Therapeutic use of an Anti-CD24 antibody

Abstract

The present invention relates to anti-CD24 antibodies for use in the treatment of myelofibrosis, thrombosis, essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia (JMML).

Description

THERAPEUTIC USE OF AN ANTI-CD24 ANTIBODYFIELD OF THE INVENTION

[0001] The present invention relates to a new therapeutic use of antibodies that bind to CD24 (referred to herein as anti-CD24 antibodies).BACKGROUND OF THE INVENTION

[0002] Myeloproliferative neoplasms (MPNs) are clonal haematopoietic stem cell disorders characterised by myeloid progenitor proliferation in the bone marrow. Polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are Philadelphia- chromosome negative classic MPNs defined as per the World Health Organization (WHO) 2016 classification and diagnostic criteria.Myelofibrosis is an uncommon type of bone marrow cancer that disrupts the normal production of blood cells in the body. Myelofibrosis causes extensive scarring of the bone marrow, leading to severe anemia that can cause weakness and fatigue. Bone marrow scarring can also cause a reduced number of platelets, which in turn can increase the risk of bleeding. Myelofibrosis is considered to be a chronic leukaemia and can happen on its own (“primary myelofibrosis”) or it can develop from another bone marrow disorder (secondary myelofibrosis). The latter can often arise in patients suffering with other myeloproliferative neoplasms, such as polycythemia vera (PV) and / or essential thrombocythemia (ET). Currently, there is no curative treatment for MPN, with the exception of HSC transplantation, which can only be performed in a minority of subjects due to its toxicity in this patient population. In addition, treatments for myelofibrosis often focus on relieving symptoms. There is, however, a need for new and effective treatments for myelofibrosis.

[0003] Thrombosis, comprising of both arterial and venous events, is recognised as a leading cause of morbidity and mortality in patients with ET and / or PV. Several patient and diseasespecific characteristics have been shown to correlate with increased thrombotic risk in MPN. Clinical risk factors include age, history of previous thrombosis, cardiovascular comorbidities, and mutation status. The pathobiology of thrombosis in MPN is multifactorial and results from a complex interplay between blood cells, endothelial cells, coagulation system and inflammatory mediators. Platelets, leukocytes, and erythrocytes exist in a hyperactive and proadhesive state. Furthermore, endothelial dysfunction, haemostatic elements and cytokine dysregulation are implicated in thrombogenesis. MPN driver mutations enhance the thrombotic environment, while the chronic inflammatory state further promotes clonal expansion. Much of the current treatment paradigms for PN focus on primary prevention of thrombosis with antiplatelet agents andcytoreduction. There is therefore a need for improved treatments for thrombosis, especiaiiy in patients with PV or ET.

[0004] There is also a need for improved treatments for essential thrombocythemia (ET) and juvenile myelomonocytic leukaemia (JMML). JMML is a highly aggressive subtype of childhood myeloproliferative neoplasm and is characterised by GM-CSF hypersensitivity and excessive proliferation of cells of the myeloid lineage. The malignancy is definitively caused by RAS- pathway activating mutations, most typically in PTPN11 , which encodes the SHP2 phosphatase. SHP2 is responsible for implementing multiple phagocytosis checkpoints such as CD47 and CD24, whose binding to their cognate receptors in a macrophage inhibits phagocytosis. In addition to SHP2 itself [Sun et al. Leukemia, volume 32, pagesl 246-1249 (2018], CD47 has recently been claimed to be druggable based on its enhanced expression in JMML [Jun et al. Klin Padiatr 2022; 234(03): 180; Wang et al. Klin Padiatr 2023; 235(03): 20], Our preliminary data analysis has found that CD24 is also highly expressed in JMML patients and has a strong correlation with MPO expression, a marker for excessive myeloid progenitors.

[0005] The present invention was devised with the foregoing in mind.SUMMARY OF THE INVENTION

[0006] The present invention resides in the recognition that CD24 activity is implicated in both myelofibrotic and thrombotic events and that anti-CD24 antibodies have the potential to treat these conditions, particularly in patients with myeloproliferative neoplasms (MPNs).

[0007] Thus, in a first aspect, the present invention provides an anti-CD24 antibody for use in the treatment of myelofibrosis and / or thrombosis.

[0008] In another aspect, the present invention provides an anti-CD24 antibody for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm.

[0009] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD24 antibody and one or more pharmaceutically-acceptable excipients, wherein the pharmaceutical composition is for use in the treatment of myelofibrosis and / or thrombosis.

[0010] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD24 antibody and one or more pharmaceutically-acceptable excipients, wherein the pharmaceutical composition is for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm.

[0011] In another aspect, the present invention provides the use of an anti-CD24 antibody in the manufacture of a medicament for use in the treatment of myelofibrosis and / or thrombosis.

[0012] In another aspect, the present invention provides the use of an anti-CD24 antibody in the manufacture of a medicament for use the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm.

[0013] In another aspect, the present invention provides a method of treating myelofibrosis and / or thrombosis, the method comprising administering to a subject a therapeutically effective amount of an anti-CD24 antibody or a pharmaceutical composition comprising an anti-CD24 antibody and one or more pharmaceutically-acceptable excipients.

[0014] In another aspect, the present invention provides a method of treating myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm, the method comprising administering to a subject a therapeutically effective amount of an anti-CD24 antibody and one or more pharmaceutically-acceptable excipients.

[0015] Features, including optional, suitable, and preferred features in relation to one aspect of the invention may also be features, including optional, suitable and preferred features in relation to any other aspect of the invention.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0016] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0017] It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a disease or condition. “T reating” or “treatment” therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the disease or condition developing in a subject that may be afflicted with or predisposed to the disease or condition, but does not yet experience or display clinical or subclinical symptoms of the disease or condition, (2) inhibiting the disease or condition, / .e., arresting, reducing or delaying the development of the disease or condition or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease or condition, / .e., causing regression of the disease or condition or at least one of its clinical or subclinical symptoms.

[0018] A “therapeutically effective amount” means the amount of the compound that, when administered to a subject for treating a disease or condition referred to herein, is sufficient to effect such treatment for the disease or condition. The "therapeutically effective amount" will vary depending on the form of the compound (e.g. the salt form), the disease or condition concerned and its severity, as well as the age, weight, etc., of the subject to be treated.

[0019] The term "subject" is used herein to mean a warm blooded mammal. Thus, the compound of the present invention may be used for human and / or veterinary applications. In a particular embodiment, the subject is a human.

[0020] As used herein, the term "antibody" is intended to denote an immunoglobulin molecule that possesses a "variable region" antigen recognition site. The term "variable region" is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region comprises a "hypervariable region" whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "Complementarity Determining Region" or "CDR" (i.e. , typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain) and / or those residues from a "hypervariable loop" (i.e., residues 26-32 (LI), 50- 52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelid antibodies, single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-id) antibodies (including, e.g., anti-id and anti-anti-ld antibodies to antibodies of the invention). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, lgG2, IgGs, lgG4, IgAi and lgA2) or subclass.

[0021] As used herein, the term "antigen binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's CDR and optionally the framework residues that comprise the antibody's "variable region" antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv),and mutants thereof, naturally occurring variants, and fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.).

[0022] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0023] Human, chimeric or humanized antibodies are particularly preferred for in vivo use in humans.

[0024] A " chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101 , and 5,585,089, the contents of each of which are incorporated herein in their entirety), veneering or resurfacing (EP 592,106; EP 519,596, the contents of each of which are incorporated herein by reference), and chain shuffling (U.S. Pat. No. 5,565,332, the contents of which are incorporated herein by reference.

[0025] As used herein, the term "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor." Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e. , at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non- human. The donor antibody may be referred to as having been "humanized," by the process of "humanization," because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an FcyRIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations).

[0026] An “anti-CD24 antibody” is an antibody as defined herein (and preferably a “humanized antibody” as defined herein) that binds to an epitope of the CD24 polypeptide. As used herein “a CD24 polypeptide” refers to a glycosylphosphatidylinositol (GPI)-anchored cell surface protein having 31 amino acids with 16 potential O- and N-glycosylation sites (in the human). Human CD24 is encoded by the CD24 gene, and is first expressed as an 80 amino acid precursor (Accession No. ACI46150).Treatment of myelofibrosis, thrombosis, essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia (JMML)

[0027] Cluster of differentiation 24 (CD24, UniProt: P25063), also known as Heat Stable Antigen (HSA) or Small Cell Lung Carcinoma Cluster 4 Antigen, is a 32-amino acid mucin-like glycosylphosphatidylinositol (GPI)-anchored molecule, which is heavily glycosylated and anchored via a GPI link to the cell surface. CD24 can interact with Sialic Acid Binding Ig Like Lectin 10 (Siglec-10) expressed on innate immune cells (e.g., macrophages) so as to diminish damaging inflammatory responses to infection, sepsis, liver damage, and chronic graft versus host disease (Pirruccello et al., J. Immunol. 136, 3779-3784 (1986); Chen et al., Glycobiology 9, 800-806 (2014); Chen et al., Cell 152(3), 467-478; Chen GY et al. Nature Biotechnology 29, 428-435 (2011); Chen GY et al. Science 323 (5922), 1722-1725 (2009): and Toubai T et al.Blood 123(22), 3512-3513 (2014)). Binding of CD24 to Siglec-10 induces an inhibitory signalling cascade mediated by SHP-1 and / or SHP-2 phosphatases associated with the two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic tail of Siglec-10, thereby blocking toll-like receptor (TLR)-mediated inflammation and the cytoskeletal rearrangement required for cellular engulfment by macrophages (Crocker PR et al., Nature Reviews Immunology. 7, 255-266 (2007); Abram CL et al., J. Leuko.c Biol. 102(3), 657-675 (2017) and Dietrich J el al., J. Immunol. 166(4), 2514-2521 (2001) 12-14).

[0028] Physiologically, the CD24 protein is expressed mainly on hematopoietic subpopulations of B-lymphocytes and neutrophils, various epithelial cells, muscle and neural cells. It plays a crucial role in cell selection and maturation during hematopoiesis and is expressed during the embryonic period, on developing neural and pancreatic cells. In addition, CD24 is a potentialligand for P-selectin which functions as an adhesion molecule that enhances platelets aggregation.

[0029] Overexpression of CD24 has been observed in various cancer cells based on immunohistochemical studies, including without limitation, breast cancer (85%), rectal cancer (84%), ovarian cancer (83%), pancreatic cancer (72%), bladder cancer (62%), cholangiocarcinoma (51%), prostatic cancer (48%), and small cell lung cancer (45%). A metaanalysis of 28 studies revealed that CD24 is overexpressed in 68% of human cancers, and CD24 expression was correlated with a higher self-renewal ability, more metastases, and a poor prognosis (Krisiansen G. et al., Am J Pathol, 161: 1215-1221, 2002; Krisiansen G. et al, Clin cancer Res, 9:4906-4913. 2003: Krisiansen G. et al., Br J Cancer, 88:231-236, 2003;Kristiansen G. et al., Prostate, 58:182-192, 2004; Hocob J. et al., Pancreatology, 4:454-460, 2004; Samuel E D, BioMed Central, 3:3-15, 2004: Min-Cheng S. et al., Cancer letter, 1-6, 2005 and Lee et al., Oncol. Rep. 2009, 22, 1149-1156).

[0030] The present inventors have surprisingly found that anti-CD24 antibodies have utility for the treatment of myelofibrosis and / or thrombosis, particularly in patients having myeloproliferative neoplasms. In addition, the present inventors have surprisingly found that anti-CD24 antibodies have utility for the treatment of thrombocythemia (ET) and / or Juvenile myelomonocytic leukaemia (JMML).

[0031] Thus, the present invention provides, in a first aspect, an anti-CD24 antibody for use in the treatment of myelofibrosis, thrombosis, essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia (JMML).

[0032] In a particular embodiment, the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm. For the avoidance of doubt, references to “a patient having a myeloproliferative neoplasm” refers to a patient that has been diagnosed as suffering from a myeloproliferative neoplasm. The term “myeloproliferative neoplasm” is well-known in the art. Particular examples of myeloproliferative neoplasms are primary myelofibrosis (PMF), polycythemia vera (PV) and / or essential thrombocythemia (ET).

[0033] In a particular embodiment, the anti-CD24 antibody is for use in the treatment of thrombosis.

[0034] In an alternative embodiment, the anti-CD24 antibody is for use in the treatment of myelofibrosis. Myelofibrosis may be primary myelofibrosis or secondary myelofibrosis. As noted above, primary myelofibrosis (PMF) is a recognised myeloproliferative neoplasm. Secondary myelofibrosis can arise in patients that have other myeloproliferative neoplasms, for example, polycythemia vera (PV) and / or essential thrombocythemia (ET).[003S] In a particular embodiment of the invention, the anti-CD24 antibody is for use in the treatment of primary myelofibrosis.

[0036] In a further embodiment of the invention, the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis (PMF), polycythemia vera (PV) and / or essential thrombocythemia (ET). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera (PV) and / or essential thrombocythemia (ET). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient has primary myelofibrosis (PMF). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient has polycythemia vera (PV). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient has essential thrombocythemia (ET).

[0037] In a further embodiment of the invention, the anti-CD24 antibody is for use in the treatment of myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis (PMF), polycythemia vera (PV) and / or essential thrombocythemia (ET). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera (PV) and / or essential thrombocythemia (ET). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of secondary myelofibrosis in a patient has polycythemia vera (PV). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of secondary myelofibrosis in a patient has essential thrombocythemia (ET).

[0038] In a further embodiment of the invention, the anti-CD24 antibody is for use in the treatment of thrombosis in a patient having a myeloproliferative neoplasm, optionally selected from primary myelofibrosis (PMF), polycythemia vera (PV) and / or essential thrombocythemia (ET). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of thrombosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera (PV) and / or essential thrombocythemia (ET). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of thrombosis in a patient has primary myelofibrosis (PMF). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of thrombosis in a patient has polycythemia vera (PV). In yet another embodiment of the invention, the anti-CD24 antibody is for use in the treatment of thrombosis in a patient has essential thrombocythemia (ET).

