Inhibitors of EBV + b cell migration for treatment and / or prevention of EBV associated disease

Abstract

The present invention relates to an inhibitor of EBV-infected B cell (EBV+ B cell) migration for use in treating and / or preventing an EBV associated disease in a subject, and to methods, kits, and uses related thereto.

Description

[0001] Inhibitors of EBV+B cell migration for treatment and / or prevention of EBV associated disease

[0002] The present invention relates to an inhibitor of EBV-infected B cell (EBV+B cell) migration for use in treating and / or preventing an EBV associated disease in a subject, and to methods, kits, and uses related thereto.

[0003] The principal role of B cells (B lymphocytes) in the immune system is the production of antigen-specific antibodies upon their activation. Activation requires that the B cell-receptor (BCR) on the surface of the B cell becomes bound to its cognate antigen. This activation of the BCR leads to activation of the B cell, which undergoes maturation and clonal expansion, which may involve migration of B cells between tissues, after which part of the cells produced this way becomes plasma cells producing antibodies specific for said antigen.

[0004] The oncogenic Epstein-Barr virus (EBV) belongs to the family of gammaherpesviruses that can infect human B lymphocytes latently. The EBV establishes lifelong persistent B cell infections in more than 90% of the human population (Kieff and B. Rickinson. (2006), Epstein-Barr virus and its replication, In D. M. Knipe and P. M. Howley (ed.), Fields virology, 5th ed. Lippincott- Raven, Philadelphia, PA: 2603-2654; Kiippers, R. (2003), Nat. Rev. Immunol. 3:801-812). In healthy individuals, the majority of EBV infected B cells shows limited viral gene expression and a resting phenotype. The terminal differentiation of latently infected cells into plasma cells leads to virus reactivation, production, and reinfection of B cells (Laichalk, L. L., and D. A. Thorley-Lawson. (2005), J. Virol. 79: 1296-1307). The expression of all viral latency genes causes growth transformation and the proliferation of infected B cells, which is reflected by the outgrowth of EBV-transformed lymphoblastoid B cell lines in vitro and by the association of EBV with a variety of B cell lymphoproliferative diseases, including different types of lymphoma, in vivo. EBV infection is controlled by T cells, as indicated by an increased incidence of EBV-associated malignancies in patients with congenital or iatrogenically induced T-cell dysfunction and by the successful treatment of EBV-associated posttransplant lymphoproliferative disease in hematopoietic stem cell transplant recipients by the infusion of polyclonal EBV-specific T-cell lines (Rooney, C. M., et al., (1998), Blood 92: 1549-1555). Besides inducing B cell lymphoproliferation, EBV has been detected in lymphomas and in tumors of epithelial or mesenchymal origin such as nasopharyngeal carcinoma or leiomyosarcoma, which is why EBV was classified as a class I carcinogenic agent by the WHO International Agency for Research on Cancer (IARC).

[0005] Extravasation of immune cells such as B cells from the blood into tissues is a multi-step process. In general, the immune cell is apprehended from the bloodstream by selectins (e.g., E-selectin, P-selectin) located on endothelial cells, which interact with leukocyte glycoproteins, such as P- selectin glycoprotein ligand-1 (PSGL-1). This weak and transient interaction results in the tethering and rolling of the immune cell along the vessel wall. Subsequent firm adhesion of the leukocyte to the inflamed endothelial cells halts the immune cell, which is mediated by cellular adhesion molecules (CAMs), such as immunoglobulin family members, cadherins, or integrins. For example, leukocytes express integrins, such as lymphocyte function-associated 1 (LFA-1) or very late activation antigen-4 (also known as a4pi or VLA-4) that respectively bind to endothelial vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Other examples of endothelial CAMs are the melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM). Chemokines are also essential regulators of the transendothelial migration of immune cells since they enhance the affinity of leukocyte integrins to bind strongly to endothelial CAMs. Following this firm adhesion, leukocytes can cross the endothelium via paracellular or transcellular migration. (Rodriguez-Mogeda et al. Biomolecules 2022, 12(6), 800)

[0006] To prevent or treat EBV associated diseases, efforts have been made to develop a vaccine to prevent or clear EBV infection. However, first clinical trials with a peptide vaccine show it does not prevent EBV infection in EBV-negative transplant recipients (Rees L, et al. (2009), Transplantation 88(8): 1025-9).

[0007] Thus, there is a need for improved methods to treat and prevent EBV infection and associated diseases. The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned need. The technical problem is solved by the means and methods of the present invention, with the features of the independent claims. Preferred embodiments, which might be realized in an isolated fashion or in any arbitrary combination are listed in the dependent claims. In accordance, the present invention relates to an inhibitor of EBV-infected B cell (EBV+B cell) migration for use in treating and / or preventing an EBV associated disease in a subject.

[0008] In general, terms used herein are to be given their ordinary and customary meaning to a person of ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or customized meaning. As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e. are equivalent to "comprising at least one". In accordance, expressions relating to one item of a plurality, unless otherwise indicated, preferably relate to at least one such item, more preferably a plurality thereof; thus, e.g. contacting "a cell" with a compound relates to contacting at least one cell, preferably to contacting a multitude of cells.

[0009] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

[0010] The methods specified herein below, preferably, are in vitro methods; however, some methods, in particular methods of treatment and / or prevention preferably are performed in vivo. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but preferably are performed in the indicated sequence; also, one or more, preferably all, of said steps may be assisted or performed by automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned above.

[0011] As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, more preferably ± 10%, most preferably ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Preferably, a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified component(s).

[0012] The degree of identity (e.g. expressed as "%identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment. The percentage is calculated by determining, preferably over the whole length of the polynucleotide or polypeptide, the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (e.g. BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. More preferably, the Basic Local Alignment Search Tool (BLAST) implementation is used with default parameter values for alignment. In the context of biological sequences referred to herein, the term "essentially identical" indicates a %identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The aforesaid applies to the term "essentially complementary" mutatis mutandis.

[0013] The term "fragment" of a biological macromolecule, preferably of a polynucleotide or polypeptide, is used herein in a wide sense relating to any sub-part, preferably subdomain, of the respective biological macromolecule comprising the indicated sequence, structure and / or function. Thus, the term includes sub-parts generated by actual fragmentation of a biological macromolecule, but also sub-parts derived from the respective biological macromolecule in an abstract manner, e.g. in silico. Thus, as used herein, an Fcor Fab fragment, but also e.g. a singlechain antibody, a bispecific antibody, and a nanobody may be referred to as fragments of an immunoglobulin.

[0014] Unless specifically indicated otherwise herein, the compounds specified, in particular the polynucleotides and polypeptides, may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further sequences, carrier molecules, retardants, and other excipients. In particular, polypeptides as specified may be comprised in fusion polypeptides comprising further peptides, which may serve e.g. as a tag for purification and / or detection, as a linker, or to extend the in vivo half-life of a compound. The term “detectable tag” refers to a stretch of amino acids which are added to or introduced into the fusion polypeptide; preferably, the tag is added C- or N- terminally to the fusion polypeptide. Said stretch of amino acids preferably allows for detection of the polypeptide by an antibody which specifically recognizes the tag; or it preferably allows for forming a functional conformation, such as a chelator; or it preferably allows for visualization, e.g. in the case of fluorescent tags. Preferred detectable tags are the Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST-tag or a fluorescent protein tag, e.g. a GFP-tag. These tags are all well known in the art. Other further peptides preferably comprised in a fusion polypeptide comprise further amino acids or other modifications which may serve as mediators of secretion, as mediators of blood-brain-barrier passage, as cell-penetrating peptides, and / or as immune stimulants. The term "protein" is understood by the skilled person; preferably, the protein comprises at least one amino acid chain, i.e. a polypeptide as specified herein below, more preferably comprises a multitude of polypeptides. Thus, the protein may be a multimer, e.g. a dimer, a trimer, or the like, wherein the polypeptides in the multimer may be connected covalently, e.g. by a disulfide bridge, or non-covalently, e.g. by ionic interactions, hydrophobic interactions, and / or van der Waals interactions. The protein may consist of identical polypeptides, e.g. may be a homodimer, or may comprise at least two non-identical polypeptides, e.g. may be a heterodimer. More preferably, the protein as specified comprises all structural components as indicated comprised in one continuous covalent polypeptide chain, thus, the protein preferably is or is comprised in a fusion polypeptide.

[0015] The term “polypeptide”, as used herein, refers to a molecule consisting of a multitude of amino acids that are covalently linked to each other by peptide bonds. Polypeptides consisting of less than 20 amino acids covalently linked by peptide bonds may also be referred to as "peptides". Preferably, the polypeptide comprises of from 20 to 1000, more preferably of from 50 to 500, still more preferably of from 100 to 500, most preferably of from 250 to 400 amino acids. The polypeptide may also be comprised in a fusion polypeptide, i.e. may comprise amino acid sequences in addition to those specifically indicated. Also, the polypeptide may comprise additional, non-peptidic structures, such as at least one glycosylation, lipid conjugation, and the like. Thus, unless specifically indicated otherwise, reference to specific polypeptides herein preferably includes polypeptide variants.

[0016] As used herein, the term "polypeptide variant" relates to any chemical molecule comprising at least one polypeptide as specified herein, having the indicated activity, but differing in structure from said specific polypeptide. Preferably, the polypeptide variant comprises a polypeptide having a contiguous amino acid sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, of the amino acid sequence of the polypeptide specifically indicated. Moreover, it is to be understood that a polypeptide variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still, preferably, at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, identical with the amino acid sequence of the specific polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and as described herein above. Polypeptide variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Moreover, the polypeptide variants referred to herein include fragments of the specific polypeptides or the aforementioned types of polypeptide variants as long as these fragments and / or variants have the biological activity as specified. Such fragments may be or may be derived from, e.g., degradation products or splice variants of the polypeptides. Further included are variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation, or myristyl ation, by including non-natural amino acids, and / or by being peptidomimetics.

[0017] The term "Epstein-Barr virus", which may be abbreviated as "EBV", is known to the skilled person and has been specified herein above. Methods for identifying EBV and diagnosing EBV infection in a subject are textbook knowledge and have been reviewed e.g. by Abusalah et al. (2020), Pathogens 9:226. EBV particles comprise the EBV structural proteins (e.g., capsid, tegument, and glycoproteins) known to the skilled artisan, as well as an EBV DNA genome, and a host derived membrane comprising the glycoproteins. Reference EBV genome sequences are available from databases, e.g. EBV type 1 : Genbank Acc No: NC_007605.1; de Jesus O et al. (2003) J. Gen. Virol. 84, 1443-1450 and EBV type 2: Genbank Acc No: NC_009334.1, Dolan A et al. (2006) Virology Vol.350, 164-170). As referred to herein, the term EBV preferably includes any and all EBV strains infecting at least one subject and / or infecting at least one cultured B cell line or lymphoblastoid cell line, preferably relates to an EBV strain infecting at least one subject. More preferably, the term EBV relates to an EBV strain identified in a sample of a subject.

