WO2024256527A1 - Use of a cxcl 10 inhibitor for the treatment of transplant vasculopathy - Google Patents

Abstract

Transplantation is a surgical operation to replace a failed organ or tissue with a healthy organ or tissue, from a donor to a recipient. It is a common remedy in case of failure of an organ or tissue as failure to replace the failed organ or tissue usually condemns the patient or forces him to undergo extensive treatment. Every recipient is at risk of transplant rejection. While significant progress has been made to control the immune reaction of the recipient against the donor transplant, little is known about how to prevent chronic rejection. The Inventors herein demonstrate that administration of a CXCL10 inhibitor in vivo in a mouse model of transplant vasculopathy decreases transplant vasculopathy after aortic allograft. Accordingly, the present invention relates to a method of treating a subject at risk or suffering from transplant vasculopathy comprising administering to said subject a therapeutically effective amount of a CXCL10 inhibitor.

Description

USE OF A CXCL 10 INHIBITOR FOR THE TREATMENT OF TRANSPLANT VASCULOPATHY

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular angiology.

BACKGROUND OF THE INVENTION:

Transplantation is a surgical operation to replace a failed organ or tissue with a healthy organ or tissue, from a donor to a recipient. It is a common remedy in case of failure of an organ or tissue as failure to replace the failed organ or tissue usually condemns the patient or forces him to undergo extensive treatment.

Every recipient is at risk of transplant rejection. Transplant rejection is a set of local and general reactions that the recipient's body may develop towards the transplant. Transplant vasculopathy (TV) is the primary cause of transplant loss after transplantation. Histologically, the transplanted organ develops a chronic vasculopathy specific to the transplanted organ (“transplant vasculopathy”). The lesions of transplant vasculopathy are typically diffuse and circumferential with concentric hyperplasia of the intima, respecting the internal elastic limit. Mechanistically, the Major Histocompatibility Complex (MHC) on the surface of cells allows the recognition of "self¹ by the immune system. As the MHC differs from one individual to another, the donor's MHC is generally considered foreign by the recipient's immune system, which triggers a defence reaction. It is therefore necessary to ensure maximum proximity of MHCs between donor and recipient before transplantation, but a perfect match is rarely achieved due to the complexity and variability of the MHC.

While significant progress has been made to control the immune reaction of the recipient against the donor transplant, little is known about how to prevent chronic rejection. The present study focuses on the evaluation of CXCL 10 as a therapeutic target to prevent transplant vasculopathy.

SUMMARY OF THE INVENTION:

The invention is defined by the claims. In particular, the present invention relates to a method of treating a subject at risk or suffering from transplant vasculopathy comprising administering to said subject a therapeutically effective amount of a CXCL 10 inhibitor. DETAILED DESCRIPTION OF THE INVENTION:

Here, the Inventors demonstrate that the administration of a CXCL10 inhibitor in vivo in a mouse model of transplant vasculopathy decreases transplant vasculopathy after aortic allograft.

A first object of the present invention relates to a method of treating a subject at risk or suffering from transplant vasculopathy comprising administering to said subject a therapeutically effective amount of a CXCL10 inhibitor.

As used herein, the term “transplant vasculopathy” refers to an occlusive blood vessel disease caused by a transplant. In particular, transplant vasculopathy is a special form of blood vessel disease that differs from sclerosis in non-transplant patients in that the lesions of transplant vasculopathy are diffuse and circumferential with concentric hyperplasia of the intima. The vascular lumen is replaced by an accumulation of smooth muscle cells and connective tissue, leading to a loss of blood flow within the transplant. In some embodiments, the subject is at risk of transplant vasculopathy. In some embodiments, the subject suffers from transplant vasculopathy.