[0039] In a further embodiment, the anti-CD24 antibody is for use in the treatment of essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia (JMML). In a particular embodiment, the anti-CD24 antibody is for use in the treatment of essential thrombocythemia (ET). In a further particular embodiment, the anti-CD24 antibody is for use in the treatment ofjuvenile myelomonocytic leukaemia (JMML). The present inventors have observed that CD24 is upregulated in JMML patients and this upregulation exhibited a strong correlation with the expression of myeloperoxidase (MPO), which is involved in the activation of TGF-beta and the development of myelofibrosis. GSEA analysis also revealed a significant enrichment of the SHP2 pathway in JMML patients, which is responsible for the transduction of CD24 signalling. For these reasons, anti-CD24 antibodies are also potentially useful therapeutic agents for the treatment of JMML.

[0040] In a further aspect, the present invention provides the use of an anti-CD24 antibody in the manufacture of a medicament for use in the treatment of:(i) myelofibrosis and / or thrombosis;(ii) thrombosis;(iii) myelofibrosis;(iv) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(v) myelofibrosis in a patient having a myeloproliferative neoplasm;(vi) thrombosis in a patient having a myeloproliferative neoplasm;(vii) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(viii) primary myelofibrosis;(ix) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(x) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(xi) secondary myelofibrosis in a patient that has polycythemia vera;(xii) secondary myelofibrosis in a patient that has essential thrombocythemia;(xiii) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(xiv) thrombosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(xv) thrombosis in a patient that has primary myelofibrosis;(xvi) thrombosis in a patient has polycythemia vera;(xvii) thrombosis in a patient has essential thrombocythemia;(xviii) essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia (JMML);(xix) essential thrombocythemia (ET); or(xx) juvenile myelomonocytic leukaemia (JMML).

[0041] In a further aspect, the present invention provides a method of treating:(i) myelofibrosis and / or thrombosis;(ii) thrombosis;(iii) myelofibrosis;(iv) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(v) myelofibrosis in a patient having a myeloproliferative neoplasm;(vi) thrombosis in a patient having a myeloproliferative neoplasm;(vii) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(viii) primary myelofibrosis;(ix) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(x) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(xi) secondary myelofibrosis in a patient that has polycythemia vera;(xii) secondary myelofibrosis in a patient that has essential thrombocythemia;(xiii) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(xiv) thrombosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(xv) thrombosis in a patient that has primary myelofibrosis;(xvi) thrombosis in a patient has polycythemia vera; ;(xvii) thrombosis in a patient has essential thrombocythemia;(xviii) essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia(JMML);(xix) essential thrombocythemia (ET); or(xx) juvenile myelomonocytic leukaemia (JMML). the method comprising administering to a patient in need thereof a therapeutically effective amount of an anti-CD24 antibody.The anti-CD24 antibody

[0042] In reference to the data presented in the example section herein, it will be appreciated that the binding of the anti-CD24 antibodies to an epitope of CD24 inhibits / diminishes the activity of CD24 and results in the observed therapeutic utility for the treatment of myelofibrosis and / or thrombosis.

[0043] It will be appreciated that the precise nature of the antibody is not critical as long as it is capable of binding to an epitope of CD24. Any suitable anti-CD24 antibody known in the art could therefore be used in the present invention. Suitably, the antibody is a humanized antibody, as defined herein.

[0044] It will also be appreciated that the term “antibody”, as defined herein, includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelid antibodies, single chain antibodies, disulfide- linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-id) antibodies (including, e.g., anti-id and anti-anti-ld antibodies to antibodies of the invention).

[0045] Many examples of anti-CD24 antibodies are known in the art and some of these have been used clinically. Particular examples include:GB-7011 (see, for example, https: / / genechembio.com / pipeline.html and https: / / synapse.patsnap.com / drug / cf3dd7f466974d92b9038a50e5c0dd72);ALB9 (commercially available - e.g. Thermo Fisher - Catalogue # MA1-91384; Beckman Coulter - Product No:A87785);SWA11 (commercially available - e.g. My Bio Source - Catalogue MBS1564324; see also Laboratory Investigation, volume 90, pagesl 102-1116 (2010); Int. J. Mol. Sci. 2019, 20(3), 660; BMC Clinical Pathology 2015, 15:19 (DOI: 10.1186 / s12907-015-0019-z); W02009063461);SN3 (commercially available, e.g. Thermo Fisher - Catalogue #MA5- 11833Kristiansen, G., Machado, E., Bretz, N. et al. Molecular and clinical dissection of CD24 antibody specificity by a comprehensive comparative analysis. Lab Invest 90, 1102-1116 (2010) (https: / / doi.Org / 10.1038 / labinvest.2010.70));G7 (commercially available - e.g. He et al. A novel antibody targeting CD24 and hepatocellular carcinoma in vivo by near-infrared fluorescence imaging. Immunobiology. (2015); 220(12):1328- 36; and J. Pers. Med. 2022, 12, 1235. (https: / / doi.org / 10.3390 / jpm12081235);IMM-47 and IMM-4701 (WO-2023061064, WO-2023061084);1H5C4 (commercially available - e.g. Fisher Scientific - Product code 16870356 (Proteintech 67627-1 -IG); https: / / www.fishersci.co.uk / shop / products / cd24-mouse-anti-human-unconjugated- clone-1 h5c4-proteintech-2 / p-7223200);2Q1282 (commercially available - e.g. Santa Cruz Biotechnology - Catalogue # sc-70598 / sc- 539299 / SC-541390; https: / / uk01.l.antigena.com / l / pxN8acokp43sfEX7nD_mrKH0s2vWzyRF8vOEqCUO2row2WS_f3 59dPOSKdcFqsbijqcJK13qW9GFO~s4Z1RleWXa8pEpzHlvG8U3Pk6jjfjD99-kyfaCkAKqKj- ogM oa 1 fG RgZm 1 kjzO6CuhtSS5YVvp) :32D12 (commercially available - e.g. https: / / www.lsbio.com / antibodies / cd24-antibody-clone- 32d12-flow-ihc-ls-c783035 / 809571?trid=247);3B6 (commercially available - e.g. https: / / uk01.l.antigena.com / l / pyNnqtgk8W02CRnBvelMZjprpTol7E4ctBzl6ffHxce7THRdMt2mbcr Z6alGY3AsOcxs9efmlRHQFH34tlhAyBdm-k~NwwPB5UmMGRo4WSHMq8j54xPfKUUEfS0gk- DAgoP2YBIoThtrgJILBWADXOihV2yE);4F4E10 (commercially available - e.g. https: / / uk01J.antigena.eom / l / EsDX9cZmfZk7UISH8BYN2fCp2DBXWXsihLJs6aQ~DFjZVar1V17 16Jpuu~2JrH15HdYR~tHYRiMtuwTWDRHV2xF1vm_csXRbRYIQrn3GRM5F30Oj8bgYBpt24T CUaGrcXb4lwSBQQSflXAwHlsbXqbw9LtFi~pLukZqOcNmGqSQbwrbxSdofrFOiL7aL)B3298M (commercially available - e.g. https: / / uk01J.antigena.eom / l / rf8bTc5hTjjlQ9Btczv5oOr2VyGgpfjMxBBiXvrVpim28detJHH~DWI M2BpV6Qla6Ke8eRPhyuYq2oLRDE~CnKfeS2X20~tAdK6NP5- n1SkQeXadgh6oWVqLHKNbogXz-W7sq5gkE2s5UtizTYZM0my4Trmd1LSHN0JnTh)BA-1 (commercially available - e.g. https: / / www.lsbio.com / antibodies / cd24-antibody-clone-ba-1- flow-ls-c134342 / 138023?trid=247)DMC213 (commercially available - e.g. https: / / www.dimabio.com / product / Anti-CD24-antibody- DMC213-lgG1-Chimeric-mAb)DMC217 (commercially available - e.g. https: / / www.dimabio.com / product / Anti-CD24-antibody- DMC217-lgG1-Chimeric-mAb)EPR19925 (commercially available - e.g. https: / / www.abcam.com / products / primary- antibodies / cd24-anti body-epr19925-ab202073.html)MAB5247 (commercially available - e.g. https: / / www.rndsystems.com / products / human-cd24- antibody-ml5_mab5247?utm_source=biocompare&utm_medium=referral&utm_campaign=product_MAB 5247&utm_term=primaryantibodies&utm_content=editorial)ML5 (commercially available - e.g. https: / / www.novusbio.com / products / cd24-antibody- ml5_nb100-77903)QA21A28 (commercially available - e.g. https: / / www.biolegend.com / en-gb / products / purified- anti-human-cd24-recombinant-antibody-23280)S8230.8 (commercially available - e.g. https: / / www.creative-diagnostics.com / Anti-CD24- Antibody- 100867- 144. htm)W20001B (commercially available - e.g. https: / / www.biolegend.com / en-gb / products / purified- anti-human-cd24-antibody-21986) as well as:Rabbit Anti-CD24 Polyclonal Antibody (MRO-1739-CN),Human Anti-CD24 Recombinant Antibody, scFv Fragment (TAB-007LC-S(P)),Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10),Human Anti-CD24 Recombinant Antibody VS-0723-WK217,Human Anti-CD24 Recombinant Antibody VS-0723-WK207,Human Anti-CD24 Recombinant Antibody Fab Fragment TAB-007LC-F(E));Human Anti-CD24 Recombinant Antibody (TAB-007LC),Mouse Anti-CD24 Recombinant Antibody (TAB-006LC) (TAB-006LC),Human Anti-CD24 Recombinant Antibody; scFv Fragment (TAB-007LC-S(P)),Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10) (VS3-XY243),Mouse Anti-CD24 Recombinant Antibody; scFv Fragment (TAB-006LC-S(P)), andMouse Anti-CD24 Recombinant Antibody; Fab Fragment (TAB-006LC-F(E)) (TAB-006LC-F(E)), all of which are commercially available from Creative Biolabs (www.creativebiolabs.net).

[0046] Particular antibodies of interest include: ATG-31 (WO 2022 / 170280); ALB9; SN3; IMM- 47, IMM-4701 and SWA-11.

[0047] Methods of generating antibodies are well known in the art. Antibodies may be generated via any one of several methods known in the art, which methods can employ induction of in-vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi D. R. et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter G. et al., 1991. Nature 349:293-299) or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler G. et al.,1975. Nature 256:495-497; Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42; Cote R J. et al., 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030; Cole S P. et al., 1984. Mol. Cell. Biol. 62:109-120). Anti-CD24 antibodies, both polyclonal and monoclonal, suitable for use in the methods and compositions of the present invention are commercially available, for example, from Santa Cruz Biotechnology (Santa Cruz, Calif.), AbDSerotec (Kidlington, UK) and Life Span BioSciences, Inc (Seattle Wash.).

[0048] In cases where target antigens are too small to elicit an adequate immunogenic response when generating antibodies in-vivo, such antigens (haptens) can be coupled to antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or serum albumin [e.g., bovine serum albumin (BSA)] carriers (see, for example, U.S. Pat. Nos. 5,189,178 and 5,239,078], Coupling a hapten to a carrier can be effected using methods well known in the art. For example, direct coupling to amino groups can be effected and optionally followed by reduction of the imino linkage formed. Alternatively, the carrier can be coupled using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents. Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, III. The resulting immunogenic complex can then be injected into suitable mammalian subjects such as mice, rabbits, and the like. Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum. The titers of the immune serum can readily be measured using immunoassay procedures which are well known in the art.

[0049] The antisera obtained can be used directly or monoclonal antibodies may be obtained as described hereinabove.

[0050] A process for preparing antibodies targeting specific epitopes within intrinsically disordered proteins is described in PNAS, 112(32), 9902-9970 (https: / / www.pnas.Org / doi / 10.1073 / pnas.1422401112).

[0051] Antibody fragments can be obtained using methods well known in the art. [(see, for example, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, New York, (1988)]. For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.

[0052] Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As described hereinabove, an (Fab')2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. Ample guidance for practicing such methods is provided in the literature of the art (for example, refer to: Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647; Porter, R R., 1959. Biochem. J. 73:119-126). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0053] As described hereinabove, an Fv is composed of paired heavy chain variable and light chain variable domains. This association may be noncovalent (see, for example, Inbar et al., 1972. Proc. Natl. Acad. Sci. USA. 69:2659-62). Alternatively, as described hereinabove the variable domains can be linked to generate a single chain Fv by an intermolecular disulfide bond, or alternately, such chains may be cross-linked by chemicals such as glutaraldehyde.

[0054] Preferably, the Fv is a single chain Fv.

[0055] Single chain Fv's are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains. Ample guidance for producing single chain Fv's is provided in the literature of the art (for example, refer to: Whitlow and Filpula, 1991. Methods 2:97-105; Bird et al., 1988. Science 242:423-426; Pack et al., 1993. Bio / Technology 11 :1271-77; and Ladner et al., U.S. Pat. No. 4,946,778).

[0056] Isolated complementarity determining region peptides can be obtained by constructing genes encoding the complementarity determining region of an antibody of interest. Such genes may be prepared, for example, by RT-PCR of mRNA of an antibody-producing cell. Ample guidance for practicing such methods is provided in the literature of the art (for example, refer to Larrick and Fry, 1991. Methods 2:106-10).

[0057] It will be appreciated that for human therapy or diagnostics, humanized antibodies are preferably used. Humanized forms of non human (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having — preferably minimal — portions derived from non human antibodies. Humanized antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementarity determining region of a non human species (donor antibody) such as mouse, rat or rabbit having the desired functionality. In some instances, Fv frameworkresidues of the human antibody are replaced by corresponding non human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanized antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321 :522- 525; Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr. Op. Struct. Biol. 2:593-596).

[0058] Methods for humanizing non human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non human. These non human amino acid residues are often referred to as imported residues which are typically taken from an imported variable domain. Humanization can be essentially performed as described (see, for example: Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988. Science 239:1534-1536; U.S. Pat. No. 4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions. Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non human species. In practice, humanized antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. In one embodiment of the present invention, the anti-CD24 antibody is a humanized anti-SWA11 antibody, wherein the scFv portion (SEQ ID NO: 21) is produced as described herein.