[0018] The term "B cell", for which also the term "B lymphocyte" may be used, relates to a type of lymphocyte known to the skilled person. B cells may e.g. be identified by cell surface markers essentially specific for B cells (B cell markers). B cell marker expression may be dependent on B cell development; e.g. CD22 is known as a marker of pro-B, pre-B, and immature B cells, while CD 19 is a marker of mature B cells, including antibody-secreting plasmablasts.

[0019] In view of the description herein, the term "EBV-infected B cell", which may be abbreviated as "EBV+B cell" is self-evident. The term preferably includes any and all B cells expressing at least one EBV gene, e.g. at least one EBV-encoded RNA (EBER), latent membrane protein 1 (LMP-1) or 2 (LMP-2), and / or Epstein-Barr nuclear antigen 1 (EBNA-1). Thus, an EBV+B cell may be identified by detecting expression of said at least one EBV gene. Thus, the EBV+B cell may be a lyrically infected B cell, i.e. a B cell producing EBV particles, or may be a latently infected B cell, i.e. a B cell expressing a very limited set of EBV genes and not producing EBV particles; said latent EBV-infected B cell preferably is proliferating actively. The EBV+B cell preferably is a B cell in a subject, in particular in a subject's blood and / or brain. In case the subject is an organ graft donor or organ graft recipient, the EBV+cell may be a B cell within said organ grafted or to be grafted.

[0020] The term "migration" is understood by the skilled person and includes any type of active locomotion; in the context of the instant description, migration is cell migration, in particular of B cells, preferably of EBV+B cells. Mechanisms of cell migration are known in the art. Preferably, migration is extravasation of B cells, preferably of EBV+B cells, from the bloodstream to at least one adjacent tissue or organ. Cell migration can be measured e.g. by determining the trajectory of cells through a collagen mesh according to methods in principle known in the art and as described herein in the Examples. As the skilled person understands, said migration does not necessarily have to be strictly directional; nonetheless, migration preferably has a directional component, i.e. preferably causes a cell to overall move into a direction. Thus, determining cell migration may comprise determining the velocity of a candidate cell along a trajectory, which may be compared to a control cell known to be not migrating, e.g. a non-EBV infected B cell. In view of the description herein above, determining cell migration does not necessary have to, but may, comprise determining movement of a cell in a certain direction. Thus, determining cell migration may also be performed in the form of a transwell cell migration assay, which is known in the art. Preferably, migration is augmented by B cells, preferably EBV+B cells, having a density of at least 3x 10E4 cells / ml and / or by including CCL5, CCL4, CCL3, and / or IL-10 in the medium surrounding said cells.

[0021] As used herein, the term "migration factor" relates to each and every compound modulating, preferably increasing, B cell migration, preferably EBV+B cell migration. The migration factor may be a compound produced by a cell of a subject, such as by a B cell, preferably an EBV+B cell, e.g. a product of gene expression, or may be a compound comprised in a medium surrounding said cell, preferably in a bodily fluid of a subject. Thus, the migration factor may be a signaling molecule, preferably is CCL4, CCL3, and / or IL-10. Preferably, the migration factor is a gene product of a B cell, more preferably of an EBV+B cell. More preferably the migration factor is selected from the list consisting of Protein tyrosine kinase 2 beta (PTK2B, Reference sequence Genbank Acc. No. NP_004094.3), CDC42 (Reference sequence Genbank Acc. No. NP_001034891.1), ROCK (Reference sequence Genbank Acc. No. NP_005397.1), Phosphoinositide 3-kinase (PI3K), CCL4 (Reference sequence Genbank Acc. No. NP_996890.1), and CCR1 (Reference sequence Genbank Acc. No. NP_001286.1), or is a polypeptide variant as specified herein above of any of the aforesaid. As the skilled person is aware of, CCL4, CCL3, and IL- 10 can be inhibited by neutralizing antibodies, which are commercially available. CCL4 release can be inhibited with pirtobrutinib (5-amino-3-[4-[[(5- fhioro-2-methoxybenzoyl)amino]methyl]phenyl]- 1 -[(2S)- 1 , 1 , 1 -trifluoropropan-2-yl]pyrazole- 4-carboxamide; CAS No. 2101700-15-4).

[0022] The term "EBV-associated disease", as used herein, includes any and all diseases which are caused or exacerbated by the presence of EBV+B cells in the afflicted subject. Thus, an EBV associated disease may in particular be a disease for which EBV infection is a known risk factor or cause. As the skilled person will understand, in some diseases for which EBV is a risk factor, EBV may be only one of several possible causative agents leading to disease. In such case, the EBV associated disease preferably is the EBV associated form of said disease. As the case may be, there may be also other diseases in which it is known that EBV is a risk factor, but it may be unknown which other factors contribute to disease; in such cases, the skilled person will accept that treatment and prevention as described herein may not be successful in each and every disease case. Nonetheless, it may be satisfactory that, overall, the morbidity and / or severity of disease can be reduced.

[0023] As is known in the art, EBV infection is associated with a variety of diseases, which may be benign, such as infectious mononucleosis, or malignant, such as a lymphoma, in particular EBV associated lymphoma, preferably Burkitt's lymphoma or PTLD associated lymphoma. Preferably, the EBV associated diseases is a benign disease. More preferably, the EBV associated disease is infectious mononucleosis, post-transplant lymphoproliferative disease (PTLD), multiple sclerosis (MS), EBV-associated chronic fatigue syndrome, or is presence of EBV or increased EBV load in a subject, preferably in a transplant donor and / or a transplant receptor. Also preferably, the EBV associated diseases is a malignant disease, in particular a lymphoma, preferably PTLD associated lymphoma. All of the aforesaid diseases and methods for diagnosing them are known in the art. The term "inhibitor" is known to the skilled person to relate to any compound causing the relevant activity, preferably B cell migration, to decrease, preferably significantly, compared to the activity in the absence of said inhibitor. Preferably, said inhibition is an inhibition by at least 20%, more preferably at least 50%, even more preferably at least 75%, even more preferably at least 90% of the value of an activity parameter in the absence of said inhibitor. The inhibition may, however, also be an inhibition by a factor of at least two, preferably at least five, more preferably at least ten, or most preferably an essentially complete inhibition. Thus, inhibition may, also be complete abolishment of an activity such as B cell migration present in the absence of said inhibitor. The effect of the inhibitor may be temporary, e.g. short-term over a time frame of hours or days, or long-lasting, e.g. over weeks or months, or may be permanent, in particular depending on the specific choice of the inhibitor. Preferably, said effect is temporary and lasts for of from 2h to 6 months, preferably of from 4h to 1 months, more preferably of from 6h to 1 week, most preferably of from 12h to 2d. The effect of the inhibitor may be local, i.e. topical at a site of administration, or may be systemic, e.g. after systemic administration of the inhibitor.

[0024] As used herein, the term "inhibitor of EBV+B cell migration" relates to each and every inhibitor as specified herein above causing EBV+B cell migration to decrease compared to the EBV+B cell migration in the absence of said inhibitor. Thus, the inhibitor of EBV+B cell migration preferably reduces the activity of at least one of the migration factors specified herein elsewhere. Also preferably, the inhibitor of EBV+B cell migration reduces the amount of at least one migration factor in an EBV+B cell and / or the amount of at least one migration factor secreted by an EBV+B cell. Preferably, the inhibitor of EBV+B cell migration is a small molecule inhibitor, an immunoglobulin or binding fragment thereof, a peptide aptamer, a polynucleotide aptamer, an anticalin, or a Designed Ankyrin Repeat Protein. The inhibitor of EBV+B cell migration may be an inhibitor of B cell migration, i.e. does not necessarily have to be specific for EBV+ B cells. Nonetheless, the inhibitor of EBV+B cell migration preferably is lymphocyte specific, more preferably is B cell specific, most preferably is EBV+B cell specific. In the context of inhibition of cell migration, "specific inhibition" preferably is an at least 2fold, more preferably at least 4fold, still more preferably at least lOfold stronger inhibition of the target cell compared to a non-target cell.

[0025] Preferably, the inhibitor of EBV+B cell migration is a small molecule inhibitor. Small molecule inhibitors of the migration factors referred to herein are known in the art, e.g. Defactinib (N- methyl-4-[[4-[[3-[methyl(methylsulfonyl)amino]pyrazin-2-yl]methylamino]-5- (trifluoromethyl)pyrimidin-2-yl]amino]benzamide, CAS No. 1073154-85-4) as an inhibitor of PTK2B; ZCL278 (CAS No. 587841-73-4) as an inhibitor of CDC42; Y27632 ((lR,4r)-4-((R)- l-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide; CAS No. 146986-50-7) as an inhibitor of ROCK; ZSTK474 (2-(2-Difluoromethylbenzimidazol-l-yl)-4,6-dimorpholino- 1,3, 5 -triazine, CAS No. 475110-96-4), Leniolisib (CAS No. 1354690-24-6), and Duvelisib (8- Chloro-2-phenyl-3-[(lS)-l-(3H-purin-6-ylamino)ethyl]-l(2H)-isoquinolinone, CAS No. 1201438-56-3) as inhibitors of PI3K; and AZD4818 ((S)-2-(2-chloro-5-(3-(5-chloro-3H- spiro[benzofuran-2,4'-piperidin]-l'-yl)-2-hydroxypropoxy)-4-(methylcarbamoyl)phenoxy)-2- methylpropanoic acid, CAS No. 1003566-93-5) and BX471 (N-[5-chloro-2-[2-[(2R)-4-[(4- fluorophenyl)methyl]-2-methyl-l-piperazinyl]-2-oxoethoxy]phenyl]-urea, CAS No. 217645- 70-0) as inhibitors of CCR1. Thus, the inhibitor ofEBV+B cell migration preferably comprises, more preferably is, Defactinib, ZCL278, Y27632, ZSTK474, Leniolisib, Duvelisib, and / or AZD4818. Also preferably, the small molecule inhibitor of EBV+B cell migration is an inhibitor of PTK2B and is Defactinib; or is an inhibitor of CDC42 and is ZCL278; or is an inhibitor of ROCK and is Y27632; or is an inhibitor of PI3K and is ZSTK474, Leniolisib, or Duvelisib, or is an inhibitor of CCR1 and is AZD4818 and / or BX471. More preferably, the small molecule inhibitor of EBV+B cell migration is an inhibitor of PTK2B and most preferably is Defactinib.