As used herein, the term “transplant” refers to a surgical procedure in which an organ or living tissue (i.e. a transplant) is partially or totally transferred from a donor to a recipient. In some embodiments, the transplant is kidney transplant, liver transplant, heart transplant, heart valves transplant, vascular tissue transplant (e.g. veins, arteries), lung transplant, pancreas transplant, intestine transplant, spleen transplant, uterus transplant, skin transplant, face transplant, corneal transplant, bone transplant, bone marrow transplant, tendon transplant or ligament transplant. In some embodiments, the transplant is an artery transplant. In some embodiments, the transplant is an aortic transplant. In some embodiments, the transplant is an allograft transplant (i.e. transplant to a genetically non-identical member of the same species). In some embodiments, the transplant is an autograft transplant (i.e. transplant wherein the donor and recipient are the same person). In some embodiments, the transplant is an isograft transplant (i.e. transplant to a genetically identical recipient, like a twin). In some embodiments, the transplant is a xenograft transplant (i.e. transplant from one species to another).

As used herein, the term “subject”, “patient”, “donor” or “recipient” denotes a mammal, such as a rodent, a feline, a canine, a pig, a beef, a cow and a primate. Particularly, the subject according to the invention is a human. In some embodiments, the subject is going to undergo a transplant. In some embodiments, the subject has undergone a transplant.

In some embodiments, the subject is at risk from transplant rejection. In some embodiments, the subject suffers from transplant rejection. As used herein, the term “rejection” refers to a mechanism occurring when the recipient’s immune system impairs or destroy the function of the transplant. Transplant rejections are typically classified into three groups: hyperacute, acute and chronic, based upon the mechanisms for rejection. In some embodiments, the transplant rejection is an hyperacute transplant rejection. Hyperacute rejection occurs within minutes or hours after transplantation typically when antigens are completely unmatched between the donor and recipient. In some embodiments, the transplant rejection is an acute transplant rejection. Acute rejection occurs days or weeks after transplantation. In some embodiments, the transplant rejection is a chronic transplant rejection. Chronic rejection occurs months to years after transplantation, when the transplant is slowly damaged by the constant immune response of the recipient through transplant vasculopathies and interstitial inflammation. As example, transplant rejection can be diagnosed with clinical signs (e.g. blood sugar, urine released, shortness of breath, skin color), biopsy of the transplant, or tests (e.g. scan, x-ray, echography, arteriography, ultrasounds, lab tests).

As used herein, the term "risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population.

As used herein, the terms “treating”, “treatment” or “therapy” 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 subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The term encompasses reduction in the severity of symptoms due to transplant vasculopathy. In some embodiments, the term “treatment” also refers to the preventive treatment of the transplant vasculopathy in a subject at risk. The treatment may be administered to a subject having a medical disorder or a subject likely to suffer from 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 therapeutic 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 regular intervals, 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 [e.g., pain, disease manifestation, etc.]).

As used herein, the term “efficient” denotes a state wherein the administration of one or more drugs to a subject permit to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or to prolong the survival of a subject beyond that expected in the absence of such treatment. 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 coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

As used herein, the term “CXCL10” or “C-X-C motif chemokine ligand 10” refers to a cytokine belonging to the CXC chemokine family and encoded by the CXCL10 gene (Entrez: 3627; Ensembl: ENSG00000169245). An exemplary amino acid sequence for CXCL10 is represented in SEQ ID NO:1.

SEQ ID NO : 1 >sp | P02778 | CXL10_HUMAN 0S=Homo sapiens OX=9606 GN=CXCL10 PE = 1 SV=2 MNQTAI LI CC LI FLTLSGIQ GVPLSRTVRC TCI S I SNQPV NPRSLEKLE I I PASQFCPRV EI IATMKKKG EKRCLNPESK AIKNLLKAVS KERSKRS P

As used herein, the term “CXCL10 inhibitor” refers to a molecule that partially or fully blocks, inhibits or neutralizes a biological activity or expression of CXCL10.

As example, CXCL10 inhibitors include, but are not limited to, DT390-IP-10, BMS- 936557 (Eldelumab, Bristol-Myers Squibb), JT-02 (Jyant Technologies), NI-0801 (Novimmune SA), or MDX-1100 (Medarex). In some embodiments, the CXCL10 inhibitor is not a statin. In one embodiment, the CXCL10 inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In one embodiment, the inhibitor according to the invention (i.e. CXCL10 inhibitor) is an antibody. Antibodies directed against CXCL10 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against CXCL10 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-CXCLIO single chain antibodies. Compounds useful in practicing the present invention also include anti-CXCLIO antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to CXCL10. Humanized anti- CXCLIO antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). In the context of the present invention, all CDRs are defined with Kabat numbering scheme.