[0059] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [see, for example, Hoogenboom and Winter, 1991. J. Mol. Biol. 227:381; Marks et al., 1991. J. Mol. Biol. 222:581; Cole et al., “Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss, pp. 77 (1985); Boerner et al., 1991. J. Immunol. 147:86-95). Humanized antibodies can also be made by introducing sequences encoding human immunoglobulin loci into transgenic animals, e.g., into mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed in such animals which closely resembles that seen in humans in all respects, including gene rearrangement, chain assembly, and antibody repertoire. Ample guidance for practicing such an approach is provided in the literature of the art (forexample, refer to: U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., 1992. Bio / Technology 10:779-783; Lonberg et al., 1994. Nature 368:856-859; Morrison, 1994. Nature 368:812-13; Fishwild et al., 1996. Nature Biotechnology 14:845-51; Neuberger, 1996. Nature Biotechnology 14:826; Lonberg and Huszar, 1995. Intern. Rev. Immunol. 13:65-93).

[0060] It will be appreciated that targeting of particular compartment within the cell can be achieved using intracellular antibodies (also known as “intrabodies”). These are essentially SCA to which intracellular localization signals have been added (e.g., ER, mitochondrial, nuclear, cytoplasmic). This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors and to inhibit a protein function within a cell (See, for example, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141 ; Deshane et al., 1994, Gene Ther. 1: 332-337; Marasco et al., 1998 Human Gene Ther 9: 1627-42; Shaheen et al., 1996 J. Virol. 70: 3392-400; Werge, T. M. et al., 1990, FEBS Letters 274:193-198; Carlson, J. R. 1993 Proc. Natl. Acad. Sci. USA 90:7427-7428; Biocca, S. et al., 1994, Bio / Technology 12: 396-399; Chen, S-Y. et al., 1994, Human Gene Therapy 5:595- 601 ; Duan, L et al., 1994, Proc. Natl. Acad. Sci. USA 91 :5075-5079; Chen, S-Y. et al., 1994, Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al., 1994, J. Biol. Chem. 269:23931- 23936; Mhashilkar, A. M. et al., 1995, EMBO J. 14:1542-1551; PCT Publication No. WO 94 / 02610 by Marasco et al.; and PCT Publication No. WO 95 / 03832 by Duan et al.).

[0061] To prepare an intracellular antibody expression vector, the cDNA encoding the antibody light and heavy chains specific for the target protein of interest are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the marker. Hybridomas secreting anti-marker monoclonal antibodies, or recombinant monoclonal antibodies, can be prepared using methods known in the art. Once a monoclonal antibody specific for the marker protein is identified (e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library), DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques. For hybridoma derived antibodies, light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening. For recombinant antibodies, such as from a phage display library, cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process and the nucleotide sequences of antibody light and heavy chain genes are determined. For example, many such sequences are disclosed in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242 and in the “Vbase” human germline sequence database. Once obtained, the antibody lightand heavy chain sequences are cloned into a recombinant expression vector using standard methods.

[0062] For cytoplasmic expression of the light and heavy chains, the nucleotide sequences encoding the hydrophobic leaders of the light and heavy chains are removed. An intracellular antibody expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH / CH1 region of the heavy chain such that a Fab fragment is expressed intracellularly. In another embodiment, the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker [e.g., (Gly4Ser)sand expressed as a single chain molecule. To inhibit marker activity in a cell, the expression vector encoding the intracellular antibody is introduced into the cell by standard transfection methods, as discussed hereinbefore.

[0063] Once antibodies are obtained, they may be tested for activity, for example via ELISA.Pharmaceutical Compositions

[0064] The present invention further provides a pharmaceutical composition comprising an anti- 0024 antibody and one or more pharmaceutically-acceptable excipients, wherein the pharmaceutical composition is for use in the treatment of:(i) myelofibrosis and / or thrombosis;(ii) thrombosis;(iii) myelofibrosis;(iv) primary myelofibrosis;(v) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(vi) myelofibrosis in a patient having a myeloproliferative neoplasm;(vii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(viii) thrombosis in a patient having a myeloproliferative neoplasm;(ix) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(x) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(xi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(xii) secondary myelofibrosis in a patient having polycythemia vera;(xiii) secondary myelofibrosis in a patient having essential thrombocythemia;(xiv) essential thrombocythemia (ET) and / or juvenile myelomonocytic leukaemia (JMML);(xv) essential thrombocythemia (ET); or(xvi) juvenile myelomonocytic leukaemia (JMML).

[0065] The pharmaceutical compositions of the invention may be in a form suitable for parenteral administration (for example as a sterile aqueous solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing).

[0066] The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art.

[0067] An effective amount of an anti-CD24 antibody of the present invention for use in the therapy as defined above is an amount sufficient to effectively treat the myelofibrosis or thrombosis in the patient concerned.

[0068] The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for parenteral administration to humans will generally contain, for example, from 50mcg to 0.5 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.

[0069] The size of the dose for therapeutic will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. An anti-CD24 antibody may be administered at a daily dose in the range of, for example, 0.1 mg / kg to 30 mg / kg body weight will generally be used.Routes of Administration

[0070] Typically an anti-CD24 antibody will be administered by parenteral administration, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.Combination Therapies

[0071] An anti-CD24 antibody may be used as a sole therapy or may involve therapy with one or more additional therapeutic agents.

[0072] Thus, in another aspect, the present invention provides an anti-CD24 antibody for use as defined hereinbefore in combination with one or more additional therapeutic agents.

[0073] In another aspect, the present invention provides the use of an anti-CD24 antibody as defined herein in the manufacture of medicament for use as defined hereinbefore in combination with one or more additional therapeutic agents.

[0074] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compound of this invention within the dosage range described hereinbefore and the other pharmacologically-active agent within its approved dosage range.

[0075] According to a further aspect of the invention there is provided a combination suitable for use in the treatment of myelofibrosis and / or thrombosis as defined hereinbefore comprising an anti-CD24 antibody, as defined herein, or a pharmaceutically acceptable salt or solvate thereof, and one or more additional therapeutic agents.

[0076] Herein, where the term “combination” is used, it is to be understood that this refers to simultaneous, separate or sequential administration. In one aspect of the invention “combination” refers to simultaneous administration. In another aspect of the invention “combination” refers to separate administration. In a further aspect of the invention “combination” refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination.

[0077] According to a further aspect of the invention there is provided a pharmaceutical composition for use in the treatment of myelofibrosis and / or thrombosis which comprises an anti-CD24 antibody and one or more additional therapeutic agents, and a pharmaceutically acceptable diluent or carrier.Patient selection

[0078] The present invention relates to the use of anti-CD24 antibodies for use in the treatment of the conditions noted above. In embodiments of the invention, the anti-CD24 antibodies are particularly effective at treating the conditions defined herein in patients in which the myeloproliferative neoplasm comprises a mutation in the JAK2 gene. In another embodiment of the invention, the anti-CD24 antibodies are particularly effective at treating the conditionsdefined herein in patients in which the myeloproliferative neoplasm comprises elevated levels of CD24 expression.

[0079] In an embodiment, the present therefore provides an anti-CD24 antibody for use in a method of treatment of a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; the method comprising:(i) determining, from a biological sample obtained from the patient, whether the myeloproliferative neoplasm comprises a mutation in the JAK2 gene and / or elevated levels of CD24 expression; and(ii) if there is a mutation in the JAK2 gene and / or elevated levels of CD24 expression in the myeloproliferative neoplasm, administering to said patient a therapeutically effective amount of an anti-CD24 antibody.

[0080] Any suitable biological sample may be used in order to assess the JAK2 mutation status and / or the levels of CD24 expression in the myeloproliferative neoplasm. In some embodiments, the JAK2 mutation status and / or the levels of CD24 expression in the myeloproliferative neoplasm can be assessed by obtaining a blood sample and analysing the cells (e.g. white blood cells such as neutrophils) for the presence of a JAK2 mutation and / or elevated levels of CD24 expression.

[0081] In some embodiments, the biological sample is a blood sample. The determination of whether the myeloproliferative neoplasm comprises a mutation in the JAK2 gene and / orelevated levels of CD24 expression can suitably be carried out by collecting a blood sample from the patient and testing the sample to determine whether blood cells present in the sample comprise a mutation in the JAK2 gene and / or elevated levels of CD24 expression in granulocytes / neutrophils. Alternatively, the presence of a mutation in the JAK2 gene can be determined by analysing a sample of nucleic acid derived from a blood sample. Suitably, therefore, the method comprises:(i) determining from a biological sample obtained from the patient whether the blood cells comprise a mutation in the JAK2 gene and / or elevated levels of CD24 expression in granulocytes / neutrophils; and(ii) if there is a mutation in the JAK2 gene and / or elevated levels of CD24 expression in the patient’s neutrophils, administering to said patient a therapeutically effective amount of an anti-CD24 antibody.

[0082] In a further embodiment, the anti-CD24 antibody is for use in a method of treatment of a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; in which the myeloproliferative neoplasm possess a JAK2 mutation and / or elevated levels of CD24 expression, the method comprising the administration to a patient in need thereof a therapeutically effective amount of an anti-CD24 antibody.

[0083] The present invention further provides a method for selecting a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; for treatment with an anti-CD24 antibody, the method comprising assessing the JAK2 mutation status of myeloproliferative neoplasm and / or whether the myeloproliferative neoplasm have elevated CD24 expression, wherein if the myeloproliferative neoplasm are JAK2 mutation positive and / or have elevated CD24 expression, the patient is selected for treatment with an anti-CD24 antibody.

[0084] The present invention further provides a method for selecting a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; the method comprising:(i) determining whether the myeloproliferative neoplasm possesses a JAK2 mutation and / or elevated levels of CD24 expression; and(ii) administering to a patient whose myeloproliferative neoplasm possesses a JAK2 mutation or elevated levels of CD24 expression a therapeutically effective amount of an anti-CD24 antibody.

[0085] Suitably, whether the myeloproliferative neoplasm possess a JAK2 mutation and / or elevated levels of CD24 expression is determined by determining whether blood cells ina blood sample obtained form the patient comprise a mutation in the JAK2 gene and / or whether elevated levels of CD24 are present in granulocytes / neutrophils. If the blood cells possess a JAK2 mutation and / or elevated levels of CD24 are present in granulocytes / neutrophils, a therapeutically effective amount of an anti-CD24 antibody can then be administered.

[0086] Suitably, therefore, the method comprises:(i) determining whether the blood cells from the patient possess a JAK2 mutation and / or elevated levels of CD24 expression in granulocytes / neutrophils; and(ii) administering to a patient whose blood cells possess a JAK2 mutation and / or elevated levels of CD24 expression in granulocytes / neutrophils, a therapeutically effective amount of an anti-CD24 antibody.

[0087] The JAK2 mutation may be any known JAK2 mutation. In an embodiment, the JAK2 mutation is JAK2V617F.

[0088] Methods for detecting JAK2 mutations are well known in the art (see, for example, A. J. Bench, E. J. Baxter, A. R. Green, Methods for detecting mutations in the human JAK2 gene. Methods Mol Biol 967, 115-131 (2013)).

[0089] Levels of CD24 expression may also be determined by techniques well known in the art. For example, CD24 expression in isolated granulocytes could be measured by qPCR or by flow cytometry techniques.

[0090] In another aspect, the invention provides proves a JAK2 mutation diagnostic test for use in a method of selecting a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; for treatment with an anti-CD24 antibody.

[0091] Suitably, the test comprises obtaining a biological sample from the patient (e.g. a blood sample) and testing the sample for JAK2 mutation status using the JAK2 mutation diagnostic test. The test may further comprise the step of isolating white blood cells from the sample, e.g. B-lymphocytes or neutrophils, and testing the JAK2 mutation status of these cells.The use of CD24 expression as a marker of the response to therapy with a JAK inhibitor

[0092] The data presented herein also demonstrates that CD24 expression is reduced by administration of a JAK (e.g. JAK1 and / or JAK2) inhibitor therapy, e.g. ruxolitinib. Thus, monitoring CD24 expression levels (e.g. in neutrophils / granuloctyes in a blood sample) provides a surrogate marker of the monitoring the effect of JAK inhibitor therapy. The levels of CD24 expression (e.g. in neutrophils / granuloctyes) can be determined by the techniques noted hereinbefore.

[0093] The present invention therefore further provides the use of CD24 expression as a marker of the response to JAK inhibitor therapy.