[0026] Preferably, the inhibitor of EBV+B cell migration is an immunoglobulin. As used herein, the term "immunoglobulin" relates to any polypeptide or fragment thereof from the class of polypeptides known to the skilled person under this designation and comprising at least one antigen binding site. Preferably, the immunoglobulin is a soluble immunoglobulin from any of the classes IgA, IgD, IgE, IgG, or IgM, or a fragment comprising at least one antigen binding site derived thereof. Also comprised as immunoglobulins are a bispecific immunoglobulin, a synthetic immunoglobulin, an immunoglobulin fragment, such as Fab, Fvor scFvfragments etc., a single chain immunoglobulin, and a nanobody. Further included are chemically modified derivatives of any of the aforesaid, e.g. PEGylated derivatives, as well as fusion proteins comprising any of the aforesaid immunoglobulins and fragments thereof. The immunoglobulin may be a human or humanized immunoglobulin, a primatized, or a chimerized immunoglobulin or a fragment thereof as specified above. Preferably, the immunoglobulin is a polyclonal or a monoclonal immunoglobulin, more preferably a monoclonal immunoglobulin or a fragment thereof as specified above. Preferably, the immunoglobulin of the present invention shall specifically bind (i.e. does not cross react with other polypeptides or peptides) to at least one of the migration factors as specified herein. Specific binding can be tested by various well known techniques. In view of the description herein above, the skilled person understands that the immunoglobulin is an inhibitory immunoglobulin, i.e. an immunoglobulin binding, preferably specifically, to at least one migration factor and thereby inhibiting the modulating activity of the migration factor on EBV+B cell migration, e.g. by inhibiting interaction of the migration factor with its cognate interaction target in or on the surface of a cell. CCR1 is known to respond to CCL3 and CLL4 stimulation. Immunoglobulins against target polypeptides can be prepared by well-known methods e.g. using a purified protein or a suitable fragment derived therefrom as an antigen.

[0027] Preferably, the inhibitor of EBV+B cell migration is an RNAi agent. As used herein, the term “RNAi agent” refers to an shRNA, an siRNA agent, or an miRNA agent as specified below, causing expression of at least one of the migration factors referred to herein in a cell to decrease compared to a control target cell. The RNAi agent is of sufficient length and complementarity to stably interact with the target RNA, i.e. it comprises at least 15, at least 17, at least 19, at least 21, at least 22 nucleotides complementary to the target RNA. By "stably interact" is meant interaction of the RNAi agent or its products produced by the cell with a target RNA, e.g., by forming hydrogen bonds with complementary nucleotides in the target RNA under physiological conditions. As the skilled person understands, the RNAi agent may also be a chemical derivative of a polynucleotide, e.g. a morpholino.

[0028] The term “siRNA agent” as meant herein encompasses: a) a dsRNA consisting of at least 15, at least 17, at least 19, at least 21 consecutive nucleotides base-paired, i.e. forming hydrogen bonds with complementary nucleotides, b) a small interfering RNA (siRNA) molecule or a molecule comprising an siRNA molecule. The siRNA is a single-stranded RNA molecule with a length, preferably, greater than or equal to 15 nucleotides and, preferably, a length of 15 to 49 nucleotides, more preferably 17 to 30 nucleotides, and most preferably 17 to 30 nucleotides, preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. According to the present invention, the term "molecule comprising an siRNA molecule" includes RNA molecules from which an siRNA is processed by a cell, preferably by a mammalian cell. Thus, a molecule comprising an siRNA molecule, preferably, is a small hairpin RNA, also known as shRNA. As used herein, the term "shRNA" relates to a, preferably artificial, RNA molecule forming a stem-loop structure comprising at least 10, preferably at least 15, more preferably at least 17, most preferably at least 20 nucleotides base-paired to a complementary sequence on the same mRNA molecule (“stem”), i.e. as a dsRNA, separated by a stretch of non-base-paired nucleotides (“loop”), c) a polynucleotide encoding a) or b), wherein, preferably, said polynucleotide is operatively linked to an expression control sequence. Thus, the function of the siRNA agent to inhibit expression of the target gene can preferably be modulated by said expression control sequence. Preferred expression control sequences are those which can be regulated by exogenous stimuli, e.g. the tet operator, whose activity can be regulated by tetracycline, or heat inducible promoters. Alternatively or in addition, one or more expression control sequences can be used which allow cell type specific, e.g. B cell specific, expression of the siRNA agent. siRNAs against the migration factors referred to herein, e.g. PTK2B, are commercially available.

[0029] It is, however, also contemplated that the RNAi agent is an miRNA agent. An “miRNA agent” as meant herein encompasses: a) a pre-microRNA, i.e. a mRNA comprising at least 30, at least 40, at least 50, at least 60, at least 70 nucleotides base-paired to a complementary sequence on the same mRNA molecule (“stem”), i.e. as a dsRNA, separated by a stretch of non-base-paired nucleotides (“loop”), b) a pre-microRNA, i.e. a dsRNA molecule comprising a stretch of at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 base-paired nucleotides formed by nucleotides of the same RNA molecule (stem), separated by a loop, c) a microRNA (miRNA), i.e. a dsRNA comprising at least 15, at least 17, at least 18, at least 19, at least 21 nucleotides on two separate RNA strands, d) a polynucleotide encoding a) or b), wherein, preferably, said polynucleotide is operatively linked to an expression control sequence as specified above.

[0030] Thus, preferably, the inhibitor of EBV+B cell migration is an anti-PTK2B shRNA or siRNA, an anti-CDC42 shRNA or siRNA, an anti-ROCK shRNA or siRNA, an anti-PI3K shRNA or siRNA, an anti-CCL4 shRNA or siRNA, and / or an anti-CCRl shRNA or siRNA.

[0031] Also preferably, the inhibitor of EBV+B cell migration comprises at least one, preferably two, gRNAs, i.e. preferably CRISPR / Cas targeting oligonucleotides, targeting a gene encoding a migration factor. The CRISPR / Cas system has been known for several years as a convenient system for inducing knock-out mutations, i.e. deletions, preferably of chromosomal genes. The skilled person knows how to design appropriate oligonucleotides, which are, preferably, expressed from a vector, to induce deletion of a DNA sequence of interest. Preferably, said deletion is a partial deletion, more preferably deletion of a portion of the gene essential for function; most preferably said deletion is a complete deletion of at least the whole coding region.

[0032] Preferably, the inhibitor of EBV+B cell migration is an aptamer. As used herein, the term "aptamer" relates to a polynucleotide or polypeptide binding specifically to a target molecule by virtue of its three-dimensional structure. Preferably, the aptamer specifically interacts with a migration factor as specified for the immunoglobulins described herein above. Preferably, the aptamer is a peptide aptamer. Peptide aptamers, preferably, are peptides comprising 8-80 amino acids, more preferably 10-50 amino acids, and most preferably 15-30 amino acids. They can e.g. be isolated from randomized peptide expression libraries in a suitable host system like baker’s yeast (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998). A peptide aptamer, preferably, is a free peptide; it is, however, also contemplated that a peptide aptamer is fused to a polypeptide serving as “scaffold”, meaning that the covalent linking to said polypeptide serves to fix the three-dimensional structure of said peptide aptamer to a specific conformation.

[0033] Preferably, the inhibitor of EBV+B cell migration is an anticalin. As used herein, the term "anticalin" relates to an artificial polypeptide derived from a lipocalin specifically binding a migration factor as specified for the immunoglobulins described herein above. Similarly, a "Designed Ankyrin Repeat Protein" or "DARPin", as used herein, is an artificial polypeptide, comprising several ankyrin repeat motifs, specifically binding a migration factor as specified for the immunoglobulins described herein above.

[0034] Preferably, the inhibitor of EBV+B cell migration is comprised in a pharmaceutical composition, said pharmaceutical composition preferably further comprising a pharmaceutically acceptable carrier. The term “pharmaceutical composition”, as used herein, thus relates to a composition comprising the inhibitor of EBV+B cell migration in a pharmaceutically acceptable form and, optionally, a pharmaceutically acceptable carrier. The compounds of the present invention can be formulated as pharmaceutically acceptable salts. Acceptable salts comprise acetate, HC1, sulfate, chloride, and the like. The pharmaceutical compositions are, preferably, administered topically or systemically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, or parenteral administration as well as inhalation. Preferably, the pharmaceutical composition of the present invention is administered via a parenteral route, preferably by intravenous injection. However, polynucleotide compounds may also be administered in a gene therapy approach by using viral vectors, viruses or liposomes, and may also be administered topically, e.g. as an ointment. Moreover, the compounds can be administered in combination with other drugs either in a common pharmaceutical composition or as separated pharmaceutical compositions wherein said separated pharmaceutical compositions may be provided in form of a kit of parts. The compounds are, preferably, administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, either a solid, a gel or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The diluent(s) is / are preferably selected so as not to affect the biological activity of the inhibitor of EBV+B cell migration and potential further pharmaceutically active ingredients. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0035] A therapeutically effective dose refers to an amount of the compounds to be used in a pharmaceutical composition of the present invention which prevents, ameliorates or treats a condition referred to herein. Therapeutic efficacy and toxicity of compounds can be determined by standard pharmaceutical procedures in cell culture or in experimental animals, e.g., by determining the ED50 (the dose therapeutically effective in 50% of the population) and / or the LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50. The dosage regimen will be determined by the attending physician, preferably taking into account relevant clinical factors and, preferably, in accordance with any one of the methods described elsewhere herein. As is well known in the medical arts, a dosage for any one patient may depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 1 pg to 10000 pg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example, preferably from one to four times, more preferably two or three times.

[0036] Preferably, the pharmaceutical composition comprises at least one further pharmaceutically active compound, i.e. preferably is a combined preparation. The term “combined preparation” as referred to in this application preferably comprises all pharmaceutically active compounds in one preparation so that all compounds are administered simultaneously and in the same way. Also preferably, the combined preparation comprises at least two physically separated preparations for separate administration, wherein each preparation contains at least one pharmaceutically active compound. The latter alternative is preferred in cases where the pharmaceutically active compounds of the combined preparation have to be administered by different routes, e.g. parenterally and orally, due to their chemical or physiological properties. Preferably, the at least two separated preparations are administered simultaneously. This means that the time frames of the administration of the preparations overlap. Also preferred is the sequential administration of the at least two preparations, whereas the administration of the single preparations shall occur in time frames which do not overlap. Preferably, the at least two preparations are administered in a time interval of 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 1 day or 2 days, preferably within 1 hour, more preferably simultaneously, most preferably in a combined preparation comprising all pharmaceutically active compounds.