Monoclonal antibodies that are CXCL10 inhibitors are well known in the art and includes as example those described in the international patent applications W02005/058815, W02008/106200, WO2012/149320, WO2017/095875 or WO2018/112264. As example, antibodies having specificity for CXCL10 includes, but are not limited to, eldelumab (see e.g. NCT01294410), JT-02 (Jyant Technologies), NI-0801 (see e.g. NCT01430429) or MDX1100 (see e.g. NCT01017367).

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 2 and a VL domain that consists of the sequence as set forth in SEQ ID NO:6. According to this embodiment, the VH-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:3 (SYGM), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:4 (VIWFGSIKYYADSVKG) and the VH-CDR3 of the anti- CXCLIO antibody is defined by SEQ ID NO:5 (EGAGS SLYYYGMDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:7 (RASQSVSSGHL), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:8 (GASRAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:9 (QYGSSPYT).

SEQ ID NO : 2 > VH domain o f an anti-CXCLI O antibody ( FR1-CDR1- FR2 -CDR2 - FR3-CDR3- FR4 ) QVQLVESGGGVVQPGRSRLSCAASGFTFSSYGMHVRQAPQKGLEWVAVIWFGS IKYYADSVKGRFTI SD NSKNTLYLQNNSLRAETAVYYCAREGAGSSLYYYGMDVWGQGTTVTVSS SEQ ID NO: 6 > VL domain of an anti-CXCLIO antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4) EIVLTQSPGTLSLSPGEATLSCRASQSVSSGHLAYQQKPGQAPRLLIYGASRATGIPGRFSGSGSGTDT LTISRLEPEDFAVYYCQYGSSPYTFGQGTKLEI

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 10 and a VL domain that consists of the sequence as set forth in SEQ ID NO: 14. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:11 (TYGM), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO: 12 (VIWYGSDKYYADSVKD) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO: 13 (NIVADVAFL). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:15 (RASQSVSSYL), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:16 (DASNAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:17 (QRSNPPLT).

SEQ ID NO: 10 > VH domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QEQLVESGGNVVQPGRSRLSCAASGFTFSTYGMHVRQAPGKGLEWVAVIWYGSDKYYADSVKDRFTVSD NSKNTLYLQMNSLRAETAVYYCARNIAVADVAFLWGQGTMVTVSS

SEQ ID NO: 14 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EIVLTQSPAILSLSPGEATLSCRASQSVSSYLAWQQKPGQAPRLLIYDASNATGIPARFSGSGSGTDFL TISSLEPEDFAYYCQRSNPPLTFGGGTKVEI

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 18 and a VL domain that consists of the sequence as set forth in SEQ ID NO:22. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:19 (NCGMH), the VH-CDR2 of the anti- CXCLIO antibody is defined by SEQ ID NQ:20 (LIGYDGINEYYADSVKG) and the VH- CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:21 (DWPEGYYNGMDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:23 (RASQSVSSSYLA), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:24 (GASSRAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:25 (QQYGSSPPFT).

SEQ ID NO: 18 > VH domain of an anti-CXCLIO antibody ( FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QVQLVESGGGVVQPGRSLRLSCAASGFTFSNCGMHWVRQAPGKGLEWVALIGYDGINEYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVFYCARDWPEGYYNGMDVWGQGTTVTVSS

SEQ ID NO: 22 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPPFTFGPGTKVDIK

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:26 and a VL domain that consists of the sequence as set forth in SEQ ID NO:30. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:27 (SYWI), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:28 (VISPDSDTRYSPSFQG) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:29 (GYCSGGSCYFFQY). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:31 (RASQGISSALA), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:32 (DASSLES) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:33 (QQPDSPPHT).