[0094] The present invention also provides a method of monitoring therapy with a JAK inhibitor, the method comprising administering a JAK inhibitor to a subject and measuring the CD24 expression levels, optionally over time or at predetermined times (e.g. in neutrophils / granuloctyes in a blood sample).BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The invention is described further in reference to the accompanying Figures in which:Figure 1 shows the deficient clearance of senescent neutrophils in MPN due to CD24 don’t- eat-me signal. A-B, Frequency of BM neutrophils (A; n=7-11 mice per group) and senescent neutrophils (B; n=10-13 mice per group) in WT mice and MPN mouse models of essential thrombocythemia (ET) or polycythemia vera (PV). C-D, Flow cytometry plots (C) and normalized frequency (D) of WT or MPN macrophages phagocytosing neutrophils from WT or MPN (ET-like or PV-like) mice (n=3-9). Note that JAK2V617Fin neutrophils (but not macrophages) reduces efferocytosis. E, Frequency of circulating senescent neutrophils from MPN patients (n=14) and healthy controls (HCs; n=9). F, Correlation of JAK2' / 677Fvariant allele frequency (VAF) in circulating granulocytes and the frequency of senescent neutrophils in the peripheral blood of MPN patients (n=8). G, Frequency of human phagocytic macrophages in co-culture with granulocytes from MPN patients or healthy controls (HC; n=4-5 per group). H, Supervised analyses of human blood granulocytes microarray datasets from MPN patients (PV, n=28; ET, n=47; myelofibrosis, MF, n=18) and age-matched HCs (n=11). I-J, CD24 (I) or CD47 (J) protein expression in blood neutrophils from MPN patients (I, n=17; J, n=7) and HCs (I, n=10; J, n=9). K- L, CD24 (K) and CD47 (L) mRNA expression in granulocytes from human MPN patients transformed into AML (n=8), compared with HCs (n=15). M, BM CD24 protein expression in senescent neutrophils from WT (n=9), ET-like (n=6), PV-like (n=6) or post-ET myelofibrosis (MF; n=5) mice. ET or PV mice were analyzed at early stage of disease (age 13-16w), post-ET MF at late stage (age 20-24w). N, Frequency of human phagocytic macrophages in co-culture with labelled MPN granulocytes (green), treated 3h with anti-CD24 (clone SN3) or IgG antibodies (n=3). O, Frequency of WT macrophages phagocytosing DsRed+neutrophils (red) from DsRed control mice (n=3) or ET-like mice (compound DsRed;Vav-Cre;JAK2' / 677 / =mice; n=4) treated 3h with anti-CD24 antibody or control IgG. P, Frequency of BM WT (n=4) or ET-like (n=5-7) neutrophils (top), mirroring macrophages phagocytosing WT (n=3) or ET neutrophils (n=3-4), 24h after adoptive transfer of the same number of JAK2' / 677F-mutated / unmutated senescent neutrophils into WT mice treated with anti-CD24 blocking antibody or control IgG (see Extended Data Figure 2J for experimental design).Figure 2 shows GM-CSF induces CD24 expression in senescent neutrophil via JAK2- STAT5 signalling. A, Correlation of JAK2V617Fvariant allele frequency (VAF) in circulating granulocytes and CD24 protein expression in peripheral blood neutrophils from MPN patients (n=16). B, CD24 expression in senescent neutrophils isolated from MPN mice after 16h culture with GM-CSF (n=6), G-CSF (n=4), M-CSF (n=4), IL-1 (n=3) or medium only (n=5). C-D, CD24 expression (C) and frequency of senescent neutrophils (D) from MPN patients after 16h culture with GM-CSF or vehicle, in presence / absence of JAK inhibitor ruxolitinib (n=4-5 per group). E-F,Frequency of senescent neutrophils (E, n=3 per group) and CD24 expression (F) from ET-like mice after 16h culture with GM-CSF (n=5), ruxolitinib (n=6), STAT5 inhibitor (AC-4-130, n=3) or vehicle (n=6). G, CD24 expression in senescent neutrophils isolated from WT or Mx1- Cre;STAT5fl / fl mice (induced with pIpC), after 16h culture with GM-CSF (n=4-5 per group). H, CD24 expression (fold change) in BM senescent neutrophils from ET-like mice treated for 2.5w with ruxolitinib (twice weekly; n=10), compared with control vehicle (n=6). I, CD24 protein expression in blood neutrophils from MPN patients receiving JAK inhibitor ruxolitinib treatment compared with healthy control or MPN patients treated with other drugs (n=4-5 per group). J, Quantification of phagocytic macrophages co-cultured with DsRed+neutrophils from ET-like mice pre-treated with GM-CSF, alone or in combination with ruxolitinib or anti-CD24 blocking antibody (n=4-5). K-L, Frequency of neutrophils (K) or senescent neutrophils (L) in the BM of LysM- Cre;JAK2V617Fmice (n=6) or WT mice (n=5-8). M-N, CD24 expression in (M) senescent neutrophils freshly isolated from LysM-Cre;JAK2V617Fmice or WT mice, or (N) analysed 16h after incubation with GM-CSF or vehicle (n=4).Figure 3 shows CD24 inhibition reduces emperipolesis and neutrophil aggregation with platelets and improves thrombocytosis in MPN. A-B, Quantification of immunofluorescence of CD41+megakaryocytes and DsRed+neutrophils isolated from control mice or MPN mice, 24h after adoptive transfer into WT recipients (see Extended Data Figure 2J for experimental design). A, Minimal distance between megakaryocytes and DsRed+neutrophils. Each dot is a pair (WT, n=170; ET, n=82; PV, n=192). B, Frequency of megakaryocytes manifesting emperipolesis of DsRed+neutrophils. C, Quantification of immunofluorescence of CD41+megakaryocytes and Ly6G+neutrophils (emperipolesis) in the BM of WT (n=9), ET-like (n=8) or PV-like (n=12) mice. D, Frequency of CD11b+F4 / 80+macrophages among BM Ly6G' cells from WT mice (n=7) or ET- like mice (n=8). E-F, Quantification of immunofluorescence of CD169+macrophages or VWF- TdTomato* megakaryocytes showing emperipolesis of neutrophils 24h after treatment with clodronate liposomes, to deplete macrophages, or vehicle (n=3 per group). G, Circulating platelet counts after repeated adoptive transfer of same number of WT or JAK2V617Fsenescent neutrophils into WT mice (n=9 per group). H, Frequency of neutrophil-platelet aggregates in the bloodstream of human MPN (n=10), compared with healthy controls (HCs; n=6). I, Correlation between CD24 protein expression in human blood neutrophils and the frequency of blood neutrophil-platelet aggregates (HC, light gray dots; MPN, dark gray dots; n=15). J, N, Q, T, Therapeutic effects of CD24 blockade in mouse model of essential thrombocythemia (ET). WT mice were lethally irradiated, transplanted with BM cells from ET-like mice and treated 9-10w after transplantation with anti-CD24 blocking antibody, or control IgG, for 7w or 11w (see Extended Data Figure 3C). K, O, R, U, Therapeutic effects of CD24 blockade in mouse model of polycythemia vera (PV). WT mice were lethally irradiated, transplanted with BM cells from PV-like mice 8w after polykpolyC induction and treated for 11w with anti-CD24 blocking antibody, or control IgG, starting 10w aftertransplantation (see Extended Data Figure 3C ). L, M, P, S, V, Genetic deletion of CD24 in mouse model of essential thrombocythemia (ET). WT mice were lethally irradiated, transplanted with BM cells from compound Vav-Cre;JAK2V617FCd24' / ' mice or control Vav-Cre;JAK2V617Fmice and were analyzed 18w after transplantation (see Extended Data Figure 3B). J-M, Frequency of neutrophilplatelet aggregates in blood (J, n=5-7; L, n=6-7) or BM samples (K, n=4-7; M, n=7). N-P, Frequency of BM senescent neutrophils (n=4-7). Q-S, Quantification of immunofluorescence of CD41+megakaryocytes and Ly6G+neutrophils (emperipolesis; n=4-7). T-V, Circulating platelets (T, II) before and 11 weeks after treatment with anti-CD24 blocking antibody or control IgG, in ET-like mice (T, n=3-5) or PV-like mice (II; n=6-7); or (V) 18w after transplantation of WT mice with BM cells from compound Vav-Cre;JAK2V617FCd24'' mice, or control Vav-Cre;JAK2V617Fmice (n=6-7)Figure 4 shows CD24 blockade prevents TGF- activation and myelofibrosis in MPN models. A, Active TGF-p concentration in endosteal or central BM of WT or ET-like mice (n=7- 9). B, Active TGF-p concentration in endosteal or central BM 18w after transplantation of WT mice with BM cells from compound Vav-Cre;JAK2V617FCd24'' mice or control Vav-Cre;JAK2V617Fmice (n=7). C-D, Active TGF-p concentration in endosteal or central BM of (C) ET-like mice treated for 7w with anti-CD24 blocking antibody, or control IgG (n=5-8), and (D) PV-like mice treated for 11wwith anti-CD24 blocking antibody, or control IgG (n=7-8). E-F, BM Gdmdri staining of reticulin fibers of MPN models of polycythemia vera (E, PV) or essential thrombocythemia (F, ET) treated for 11w with anti-CD24 blocking antibody, or control IgG (n=5-7). Scale bar, 40pm. G, BM Gbmbri staining of reticulin fibers 18w after transplantation of WT mice with BM cells from compound Vav-Cre;JAK2V617FCd24'' mice, or control Vav-Cre;JAK2V617Fmice (n=5-7). Scale bar, 40pm. H-J, Myelofibrosis grade in mice in (E-G). K-L, Osteosclerosis (K) and quantification (L) of bone per BM area in ET-like mice treated for 11w with anti-CD24 blocking antibody, or control IgG (n=6-7). Scale bar, 100pm. M-N, Spleen weight (M) and representative pictures (N) in hematopoietic chimeras with / without CD24 deletion 18w after transplantation of WT mice with BM cells from compound Vav-Cre;JAK2V617FCd24~ / ~ mice, or control Vav-Cre;JAK2V617Fmice (n=7-9).Extended Data Figure 1 shows senescent neutrophils accumulate in MPN BM due to defective efferocytosis. A, Schematic illustration of endosteal and central BM cell extraction. B, I, J, Gating strategy of (B) mouse BM neutrophils and senescent neutrophils, (I) phagocytic macrophages, and (J) human blood neutrophils and senescent neutrophils. C-E, Analysis of senescent (CD62L10) and young (CD62Lhi) neutrophils. Expression of (C) CD11 b or (D) CD45 in CD62L10or CD62LhiMPN neutrophils (n=8-9). E, Frequency of forward side scatter (FCS) of CD62L10or CD62LhiMPN neutrophils (n=8-9). F-G, Frequency of (F) total or (G) senescent neutrophils in WT mouse circulation and BM niches close to the bone surface (endosteal) or further away from bone (central) (n=3-4 mice). H, Experimental workflow for in vitro phagocytosisassay of WT or MPN macrophages co-cultured with WT or MPN (ET-like or PV-like) senescent neutrophils treated with anti-CD24 blocking antibody or control IgG (created in BioRender.com).Extended Data Figure 2 shows high CD24 expression in JAK2V617Fneutrophils and pathogenic interactions of neutrophils with megakaryocytes in MPN. A, Principal Component Analysis of RNAseq from JAK2' / 677F-mutated HEL cells showing clustering of cells cultured with vehicle (con) or after prolonged treatment with the JAK inhibitors ruxolitinib or fedratinib. B-C, Supervised analysis from dataset in (A) shows high CD24 mRNA expression in HEL cells resistant to the JAK inhibitors ruxolitinib (B) or fedratinib (C), compared to control HEL cells. D, BM CD47 protein expression in senescent neutrophils from WT (n=6) or ET-like (n=7) mice. E-F, Human phagocytic macrophages (fold change) in co-culture with labelled granulocytes from MPN patients (E) or labelled HEL (JAK' / 677F) cells (F), treated 3h with anti-CD24 antibody (clone ATG-031) or JAK2 inhibitor ruxolitinib. The efferocytosis of human MPN neutrophils or HEL (JAK' / 677F) cells by human macrophages was increased mainly after CD24 blockade (ATG-031) compared to ruxolitinib. G-H, Frequency (G) and flow cytometry plots (H) of AnnexinV* apoptotic WT or MPN neutrophils after 3h culture with anti-CD24 antibody or control IgG (n=3-4 mice per group). I, Frequency of AnnexinV* apoptotic MPN neutrophils after 22h culture with primary anti- CD24 antibody, control IgG or with crosslinked primary-secondary antibodies (n=3 mice per group). Primary and secondary antibodies pre-incubated before addition to cells at a 2: 1 ratio to ensure efficient cross-linking of primary antibody. J, Experimental workflow of adoptive transfer. WT mice were injected with WT or JAK2' / 677FDsRed+neutrophils (i.v.) and anti-CD24 blocking antibody or control IgG (i.p.) and were sacrificed 24h later (created in BioRender.com). K, CFS2RA mRNA expression in blood granulocytes from MPN patients including essential thrombocythemia (ET, n=25), polycythemia vera (PV, n=23) and primary myelofibrosis (PMF, n=7), compared with healthy controls (n=8). L, Quantification of in vitro emperipolesis of WT megakaryocytes co-cultured with WT or JAK2 ' / 677Fneutrophils (n=5-6 per group). M, Experimental workflow for in vitro emperipolesis assay of WT megakaryocytes with freshly sorted CD62Lhi9hyoung neutrophils or CD62L|OWsenescent neutrophils isolated from JAK2V617Fp-actin-DsRed mice (created in BioRender.com). N, Quantification of in vitro emperipolesis of VWF-eGFP+megakaryocytes with DsRed+CD62Lhi9hyoung or CD62L|OWsenescent neutrophils (n=3).Extended Data Figure 3 shows CD24 blockade prevents myelofibrosis in MPN. A, Experimental workflow of repeated adoptive transfer of WT or JAK2' / 677Fsenescent neutrophils (created in BioRender.com). B, Experimental workflow of non-competitive transplantation of BM cells from ET CD24+ / +or ET CD24'' mice into WT recipients (created in BioRender.com). C, Experimental workflow of chronic treatment of ET-like or polycythemia vera (PV)-like MPN mice with anti-CD24 blocking antibody or control IgG (created in BioRender.com). D, Flow cytometry plots of BM CD11b+Ly6G+CD62L10senescent neutrophils from chimeric ET miceproficient / deficient for CD24 expression in hematopoietic cells. E, Frequency of BM senescent neutrophils from WT mice treated with anti-CD24 blocking antibody or control IgG for 7w (n=3-4). F, Gdmdri staining of reticulin fibrosis in BM section from ET-like mice treated with anti-CD24 blocking antibody or control IgG for 11w. Scale bar, 50pm. G, Model illustrating the interpretation of results: senescent neutrophil accumulation in MPN is not only due to their excessive production, but also to their defective clearance caused by JAK-STAT-dependent upregulation of CD24, which prevents their normal phagocytosis by macrophages. Consequently, neutrophils exhibit pathogenic interactions with megakaryocytes and platelets in MPN, which can be prevented by pharmacological blockade or genetic deletion of CD24 (created in BioRender.com).Examples

[0096] The invention will now be illustrated in the following Examples.MethodsHuman studies

[0097] All centers had appropriate research and ethical approval; patients gave their written informed consent. Samples were derived from the Cambridge Biobank and patients attending outpatient clinics at Addenbrooke’s Hospital (UK), under the clauses of the Causes of Clonal Haematological Disorders project, which had regional ethical approval from the Eastern Multiregion Ethics Committee and local research and ethical approval at participating UK hospitals. Both male and female patients were included in the different clinical studies. Differences regarding sex were not investigated in this study. The information on the disaggregation between sex and gender was not collected.Mouse strains

[0098] All experiments using mice followed protocols approved by the Animal Welfare Ethical Committee, according to the United Kingdom Home Office regulations (PPL P0242B783 and PP1036410). Mice were housed in specific pathogen-free facilities in individually ventilated cages under 12 h light- dark cycles and controlled temperature (19-23 °C) and humidity (55 ± 10%) with free access to standard rodent chow (SafeDiet R105-25). The humane endpoint was defined by the project license in accordance with the Home Office regulations as a 15% loss of maximal body weight; all mice were euthanized before or on reaching this stage. B6.FVB-Tg(Acta2- DsRed)1 Rkl / J (stock no. 31159, The Jackson Laboratory), B6.129S6- Stat5btm1MamStat5atm2Mam / Mmjax (stock no. 032053, The Jackson Laboratory), LysM-Cre (B6.129P2-Lyz2tm1 (cre)lfo / J) (stock no. 4781 , The Jackson Laboratory), Vwf-TdTomato, Vwf- eGFP, Vav-Cre;JAK2' / 677F, Mx1-Cre;JAK2' / 677F, C57BL / 6-Cd24atm1 Pjln / Mmmh (stock no. 41412-MU, MMRRC) and congenic B6.SJL-PtprcaPepcb / BoyJ (CD45.1), CD45.2 C57BL / 6 mice (Charles River Laboratories) were used in this study. In double transgenic Mx1-Cre;JAK2' / 677 / =mice, Cre activation was induced through intraperitoneal injection of 300 pg / mouse polyinosinepolycytosine (pIpC; catalog no. P1530, Sigma-Aldrich) as a single dose at age 8-9 weeks old, and analysed 6-8 weeks after induction (for advanced myelofibrosis, mice were analysed 15-17 weeks after induction). In double transgenic Mx1-Cre; Stat5a / 5b mice, (both Mx1-Cre;WT or Mx1- Cre;hemi) Cre activation was induced through 6 intraperitoneal injection of pIpC (16mg / kg) and mice were culled after 6x pIpC injection. Mice were sacrificed and cells were harvested in the same timeframe in the morning (8-10AM) to account for the results of the clearance, which occurs preferentially at night.