[0037] In a preferred embodiment, the further pharmaceutically active compound in the combined preparation is at least one nonsteroidal anti-inflammatory drug (NSAID) and / or acetaminophen (Paracetamol, N-(4-Hydroxyphenyl)-acetamid, CAS No. 103-90-2). NSAIDs are known in the art; preferably, the NS AID is ibuprofen ((RS)-2-(4-(2-Methylpropyl)phenyl)propanoic acid, CAS NO. 15687-27-1), naproxen ((+)-(S)-2-(6-Methoxynaphthalen-2-yl)propanoic acid, CAS No. 22204-53-1), or ketorolac ((±)-5-benzoyl-2,3-dihydro-lH-pyrrolizine-l-carboxylic acid, CAS No. 74103-06-3), preferably R-ketorolac ((+)-ketorolac, (lR)-5-benzoyl-2,3- dihydro-lH-pyrrolizine-1 -carboxylic acid, CAS No. 66635-93-6), all preferably at usual doses known to the skilled person.

[0038] The terms "treating" and “treatment” refer to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to said diseases or disorders. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein, or an identifiable subgroup thereof, can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well-known statistic evaluation tools, e.g., as described herein above. Preferably, treating comprises reducing the number of EBV+B cells in at least one tissue or organ of said subject and / or comprises reducing the number of EBV+B cells in said subject. Also preferably, treating comprises reducing the number of B cells in at least one tissue or organ of said subject, in particular in the brain, more preferably comprises reducing the number of CDl lc+T-bet+B cells in the brain of said subject; in such case, the subject preferably is a subject suffering from MS.

[0039] Preferably, treating infectious mononucleosis comprises reducing the number of mononuclear cells in a subject, reducing EBV load in said subject, reducing transmission risk of EBV infection from a subject, and / or a reduction of the number and / or severity of symptoms associated with infectious mononucleosis, such as fever, pharyngitis, lymphadenopathy, headache, and the like. Also preferably, treating post-transplant lymphoproliferative disease (PTLD) comprises prevention of B cell proliferation in a subject, reduction of B cell numbers in a subject, and / or reduction of the number and / or severity of symptoms associated with PTLD, such as fever, in particular periodic fever, weight loss, and / or general weakness. Also preferably, treating multiple sclerosis (MS) comprises prevention or reduction in severity of MS relapses, deceleration of MS progression, and / or reduction of the number and / or severity of symptoms associated with MS, in particular numbness, tingling, Lhermitte sign, slurred speech, unsteady gait and / or inability to walk. Thus, treating MS may comprise achieving stable disease. Also preferably, treating EBV-associated chronic fatigue syndrome comprises improvement of severity and / or number of fatigue episodes. Also preferably, treatment of presence of EBV or increased EBV load a subject, preferably in a transplant donor and / or a transplant receptor, comprises reducing the number of EBV+B cells in a subject; as the skilled person will understand, said reduction preferably is before transplantation in a transplant donor, and is after transplantation in the transplant receptor. Thus, a transplant donor may be preferably be treated before donating the transplant; in such case, treatment may also be prophylactic or depleting; so preferably the treatment comprises administration of said inhibitor of EBV+B cell migration to a transplant donor, preferably to prevent or reduce transmission of EBV+B cells via a transplant donated by said transplant donor. Also, a transplant recipient may preferably be treated after receiving the transplant, wherein said treatment may be therapeutic, i.e. to reduce EBV+B cell numbers to acceptable or normal levels, or may be prophylactic, i.e. to avoid or reduce an increase in EBV+B cell numbers in the first place.

[0040] The term “preventing” refers to retaining health with respect to the diseases or disorders referred to herein for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the amount of the preparation, e.g. the inhibitor of EBV+B cell migration, which has been administered and individual factors of the subject discussed elsewhere in this specification. It is to be understood that prevention may not be effective in all subjects treated with a preparation according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context which normally, i.e. without preventive measures according to the present invention, would develop a disease or disorder as referred to herein, or would experience more severe disease. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well-known statistic evaluation tools discussed elsewhere in this specification. It will be understood by the skilled person that there may be cases in which treatment and prevention according to common use of the terms cannot be clearly differentiated; e.g. prevention of an MS relapse may be part of an MS treatment. Preferably, preventing comprises preventing an increase of the number of EBV+B cells in at least one tissue or organ of a subject and / or comprises preventing a spread of an EBV infection to at least one previously non-infected tissue.

[0041] Also preferably, preventing comprises preventing an increase of the number of B cells in at least one tissue or organ of said subject, in particular in the brain, more preferably comprises preventing an increase of the number of CD1 lc+T-bet+B cells in the brain of said subject; in such case, the subject preferably is a subject known or suspected to suffer from MS or to be at an increased risk compared to the general population to develop MS. Further preferably, the EBV associated disease is PTLD and preventing is preventing onset of PTLD. Further, in case the EBV associated disease is MS or EBV-associated chronic fatigue syndrome, preventing may also be reducing the probability of occurrence of said MS or EBV-associated chronic fatigue syndrome.

[0042] As detailed elsewhere herein, preventing may also comprise reducing the risk of occurrence of EBV associated disease, be it benign or malignant EBV associated disease. Preferably, preventing comprises reducing the risk of occurrence of malignant EBV associated disease.

[0043] In a preferred embodiment, treating and / or preventing comprises administering to said subject an inhibitor of migration factor PTK2B and an inhibitor of migration factor CCR1, preferably wherein said inhibitor of migration factor PTK2B is defactinib and / or wherein said inhibitor of migration factor CCR1 is BX471, more preferably wherein said inhibitor of migration factor PTK2B is defactinib and wherein said inhibitor of migration factor CCR1 is BX471. Preferably, said inhibitor of migration factor PTK2B and said inhibitor of migration factor CCR1 are for simultaneous or for sequential administration, and / or are for separate or for combined administration.

[0044] In a preferred embodiment, "simultaneous administration", as used herein, relates to an administration wherein the pharmaceutically active compounds as specified are administered at the same time, i.e., preferably, administration of the pharmaceutically active compounds starts within a time interval of less than 15 minutes, more preferably, within a time interval of less than 5 minutes. Most preferably, administration of the pharmaceutically active compounds starts at the same time, e.g. by swallowing a tablet comprising the pharmaceutically active compounds, or by swallowing a tablet comprising one of the pharmaceutically active compounds and simultaneous injection of the second compound, or by applying an intravenous injection of a solution comprising one pharmaceutically active compound and injecting second compound in different part of the body. Conversely, "sequential administration", as used herein, relates to an administration causing plasma concentrations of the pharmaceutically active compounds in a subject enabling the synergistic effect of the present invention, but which, preferably, is not a simultaneous administration as specified herein above. Preferably, sequential administration is an administration wherein administration of the pharmaceutically active compounds, preferably all pharmaceutically active compounds, starts within a time interval of 1 or 2 days, more preferably within a time interval of 12 hours, still more preferably within a time interval of 4 hours, even more preferably within a time interval of one hour.

[0045] In a preferred embodiment, "separate administration", as used herein, relates to an administration wherein at least two of the pharmaceutically active compounds of the present invention are administered via different routes and / or at different parts of the body of a subject. E.g. one compound may be administered by enteral administration (e.g. orally), whereas a second compound is administered by parenteral administration (e.g. intravenously). Preferably, in such case at least two physically separated preparations are provided for separate administration, wherein each preparation contains at least one pharmaceutically active compound; said alternative may be preferred e.g. in cases where the pharmaceutically active compounds of the combined preparation have to be administered by different routes, e.g. parenterally and orally, due to their chemical or physiological properties. Conversely, "combined administration" relates to an administration wherein the pharmaceutically active compounds of the present invention are administered via the same route, e.g. orally or, preferably, intravenously. Thus, in such case, a preparation may be administered comprising at least two, preferably all, pharmaceutically active compounds in a single preparation.

[0046] The term "subject", as used herein, relates to a mammal, such as a goat, a sheep, a cattle, a pig, a horse, a dog, a cat, a hamster, a rat, a mouse, an alpaca, a guinea pig, a rabbit, a hare, or to a human, preferably to a livestock or experimental animal. More preferably, the subject is a human. Preferably, the subject is known or suspected to be suffering, preferably at the time of administration of a treatment as specified herein, from an EBV associated disease. Also preferably, the subject is known or suspected, at the time of prevention, to be at an increased risk of developing an EBV associated disease. Thus, the subject may in particular be a subject planned to donate or receive an organ graft and / or a subject suffering from MS. Advantageously, it was found in the work underlying the present invention that EBV+B cell migration can be inhibited by the migration inhibitors described herein. Moreover, it was found that inhibiting EBV+B cell migration reduces or completely abolishes EBV spread through organs of an infected subject, and that preventing said migration abolishes EBV-induced B cell proliferation.

[0047] The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

[0048] In view of the above, the present invention preferably relates to defactinib for use in treating and / or preventing an EBV associated disease in a subject, to AZD4818 for use in treating and / or preventing an EBV associated disease in a subject, and / or to BX471 for use in treating and / or preventing an EBV associated disease in a subject. In a preferred embodiment, the invention relates to an inhibitor of migration factor PTK2B for use in treating and / or preventing an EBV associated disease in a subject, wherein said treating and / or preventing comprises administration of an inhibitor of migration factor CCR1; and the present invention relates to an inhibitor of migration factor CCR1 for use in treating and / or preventing an EBV associated disease in a subject, wherein said treating and / or preventing comprises administration of an inhibitor of migration factor PTK2B.

[0049] The present invention also relates to a use of an inhibitor of EBV-infected B cell (EBV+B cell) migration for the manufacture of a medicament for treating and / or preventing an EBV associated disease; and to a method for treating and / or preventing an EBV associated disease in a subject, said method comprising (a) administering at least one inhibitor of EBV+B cell migration to said subject, and (b) thereby treating and / or preventing said EBV associated disease in said subject.

