according to the recommendations of the International Society for Extracellular Vesicles (Thery et al. - see reference above). This technique makes it possible to estimate the hydrodynamic diameter and the concentration of extracellular vesicles isolated in PC Vs by video analysis of their light diffraction capacity and their Brownian motion. Five 40-second videos were captured and analyzed for each sample diluted at the concentration recommended by the manufacturer (1 to 5 xl08 particles/mL, dilution varying between 1:500 and 1:1000 depending on the samples) in PBS without particles (Gibco). Sample analysis was standardized by auto-sampler overnight. The 158 samples (corresponding to the two timepoints for each patient) were passed through a 96-well half-plate (Greiner Bio-One 96- well ImL) or 48 samples at a time. The parameters used corresponded to the routine settings by the MSCMed laboratory and are as follows: camera level 16 and detection threshold 5.

Results

Among the patients of the national COLT cohort, the circulating EVs-HLA-G+ of lung transplant patients (n=78) were quantified in a plasma blood sample 12 months posttransplantation (M12). The results show that in patients with the BOS type CLAD form, the plasma level of EVs-HLA-G+ is significantly lower compared to that of patients stable at M12 (mean value of EVs-HLA-G+ of 38 .7 versus 23.7 ng/mL, difference - 12.7 95% CI [-23.48; -1.89], p value = 0.02) (Figure 2). Among all patients, mean plasma level of EVs- HLA-G+ of BOS patients at M12 was lower to that of all other patients (Stable and RAS patients; BOS: 23.35 ng/ml versus STA + RAS: 38.97 ng/ml, difference between means 15.62 + 5.24, p=0.004).

The inventors then studied the predictive role of the plasma concentration of EVs-HLA-G+ on survival without BOS and CLAD (Figure 3) and graft survival (Figure 4).

The establishment of a threshold for the assay of EVs-HLA-G+ (in order to distinguish 2 populations “HLA-GEV High” and “HLA-GEV LOW”) is not known in the context of TxP. Derived from a study showing the predictive role on tumor progression in breast cancer of the plasma level of EVs-HLA-G+ at the threshold of 15 ng/ml (Konig et al., 2016), the inventors studied survival without CLAD according to this threshold of 15 ng/ml. They also studied CLAD-free survival and graft survival according to 25% quartile values. The 25% IQR value for the whole transplant population at M12 was 21.3 ng/mL and was taken to classify HLA-GEv-High versus HLA-GEV-LOW patients.

The inventors observed a higher freedom from BOS among the 78 patients of this cohort with high EVs-HLA-G+ values at M12 by taking two different thresholds (> 21.3 ng/ml (p=0.019) - and > 15 ng/ml (0.0353), figure 3A).

The inventors also observed a CLAD-free survival greater than 3 years post-Tx among the 78 patients of the cohort with a EVs-HLA-G+ value >21.3 ng/ mL (25% IQR) at M12 (p=0.0017). A CLAD-free survival greater than 3 years post-Tx was observed among the 76 stable and CLAD transplanted patients (i.e. all the transplanted patients in the study) with a EVs-HLA-G+ value >15ng/mL at M12 (p= 0.0045) (Figure 3B).

By taking the threshold at 21.3ng/mL for the 78 patients of the cohort, a significantly higher graft survival was observed among the patients with an EVs-HLA-G+ level >21.3ng/mL versus those with EVs-HLA-G+ <21.3ng/mL (p = 0.037, Figure 4).

In conclusion, the inventors were able to show that the circulating EVs-HLA-G+ of lung transplant patients at 12 months represented an efficient biomarker of the likelihood of occurrence of bronchiolitis obliterans syndrome in said patients. In particular, it was demonstrated that decreased extracellular vesicles expressing HLA-G plasma levels at 12- month post-LTx was associated with an increased risk of BOS onset at 3 years.