[0099] Both female and male mice were used in the different experiments; possible differences regarding sex were not investigated in this study. Mice with similar blood counts were randomly allocated to control or treatment groups.Bone marrow transplantation

[0100] Age-matched, CD45.2 C57BL / 6J mice (8-12 weeks old) were used as recipients in the BM transplantation assays. Recipients were subjected to lethal irradiation (12 Gy wholebody irradiation, split dose 6.0 + 6.0 Gy, 3h apart) before injection. For competitive transplantation, 0.2x106BM cells from a CD45.2 donor Mx1-Cre;JAK2V617F(8 weeks after pIpC injection) and 1.8x106BM cells from CD45.1 donor were injected into the tail vein of wild-type mice. For noncompetitive transplantation, 2 x 106BM cells from Vav-Cre;JAK2V617For Vav-Cre; JAK2' / 677 / 7CD24' / _were injected into wild-type mice. Blood counts (WBC, RBC and platelets) and spleen weight were analysed from donors before transplantation to determine the disease stage.In vivo treatmentsFirst, we confirmed the disease stage before treating the transplanted mice based on their higher levels of blood platelets (ET-like) or RBC (PV-like) 9w post transplantation. PV mice were treated intraperitoneally two times a week for 11 weeks, and ET mice were treated two times for 7 or 11 weeks, with an anti-mouse CD24 mAb (clone M1 / 69, catalog no. BE0360, BioXCell) or rat lgG2b Isotype control (catalog no. BE0090, BioXCell) at a dose of 200 pg / mouse. Mice were sacrificed 1 day after the last antibody injection. Ruxolitinib treatment of ET mice: Ruxolitinib (Jakavi, Novartis) was resuspended in polyethylene glycol (catalog no. 202371-500g, Sigma-Aldrich) for administration to mice. 8-9 weeks post-transplantation, mice were treated with ruxolitinib (70 mg kg— 1 ) or vehicle once daily, two times weekly for 2.5 weeks. Murine neutrophil isolation

[0101] Bone marrow cells were harvested from 13- to 19-week-old WT, ET-like or PV-like mice. Femurs and tibiae were isolated and cleaned of muscles. Bones were then crushed with a mortar and pestle and filtered through a 40pm cell strainer. RBCs were removed using RBC LysisBuffer (catalog.no 420301 , BioLegend). BM cells were resuspended in 200 pl MACS buffer (2 mM EDTA, 0.5% bovine serum albumin in PBS) and neutrophils were isolated following the manufacturer’s protocol (catalog no.130-097-658, Miltenyi). The purity was >94% as determined by flow cytometry. To induce neutrophil ageing ex vivo, isolated neutrophils were incubated at 37°C for 16-18 hours.Mononuclear cells and granulocyte isolation from human peripheral blood

[0102] Mononuclear cells and granulocyte isolation from human peripheral blood from each healthy control or MPN patient, 18 mL of peripheral venous blood was collected using a 21- G multi-fly needle to avoid lysing cells into S-monovette EDTA vacutainers (S-monovette 9ml K3E REF: 02.1066.001 , 1.6mg EDTA / ml). Peripheral blood was then layered slowly onto 20ml Lymphoprep and centrifuged at 1800 rpm for 30 min with brake off. The plasma layer was removed (to within 0.5cm above the MNC layer) and the MNC layer was carefully taken up using a plastic pastette and transferred into a fresh falcon tube, at this point the MNCs were placed into a known volume with PBS / 0.5%BSA to enable cell counts and viability to be recorded. The MNCs were then frozen until use for macrophage differentiation. To isolate granulocytes, the remainder of lymphoprep was removed (to within 0.5-1.0 cm of the red cell layer; containing granulocytes). Cold red blood cell (RBC) lysis solution was added to the red cell layer for 5 minutes and then centrifuged at 1800rpm for 5 mins (RBC lysis step was repeated twice with the second step exposing the red cell pellet to 3 minutes of RBC lysis). The pellet was resuspended in a known volume of PBS / 0.5%BSA to enable cell counts and viability to be recorded. Granulocytes were isolated from individuals diagnosed with ET or PV and carrying the JAK2V617Fmutation (The human JAK2 allele burden in granulocytes was detected as described previously(l).Cell Culture: HEL JAK2V617Fcell line

[0103] The MPN cell line HEL was obtained from the American Type Culture Collection (ATCC; http: / / www.atcc.org, Manassas, VA). HEL cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (penicillin, 100 I.U. / ml; streptomycin, 100 pg / ml). Cells were cultured at 37 °C with 5% CO2 in a water-jacketed incubator. HEL cells were used for phagocytosis (see” Human in vitro phagocytosis assay” section).Adoptive transfer of BM neutrophils

[0104] WT mice aged 8-12 weeks were divided into two-three recipient groups. Isolated DsRed+ BM WT or JAK2' / 677Fneutrophils were aged ex vivo. Senescent neutrophils were resuspended in PBS, and same number (5 x 106) of cells were adoptively transfused each time to recipient mice by tail intravenous injection. For short adoptive transfer, mice recipient were transfused (intravenously) one time with either WT or JAK2' / 677Fneutrophils, injected intraperitoneally with IgG or anti-CD24 antibody (200pg per mouse) and sacrificed 24h later.Bones were collected for flow cytometry and histology analysis. For chronic adoptive transfer, recipient mice were transfused (intravenously) with either WT or JAK2V617Fsenescent neutrophils on day 0, 7, 14 and 21 , and were bled on day 5, 12, 19 and 23, to monitor circulating platelet counts.In vitro stimulation

[0105] BM isolated neutrophils (1 x 106) from WT, MPN, Mx1-Cre;STAT5fl / fl or their control (treated with pIpC) mice were seeded on 12-well plate in RPMI-1640 culture media (supplemented with 10%FBS and 1 % penicillin-streptomycin, and then stimulated with either PBS, GM-CSF (100ng / ml), G-CSF (100ng / ml), M-CSF (100ng / ml), IL-1 (100ng / ml), Ruxolitinib (0.5 pM) or STAT5 inhibitor (AC-4-130, 5 pM). Cultured neutrophils were harvested 16-18h post treatment, then washed, stained and analysed by flow cytometry.BM cell extraction, flow cytometry and cell sorting

[0106] Hematopoietic cell isolation from BM or PB was performed as described previously(5). The marrow was flushed and the bones crushed with a mortar and pestle (catalog no. 10656405, Fisher), in PBS and filtered through a 40-pm strainer (catalog no. 542040, Greiner Bio-One). PB or BM cell suspension was depleted of red blood cells using the RBC lysis buffer for 8 min at 4 °C (catalog no. 420301 , BioLegend), washed and numerated using trypan blue (catalog no. 10593524, Fisher). Cells were incubated with the appropriate dilution (2-5 mg ml-1) of fluorescent antibody conjugates. DAPI (catalog no. D9542, Sigma-Aldrich) was added to discriminate dead cells. Samples were analyzed with an LSRFortessa flow cytometer (BD Biosciences) or sorted (FACSAria, BD Biosciences) equipped with the FACSDiva Software (BD Biosciences). The following antibodies were used for the staining of the mouse hematopoietic cells in different panels: anti-mouse CD11b (clone M1 / 70, catalog no. 101216, 101236, 553309, 101211 BioLegend); anti-mouse Ly6G (clone, 1A8, catalog no. 127624, BioLegend); anti-mouse CD62L (clone MEL-14, catalog no. 553152, BD Pharmigen); anti-mouse CD24 (clone 30-FI, catalog no. 138503 or clone M1 / 69, catalog no. 101821 , BioLegend); anti-mouse CD47 (clone miap301 , catalog no. 127503, BioLegend); anti-mouse F4 / 80 (clone BM8, catalog no. 11-4801- 82, Invitrogen, eBioscience or catalog no. 123110, BioLegend ); anti-mouse CD41 (clone MWReg30, catalog no. 133904, Biolegend); anti-mouse NK1.1 (catalog no. 108723, BioLegend); anti-mouse CD45.2 (clone 104, catalog no. 109847, BioLegend or 25-0454-U100, TONBO biosciences); anti-mouse CD45.1 (clone A20, catalog no. 60-0453-U100, TONBO biosciences); streptavidin (catalog no. 405241 , BioLegend). The following antibodies were from BioLegend, used for the staining of human neutrophils and platelets in healthy donors or MPN patients and for human macrophages: anti-human CD11b (catalog no. 301343), anti-human CD16 (catalog no. 302017); anti-human CD62L (catalog no. 304809), anti-human CD24 (clone ML5, catalog no. 311105); anti-human CD47 (catalog no. 323106); anti-human CD66b (catalog no. 305117); anti-human CD41 (catalog no. 303703); anti-human CD45 (catalog no. 368531); anti-human CD14 (catalog no.. 301806). For flow cytometry staining, we used the fluorescence minus one (FMO) as a negative control, which contains all the fluorochromes except for the one that is being measured. All flow cytometry data were analysed using Kaluza software. The expression levels of proteins (e,g CD24 or CD47) are quantified and presented as “geometric mean”, which is the mean of the fluorescence intensity. Representative flow cytometry gating strategy, plots or histograms were created by Kaluza or Flowjo software.Apoptosis analysis

[0107] Annexin V / DAPI staining. Isolated BM neutrophils from WT or MPN mice were cultured in RPMI media (1 x 106cells / ml) and treated for 3hr with either 10 pg / ml rat anti-mouse CD24 mAb (clone M1 / 69) or 10 pg / ml isotype antibody (rat lgG2b) from BioXcell. After 3hr, cells were collected for staining. For cross-linking CD24, isolated BM neutrophils from MPN mice were cultured and treated for 22hr with primary anti-CD24 antibody, control IgG or with crosslinked primary-secondary antibodies. Primary and goat anti-rat secondary (catalog no. 112-005-003; Jackson ImmunoResearch) antibodies pre-incubated before addition to cells at a 2 : 1 ratio to ensure efficient cross-linking of primary antibody and to ensure no excess secondary antibody present in the culture. Isolated neutrophils were stained first with cell surface markers (CD11b, Ly6G), and then resuspended in Annexin V binding buffer. Apoptosis was measured using FITC AnnexinV (catalog no. 640945, BioLegend) and DAPI, and cells were analysed by flow cytometry.Human in vitro phagocytosis assay

[0108] Human macrophage generation and stimulation: Peripheral mononuclear cells (PBMCs) were obtained from the buffy coats of healthy donors by the Human Research and Human Tissue Authorities. PBMCs were then pelleted by centrifugation at 300 g for 5 mins, resuspended in a RPMI-1640 medium consisting of 20% FBS and 10% DMSO, and cryopreserved until use. Monocytes were isolated from PBMCs through adherence to plastic and then differentiated into macrophages by 7-9 days of culture in IM DM + 10% human AB serum (Sigma, #H5667). Macrophage differentiation was induced by adding 25ng / mL of human M-CSF (Peprotech, catalog no. 300-25) on day 1. Half of the medium was replaced on day 4, and from that day onwards, three human cytokines (20ng / mL IL-4, 50ng / mL IL-10, and 50ng / mL TGF-pi) from Peprotech (catalog no. 200-04, 200-10, and 100-21, respectively) were added daily. Additionally, two PBS washes followed by complete medium replacement were performed one day before co- culture.

[0109] Human granulocytes preparation: Fresh granulocytes from healthy donors or MPN patients were obtained alongside the PBMCs above and immediately cultured upon receipt in RPMI-1640 + 10% FBS overnight prior to co-culture, to induce their aging.