[0050] The present invention moreover relates to a method for identifying a compound for treating and / or preventing EBV-associated disease, the method comprising

[0051] (A) administering a candidate compound to EBV+B cells,

[0052] (B) determining migration of the EBV+B cells of (A); and

[0053] (C) based on determining step (B), identifying a compound for treating and / or preventing EBV- associated disease. The term "candidate compound" is a broad term including all chemical compounds for which the skilled person may assume that they could be effective in treating and / or preventing EBV- associated disease. Preferably, said candidate compound is a macromolecule, preferably from a group of macromolecules as specified herein above as inhibitors of EBV+B cell migration. More preferably, the candidate compound is a small molecule compound, preferably with a molecular mass of less than 1000 Da, more preferably less than 750 Da. Preferably, the candidate compound is comprised in a compound library. The candidate compound preferably is a chemical compound known to have pharmaceutically acceptable properties, i.e. in particular not being toxic to a subject. The candidate compound preferably is a known inhibitor of one of the migration factors described elsewhere herein, and more preferably has successfully passed at least one phase I clinical trial. Also preferably, the candidate compound is a known inhibitor of at least one of the migration factors described elsewhere herein under clinical investigation and / or in clinical use for treatment of a non-EBV associated disease.

[0054] As used herein, the term "determining" relates to providing the indicated information, in particular a value of a parameter such as average migration velocity, a migration vector, number or percentage of cells having successfully migrated a predetermined distance, e.g. in a transwell assay, or the like. Preferred are parameters as determined in the Examples provided herein below. Appropriate methods are known in the art and include in particular the methods described herein in the Examples. Thus, the term "determining migration" refers to semi quantitative or quantitative determination of at least one parameter correlating with migration activity of cells.

[0055] The method comprises step (A) administering a candidate compound to EBV+B cells. The term "administering" is understood to be a broad term including each and every method causing the candidate compound to come into contact with the EBV+B cells. Thus, in case the method is an in vitro method, administering may be a simple addition of the candidate compound to the growth medium used for the EBV+B cells. In in vitro methods, administration may comprise formulation of the candidate compound as a pharmaceutically acceptable formulation, preferably as specified herein above. In case the method is an in vivo method performed on an experimental animal, said experimental animal is preferably sacrificed after the method is performed, preferably after step (B) was performed. Preferably, said method is not performed on a human subject. Preferably, in steps (A) and / or (B), the EBV+B cells are maintained at a cell density of at least 3xlOE4 cells / ml and / or are maintained in a medium comprising CCL4, CCL3, CCL5, and / or IL-10. Preferably, the CCL4 concentration in said medium is of from 0.4 ng / ml to 40 ng / ml, more preferably of from 1 ng / ml to 16 ng / ml, still more preferably of from 2 ng / ml to 8 ng / ml, most preferably about 4ng / ml; also preferably, the CCL3 concentration in said medium is of from 0.2 ng / ml to 20 ng / ml, more preferably od from 0.5 ng / ml to 8 ng / ml, still more preferably of from 1 ng / ml to 4 ng / ml, most preferably about 2 ng / ml; also preferably, the CCL5 concentration in said medium is of from 10 pg / ml to 1 ng / ml, more preferably of from 25 pg / ml to 400 pg / ml, still more preferably of from 50 pg / ml to 200 pg / ml, most preferably about 100 pg / ml; also preferably, the concentration of IL- 10 in said medium is of from 1 ng / ml to 100 ng / 1, more preferably of from 2.5 ng / ml to 40 ng / ml, still more preferably of from 5 ng / ml to 20 ng / ml, most preferably about 10 ng / ml. Thus, preferably, EBV+ B cells are maintained at a cell density of at least 3xlOE4 cells / ml and / or are maintained in a medium comprising CCL4 at a concentration of 4 ng / ml, CCL3 at a concentration of 2 ng / ml, CCL5 at a concentration of 100 pg / ml, and / or IL- 10 at a concentration of 10 ng / ml.

[0056] Preferably, step (A) includes an incubation step allowing the candidate compound to exert its effect(s), if any, on the cell. Said incubation step may be for of from 15 min to 7 days, preferably of from 30 min to 2 days, more preferably of from Ih to 24h.

[0057] The method comprises step (B) determining migration of the EBV+B cells of step (A); methods for determining migration of EBV+B cells have been discussed herein elsewhere. In in vivo methods, homing of B cells and / or spread of EBV and / or of EBV+B cells to initially uninfected tissues may be determined, which may require taking samples of tissue or of whole organs from an experimental animal.

[0058] The method comprises step (C) based on determining step (B), identifying a compound for treating EBV-associated disease. Preferably, a compound for treating EBV associated disease is identified if inhibition of EBV+B cell migration is determined in step (B). The identification may be possible based on the result obtained in step (B) for the cells to which the candidate compound was administered alone, e.g. in case essentially absence of migration is determined. Preferably, however, the method comprises at least one positive control, i.e. cells contacted with a known inhibitor of EBV+B cell migration, e.g. defactinib; and / or at least one negative control, i.e. cells kept under normal growth conditions. In some cases, it may be necessary to compare the results of step (B) to a reference in order to make the identification in step (C). Thus, the method may comprise further step (Bl) comparing migration determined in step (B) to a reference. Such a reference preferably is a cell for which it is known whether migration is inhibited or not. Thus, the reference may be the aforesaid negative control and / or the aforesaid positive control. As the skilled person will understand, in case the reference is a negative control (i.e. an untreated cell), any parameter value indicating migration less that the reference preferably is indicative of a compound for treating and / or preventing EBV-associated disease; and in case the reference is a positive control as specified herein above, e.g. a cell treated with a known inhibitor of EBV+B cell migration, any parameter value indicating migration similar to or less that the reference preferably is indicative of a compound for treating and / or preventing EBV-associated disease.

[0059] The aforesaid method may also comprise further steps, e.g. further controls, such as viability controls, controls of other cell functions, or administration of the candidate compound to other cell types, in order to evaluate specificity of the effect of the candidate compound on EB V+B cell migration. Preferably, a candidate compound specifically inhibiting migration of EBV+B cell migration is selected as compound for treating and / or preventing EBV-associated disease in step (C). Thus, the identification in step (C) comprises an evaluation of the result of step (B), but may also comprise evaluation of results from further steps, in particular control and / or reference experiments.

[0060] The present invention further relates to a kit comprising at least one inhibitor of EBV-infected B cell (EBV+B cell) migration and at least one nonsteroidal anti-inflammatory drug (NS AID) and / or acetaminophen.

[0061] The term “kit”, as used herein, refers to a collection of the aforementioned compounds, which may or may not be packaged together. Preferably, the inhibitor of EBV+B cell migration is comprised in a composition, preferably as a medicament, in the kit. The components of the kit may be comprised in a housing, wherein the housing may be any kind of container and / or packaging deemed appropriate by the skilled person. The components of the kit may be comprised by separate vials (i.e. as a kit of separate parts) or provided in a single vial. Moreover, it is to be understood that the kit, preferably, is to be used for practicing the treatment and / or prevention referred to elsewhere herein. It is, preferably, envisaged that all components are provided in a ready-to-use manner. Further, the kit, preferably, contains instructions for carrying out said methods. The instructions can be provided by a user's manual in paper or electronic form. In addition, the manual may comprise instructions for administration and / or dosage instructions for carrying out the aforementioned treatment and / or prevention using the kit. Preferably, the kit comprises a diluent and / or a means of administration. Appropriate diluents are known to the skilled person; means of administration are all means suitable for administering the inhibitor of EBV+B cell migration to a subject. The means of administration may include a delivery unit for the administration of the compound and a storage unit for storing said compound until administration. However, it is also contemplated that the means of the current invention may appear as separate devices in such an embodiment and are, preferably, packaged together in said kit. Preferred means for administration are those which can be applied without the particular knowledge of a specialized technician. Preferably, the means for administration is a syringe, more preferably with a needle, comprising the compound or composition of the invention. Also preferably, the means for administration is an intravenous infusion (IV) equipment comprising the compound or composition. Further preferably, the means for administration is an inhaler comprising the compound of the present invention, wherein, more preferably, said compound is formulated for administration as an aerosol.

[0062] The kit further comprises at least one nonsteroidal anti-inflammatory drug (NSAID) and / or acetaminophen, all as described herein above.

[0063] The present invention furthermore relates to a use, preferably in vitro use, of an inhibitor of EBV-infected B cell (EBV+B cell) migration for inhibiting EBV+B cell migration, for preventing B cell transformation, and / or for killing EBV+B cells.

[0064] In view of the above, the following embodiments are particularly envisaged:

[0065] Embodiment 1 : An inhibitor of EBV-infected B cell (EBV+B cell) migration for use in treating and / or preventing an EBV associated disease in a subject.

[0066] Embodiment 2: The inhibitor of EBV+B cell migration for use of embodiment 1, wherein said inhibitor of EBV+B cell migration is an inhibitor of a migration factor selected from the list consisting of Protein tyrosine kinase 2 beta (PTK2B), CDC42, ROCK, Phosphoinositide 3-kinase (PI3K), CCL4, and CCR1. Embodiment 3: The inhibitor of EBV+B cell migration for use of embodiment 1 or 2, wherein said inhibitor of EBV+B cell migration reduces the activity of at least one of said migration factors.

[0067] Embodiment 4: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 3, wherein said inhibitor of EBV+B cell migration is a small molecule inhibitor, an immunoglobulin or binding fragment thereof, a peptide aptamer, a polynucleotide aptamer, an anticalin, or a Designed Ankyrin Repeat Protein.

[0068] Embodiment 5: The inhibitor of EBV+B cell migration for use of any one of embodiments 2 to 4, wherein said inhibitor of PTK2B is Defactinib.

[0069] Embodiment 6: The inhibitor of EBV+B cell migration for use of any one of embodiments 2 to 4, wherein said inhibitor of CDC42 is ZCL278.

[0070] Embodiment 7: The inhibitor of EBV+B cell migration for use of any one of embodiments 2 to 4, wherein said inhibitor of ROCK is Y27632.

[0071] Embodiment 8: The inhibitor of EBV+B cell migration for use of any one of embodiments 2 to 4, wherein said inhibitor of PI3K is ZSTK474, Leniolisib, or Duvelisib. Embodiment 9: The inhibitor of EBV+B cell migration for use of any one of embodiments 2 to 4, wherein said inhibitor of CCL4 is a neutralizing antibody.

[0072] Embodiment 10: The inhibitor of EBV+B cell migration for use of any one embodiments

[0073] 2 to 4, wherein said inhibitor of CCR1 is AZD4818 or BX471 .