SEQ ID NO: 26 > VH domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EVQLVQSGAEVKKPGESKISCKGSGYNFPSYWIGVRQMPGKGLEWMGVISPDSDTRYSPSFQGQVTISD KSISTAYLQWSSLKASTAMYYCARGYCSGGSCYFFQYWGQGTLVTVSS

SEQ ID NO: 30 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQPDSPPHTFGGGTKVEIK

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:34 and a VL domain that consists of the sequence as set forth in SEQ ID NO:38. According to this embodiment, the VH-CDR1 of the anti- CXCL10 antibody is defined by SEQ ID NO:35 (NNGM), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:36 (VIWFGMNKFYVDSVKG) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:37 (EGDGSGIYYYGMDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:23 (RASQSVSSSYLA), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:24 (GASSRAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:39 (QQYGSSPIFT).

SEQ ID NO: 34 > VH domain of an anti-CXCLIO antibody ( FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QMQLVESGGGVVQPGRSRLSCTASGFTFSNNGMHVRQAPGKGLEWVAVIWFGMNKFYVDSVKGRFTISD NSKNTLYLEMNSLRAETAIYYCAREGDGSGIYYYGMDVWGQGTTVTVSS

SEQ ID NO: 38 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:40 and a VL domain that consists of the sequence as set forth in SEQ ID NO:43. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:11 (TYGM), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:41 (IIWFGSNEDYAASVKG) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:42 (EGDGSSLYYYGMDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:44 (RASQSISSGYLAYQQK), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:45 (RAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:46 (QYGSSPT).

SEQ ID NO: 40 > VH domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QVQLVESGGGVVQPGRSRLSCTASGFTFSTYGMHVRQAPGKGLEWVAIIWFGSNEDYAASVKGRFTISD NSKNTLYLQMNSLRAETAVYYCAREGDGSSLYYYGMDVWGQGTTVTVSS

SEQ ID NO: 43 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EVVLTQS PGTLSLS PGEATLSCRASQS I SSGYLAYQQKPGQAPRLLIYGASRATGI PDRFSGSGSGTDT LTI SRLE PEDFAVYYCQYGSS PTFGGTKVE IK

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:47 and a VL domain that consists of the sequence as set forth in SEQ ID NO:50. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:48 (NSAMH), the VH-CDR2 of the anti- CXCLIO antibody is defined by SEQ ID NO:49 (LIPFDGYNKYYADSVKG) and the VH- CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:51 (EGGYTGYDGGFDY). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:15 (RASQSVSSYL), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO: 16 (DASNAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:52 (QRSNWPPYT).

SEQ ID NO : 47 > VH domain o f an anti-CXCLI O antibody ( FR1 -CDR1- FR2 - CDR2 - FR3- CDR3- FR4 ) QVQLVESGGGVVQPGRSLRLSCAASGFTFSNSAMHWVRQAPGKGLEWVALI PFDGYNKYYADSVKGRFT I SRDNSKNTLYLQMNSLRAEDTAVYYCAREGGYTGYDGGFDYWGQGI LVTVSS

SEQ ID NO : 50 > VL domain o f an anti-CXCLI O antibody ( FR1 -CDR1- FR2 - CDR2 - FR3- CDR3- FR4 ) E IVLTQS PATLSLS PGEATLSCRASQSVSSYLAWQQKPGQAPRLLIYDASNATGI PARFSGSGSGTDFL TI SSLE PEDFAVYYCQRSNWPPYTFGQGTKLE I

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:53 and a VL domain that consists of the sequence as set forth in SEQ ID NO:57. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:54 (NSGMH), the VH-CDR2 of the anti- CXCLIO antibody is defined by SEQ ID NO:55 (VIDYDGIIQYYADSVKG) and the VH- CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:56 (ERGTHYYGSGSFDY). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:58 (RASQGISSWL), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:59 (AASSQS) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NQ:60 (QYNSYPPT). SEQ ID NO: 53 > VH domain of an anti-CXCLIO antibody ( FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QVQLVESGGGVVQPGRSLRLSCAASGFTFSNSGMHWVRQAPGKGLEWVAVIDYDGI IQYYADSVKGRFT ISRDNSKNTLYLQINSLRAEDTAVYYCATERGTHYYGSGSFDYWGQGTLVTVSS

SEQ ID NO: 57 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) DIQMTQSPSSLSASVGDVTITCRASQGISSWLAWQQKPEKAPKSLIYAASSQSGVPSRFSGSGSGTDFL TISSLQPEDFATYYCQYNSYPPTFGGGTKVEIK

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:61 and a VL domain that consists of the sequence as set forth in SEQ ID NO:65. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:62 (TYGMH), the VH-CDR2 of the anti- CXCLIO antibody is defined by SEQ ID NO:63 (VISYDGIIKHYADSVKG) and the VH- CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:64 (DSSSWYVYFDY). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:66 (RASQSVSSYV), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO: 16 (DASNAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:67 (QRSNSPPWT).