[0110] Phagocytosis assay: On the day of assay, the number of macrophages per well was counted with a haemocytometer. Senescent granulocytes or HEL cells were labelled by incubation with 5pM Cell-Tracker Green CMFDA (catalog no. C7025, Invitrogen) for 20 min in the dark at 37°C and washed with RPMI serum-free medium. Where indicated, 10pg of anti-human CD24 antibodies (clone SN3, catalog no. NB100-64861 , Novus or clone ATG-031 , catalog no. HY-P99176, MCE), mouse lgG1 isotype control (catalog.no BE0083, BioXcell) or Ruxolitinib (0.5 pM) was added to macrophage cultures immediately before co-culture. Then, four times the number of granulocytes or HEL cells were added to each macrophage culture to achieve a 4:1 target-to-effector cell ratio. Phagocytic macrophages were analysed 3h post co-culture (either by flow cytometry (Extended data Figure 2E-F) or by live cell imaging on an Incucyte system (Figure 1G, N)). Phagocytic (CMFDA positive) macrophages were manually quantified with at least two randomly selected fields of view for each condition by Imaged software (live imaging).Mouse in vitro Phagocytosis assay

[0111] Macrophage generation and stimulation: bone marrow cells were harvested from 10- to 15-week-old WT mice. Femurs and tibiae were isolated and cleaned of muscles. Bones were then crushed with a mortar and pestle, and a uniform cell suspension was made by extensive passage of marrow through a 40pm cell strainer. RBCs were removed using RBC Lysis Buffer (catalog.no 420301 , BioLegend). Bone marrow macrophages were generated by culturing the BM cells in DMEM / F-12, HEPES (catalog.no 11330032, Gibco) with 10% FBS and 25 ng / mL murine M-CSF (catalog.no 315-02, Peprotech,). On day 4, cells were detached and replated in a 12-well plate with 105 cells per well with 1 ml medium and M-CSF and were kept until use on day 7.

[0112] Neutrophil preparation: one day before the phagocytosis assay, neutrophils were isolated from WT or MPN mice (as described above in “Neutrophil isolation” section) and cultured in RPMI- 1640+10%FBS overnight prior to co-culture.

[0113] On the day of assay, the number of macrophages per well was counted with a haemocytometer. Senescent neutrophils were labelled by incubation with 5pM Cell-Tracker Green CMFDA (catalog no. C7025, Invitrogen) for 20 min in the dark at 37°C and washed with RPMI serum-free medium. Where indicated, 10pg of anti-mouse CD24 mAb (clone M1 / 69, catalog no. BE0360, BioXCell) or rat lgG2a isotype control (catalog.no BE0089, BioXcell) was added to macrophage cultures immediately before co-culture. Then, senescent neutrophils were added to each macrophage culture to achieve a 4:1 target-to-effector cell ratio. Phagocytic macrophages were analysed 3hr post co-culture (either by flow cytometry (Figure 1C-D, P) or by live cell imaging on an Incucyte system (Figure 10; Figure 2J)). Phagocytic (CMFDA positive) macrophages were quantified with at least two randomly selected fields of view for each condition (live imaging).Mouse in vitro emperipolesis assay

[0114] Megakaryocyte generation and stimulation: bone marrow cells were harvested from 10- to 15-week-old vWF-eGFP mice. Femurs and tibiae were isolated and cleaned of muscles. Bones were then crushed with a mortar and pestle, and a uniform cell suspension was made by extensive passage of marrow through a 40|jm cell strainer. RBCs were removed using RBC Lysis Buffer (catalog.no 420301 , BioLegend). Bone marrow cells were incubated with biotin-linage Cocktail for 20 min on ice and washed with magnetic buffer (PBS+0.5%BSA+2mM EDTA). Cells were incubated with BDIMag StrepParticle Plus and magnetic buffer for 30 mins on ice. Next, magnetic buffer was added to depleted cells on magnet for 5 min in 4°C. Lineage negative cells were cultured in StemSpan H3000 / Cellgro medium with 50ng / ml TPO for 4-5 days.

[0115] Neutrophil preparation: one day before the emperipolesis assay, neutrophils were isolated from DsRed WT or MPN mice (as described above in “Neutrophil isolation” section) and cultured in RPMI-1640+10%FBS overnight prior to co-culture.

[0116] On the day of assay, the number of megakaryocytes per well was counted with a haemocytometer. Senescent neutrophils were added to each megakaryocyte culture to achieve a 4:1 neutrophil-to-megakaryocyte ratio. Emperipolesis of neutrophils were imaged 3hr post coculture by confocal microscopy and were quantified with at least two randomly selected fields of view for each condition by using Imaged software. Emperipolesis quantification: for each area, 30-70 megakaryocytes were counted. Results are expressed as percentage of megakaryocytes with emperipolesis (presence of neutrophils within the megakaryocytes was indicated as emperipolesis).In vivo macrophage depletion

[0117] 12-15 weeks Vwf-tdTomato or MPN mice were injected into the tail vein with PBS- liposome control or clodronate liposomes (250|jl / mouse, catalog no. CP-005-005, LOPOSOMA research) and sacrificed 24h later. Femurs were collected for histology.Histology of mouse bones

[0118] Femurs and tibias were collected, cleaned and put in PBS, 2% paraformaldehyde (PFA) (Sigma-Aldrich) overnight. For cryosectioning and immunostaining, bones were washed once with PBS, decalcified in 250 mM EDTA and PBS for 14 days at 4 °C, put in 15% sucrose and PBS for 24 h then in 30% sucrose and PBS for another 24 h, and embedded with OCT (catalog no. 12678646, Thermo Fisher Scientific) in plastic cryomolds (catalog no. 4557, Sakura). The samples were stored at -80 °C and sections (12 pm) were obtained using a cryostat (Leica

[0119] Biosystems). Alternatively, after fixation bones were embedded in paraffin and 3- pm-thick sections were stained with reticulin for conventional fibrosis and osteosclerosisevaluation. Images were acquired with a microscope (Zeiss Apotome) using 10x, 20x and 40x objectives and analysed with Imaged. Osteosclerosis quantification was performed using the NDP.view2 software (Hamamatsu).Immunofluorescence of cryosections

[0120] Immunofluorescence staining of cryosections was performed as described previously12using femurs, with minor modifications. Briefly, cryosections of 12 pm were with Tris- NaCI-blocking (TNB) buffer (0.1 M Tris-HCI, pH 7.5, 0.15 M NaCI, 0.5% blocking reagent, PerkinElmer) at RT, for 1 hour. Samples were incubated with conjugated antibodies: anti-Ly6G (1 :200, catalog no. 127610, BioLegend); anti-CD41 (1 :200, catalog no. 133904, BioLegend); anti- CD169 (1 :200, catalog no. 142419); anti-CD62L (1 :200, catalog no. 104419, BioLegend) diluted in PBS overnight at 4 °C. Samples were rinsed with PBS four to five times for 10 min. Stained tissue sections were counterstained for 10 min with 5 mM DAPI in PBS and rinsed with PBS. For sections, slides were mounted in mounting medium (catalog no. S3023, DAKO). Images were acquired with a confocal microscope (Stellaris or ZEISS 980) using 10x, 20* and 40* objectives and analysed with Imaged. At least two independent and randomly selected BM areas in the diaphysis were imaged as stacks and analysed per sample. To quantify emperipolesis, MKs number (with or without neutrophils) was counted and normalised to the BM area7.Active TGF-p quantification

[0121] Mouse active TGF-p or GM-CSF protein amount were measured in BM samples using commercial ELISA reagents, following the manufacturer’s protocol (catalog no.437707, BioLegend, R&D Systems; respectively). Total protein content in endosteal and central BM fluids was quantified by BSA assay. Active TGF-p and GM-CSF concentration in the BM fluids was normalized per protein content.Data processing

[0122] The oligonucleotide microarrays data of neutrophils from MPN patients15was downloaded from Gene Expression Omnibus with reference series number GSE54646. The differential expression analysis was performed using limma16. Genes with P value less than 0.1 are considered as differentiated expressed genes. Raw reads were aligned by HISAT2 using Genome Reference Consortium Human Build 3818, followed by gene read count quantification using FeatureCounts19. Count reads were normalised by DeSeq220.

[0123] To investigate the transcriptomic discrepancy between the neutrophils from CD24- high MPN patients (with top 25% CD24 expression level in the cohort) and CD24-low MPN patients (with bottom 25% CD24 expression level in the cohort), gene set enrichment analyses (GSEA) were performed using a weighted statistic, ranking by signal to noise ratio, 1000 geneset permutations, and molecular signature databases (Hallmarks, Gene Ontology andReactome pathway databases)21. In parallel, gene set variation analysis (GSVA), a sample- wise gene set enrichment analysis22was used to score the pathway activity of each myelofibrosis patient. The statistical relationship of pathway activity for each patient with the genes of interest was evaluated using Spearman's rank correlation.Statistics, reproducibility and analysis

[0124] Statistical analyses and graphics were carried out with Prism (GraphPad Software). Datasets were compared using different tests described in the legends. P values less than 0.05 were considered statistically significant. Data distribution was assumed to be normal, but this was not formally tested. No statistical method was used to predetermine sample size, but our sample sizes are similar to those calculated for similar experiments in previous publications. No animals or data points were excluded from the analyses.ResultsSenescent neutrophils accumulate in MPN BM due to defective efferocytosis

[0125] First, we measured WT senescent neutrophils in mouse circulation and BM niches close to the bone surface (endosteal) or further away from bone (central; Extended Data Figure 1A). Senescent neutrophils were characterized as Ly6Gh7CD62Llo cells (2). MPN senescent neutrophils presented high expression of CD11b and CD45 and reduced forward scattering properties, which reflect cell size changes during aging (Extended Data Figure 1 B-E). As previously described (2), senescent neutrophils were more abundant in circulation during the day, whereas they peaked at night in the BM, where they home to be cleared by macrophages (3); it was noted that senescent neutrophils accumulate in the endosteal BM, which appears to be their normal clearance site in the BM (Extended Data Figure 1 F-G).

[0126] To study the homeostatic clearance of mutant neutrophils in MPN, mice carrying the JAK2V617Fmutation driven by Mx1-Cre or Vav1-Cre were used, which respectively develop PV- like or ET-like MPN (4). During daytime, BM neutrophils were 20%-increased, and senescent neutrophils were 2-3-fold higher, in the endosteal BM of MPN mice, compared with WT mice (Figure 1A-B). It was then evaluated whether the accumulation of senescent neutrophils in MPN BM is solely caused by their overproduction, or also due to altered efferocytosis. BM nucleated cells were differentiated in vitro into macrophages and co- cultured with BM neutrophils aged ex vivo and fluorescently labelled (Extended Data Figure 1 H-I). Senescent neutrophil accumulation in MPN BM was explained by their 2-3-fold-decreased phagocytosis; interestingly, JAK2' / 677 / =mutation in macrophages did not affect their phagocytic function, suggesting a dominant role of JAK2' / 677Fin neutrophils (Figure 1C-D). These findings were validated in human MPN: circulating senescent neutrophils were high in MPN patients (Figure 1 E; Extended Data Figure 1J). Thefrequency of senescent neutrophils directly correlated with JAK2' / 677Fvariant allele frequency in granulocytes (Figure 1 F), suggesting that pathogenic JAK-STAT signalling causes the accumulation of senescent neutrophils. In addition, the efferocytosis of senescent neutrophils was reduced in human MPN (Figure 1G), validating the findings in mouse models.Defective clearance of MPN neutrophils is due to high expression of CD24 “don’t-eat-me” signal

[0127] The homeostatic recognition and efferocytosis of neutrophils by macrophages rely on the balance of "eat-me” and “don’t-eat-me” signals. Supervised analyses of human granulocyte microarray datasets from 93 patients with MPN (28 PV, 47 ET, 18 MF) and 11 age-matched normal donors (5) showed in MPN granulocytes moderate CD47 mRNA expression, and comparatively higher expression of CD24 (Figure 1 H), which reportedly functions as a don’t- eat- me signal in solid tumours (6). Higher mRNA content was matched by protein abundance, since CD24 (not CD47) protein expression was high in human blood MPN neutrophils, compared with healthy controls (Figure 1 l-J). These results were confirmed in an independent MPN cohort(IO), where CD24 (not CD47) mRNA expression was found to be high in granulocytes from MPN transformed into secondary acute myeloid leukaemia (Figure 1 K-L). Furthermore, CD24 mRNAV617F expression was high in HEL human erythroleukaemia cell line carrying the JAK2 mutation (7) and resistant to the JAK inhibitors ruxolitinib and fedratinib (8) (Extended Data Figure 2A-C).

[0128] High CD24 (not CD47) protein expression was detected in endosteal BM senescent neutrophils from mouse models of PV, ET or secondary (post-ET) myelofibrosis, while central BM senescent neutrophils showed increased CD24 protein in secondary myelofibrosis (Figure 1M and Extended Data Figure 2D). These results suggest that CD24 expression in neutrophils progressively increases throughout the BM during myelofibrosis development.Blockade of CD24 improves neutrophil clearance in MPN

[0129] To investigate the possible role of CD24 as a don’t-eat-me signal for human MPN neutrophils, macrophages were differentiated in culture from human peripheral blood mononuclear cells and co-cultured with granulocytes freshly isolated from MPN patients, aged ex vivo and fluorescently stained in presence of anti-CD24 blocking antibody (clone SN3) or control IgG. Blocking CD24 increased 20% MPN neutrophil efferocytosis (Figure 1 N), confirming CD24 function as a don’t-eat-me signal for human neutrophils. These findings were confirmed using different clone of anti-CD24 blocking antibody (ATG-031 , Extended Data Figure 2E) in co-culture with human primary MPN neutrophils (Extended Data Figure 2E) or MPN-HEL cell line (Extended Data Figure 2F). In addition, CD24 blockade was more efficient than ruxolitinib increasing phagocytosis of MPN cells (Extended Data Figure 2E-F).

[0130] Similar results were observed in MPN mice, as CD24 blockade restored normal efferocytosis in MPN neutrophils (Figure 10) without altering apoptotic cell frequency (Extended Data Figure 2G-H). In hematopoietic cells, antibody-mediated CD24 crosslinking can induce apoptosis (9, 10), which is prevented by JAK2' / 677F 30. We tested CD24 crosslinking and found minimal (~5%) neutrophil apoptosis, similarly observed with isotype control (Extended Data Figure 21). These results support the role of CD24 as a "don’t-eat-me" signal in MPN neutrophils.