[0074] Embodiment 11 : The inhibitor of EBV+B cell migration for use of any one of embodiments 2 to 10, wherein said inhibitor of EBV+B cell migration reduces the amount of at least one of said migration factors in an EBV+B cell and / or the amount of at least one of said migration factors secreted by an EBV+B cell.

[0075] Embodiment 12: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 11, wherein said inhibitor of EBV+B cell migration is an RNAi agent or a gRNA.

[0076] Embodiment 13: The inhibitor of EBV+B cell migration for use of embodiment 12, wherein said RNAi agent is an shRNA, an siRNA agent, or an miRNA agent.

[0077] Embodiment 14: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 13, wherein said inhibitor of EBV+B cell migration is an anti-PTK2B shRNA or siRNA, an anti-CDC42 shRNA or siRNA, an anti-ROCK shRNA or siRNA, an anti-PI3K shRNA or siRNA, an anti-CCL4 shRNA or siRNA, or an anti-CCRl shRNA or siRNA. Embodiment 15: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 14, wherein said EBV associated disease is a benign EBV associated disease.

[0078] Embodiment 16: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 15, wherein said EBV associated disease is infectious mononucleosis, posttransplant lymphoproliferative disease (PTLD), multiple sclerosis (MS), EBV-associated chronic fatigue syndrome, or presence of EBV or increased EBV load a subject, preferably in a transplant donor and / or a transplant receptor.

[0079] Embodiment 17: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 16, wherein said use comprises administration of said inhibitor of EBV+B cell migration to a transplant donor, preferably to prevent or reduce transmission of EBV+B cells via a transplant donated by said transplant donor.

[0080] Embodiment 18: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 17, wherein said treating comprises reducing the number of EBV+B cells in at least one tissue organ of said subject and / or comprises reducing the number of EBV+B cells in said subject.

[0081] Embodiment 19: The inhibitor of EBV+B cell migration for use of embodiment 18, wherein said treating further comprises reducing the number of B cells in at least one tissue or organ of said subject.

[0082] Embodiment 20: The inhibitor of EBV+B cell migration for use of embodiment 18 or 19, wherein said treating further comprises reducing the number of CD1 lc+T-bet+B cells in the brain of said subject.

[0083] Embodiment 21 : The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 19, wherein said EBV associated disease is PTLD and wherein preventing is preventing onset of PTLD.

[0084] Embodiment 22: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 20, wherein said EBV associated disease is MS or EBV-associated chronic fatigue syndrome and wherein preventing is reducing the probability of occurrence of said MS or EBV-associated chronic fatigue syndrome.

[0085] Embodiment 23 : The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 14, wherein said EBV associated disease is a malignant EBV associated disease. Embodiment 24: The inhibitor of EBV+B cell migration for use of embodiment 23, wherein said EBV associated disease is a lymphoma, preferably a B cell lymphoma, more preferably an EBV+B cell lymphoma.

[0086] Embodiment 25: The inhibitor of EBV+B cell migration for use of embodiment 23 or 24, wherein said EBV associated disease is a PTLD associated lymphoma.

[0087] Embodiment 26: The inhibitor of EBV+B cell migration for use of any one of embodiments 23 to 25, wherein said treating comprises reducing the number of EBV+B cells in said subject.

[0088] Embodiment 27: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 26, wherein said preventing comprises reducing the risk of occurrence of said EBV associated disease.

[0089] Embodiment 28: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 27, wherein said subject is a mammal.

[0090] Embodiment 29: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 28, wherein said subject is an experimental animal.

[0091] Embodiment 30: The inhibitor of EBV+B cell migration for use of any one of embodiments 1 to 29, wherein said subject is a human.

[0092] Embodiment 31 : The inhibitor of EBV+ B cell migration for use of any one of embodiments 1 to 30, wherein said inhibitor of EBV+ B cell migration comprises an inhibitor of migration factor PTK2B and an inhibitor of migration factor CCR1.

[0093] Embodiment 32: The inhibitor of EBV+ B cell migration for use of any one of embodiments 1 to 31, wherein said inhibitor of migration factor PTK2B is defactinib and / or said inhibitor of migration factor CCR1 is BX471.

[0094] Embodiment 33 : The inhibitor of EBV+ B cell migration for use of any one of embodiments 1 to 31, wherein said inhibitor of migration factor PTK2B is defactinib and wherein said inhibitor of migration factor CCR1 is BX471.

[0095] Embodiment 34: An inhibitor of migration factor PTK2B for use in treating and / or preventing an EBV associated disease in a subject, wherein said treating and / or preventing comprises administration of an inhibitor of migration factor CCR1.

[0096] Embodiment 35: An inhibitor of migration factor CCR1 for use in treating and / or preventing an EBV associated disease in a subject, wherein said treating and / or preventing comprises administration of an inhibitor of migration factor PTK2B. Embodiment 36: The subject matter of embodiment 34 or 35, wherein said inhibitor of migration factor PTK2B is defactinib and / or wherein said inhibitor of migration factor CCR1 is BX471.

[0097] Embodiment 37: The subject matter of any one of embodiments 34 to 36, wherein said inhibtor of migration factor PTK2B is defactinib and wherein said inhibitor of migration factor CCR1 is BX471.

[0098] Embodiment 38: The subject matter of any one of embodiments 31 to 37, wherein said inhibitor of migration factor PTK2B and said inhibitor of migration factor CCR1 are for simultaneous or for sequential administration.

[0099] Embodiment 39: The subject matter of any one of embodiments 31 to 38, wherein said inhibitor of migration factor PTK2B and said inhibitor of migration factor CCR1 are for separate or for combined administration.

[0100] Embodiment 40: Defactinib, AZD4818, or BX471 for use in treating and / or preventing an EBV associated disease in a subject.

[0101] Embodiment 41 : The Defactinib, AZD4818, or BX471 for use of embodiment40, further having a feature of at least one of embodiments 1 to 39.

[0102] Embodiment 42: Use of an inhibitor of EBV-infected B cell (EBV+B cell) migration for the manufacture of a medicament for treating and / or preventing an EBV associated disease. Embodiment 43 : A method for treating and / or preventing an EBV associated disease in a subject, said method comprising

[0103] (a) administering at least one inhibitor of EBV-infected B cell (EBV+B cell) migration to said subject, and

[0104] (b) thereby treating and / or preventing said EBV associated disease in said subject.

[0105] Embodiment 44: The method of embodiment 43, wherein said inhibitor of EBV+B cell migration has a feature of any one of embodiments 1 to 39.

[0106] Embodiment 45: The method of embodiment 43 or 44, wherein step (a) comprises administering to said subject an inhibitor of migration factor PTK2B and an inhibitor of migration factor CCR1, preferably wherein said inhibitor of migration factor PTK2B is defactinib and / or wherein said inhibitor of migration factor CCR1 is BX471.

[0107] Embodiment 46: A method for identifying a compound for treating and / or preventing

[0108] EBV-associated disease, the method comprising

[0109] (A) administering a candidate compound to EBV+B cells,

[0110] (B) determining migration of the EBV+B cells of (A); and (C) based on determining step (B), identifying a compound for treating and / or preventing EBV- associated disease.

[0111] Embodiment 47: The method of embodiment 46, wherein said method comprises further step (Bl) comparing migration determined in step (B) to a reference.

[0112] Embodiment 48: The method of embodiment 46 or 47, wherein said reference is a reference from untreated EBV+B cells (negative control).

[0113] Embodiment 49: The method of any one of embodiments 46 to 48, wherein a compound for treating EBV-associated disease is identified if migration lower than the reference is identified in step (Bl).

[0114] Embodiment 50: The method of any one of embodiments 46 to 47, wherein said EBV+B cells are maintained at a cell density of at least 3xlOE4 cells / ml in step (A) and / or (B) and / or are maintained in a medium comprising CCL4, CCL3, and / or IL- 10.

[0115] Embodiment 51 : A kit comprising (i) at least one inhibitor of EBV-infected B cell (EBV+

[0116] B cell) migration and at least one nonsteroidal anti-inflammatory drug (NS AID) and / or acetaminophen and / or (ii) comprising an inhibitor of migration factor PTK2B and an inhibitor of migration factor CCR1.

[0117] Embodiment 52: The kit of embodiment 51, wherein said NSAID is ibuprofen, naproxen, or ketorolac, preferably R-ketorolac.

[0118] Embodiment 53: The kit of claim 51 or 52, wherein said inhibitor of migration factor

[0119] PTK2B is defactinib and / or wherein said inhibitor of migration factor CCR1 is BX471. Embodiment 54: Use of an inhibitor of EBV-infected B cell (EBV+B cell) migration for inhibiting EBV+B cell migration, for preventing B cell transformation, and / or for killing EBV+B cells.

[0120] Embodiment 55: The use of embodiment 54, wherein said use is an in vitro use.

[0121] Embodiment 56: The use of embodiment 54 or 55 or the inhibitor of EBV-infected B cell

[0122] (EBV+B cell) migration for use of any one of embodiments 1 to 39, wherein said inhibitor of EBV-infected B cell (EBV+B cell) migration was identified by the method according to any one of embodiments 46 to 50.

[0123] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0124] Figure Legends Fig. 1 : A) Analysis of tracks generated by migrating B cells; percentage of motile cells (left) in the indicated populations; velocity (middle) and directionality (right) of the migrating cells as given in the dot plots (n=50). Mean values and standard error are given. B) Square root time profile of the indicated cell populations (mean displacement plotted against the square root of time). Mean values and standard error are given.

[0125] Fig. 2 A) Concentration (pg / ml) of CCL3, CCL4, and CCL5 in supernatants of the indicated cell populations, determined by ELISA. Mean values and standard error are given.B) Transwell assays were performed with hIL-10, CCL5 and CCL3. EBV infected B cells were placed in one chamber and the various chemokines in the other. Numbers of cells migrating in a transwell assay as the result of chemokine attraction by the indicated chemokine; mock: migration without external chemokine stimulus. Mean values and standard error are given.

[0126] Fig. 3: A) Velocity (left) and percentage of migrating cells (right) of infected B cells seeded in a collagen matrix at low concentration (3xlOE4 cells per ml) in the presence or absence of CCL4. The same cells seeded at high concentration (3xl0E5 cells per ml) served as a positive control. Mean values and standard error are given. B) The ability of infected B cells to grow depends on their concentration. Infected B cells were seeded at decreasing concentrations, from 3xl0E5 cells / ml to 3xl0El cells / ml. The graph gives the fold change in cell concentration of the various samples after 7 days in culture. C) Number of infected B cells migrating in a transwell assay as the result of CCL4 chemokine attraction, compared to spontaneous migration of the cells without external chemokine stimulus (mock). Mean values and standard error are given.