SEQ ID NO: 61 > VH domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QVQLVDSGGGVVQPGRSLRLSCAASGFTFNTYGMHWVRQAPGKGLEWVAVISYDGIIKHYADSVKGRFT ITRDNSKNMVHLQMNSLRAEDTAVYYCARDSSSWYVYFDYWGQGTLVTVSS

SEQ ID NO: 65 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EIVLTQSPATLSLSPGEATLSCRASQSVSSYVAWQQKPGQAPRLLIYDASNATGIPARFSGSGSGTDFL TISSLEPEDFAIYYCQRSNSPPWTFGQGTKVEI

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 68 and a VL domain that consists of the sequence as set forth in SEQ ID NO:72. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:69 (NCGM), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:70 (LIGFGINEYYADSVKG) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:71 (DWPEGYYNGDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:73 (RASQSVSSSYL), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:8 (GASRAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:74 (QYGSSPPFT).

SEQ ID NO: 68 > VH domain of an anti-CXCLIO antibody ( FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QVQLVESGGGVVQPGRSRLSCAASGFTFSNCGMHVRQAPGKGLEWVALIGFGINEYYADSVKGRFTISD NSKNTLYLQMNSLRAETAVYYCARDWPEGYYNGDVWGQGTTVTSS

SEQ ID NO: 72 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EIVLTQSPGTLSLSPGEATLSCRASQSVSSSYLAYQQKPGQAPRLLIYGASRATGIPDRFSGSGSGTDT LTISRLEPEDFAVYYCQYGSSPPFTFGPGTKVDK

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:75 and a VL domain that consists of the sequence as set forth in SEQ ID NO:79. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:76 (SGDYYS), the VH-CDR2 of the anti- CXCLIO antibody is defined by SEQ ID NO:77 (NIYSGSTNYNPSLKS) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:78 (GGGTVVRGIHYYYYYGMDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:73 (RASQSVSSSYL), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:8 (GASRAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NQ:80 (QYGSSPEYT).

SEQ ID NO: 75 > VH domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) QVQLQESGPGLVKPSETSLTCTISGGSVSSGDYYSWIRQPPGKGLEWIGNIYSGSTNYNPSLKSRVTIV DTSKNQFSLKLSSVTADTAVYYCARGGGTVVRGIHYYYYYGMDVWGQGTTTVSS

SEQ ID NO: 79 > VL domain of an anti-CXCLIO antibody (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4) EIVLTQSPGTLSLSPGEATLSCRASQSVSSSYLAYQQKPGQAPRLLIYGASRATGIPDRFSGSGTDTLT ISRLEPEDFAVYYCQYGSSPEYTFGQGTKLEK In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:81 and a VL domain that consists of the sequence as set forth in SEQ ID NO:85. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NO:82 (NSGIH), the VH-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:83 (VISYDGSNKYYADSVKG) and the VH-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:84 (LRDNAEYTDY). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:86 (TGSGGSIASNYVQ), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:87 (EDNQRPS) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:88 (QSYDPLPVWV).

SEQ ID NO : 8 1 > VH domain o f an anti-CXCLI O antibody ( FR1 -CDR1- FR2 - CDR2 - FR3- CDR3- FR4 ) QVQLVESGGGWQPGRSLRLSCAASGFTFSNSGIHWVRQAPGKGLEWVAVI SYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARLRDNAEYTDYWGQGTLVTVSS

SEQ ID NO : 85 > VL domain o f an anti-CXCLI O antibody ( FR1 -CDR1- FR2 - CDR2 - FR3-CDR3- FR4 ) NFMLTQPHSVSES PGKTVTI SCTGSGGS IASNYVQWYQQRPGSS PTTVIYEDNQRPSGVPDRFSGS IDS SSNSASLTI SGLKTEDEADYYCQSYDPLPVWVFGGGTKLTVL