[0131] These findings were validated in vivo through the adoptive transfer of WT or JAK2V677F-mutated neutrophils into WT mice treated with anti-CD24 blocking antibody or control IgG (see Extended Data Figure 2J). After 24h, transferred mutant neutrophils accumulated (3- fold- increased) in the BM, which was mirrored by their reduced phagocytosis by BM macrophages; however, CD24 blockade restored the normal neutrophil frequency and phagocytosis observed in WT cells (Figure 1 P). These results confirm CD24 function as a don’t- eat-me signal for JAK2' / 677F-mutated neutrophils in vivo.GM-CSF induces CD24 expression in senescent neutrophil via JAK2-STAT5 signalling

[0132] Next, the cause of CD24 upregulation in MPN neutrophils was investigated. Given that JAK2' / 677Fleads to the constitutive activation of cytokine-regulated JAK-STAT signalling, we investigated this pathway in MPN neutrophils. CD24 expression correlated with JAK2V617Fvariant allele frequency in human granulocytes (Figure 2A), suggesting that pathogenic JAK-STAT signalling induces CD24 expression. To test this, cytokines activating the JAK-STAT pathway in MPN neutrophils were examined. Granulocyte-macrophage colony-stimulating factor (GM-CSF), which is increased in MPN patients (11, 12), doubled CD24 expression in mouse MPN neutrophils; unlike other overproduced cytokines (13-15), including JAK-STAT-dependent G-CSF, or JAK- independent cytokines like M-CSF and interleukin-1 p (Figure 2B).

[0133] GM-CSF can delay neutrophil apoptosis and upregulate CD24 expression during sepsis (9, 16), suggesting it may induce CD24 expression in MPN neutrophils via JAK-STAT signalling. We found increased GM-CSF receptor (CFS2RA) mRNA expression in human MPN granulocytes compared with healthy controls (Extended Data Figure 2K). in vitro GM-CSF treatment induced CD24 expression in human MPN neutrophils, but this was blocked by the JAK1 / 2 inhibitor ruxolitinib (Figure 2C). Furthermore, GM-CSF increased the frequency of human or mouse MPN senescent neutrophils, which was also prevented by ruxolitinib (Figure 2D-E).

[0134] The STAT requirement for CD24 upregulation in MPN neutrophils was then investigated. In vitro GM-CSF treatment induced CD24 expression in MPN mouse senescent neutrophils, but not after pre-incubation with STAT5 inhibitor (blocking signalling downstream of GM-CSF receptor (17), phenocopying the effect of ruxolitinib (Figure 2F). To confirm therequirement of STAT5 for GM-CSF-induced CD24 expression, neutrophils were isolated from conditional knockout (cKO) mice lacking STAT5 in the hematopoietic system and treated with GM-CSF. GM-CSF doubled CD24 expression in WT neutrophils, to a much larger extent than in STAT5'7' neutrophils (Figure 2G). In vivo, ruxolitinib treatment reduced CD24 expression in JAK2' / 677Fsenescent neutrophils (Figure 2H). In line with these findings, CD24 expression was lower on neutrophils from MPN patients receiving JAK inhibitor (ruxolitinib), compared with hydroxycarbamide treatment (Figure 2I). Together, these results suggest that increased GM-CSF levels in MPN induce CD24 expression in mutated neutrophils mainly via JAK2-STAT5 signalling.

[0135] To investigate the potential of cytokines activating JAK-STAT signalling to prevent MPN neutrophil efferocytosis, WT macrophages were co-cultured with GM-CSF-stimulated MPN senescent neutrophils in the presence of ruxolitinib or anti-CD24 antibody (Figure 2J). Maximal phagocytosis was achieved with the latter, consistent with ruxolitinib normalizing, but not blunting, CD24 expression in JAK2' / 677Fneutrophils (Figure 2C,F). Indeed, CD24 blockade was more efficient than ruxolitinib increasing phagocytosis (Figure 2J). These results suggest that cytokines activating JAK-STAT signalling in neutrophils prevent their efferocytosis in MPN through CD24 upregulation.JAK2V617Fmutation in neutrophils is necessary but not sufficient to decrease neutrophil clearanceWe asked whether the JAK2V617Fmutation in neutrophils is sufficient to induce CD24 expression and immune evasion. Mice carrying the JAK2V617Fmutation in myeloid cells (LysM-Cre;JAK2V617Fmice), but not in HSCs, develop erythrocytosis but do not exhibit chronic inflammation, or develop fully-blown MPN or myelofibrosis (18). We found unchanged frequencies of total or senescent BM neutrophils in LysM-Cre;JAK2V617Fmice (Figure 2K-L). CD24 expression was normal in the senescent neutrophils from these mice but was doubled after in vitro treatment with GM-CSF (Figure 2M-N), arguing for the need of inflammatory cytokines activating JAK-STAT signaling in neutrophils, besides the JAK2V617F mutation.Pathogenic interactions of neutrophils evading efferocytosis with megakaryocytes in MPN

[0136] We next asked whether cumulative senescent neutrophils in MPN might lead to abnormal microenvironmental interactions in the BM. Forthat, we adoptively transferred the same number of DsRed WT or JAK2 ' / 677Fsenescent neutrophils, to facilitate cell tracking (see Extended Data Figure 2J). 24h after transfer into WT recipients, a closer distance was evidenced between megakaryocytes and JAK2' / 677Fneutrophils, compared with WT neutrophils (Figure 3A), suggesting that senescent neutrophils are prone to interact with megakaryocytes in MPN. The engulfment of granular leukocytes within megakaryocytes (“emperipolesis”) has been noted for half a century (19). However, the underlying cellular and molecular mechanisms and theirpathophysiological implications remain unclear (20). We found that JAK2V617F(but very rarely WT) senescent neutrophils undergo emperipolesis in megakaryocytes following adoptive transfer (Figure 3B). A high frequency of megakaryocyte emperipolesis of JAK2' / 677Fneutrophils was confirmed in MPN mice (Figure 3C), correlating with overall disease burden, which is known to be higher for PV, compared with ET.

[0137] To investigate megakaryocyte-neutrophil interactions, we co-cultured BM megakaryocytes with neutrophils freshly isolated and aged ex vivo. Doubled frequency of emperipolesis was observed in megakaryocytes co-cultured with JAK2' / 677FET-neutrophils, compared with WT neutrophils (Extended Data Figure 2L). We sorted fresh (CD62Lhi) and senescent (CD62L|OW) neutrophils from DsRed MPN mice and co-cultured them with eGFP (under the regulatory elements of Von Willebrand factor (VWF) (21). Megakaryocytes (Extended Data Figure 2M). Emperipolesis was more frequently observed with senescent than fresh neutrophils (Extended Data Figure 2N). Together, these findings suggest that senescent neutrophils undergo emperipolesis when evading efferocytosis and encounter megakaryocytes after homing back to the BM.

[0138] To account for possible effects due to altered macrophages in MPN, we measured their numbers and found them reduced in MPN mice (Figure 3D), possibly contributing to reduced phagocytosis and increased emperipolesis. Supporting this possibility, emperipolesis of neutrophils increased in WT or M PN mice treated with clodronate liposomes to deplete phagocytic macrophages (Figure 3E-F), suggesting that defective neutrophil efferocytosis by BM macrophages evokes their emperipolesis in megakaryocytes.Megakaryocyte emperipolesis of neutrophils increases platelet release in MPN

[0139] Invasion of the demarcation membrane system of normal megakaryocytes by neutrophils has been suggested to enable membrane exchange between neutrophils and megakaryocytes, and to promote platelet production (22, 23). To evaluate the effect of JAK2' / 677Fneutrophils on thrombopoiesis in vivo, a chronic adoptive transfer protocol was established consisting of repeated i.v. injections of senescent neutrophils (Extended Data Figure 3A). Increased numbers of circulating platelets were detected over time in WT mice receiving JAK2' / 677Fneutrophils, compared with WT neutrophils (Figure 3G). These results suggest that megakaryocyte emperipolesis of mutant neutrophils, as a consequence of their defective clearance, may increase thrombocytosis in MPN, which is directly associated with the risk of thrombosis.CD24 expression marks activated neutrophils interacting with platelets in MPN

[0140] Activated neutrophils may interact with endothelium and platelets (24), significantly increasing the risk of thrombosis in MPN (25-28). We found increased frequency of neutrophil-platelet aggregates in human JAK2V617FMPN (Figure 3H). Moreover, the frequency of neutrophilplatelet aggregates directly correlated with human neutrophil CD24 protein (Figure 3I), suggesting that high CD24 expression marks activated neutrophils susceptible to aggregate with platelets. Therefore, we examined the effects of CD24 blockade on neutrophil-platelet interaction.Genetic deletion or pharmacological blockade of CD24 improves thrombocytosis in MPN

[0141] Chronic CD24 blockade in ET and PV mice and genetic deletion of CD24 in ET mice (Extended Data Figure 3B-C) reduced blood neutrophil-platelet aggregates and BM membrane exchange between neutrophils and megakaryocytes (Figure 3J-M), suggesting a potential improvement of thrombosis risk in MPN. Furthermore, CD24 blockade decreased senescent neutrophils in ET and PV mice, while sparing these cells in WT mice (Figure 3N-P; Extended Data Figure 3E). This was associated with decreased megakaryocyte emperipolesis of neutrophils (Figure 3Q-S). Consequently, thrombocytosis developed only in MPN mice treated with control IgG, but not in those receiving anti-CD24 blocking antibody or genetically deletion (Figure 3T-V). Taken together, these results indicate that CD24 prevents normal efferocytosis of senescent neutrophils in MPN, increasing their abnormal interactions with megakaryocytes and platelets. Similar results from CD24 conditional KO mice and those treated with anti-CD24 antibody exclude a prominent role of antibody-dependent cellular cytotoxicity. This implies that targeting CD24 could be therapeutically beneficial in improving thrombocytosis and neutrophilplatelet aggregation in preclinical MPN models.CD24 blockade reduces TGF-p, prevents myelofibrosis and improves osteosclerosis in MPN

[0142] Megakaryocyte emperipolesis of neutrophils has been associated with myelofibrosis development in disparate haematological disorders, such as MPN (20) and grey platelet syndrome (29), suggesting a possible functional relationship between both events. Myelofibrosis is driven by pro-fibrotic cytokines abundantly produced by megakaryocytes, with TGF-p being the most prominent example (30), which additionally regulates proplatelet formation(37). However, TGF- p is synthesised as a latent cytokine that is activated through proteolytic cleavage (32), suggesting the possibility that neutrophil-derived proteases might provide a causal link between emperipolesis and myelofibrosis development.

[0143] Indeed, active TGF-p concentration was increased in the BM supernatant of ET mice, compared with WT controls (Figure 4A). However, chronic CD24 blockade or genetic deletion in MPN mice halved active TGF-p (Figure 4B-D), explaining the absent myelofibrosis observed in these mice (Figure 4E-J and Extended Data Figure 3F). Excessive bone formation in the BM (osteosclerosis) is frequently observed in human myelofibrosis. CD24 blockade reduced osteosclerosis by 3-fold in MPN mice (Figure 4K-L). Moreover, myelofibrosis compromise normal BM haematopoiesis and cause extramedullary haematopoiesis in organs such as the spleen,which become abnormally enlarged. Consistent with absent myelofibrosis in MPN mice lacking CD24 (see Figure 4G, J), splenomegaly was not observed in these mice (Figure 4M-N).

[0144] Taken together, these findings reveal defective clearance of neutrophils as a cause of pathogenic microenvironmental interactions of inflammatory neutrophils with megakaryocytes and platelets, which may respectively increase myelofibrosis and thrombosis risk in MPN. Additionally, these results postulate CD24 as a candidate target for innate-immune checkpoint blockade in MPN (Extended Data Figure 3G).References1. A. J. Bench, E. J. Baxter, A. R. Green, Methods for detecting mutations in the human JAK2 gene. Methods Mol Biol 967, 115-131 (2013).2. M. Casanova-Acebes, C. Pitaval, L. A. Weiss, C. Nombela-Ameta, R. Chevre, A.G. N, Y. Kunisaki, D. Zhang, N. van Rooijen, L. E. Silberstein, C. Weber, T. Nagasawa, P. S. Frenette, A. Castrillo, A. Hidalgo, Rhythmic Modulation of the Hematopoietic Niche through Neutrophil Clearance. Cell 153, 1025-1035 (2013).3. R. C. Furze, S. M. Rankin, Neutrophil mobilization and clearance in the bone marrow. Immunology 125, 281 -288 (2008).4. R. 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Wang, Y. Hayashi, A. Yokota, Z. Xu, Y. Zhang, R. Huang, X. Yan, H. Liu, L. Ma, M. Azam, J. P. Bridges, J. A. Cancelas, T. A. Kalfa, X. An, Z. Xiao, G. Huang, Expansion of EPOR-negative macrophages besides erythroblasts by elevated EPOR signaling in erythrocytosis mouse models. Haematologica 103, 40-50 (2018).19. T. E. Larsen, Emperipolesis of granular leukocytes within megakaryocytes in human hemopoietic bone marrow. American journal of clinical pathology 53, 485- 489 (1970).20. L. Centurione, A. Di Baldassarre, M. Zingariello, D. Bosco, V. Gatta, R. A. Rana, V. Langella, A. Di Virgilio, A. M. Vannucchi, A. R. Migliaccio, Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-I (low) mice. Blood 104, 3573-3580 (2004).21. J. Carrelha, Y. Meng, L. M. Kettyle, T. C. Luis, R. Norfo, V. Alcolea, H. Boukarabila, F. Grasso, A. Gambardella, A. Grover, K. Hogstrand, A. M. Lord, A. Sanjuan-Pla, P. S. Woll, C. Nerlov, S. E. W Jacobsen, Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells. Nature 554, 106-111 (2018).22. P. Cunin, R. Bouslama, K. R. Machlus, M. Martinez-Bonet, P. Y. Lee, A. Wactor, N. Nelson-Maney, A. Morris, L. Guo, A. Weyrich, M. Sola-Visner, E. Boilard, J. E. Italiano, P. A. Nigrovic, Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasm ic neutrophils to platelets. eLife 8, (2019).23. T. Petzold, Z. Zhang, I. Ballesteros, I. Saleh, A. Polzin, M. Thienel, L. Liu, Q. Ul Ain, V. Ehreiser, C. Weber, B. Kilani, P. Mertsch, J. Gotschke, S. Cremer, W. Fu, M. Lorenz, H. Ishikawa-Ankerhold, E. Raatz, S. El-Nemr, A. Gorlach, E. Marhuenda, K. Stark, J. Pircher, D. Stegner, C. Gieger, M. Schmidt-Supprian, F. Gaertner, I. Almendros, M. Keim, C. Schulz, A. Hidalgo, S. Massberg, Neutrophil"plucking" on megakaryocytes drives platelet production and boosts cardiovascular disease. Immunity 55, 2285-2299 e2287 (2022).24. C. Cerletti, C. Tamburrelli, B. Izzi, F. Gianfagna, G. de Gaetano, Platelet-leukocyte interactions in thrombosis. Thromb Res 129, 263-266 (2012).25. F. Passamonti, E. Rumi, D. Pietra, M. G. Della Porta, E. Boveri, C. Pascutto, L. Vanelli, L. Arcaini, S. Burcheri, L. Malcovati, M. Lazzarino, M. Cazzola, Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders. Blood 107, 3676-3682 (2006).26. A. Guy, S. Favre, S. Labrouche-Colomer, L. Deloison, V. Gourdou-Latyszenok, M. A. Renault, E. Riviere, C. James, High circulating levels of MPO-DNA are associated with thrombosis in patients with MPN. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 33, 2544-2548 (2019).27. O. Wolach, R. S. Sellar, K. Martinod, D. Cherpokova, M. McConkey, R. J. Chappell, A. J. Silver, D. Adams, C. A. Castellano, R. K. Schneider, R. F. Padera, D. J. DeAngelo, M. Wadleigh, D. P. Steensma, I. Galinsky, R. M. Stone, G. Genovese, S. A. McCarroll, B. Iliadou, C. Hultman, D. Neuberg, A. Mullally, D. D. Wagner, B. L. Ebert, Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms. Sci Transl Med 10, (2018).28. A. Carobbio, A. Ferrari, A. Masciulli, A. Ghirardi , G. Barosi, T. Barbui, Leukocytosis and thrombosis in essential thrombocythemia and polycythemia vera: a systematic review and meta-analysis. Blood Adv 3, 1729-1737 (2019).29. J. A. Guerrero, C. Bennett, L. van der Weyden, H. McKinney, M. Chin, P. Nurden, Z. McIntyre, E. L. Cambridge, J. Estabel, H. Wardle-Jones, A. O. Speak, W. N. Erber, A. Rendon, W H. Ouwehand, C. Ghevaert, Gray platelet syndrome: proinflammatory megakaryocytes and alpha-granule loss cause myelofibrosis and confer metastasis resistance in mice. Blood 124, 3624-3635 (2014).30. H. Chagraoui, E. Komura, M. Tulliez, S. Giraudier, W. Vainchenker, F. 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Claims