[0127] Fig. 4: A) Percentage (left) and velocity (right) of motile infected B cells with or without exposure to a CCL4-neutralizing antibody. Mean values and standard error are given.B) Square root time profile of indicated cells upon treatment with an antibody specific to CCL4 vs. absence of said antibody (mock). Mean values and standard error are given.

[0128] Fig. 5: A) CCR1 expression on B cells stimulated with soluble CD40 and IL-4 compared to EBV-infected B cells, as determined by western blot; result of three independent experiments. Mean values and standard error are given. B) Percentage (left) and velocity (right) of motile EBV-infected B cells after exposure with the CCR1 antagonist AZD4818. Mean values and standard error are given.

[0129] Fig. 6: A) Percentage (left) and velocity (right) of motile EBV-infected B cells after treatment with the actin inhibitor latrunculin A or myosin II inhibitors BDM or Blebbistatin. Untreated cells serve as negative controls (mock). Mean values and standard error are given. B) Square root time profile of the indicated cell populations upon treatment with BDM or Blebbistatin vs. absence of said treatment (mock). Mean values and standard error are given.

[0130] Fig. 7: Effects of inhibitors of ROCK (Y27632) or CDC42 (ZCL278) (A) or PYK2 (defactinib, B) on the percentage of motile EBV-infected B cells. Mean values and standard error are given. C) Growth curve of EBV-infected B cells treated with Defactinib (3.5 pM) cells for 14 days, or in the absence of said treatment. D) Relative expression of FAK2 in EBV-infected B cells or in B cells stimulated with CD40L and IL-4 or CD40L, IL-4 and CXCL12. Mean values and standard error are given.

[0131] Fig. 8: Number of EBV DNA copies per 100 ng of spleen tissue from mice treated as described in Example 2.7. EBV-infected mice that were not treated with defactinib served as negative controls. Mean values and standard error are given.

[0132] Fig. 9: A) Curves showing EBV-infected cell growth over time in the presence of defactinib (Def.) at a 0.5 pM concentration. Infected cells were alternatively treated with BX471 (5 pM) alone or with a combination of low doses defactinib (0.5 pM) and BX471 (5 pM). Mean cell count and standard deviation is indicated (n=5 independent B cell samples). B) dose-response curve for defactinib and BX471 (n=3), together with isobolograms (ED 10 = 10% of maximal effect and ED30 = 30% of maximal effect). Statistical significance was determined using oneway analysis of variance at day 7 in A).

[0133] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0134] Example 1: Materials and Methods

[0135] 1.1 B cell isolation Peripheral blood CD19+B cells were isolated from fresh huffy coats by Ficoll density gradient followed by selection with anti-CD19 PanB Dynabeads and beads detachment, as recommended by the manufacturer (Invitrogen).

[0136] 1.2 B cell stimulation

[0137] Peripheral blood CD19+B cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Biochrom) and stimulated with 20ng / ml human recombinant IL-4 (Peprotech, Germany) and 50ng / ml soluble CD40L (Peptrotech, Germany) (sCD40L / IL4) over 5 days and subjected to subsequent assays. In other experiments we used cells that were additionally stimulated with CXC112 (sCD40L / IL4 / CXCL12). For stimulation with CXCL12 (Peptrotech, Germany) lOOng / ml of the chemokine were added to 4 days old sCD40L / IL4 stimulated B cells and analyzed 24h later.

[0138] 1.3 Virus production

[0139] For recombinant virus production, lytic replication of 293 / rM81was induced by transfection of a BZLF1 expression plasmid with or without cotransfection of a BALF4 expression plasmid, as described previously (Neuhierl B, et al. Proc Natl Acad Sci U S A 99: 15036-15041). The supernatants were collected 4 days posttransfection and filtered through a 0.45-pm filter to remove cell debris.

[0140] 1.4 Infections

[0141] Peripheral blood CD19+B were exposed to various viruses to generate new virus-transformed cell lines. LCLs were routinely cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Biochrom).

[0142] 1.5 Time lapse microscopy

[0143] For time lapse experiments, l,5xl0E5 cells were seeded on Ibidi chemotaxis slides (Ibidi GmbH, Germany) into a bovine collagen matrix with a concentration of 1.5mg / ml collagen. The slide was inserted into a 37°C heating and incubation system for the whole duration time lapse analysis. Images were acquired every minute over a period of 15 minutes.

[0144] Manual tracking was performed using the Fiji Tracking tool and presented as an overlay of dots and lines for 2D tracks. Directionalities and velocities from manual trackings were calculated using Ibidi chemotaxis and migration tool. We generated trajectory plots (“spider plots”) by setting all (x,y) coordinates of the cells’ starting points to (0,0). The data were statistically analyzed by Rayleigh test using the Ibidi chemotaxis and migration tool. We plotted the mean cell displacement by the square root of time to generate square root time profiles.

[0145] 1.6 Chemokines

[0146] Chemokines CCL3, CLL4, CCL5, hIL-10 were purchased from Peprotech (Peptrotech, Germany) and used at concentrations of 10-100ng / ml. Neutralizing antibodies against CCL4 were purchased from R&D (R&D, Minneapolis) and added at concentrations of 0.5- 1.0 pg / ml 30 minutes prior to time lapse experiments.

[0147] 1.7 Inhibitors

[0148] Cells were exposed to inhibitors 30 minutes prior to their analysis. These inhibitors were CCR1 Inhibitor ADZ4818 (CAS No. 1003566-93-5) at lOpM, Defactinib (VS-6063, Selleckchem) used at 3.5-5.0pM, Latrunculin A (CAS No. 76343-93-6) at 5pM (Cayman Chemical), BDM (2,3-Butanedione-2-monoxime, CAS No. 57-71-6, Cayman Chemical) at 50mM, (-)- Blebbistatin (CAS No. 856925-71-8, Cayman Chemical) at 50pM, Y27632 (ROCK Inhibitor, CAS No. 129830-38-2, LC Laboratories) at 5pM, ZCL278 (CDC42 Inhibitor, CAS No. 587841-73-4, Cayman Chemical) at 50pM.

[0149] 1.8 ELISA

[0150] CCL3, CCL4, CCL5 and human IL- 10 were quantified by ELISA (DuoSet ELISA, R&D) in the supernatants of lxlOE6 EBV infected B-cells, of B cells stimulated with CD40L / IL4 or of unstimulated B cells that were cultured in 1ml of RPMI / 10% FCS for 24h.

[0151] 1.9 Chemotaxis assay using Boyden chambers

[0152] The chemotaxis assays were performed using blind well chemotaxis chambers (Neuro Probe, BW100), with compartments separated by a 8 pm polycarbonate filter (Neuro Probe, PFA8). The lower compartments were filled with either 10% FCS / RPMI or chemokines (CCL3, CCL4, CCL5 and human IL-10) in 10% FCS / RPMI. The upper compartment was filled with 100 pl 10% FCS / RPMI containing 5*104cells. The chambers were incubated for 3h at 37°C / 5% CO2 and afterwards the cells in the lower compartment were counted.

[0153] 1.10 Western blotting Western blotting was performed as described before (Neuhierl B, et al. Proc Natl Acad Sci U S A 99: 15036-15041). For detection of FAK2, 50 pg of total proteins was denatured with betamercaptoethanol and loaded onto a % SDS acrylamide gel. We used antibodies against FAK2 (clone YE353, Abeam), pFAK2 (Y402) (clone MAB6210, Cell Signaling), tubulin (clone GTU-88, SIGMA), CCR1 (clone MAB145, R&D).

[0154] 1.11 Human Immune System Component Reconstitution in Mice for EBV Infection lxlOE6 B cells were exposed for two hours with M81 Virus devoid of the BZLF1 and BRLF1 genes at a concentration necessary to infect a third of these cells. The cells and the virus were injected intra-peritoneally into 6 week old NSG mice (NOD .C§,-PrkdcSCidIl2r^mlW}l). Defactinib or carrier solution (50% PG300, 5% Tween20, 40% H20, 5% DMSO) was given intra peritoneally at a dosage of 15mg / kg twice daily starting 14 days after infected cells were given. The infected mice were monitored for 6 weeks post infection and then euthanized.

[0155] 1.12 DNA extraction and EBV copy number of spleen tissue Purification of total DNA from fixed, paraffin-embedded spleen tissue was carried out with DNeasy Blood & Tissue kits (QIAGEN). Briefly, paraffin was removed by extraction with xylene. Spin-Columns were used to isolate total DNA. EBV BALF5 gene locus was amplified from total DNA by qPCR of lOOng tissue DNA. EBV copy number was calculated using a standard curve.

[0156] Example 2: Results

[0157] 2.1 EBV-infected B cells display non-random migration

[0158] EBV-infected B cells were observed using life cell imaging. EBV-infected B cells embedded in a 3D collagen mesh at intermediate cell concentration (3xl0E5 / ml) moved with a high average velocity (8 pm / min). Tracks generated by migrating B cells were recorded by time lapse imaging. Analysis of the generated racks allowed identification of mobile versus immobile cells as given in the bar graph of Fig. 1 A). While primary B cells exposed to CXCL12 did not migrate in a collagen mesh, migrating B blasts could be generated by stimulation with CD40L and IL-4 in the presence of CXCL12. These cells migrated at the same speed as EBV- infected B cells and served as a positive control for migrating B blasts. We calculated the velocity of the migrating cells and the directionality as given in the dot plots (n=50). The tracks generated by B cells were analysed by square root time profile (Fig. 1 B)). Here the mean displacement of the cells is plotted against the square root of time. Curves that grow more quickly than linear are indicative of a directed movement.

[0159] 2.2 Migration of EBV-infected cells depends on their concentration and on chemokines

[0160] We repeated the above-described experiments with EBV-infected B cells seeded at a low cell density (lxl0E5 / ml). Under these conditions, the majority of infected B cells were immobile and the few cells in motion showed short and slow trajectories. Elisa-based assays confirmed that CCL3, CCL4 and CCL5 are secreted at very high concentrations in the supernatant of EBV- infected B cells (Fig. 2A). In transwell assays performed with hIL-10, CCL5 and CCL3 as attractants, EBV infected B cells were placed in one chamber and the various chemokines in the other. The number of infected cells that migrated as the result of chemokine attraction is shown in Fig. 2B and compared to spontaneous migration of the cells without external chemokine stimulus (mock). In cell growth experiments with cells at varying densities, but in the absence of additional chemokines, higher densities favoured cell growth (Fig. 3B).