Claims

[CLAIMS]

1. An in vitro method for determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual who is a lung transplant recipient, comprising at least the steps of:

2. The in vitro method according to claim 1, wherein the individual’s sample at step (a) is selected in the group consisting of whole blood, blood plasma and blood serum, in particular is blood serum.

3. The in vitro method according to claim 1 or 2, wherein the level of EVs-HLA-G+ is measured in step (a) in a sample previously collected from the individual at least 12 months, in particular about 12 months, after said individual received the lung transplant.

4. The in vitro method according to any one of the preceding claims, wherein the individual is stable with regards to the lung transplant at least twelve months after receiving the transplant.

5. The in vitro method according to any one of the preceding claims, wherein at step (b) the reference value is the level of EVs-HLA-G+ measured in a sample previously collected from an individual who is a stable lung transplant recipient at least three years after receiving said transplant.

6. The in vitro method according to any one of the preceding claims, wherein at step (b) the reference value is a level of EVs-HLA-G+ from about 5ng/mL to about 30ng/mL, in particular from about lOng/mL to about 25ng/mL.

7. The in vitro method according to any one of the preceding claims, wherein in step (a) the level of EVs-HLA-G+ is measured by (i) isolating the EVs-HLA-G+ from the sample and then (ii) quantifying the level of EVs-HLA-G+.

8. The in vitro method according to claim 7, wherein the EVs-HLA-G+ are isolated from the sample via a size exclusion method, in particular are isolated by size exclusion chromatography.

9. The in vitro method according to claim 7, wherein the EVs-HLA-G+ level is quantified by an enzyme immunoassay.

10. The in vitro method according to claim 9, wherein the enzyme immunoassay is selected from the group consisting of Enzyme-linked immunosorbent assay (ELISA), Enzyme multiplied immunoassay technique (EMIT), Fluorescent enzyme immunoassays (FEIAs), Chemiluminescent immunoassays (CLIAs), Radioimmunoassays and in particular is an ELISA.

11. The in vitro method according to any one of claims 7 to 10, wherein the method further comprises between step (i) and step (ii), an additional control step consisting in controlling that the compounds obtained after step (i) are extracellular vesicles.

12. The in vitro method according to claim 11 , wherein the control step is completed by nanoparticle tracking analysis.

13. The in vitro method according to any one of the preceding claims, wherein when the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from an individual who is a lung transplant recipient is lower than the reference value, then said individual is more likely to develop bronchiolitis obliterans syndrome (BOS), in particular within 3 years after the lung transplant.

14. The in vitro method according to any one of the preceding claims, comprising at least the steps of:

(b) comparing the level of EVs-HLA-G+ obtained at step (a) with a reference value, wherein the reference value is the level of EVs-HLA-G+ measured in a sample previously collected from an individual who is a stable lung transplant recipient at least three years after receiving said transplant. ; and (c) determining the likelihood of occurrence of BOS in said individual based on the comparison of step (b), wherein when the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from an individual who is a lung transplant recipient is lower than the reference value, then said individual is more likely to develop bronchiolitis obliterans syndrome (BOS), in particular within 3 years after the lung transplant, wherein the individual is stable with regards to the lung transplant at least twelve months after receiving the transplant.

15. A kit for determining the likelihood of occurrence of bronchiolitis obliterans syndrome (BOS) in an individual who is a lung transplant recipient, comprising means to detect and/or quantify the level of extracellular-vesicular (EVs)-bound HLA-G (EVs-HLA-G+) in a sample previously collected from the individual.

16. The kit according to claim 15, wherein the level of extracellular- vesicular (EVs)-bound HLA- G (EVs-HLA-G+) is detected and/or quantified on the outside surface of the extracellular vesicles.

17. The kit according to any one of claims 15 or 16, wherein the means to detect and/or quantify the level of extracellular- vesicular (EVs)-bound HLA-G (EVs-HLA-G+)+) allow to maintain the structural integrity of the extracellular vesicles.