In some embodiments, the anti-CXCLIO antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:89 and a VL domain that consists of the sequence as set forth in SEQ ID NO:38. According to this embodiment, the VH-CDR1 of the anti- CXCLIO antibody is defined by SEQ ID NQ:90 (NNGMH), the VH-CDR2 of the anti- CXCLIO antibody is defined by SEQ ID NO:91 (VIWFDGMNKFYVDSVKG) and the VH- CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:92 (EGDGSGIYYYYGMDV). According to this embodiment, the VL-CDR1 of the anti-CXCLIO antibody is defined by SEQ ID NO:23 (RASQSVSSSYLA), the VL-CDR2 of the anti-CXCLIO antibody is defined by SEQ ID NO:24 (GASSRAT) and the VL-CDR3 of the anti-CXCLIO antibody is defined by SEQ ID NO:39 (QQYGSSPIFT).

SEQ ID NO : 8 9 > VH domain o f an anti-CXCLI O antibody ( FR1 -CDR1- FR2 - CDR2 - FR3- CDR3- FR4 ) QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFT ISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS SEQ ID NO : 38 > VL domain o f an anti-CXCLI O antibody ( FR1 -CDR1- FR2 - CDR2 - FR3-CDR3- FR4 ) E IVLTQS PGTLSLS PGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSG TDFTLTI SRLE PEDFAVYYCQQYGSS PI FTFGPGTKVDIK

In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to CXCL10 with any of the monoclonal antibody described above. In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to CXCL10 with a monoclonal antibody which comprises the CDRs comprised in SEQ ID NO:81 and SEQ ID NO:85. In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to CXCL10 with a monoclonal antibody which comprises the VH domain as set forth in SEQ ID NO:81 and the VL domain as set forth in SEQ ID NO:85. In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to CXCL10 with the monoclonal antibody NI-0801. In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to CXCL10 with a monoclonal antibody which comprises the CDRs comprised in SEQ ID NO:89 and SEQ ID NO:38. In some embodiments, the monoclonal antibody of the present invention cross- competes for binding to CXCL10 with a monoclonal antibody Eldelumab.

As used herein, the term “cross-competes” refers to monoclonal antibodies which share the ability to bind to a specific region of an antigen. In the present disclosure the monoclonal antibody that “cross-competes" has the ability to interfere with the binding of another monoclonal antibody for the antigen in a standard competitive binding assay. Such a monoclonal antibody may, according to non-limiting theory, bind to the same or a related or nearby (e.g., a structurally similar or spatially proximal) epitope as the antibody with which it competes. Cross-competition is present if antibody A reduces binding of antibody B at least by 60%, specifically at least by 70% and more specifically at least by 80% and vice versa in comparison to the positive control which lacks one of said antibodies. As the skilled artisan appreciates competition may be assessed in different assay set-ups. One suitable assay involves the use of the Biacore technology (e.g., by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. Furthermore a high throughput process for "binding" antibodies based upon their cross-competition is described in International Patent Application No. WO2003/48731. In some embodiments, the monoclonal antibody of the present invention retains the activity of a monoclonal antibody which comprises the CDRs comprised in SEQ ID NO:81 and SEQ ID NO:85. In some embodiments, the monoclonal antibody of the present invention retains the activity of a monoclonal antibody which comprises the VH domain as set forth in SEQ ID NO:81 and the VL domain as set forth in SEQ ID NO:85. In some embodiments, the monoclonal antibody of the present invention retains the activity of the monoclonal antibody NI-0801. In some embodiments, the monoclonal antibody of the present invention crosscompetes for binding to CXCL10 with a monoclonal antibody which comprises the CDRs comprised in SEQ ID NO:89 and SEQ ID NO:38. In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to CXCL10 with a monoclonal antibody which comprises the VH domain as set forth in SEQ ID NO:89 and the VL domain as set forth in SEQ ID NO:38. In some embodiments, the monoclonal antibody of the present invention retains the activity of the monoclonal antibody Eldelumab. Any assay well known in the art would be suitable for identifying whether the cross-competing antibody retains the desired activity. In one embodiment, the invention also provides an antibody that binds essentially the same epitope as any of the antibodies as described hereinabove.