[0161] 2.3 Migration of EBV infected cells is strongly influenced by CCL4

[0162] EBV-infected B cells were seeded in a collagen matrix at low concentration (3xlOE4 cells per ml) in the presence or absence of CCL4. Cells were subjected to live cell imaging for 20 minutes and their paths were represented by their original 2D tracks with or without alignment. The same cells seeded at high concentration (3xl0E5 cells per ml) served as a positive control; Analysis of the generated tracks allowed identification of mobile versus immobile cell and cell velocity as given in the bar graphs of Fig. 3 A. In transwell assays performed with CCL4, EBV infected B cells were placed in one chamber and the various chemokines in the other. The number of infected cells that migrated as the result of chemokine attraction is given and compared to spontaneous migration of the cells without external chemokine stimulus in Fig. 3C, showing that CCL4 induces cell migration. Reciprocally, incubation of high-density infected B cells with neutralizing antibodies specific for IL- 10, CCL4 and CCL-5 substantially reduced cell movement, with anti-CCL4 antibodies showing the strongest effects, The bar graphs of Fig. 4A show the percentage and velocity of motile infected B cells with or without exposure to a CCL4-neutralizing antibody. When EBV-infected B cells embedded in a collagen mesh were subjected to a CCL4 gradient, they clearly moved towards increasing concentrations of the chemokine. The curves in Fig. 4B show the change in the square root time profile upon treatment with an antibody specific to CCL4. 2.4 EBV-infected B cells use CCR1

[0163] Altogether, CCL4 had the strongest effect on cell migration. CCR1, a weak CCL4 receptor, was previously reported to be expressed by EBV-infected B cells, confirmed in Fig. 5A), and we found that it is preferentially expressed at the surface of the lamellipodiae (not shown). Treatment of infected cells with a CCR1 inhibitor reduced cell movement and its speed (Fig. 5B), suggesting that CCR1 is active in these cells. As CCR1 is the only CCL4 receptor on EBV- infected B cells, this suggests that CCL4 activated B cells though CCR1.

[0164] 2.5 Migration of EBV-infected B cells is dependent on the cytoskeleton

[0165] In inhibition experiments with an actin inhibitor (lantrunculin A) and myosin inhibitors BDM and Blebbistatin), it was shown that a functional cytoskeleton is required for migration of EBV- infected cells (Fig. 6).

[0166] 2.6 Migration of EBV-infected B cells is governed by the CDC42-ROCK pathway

[0167] We wished to identify signalling pathways involved in EBV-induced B cell movement and in polarization. A panel of inhibitors identified FAK2, CDC42 and to some extent the ROCK kinase as necessary for both cellular processes in EBV-infected B cells. To that end, EBV- infected B cells were treated with inhibitors of ROCK or CDC42 or PYK2 (defactinib). Results are provided in Fig. 7 A) and B) and show a strong reduction of the percentage of motile B cells after treatment. Cells treated with these inhibitors appeared rounded, showed no lamellipodiae or only in shortened form and had markedly reduced mobility. The FAK2 inhibitor defactinib further rapidly reduced cell viability, also at concentrations used in clinical trials, as shown in Fig. 7C).

[0168] In concurrence, EBV-infected B cells expressed phosphoFAK2 at higher levels than stimulated B blasts (Fig. 7D). Because defactinib, like all FAK inhibitors, inhibits to variable degrees both FAK1 and FAK2, we assessed expression of these proteins in EBV-infected B cells. Immunostains showed that these cells nearly exclusively express the FAK2 proteins, although some residual expression of the non-lymphoid kinase FAK1 was visible (not shown). Moreover, while FAK2 co-localized with CDC42 at the uropode, FAK1 was mainly perinuclear in location (not shown). Altogether, this suggests that FAK2, together with CDC42, are important mediators of polarization and movement in EBV-infected B cells.

[0169] 2.7 In vivo effect of defactinib on splenic infiltration of EBV-infected B cells Ten immunosuppressed mice were injected with human primary B cells and infectious, but replication incompetent, EB V. Half of the mice received defactinib twice daily. Six weeks after infection, animals were euthanized and spleens removed. DNA extracted from these organs were subjected to qPCR using primer and probes specific to the EBV genome to quantitate EBV in the samples. As shown in Fig. 8, splenic infiltration of EBV-infected B cells was essentially completely prevented by defactinib treatment.

[0170] 2.8 Combined treatment with defactinib and BX471

[0171] EBV-infected B cells were treated with low-dose defactinib, BX471, or a combination of low- dose defactinib and BX471, essentially as described for Fig. 7C), showing an inhibitory effect of the combined treatment even at a defactinib concentration (0,5 pM) at which defactinib alone has no detectable effect (Fig. 9A). As shown in Fig. 9B), the effect of a combined treatment with a PTK2B inhibitor (defactinib) and a CCR1 inhibitor (BX471) is synergistic. The straight lines connecting the axes in the isobolograms indicate values corresponding to an additive drug effect; since the measured values are below these lines, the effect of the combination is synergistic.

[0172] Literature

[0173] - Abusalah et al. (2020), Pathogens 9:226

[0174] - de Jesus O et al. (2003) J. Gen. Virol. 84, 1443-1450

[0175] - Dolan A et al. (2006) Virology Vol.350, 164-170

[0176] - Kieff and B. Rickinson. (2006), Epstein-Barr virus and its replication, In D. M. Knipe and P. M. - Howley (ed.), Fields virology, 5th ed. Lippincott-Raven, Philadelphia, PA: 2603-2654

[0177] - Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998

[0178] - Kiippers, R. (2003), Nat. Rev. Immunol. 3:801-812

[0179] - Laichalk, L. L., and D. A. Thorley-Lawson. (2005), J. Virol. 79: 1296-1307

[0180] - Neuhierl B, et al. Proc Natl Acad Sci U S A 99: 15036-15041

[0181] - Rees L, et al. (2009), Transplantation 88(8): 1025-9

[0182] - Rodriguez-Mogeda et al. Biomolecules 2022, 12(6), 800

[0183] - Rooney, C. M., et al., (1998), Blood 92: 1549-1555

Claims

Claims1. An inhibitor of EBV-infected B cell (EBV+B cell) migration for use in treating and / or preventing an EBV associated disease in a subject.

2. The inhibitor of EBV+B cell migration for use of claim 1, wherein said inhibitor of EBV+B cell migration is an inhibitor of a migration factor selected from the list consisting of Protein tyrosine kinase 2 beta (PTK2B), CDC42, ROCK, Phosphoinositide 3-kinase (PI3K), CCL4, and CCR1.

3. The inhibitor of EBV+B cell migration for use of claim 1 or 2, wherein said inhibitor ofEBV+B cell migration is a small molecule inhibitor, an immunoglobulin or binding fragment thereof, a peptide aptamer, a polynucleotide aptamer, an anticalin, or a Designed Ankyrin Repeat Protein.

4. The inhibitor of EBV+B cell migration for use of claim 2 or 3, wherein said inhibitor of PTK2B is Defactinib; wherein said inhibitor of CDC42 is ZCL278; wherein said inhibitor of ROCK is Y27632; wherein said inhibitor of PI3K is ZSTK474, Leniolisib, or Duvelisib; wherein said inhibitor of CCL4 is a neutralizing antibody; and / or wherein said inhibitor of CCR1 is AZD4818 or BX471.

5. The inhibitor of EBV+B cell migration for use of any one of claims 1 to 4, wherein said inhibitor of EBV+B cell migration comprises, preferably is, Defactinib, ZCL278, Y27632, ZSTK474, Leniolisib, Duvelisib, AZD4818 and / or BX471.

6. The inhibitor of EBV+ B cell migration for use of any one of claims 1 to 5, wherein said inhibitor of EBV+ B cell migration comprises an inhibitor of migration factor PTK2B and an inhibitor of migration factor CCR1.

7. The inhibitor of EBV+ B cell migration for use of any one of claims 1 to 6, wherein said inhibitor of migration factor PTK2B is defactinib and / or said inhibitor of migration factor CCR1 is BX471.

8. The inhibitor of EBV+ B cell migration for use of any one of claims 1 to 7, wherein saidinhibitor of migration factor PTK2B is defactinib and wherein said inhibitor of migration factor CCR1 is BX471.

9. The inhibitor of EBV+B cell migration for use of any one of claims 1 to 4, wherein said inhibitor of EBV+B cell migration is an RNAi agent, preferably an shRNA, an siRNA agent, or an miRNA agent; or is a gRNA.

10. The inhibitor of EBV+B cell migration for use of any one of claims 1 to 9, wherein said EBV associated disease is a benign EBV associated disease selected from the list consisting of infectious mononucleosis, post-transplant lymphoproliferative disease (PTLD), multiple sclerosis (MS), EBV-associated chronic fatigue syndrome, and presence of EBV or increased EBV load a subject, preferably in a transplant donor and / or a transplant receptor; or is a malignant EBV associated disease selected form the list consisting of a lymphoma and a PTLD associated lymphoma.

11. The inhibitor of EBV+B cell migration for use of any one of claims 1 to 10, wherein said treating comprises reducing the number of EBV+B cells in at least one tissue organ of said subject and / or comprises reducing the number of EBV+B cells in said subject.

12. The inhibitor of EBV+B cell migration for use of any one of claims 1 to 11, wherein said preventing comprises reducing the risk of occurrence of said EBV associated disease.

13. The inhibitor of EBV+B cell migration for use of any one of claims 1 to 12, wherein said subject is a human.

14. A method for identifying a compound for treating and / or preventing EBV-associated disease, the method comprising(A) administering a candidate compound to EBV+B cells,(B) determining migration of the EBV+B cells of (A); and(C) based on determining step (B), identifying a compound for treating and / or preventing EBV-associated disease.

15. The method of claim 14, wherein said method comprises further step (Bl) comparingthe migration determined in step (B) to a reference.

16. The method of claim 14 or 15, wherein said reference is a reference from untreated EBV+B cells (negative control), preferably wherein a compound for treating EBV- associated disease is identified if migration lower than the reference is identified in step(Bl).

17. A kit comprising at least one inhibitor of EBV-infected B cell (EBV+B cell) migration and at least one nonsteroidal anti-inflammatory drug (NSAID) and / or acetaminophen.

18. In vitro use of an inhibitor of EBV-infected B cell (EB V+B cell) migration for inhibiting EBV+B cell migration, for preventing B cell transformation, and / or for killing EBV+B cells.

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