CLAIMS1. An anti-CD24 antibody for use in a method of treating:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) essential thrombocythemia (ET).

2. An anti-CD24 antibody for use according to claim 1 , wherein the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm.

3. An anti-CD24 antibody for use according to claim 1 , wherein the anti-CD24 antibody is for use in the treatment of essential thrombocythemia (ET).

4. An anti-CD24 antibody for use according to claim 1 or claim 2, wherein the anti-CD24 antibody is for use in the treatment of myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm selected from:(i) primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(ii) polycythemia vera and / or essential thrombocythemia;(iii) primary myelofibrosis;(iv) polycythemia vera; or(v) essential thrombocythemia.

5. An anti-CD24 antibody for use according to claim 1 , 2 or 4, wherein the anti-CD24 antibody is for use in the treatment of myelofibrosis in a patient having a myeloproliferative neoplasm selected from:(i) primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(ii) polycythemia vera and / or essential thrombocythemia;(iii) primary myelofibrosis;(iv) polycythemia vera; or(v) essential thrombocythemia.

6. An anti-CD24 antibody for use according to claim 1 , 2 or 4, wherein the anti-CD24 antibody is for use in the treatment of secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from:(i) polycythemia vera and / or essential thrombocythemia;(ii) polycythemia vera; or(iii) essential thrombocythemia.

7. An anti-CD24 antibody for use according to claim 1 , 2 or 4, wherein the anti-CD24 antibody is for use in the treatment of thrombosis in a patient having a myeloproliferative neoplasm selected from:(i) primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(ii) polycythemia vera and / or essential thrombocythemia;(iii) primary myelofibrosis;(iv) polycythemia vera; or(v) essential thrombocythemia.

8. An anti-CD24 antibody for use according to any one of the preceding claims, the method comprising:(i) determining, from a biological sample obtained from the patient, whether the myeloproliferative neoplasm comprises a mutation in the JAK2 gene and / or elevated levels of CD24 expression in granulocytes / neutrophils; and(ii) if there is a mutation in the JAK2 gene and / or elevated levels of CD24 expression in the myeloproliferative neoplasm, administering to said patient a therapeutically effective amount of an anti-CD24 antibody.

9. An anti-CD24 antibody for use according to claim 8, wherein:(i) the biological sample comprises granulocytes / neutrophils or granulocyte / neutrophil- derived nucleic acid; and / or(ii) the biological sample is a blood sample.

10. An anti-CD24 antibody for use in a method of treatment of a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; in which the myeloproliferative neoplasm possess a JAK2 mutation and / or elevated levels of CD24 expression, the method comprising the administration to a patient in need thereof a therapeutically effective amount of an anti-CD24 antibody.

11. An anti-CD24 antibody for use according to claim 10, wherein a biological sample comprising granulocytes / neutrophils or granulocyte / neutrophil-derived nucleic acid is obtained from the pateient in order to determine whether the myeloproliferative neoplasm possess a JAK2 mutation and / or elevated levels of CD24 expression; and optionally wherein the biological sample is a blood sample.

12. An anti-CD24 antibody for use according to any one of the preceding claims, wherein the anti-CD24 antibody is selected from any one of the following: GB-7011, ALB9, SWA11, SN3, G7, ATG-31 , IMM-47, IMM-4701, 1 H5C4, 2Q1282, 32D12, 3B6, 4F4E10, B3298M, BA-1, DMC213, DMC217, EPR19925, MAB5247, ML5, QA21A28, S8230.8, W20001 B, Rabbit Anti- CD24 Polyclonal Antibody (MRO-1739-CN), Human Anti-CD24 Recombinant Antibody, scFv Fragment (TAB-007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10), HumanAnti-CD24 Recombinant Antibody VS-0723-WK217, Human Anti-CD24 Recombinant Antibody VS-0723-WK207, Human Anti-CD24 Recombinant Antibody Fab Fragment TAB-007LC-F(E)), Human Anti-CD24 Recombinant Antibody (TAB-007LC), Mouse Anti-CD24 Recombinant Antibody (TAB-006LC) (TAB-006LC), Human Anti-CD24 Recombinant Antibody scFv Fragment (TAB-007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10) (VS3-XY243), Mouse Anti-CD24 Recombinant Antibody scFv Fragment (TAB-006LC-S(P)), or Mouse Anti- CD24 Recombinant Antibody Fab Fragment (TAB-006LC-F(E)).

13. An anti-CD24 antibody for use according to any one of the preceding claims, wherein the anti-CD24 antibody is a humanized antibody.

14. A pharmaceutical composition comprising an anti-CD24 antibody and one or more pharmaceutically-acceptable excipients, wherein the pharmaceutical composition is for use in the treatment of:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; wherein the myeloproliferative neoplasm optionally comprises a JAK2 mutation and / or elevated CD24 expression.

15. A pharmaceutical composition for use according to claim 14, wherein:(i) the anti-CD24 antibody is a humanized antibody; and / or(ii) the anti-CD24 antibody is selected from any one of the following: GB-7011 , ALB9, SWA11, SN3, G7, ATG-31 , IMM-47, IMM-4701, 1 H5C4, 2Q1282, 32D12, 3B6, 4F4E10, B3298M, BA-1, DMC213, DMC217, EPR19925, MAB5247, ML5, QA21A28, S8230.8, W20001B, Rabbit Anti-CD24 Polyclonal Antibody (MRO- 1739-CN), Human Anti-CD24 Recombinant Antibody, scFv Fragment (TAB- 007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10), Human Anti-CD24 Recombinant Antibody VS-0723-WK217, Human Anti-CD24 Recombinant Antibody VS-0723-WK207, Human Anti-CD24 Recombinant Antibody Fab Fragment TAB-007LC-F(E)), Human Anti-CD24 Recombinant Antibody (TAB-007LC), Mouse Anti-CD24 Recombinant Antibody (TAB- 006LC) (TAB-006LC), Human Anti-CD24 Recombinant Antibody scFv Fragment (TAB-007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10) (VS3-XY243), Mouse Anti-CD24 Recombinant Antibody scFv Fragment (TAB-006LC-S(P)), or Mouse Anti-CD24 Recombinant Antibody Fab Fragment (TAB-006LC-F(E)).

16. The use of an anti-CD24 antibody in the manufacture of a medicament for use in the treatment of:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia;wherein the myeloproliferative neoplasm optionally comprises a JAK2 mutation and / or elevated CD24 expression.

17. The use of an anti-CD24 antibody in the manufacture of a medicament according to claim 16, wherein:(i) the anti-CD24 antibody is a humanized antibody; and / or(ii) the anti-CD24 antibody is selected from any one of the following: GB-7011 , ALB9, SWA11, SN3, G7, ATG-31 , IMM-47, IMM-4701, 1 H5C4, 2Q1282, 32D12, 3B6, 4F4E10, B3298M, BA-1, DMC213, DMC217, EPR19925, MAB5247, ML5, QA21A28, S8230.8, W20001B, Rabbit Anti-CD24 Polyclonal Antibody (MRO- 1739-CN), Human Anti-CD24 Recombinant Antibody, scFv Fragment (TAB- 007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10), Human Anti-CD24 Recombinant Antibody VS-0723-WK217, Human Anti-CD24 Recombinant Antibody VS-0723-WK207, Human Anti-CD24 Recombinant Antibody Fab Fragment TAB-007LC-F(E)), Human Anti-CD24 Recombinant Antibody (TAB-007LC), Mouse Anti-CD24 Recombinant Antibody (TAB- 006LC) (TAB-006LC), Human Anti-CD24 Recombinant Antibody scFv Fragment (TAB-007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10) (VS3-XY243), Mouse Anti-CD24 Recombinant Antibody scFv Fragment (TAB-006LC-S(P)), or Mouse Anti-CD24 Recombinant Antibody Fab Fragment (TAB-006LC-F(E)).

18. A method of treating:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; the method comprising administering to a patient in need thereof a therapeutically effective amount of an anti-CD24 antibody.

19. A method for selecting a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; for treatment with an anti-CD24 antibody, the method comprising assessing the JAK2 mutation status of myeloproliferative neoplasm and / or whether the myeloproliferative neoplasm has elevated CD24 expression, wherein if the myeloproliferative neoplasm are JAK2 mutation positive and / or has elevated CD24 expression, the patient is selected for treatment with an anti- CD24 antibody.

20. A method for selecting a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; the method comprising:(i) determining whether the myeloproliferative neoplasm possesses a JAK2 mutation and / or elevated levels of CD24 expression; and(ii) administering to a patient whose myeloproliferative neoplasm possesses a JAK2 mutation or elevated levels of CD24 expression a therapeutically effective amount of an anti-CD24 antibody.

21. A method according to any one of claims 17 to 19, wherein:(i) the anti-CD24 antibody is a humanized antibody; and / or(ii) the anti-CD24 antibody is selected from any one of the following: GB-7011 , ALB9, SWA11, SN3, G7, ATG-31 , IMM-47, IMM-4701, 1 H5C4, 2Q1282, 32D12, 3B6, 4F4E10, B3298M, BA-1, DMC213, DMC217, EPR19925, MAB5247, ML5, QA21A28, S8230.8, W20001B, Rabbit Anti-CD24 Polyclonal Antibody (MRO- 1739-CN), Human Anti-CD24 Recombinant Antibody, scFv Fragment (TAB- 007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10), Human Anti-CD24 Recombinant Antibody VS-0723-WK217, Human Anti-CD24 Recombinant Antibody VS-0723-WK207, Human Anti-CD24 Recombinant Antibody Fab Fragment TAB-007LC-F(E)), Human Anti-CD24 Recombinant Antibody (TAB-007LC), Mouse Anti-CD24 Recombinant Antibody (TAB- 006LC) (TAB-006LC), Human Anti-CD24 Recombinant Antibody scFv Fragment (TAB-007LC-S(P)), Mouse Anti-CD24 Recombinant Antibody (clone 4F4E10) (VS3-XY243), Mouse Anti-CD24 Recombinant Antibody scFvFragment (TAB-006LC-S(P)), or Mouse Anti-CD24 Recombinant Antibody Fab Fragment (TAB-006LC-F(E)).

22. A JAK2 mutation diagnostic test for use in a method of selecting a patient having a condition selected from:(i) myelofibrosis and / or thrombosis in a patient having a myeloproliferative neoplasm;(ii) myelofibrosis in a patient having a myeloproliferative neoplasm;(iii) myelofibrosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(iv) thrombosis in a patient having a myeloproliferative neoplasm;(v) thrombosis in a patient having a myeloproliferative neoplasm selected from primary myelofibrosis, polycythemia vera and / or essential thrombocythemia;(vi) secondary myelofibrosis in a patient having a myeloproliferative neoplasm.(vii) secondary myelofibrosis in a patient having a myeloproliferative neoplasm selected from polycythemia vera and / or essential thrombocythemia;(viii) secondary myelofibrosis in a patient having polycythemia vera;(ix) secondary myelofibrosis in a patient having essential thrombocythemia; or(x) essential thrombocythemia; wherein the myeloproliferative neoplasm optionally comprises a JAK2 mutation and / or elevated CD24 expression; for treatment with an anti-CD24 antibody.

23. A JAK2 mutation diagnostic test for use according to claim 22, wherein the method of selecting a patient for treatment comprises obtaining a biological sample from the patient (e.g. a blood sample) and testing the sample for JAK2 mutation status using the JAK2 mutation diagnostic test.

24. The use of CD24 expression as a marker of the response to JAK inhibitor therapy.

25. A method of monitoring therapy with a JAK inhibitor, the method comprising administering a JAK inhibitor to a subject and measuring the CD24 expression levels, optionally over time or at predetermined times during the therapy.

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