In another embodiment, the antibody according to the invention is a single domain antibody directed against CXCL10. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B- cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then, for this invention, neutralizing aptamers of CXCL10 are selected.

In one embodiment, the inhibitor according to the invention is a polypeptide. In a particular embodiment the polypeptide is an inhibitor of CXCL10 and is capable to prevent the function of CXCL10. Particularly, the polypeptide can be a mutated ligand of CXCL10, a mutated CXCL10 protein, a truncated CXCL10 protein or a similar protein without the function of CXCL10. In some embodiments, the CXCL10 inhibitor is a fusion protein. As example, the fusion protein may comprise a CXCL10 polypeptide (e.g. CXCL10 binding site) linked to a second non CXCL10 polypeptide. In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide to allow the penetration of the polypeptide in the cell. The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa). In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In another embodiment, the CXCL10 inhibitor according to the invention inhibits CXCL10 gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of CXCL10 expression in the present invention. CXCL10 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that CXCL10 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA- encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of CXCL10 gene expression in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of CXCL10 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of CXCL10 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing CXCL10. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non- cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles are provided in Kriegler, 1990 and in Murry, 1991. Preferred viruses for certain applications are the adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on . In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In another aspect, the present invention relates to a method of treating a subject at risk or suffering from transplant vasculopathy comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a CXCL10 inhibitor. In some embodiments, the pharmaceutical composition comprises a CXCL10 inhibitor and at least one further therapeutic agent. In some embodiments, the at least one further therapeutic agent is an immunosuppressive agent, a corticoid, a calcineurin inhibitor, a mTOR inhibitor, an antimetabolite and/or a glucocorticoid. In some embodiments, the at least one further therapeutic agent is selected from the list comprising or consisting of azathioprine, antithymocytes globulin, belatacept, CD52, ciclosporin, cortisone, diltiazem, everolimus, glucocorticoids, imlifidase, mycophenolic acid, mycophenolate mofetil, prednisone, pravastatine, simvastatine, sirolimus, tacrolimus, alemtuzumab, biasiliximab, certolizumab, daclizumab, eculizumab, muromonab or rituximab.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. The pharmaceutical compositions of the invention can be formulated for a topical, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

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.

FIGURES:

Figure 1. CXCLIO-neutralizing treatment protects against transplant vasculopathy after aortic transplantation in mice. Panels A-C: Mouse model of transplant vasculopathy. (A) Protocol of induction of transplant vasculopathy after aortic transplantation in mice. C57BL/6J mice (B6) are orthotopically grafted with portions of abdominal aorta from either B6 mice (B6 B6: control group) or BALB/C mice (BALB B6: transplant vasculopathy group). The animals receive daily cyclosporine A until day 14 post-surgery to prevent acute rejection of the grafts and are analyzed at day 28. (B) Histological analysis of the aortic grafts: representative Masson Trichrome-stained cross sections (scale bar = 500 pm) and quantification (n=3/group, t-test, ****p<0.0001). (C) Immunofluorescence of aortic graft cross sections. Smooth muscle marker aSMA and CXCL10 appear gray respectively on the panels above (20x magnification) and below (63x magnification). Panels D-E: Pharmacological neutralization of CXCL10 in the mouse model of transplant vasculopathy. (D) Protocol for testing the impact of a pharmacological inhibition of CXCL10 in the mouse model of transplant vasculopathy after aortic transplantation. Mice receive either an anti-CXCLIO neutralizing mAb (MAB466, R&D Systems) or a control isotype (Rat IgG2a, MAB006, R&D Systems). (E) Histological analysis of the aortic grafts: representative Masson Trichrome-stained cross sections (scale bar = 500 pm) and quantification (n=3/group, t-test, **p<0.01).

Figure 2. Inhibition of immune CXCR3 didn’t reduce transplant vasculopathy in mice. (A) Experimental protocol time-line. Aortic transplantation were performed using recipient C57BL/6J CXCR3 KO mice and donor BALB/C WT. Recipient is treated with cyclosporine A (lOmg/kg/j) for 2 weeks. Schematic representation of the histological analysis of the aortic grafts: 5 gm -thick cross-sections are cut every 100 gm along the aortic graft. (B) Representative images of histological cross-sections of aortic grafts from indicated groups stained with Masson Trichrome. (C) Quantification of transplant vasculopathy using the neointima/perimeter of internal elastic lamina (NI/LEI) of aortic graft. Data are represented as mean ± SEM. (D) Average ratio in the whole graft, regardless of the zones. Data are represented as mean ± SEM and compared with Mann-Whitney test.

EXAMPLE 1:

Material and Methods

Protocol of induction of transplant vasculopathy after aortic transplantation in mice. C57BL/6J mice (B6) are orthotopically grafted with portions of abdominal aorta from either B6 mice (B6 B6: control group) or BALB/C mice (BALB B6: transplant vasculopathy group). The animals receive daily cyclosporine A until day 14 post-surgery to prevent acute rejection of the grafts and are analyzed at day 28 (see Figure 1A).

Protocol for testing the impact of a pharmacological inhibition of CXCL10 in the mouse model of transplant vasculopathy after aortic transplantation. Mice receive either an anti- CXCL10 neutralizing mAh (MAB466, R&D Systems) or a control isotype (Rat IgG2a, MAB006, R&D Systems) (see Figure ID).

Results

The results demonstrate that administration of a CXCL10 inhibitor in vivo in a mouse model of transplant vasculopathy (Figure 1B-1C) decreases transplant vasculopathy after aortic allograft (Figure IE).

EXAMPLE 2:

We performed aortic transplants in recipient mice genetically deficient in CXCR3 (CXCL10 receptor). Experimental protocol time-line is depicted in Figure 2A. Aortic transplantation were performed using recipient C57BL/6J CXCR3 KO mice and donor BALB/C WT. Recipient is treated with cyclosporine A (lOmg/kg/j) for 2 weeks. Representative images of histological cross-sections of aortic grafts from indicated groups stained with Masson Trichrome are depicted in Figure 2B. Quantification of transplant vasculopathy using the neointima/perimeter of internal elastic lamina (NI/LEI) of aortic graft is depicted in Figure 2C. Average ratio in the whole graft, regardless of the zones is depicted in Figure 2D. Taken together, the results demonstrate that the inhibition of immune CXCR3 didn’t reduce transplant vasculopathy in mice.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating a subject at risk or suffering from transplant vasculopathy comprising administering to said subject a therapeutically effective amount of a CXCL10 inhibitor.

2. The method according to claim 1, wherein the transplant is kidney transplant, liver transplant, heart transplant, heart valves transplant, vascular tissue transplant, lung transplant, pancreas transplant, intestine transplant, spleen transplant, uterus transplant, skin transplant, face transplant, corneal transplant, bone transplant, bone marrow transplant, tendon transplant or ligament transplant.

3. The method according to claim 1, wherein the transplant is an aortic transplant.

4. The method according to any of claims 1 to 3, wherein the transplant is an allograft transplant.

5. A method of treating a subject at risk or suffering from transplant vasculopathy comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a CXCL10 inhibitor.

6. The method according to claim 5, wherein the pharmaceutical composition comprises a CXCL10 inhibitor and at least one further therapeutic agent.

7. The method according to claim 6, wherein the at least one further therapeutic agent is an immunosuppressive agent, a corticoid, a calcineurin inhibitor, a mTOR inhibitor, an antimetabolite and/or a glucocorticoid.

8. The method according to claim 6, wherein the at least one further therapeutic agent is selected from the list comprising or consisting of azathioprine, antithymocytes globulin, belatacept, CD52, ciclosporin, cortisone, diltiazem, everolimus, glucocorticoids, imlifidase, mycophenolic acid, mycophenolate mofetil, prednisone, pravastatine, simvastatine, sirolimus, tacrolimus, alemtuzumab, biasiliximab, certolizumab, daclizumab, eculizumab, muromonab or rituximab.

9. The method according to any of claims 1 to 8, wherein the CXCL10 inhibitor is an antibody having specificity for CXCL10.