Antibodies, compositions and methods of treatment

Abstract

The present invention provides anti-PSGL-1 antibodies and their therapeutic use for the prevention and / or treatment of diseases. In particular, the disease to be prevented and / or treated may be an inflammatory disease (particularly involving the accumulation of eosinophils and / or neutrophils). Treatment may be effective against asthma or COPD. The antibody may be humanized and may be defined with reference to the sequences set forth herein. The antibody may have high affinity for PSGL-1.

Description

[Technical Field]

[0001] The present invention relates to anti-human PSGL-1 antibodies. The present invention also relates to methods for treating various diseases and conditions by reducing inflammation. [Background technology]

[0002] P-selectin glycoprotein ligand-1 (PSGL-1) is a component of the innate immune system and is found on leukocytes and endothelial cells. PSGL-1 binds to members of the selectin family of adhesion molecules, including P-selectin, E-selectin, and L-selectin, but binds with the highest affinity to P-selectin. PSGL-1 also binds to major chemokines known to induce cell chemotaxis toward areas of inflammation.

[0003] PSGL-1 promotes binding between leukocytes, platelets, and the endothelium at sites of hypoxia, endothelial cell injury, and infection. Although normally a beneficial process, when activation of these cells occurs unnecessarily or excessively, PSGL-1-mediated cell-cell interactions can trigger a cascade of events culminating in the extravasation of inflammatory cells into tissues such as the lung parenchyma and bronchoalveolar spaces, resulting in pathological inflammation.

[0004] Asthma and chronic obstructive pulmonary disease (COPD) are common pulmonary inflammatory diseases characterized by excessive leukocyte infiltration into the lung parenchyma and bronchoalveolar spaces, resulting in tissue damage. Asthma is a common chronic pulmonary inflammatory disease characterized by episodes of wheezing, shortness of breath, chest tightness, and coughing, and is often accompanied by eosinophilic airway inflammation. Airway eosinophilia has been associated with asthma exacerbations and has been shown to contribute to airway remodeling. COPD is one of the leading causes of death worldwide, and few effective therapies exist. Particulate foreign matter, such as that from cigarette smoke, triggers the recruitment of inflammatory cells into the bronchial wall and endobronchial lumen, leading to progressive loss of lung function. Neutrophil accumulation at inflammatory sites in COPD may contribute to the pathophysiology of the disease by, for example, secreting proteases that cause tissue destruction or releasing mediators that further enhance or prolong the inflammatory response.

[0005] Current therapies for moderate to severe asthma include inhaled corticosteroids, inhaled long- or short-acting beta-agonists, inhaled long- or short-acting anticholinergics, leukotriene modifiers, sodium cromoglycate, theophylline, or oral corticosteroids. Five biologics are currently approved for the treatment of severe asthma, including reslizumab (for eosinophilic asthma; anti-IL-5), mepolizumab (for eosinophilic asthma; anti-IL-5), omalizumab (for allergic asthma; anti-IgE), benralizumab (for eosinophilic asthma; anti-IL-5 receptor), and tezepelumab (for severe asthma; thymic stromal lymphopoietin).

[0006] Current therapies for COPD include short-acting and long-acting beta-2 agonists, as well as inhaled corticosteroid combination therapy, theophylline, and antibiotics. While these therapies are effective in some individuals, exacerbations still occur, and no disease-modifying therapies are currently available. Neutrophil-mediated inflammation in COPD is thought to play a major role in the pathophysiology of the disease, but effective anti-inflammatory therapies remain lacking. Roflumilast, an anti-inflammatory phosphodiesterase-4 (PDE4) inhibitor, was approved by the FDA in 2011 for COPD patients. PDE4 inhibition has shown some promise, but is only effective in a subset of patients and is associated with nausea, diarrhea, headache, and weight loss.

[0007] There remains a strong unmet clinical need for therapies for pulmonary diseases such as asthma or COPD, particularly those characterized by airway eosinophilia and / or neutrophilia.

[0008] Sickle cell disease (SCD) is an inherited blood disorder characterized by the presence of an abnormal hemoglobin known as hemoglobin S (HbS). This abnormal hemoglobin causes red blood cells to assume a characteristic sickle or crescent shape. This, in turn, leads to pathological features including vaso-occlusion (when sickle-shaped red blood cells clump together and block small blood vessels) and chronic inflammation. These processes can lead to the symptoms of SCD, such as pain crises and tissue damage.

[0009] There is a strong unmet clinical need for therapies for SCD and its symptoms, such as pain crises and chronic inflammation.

[0010] Indeed, there remains an unmet need for therapies for inflammatory diseases or disorders. Summary of the Invention

[0011] According to a first aspect of the present invention, there is provided a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2.

[0012] In a preferred embodiment, the humanized anti-PSGL-1 antibody or antigen-binding fragment thereof according to the first aspect of the present invention comprises all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2.

[0013] In a second aspect, the present invention provides an anti-PSGL-1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29, or comprising a heavy chain sequence set forth in SEQ ID NO: 1 and a light chain sequence set forth in SEQ ID NO: 2.

[0014] According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising a humanized anti-PSGL-1 antibody, or an antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2, and a pharmaceutically acceptable excipient.

[0015] In a preferred embodiment, the humanized anti-PSGL-1 antibody or antigen-binding fragment thereof present in the pharmaceutical composition according to the third aspect of the present invention comprises all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2.

[0016] According to a fourth aspect of the present invention, there is provided a polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody or antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2.

[0017] According to a fifth aspect of the present invention, there is provided a polynucleotide sequence encoding a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, comprising an immunoglobulin chain comprising at least one CDR of an antibody heavy chain defined in SEQ ID NO: 1 and an immunoglobulin chain comprising at least one CDR of an antibody light chain defined in SEQ ID NO: 2.

[0018] In a preferred embodiment, a polynucleotide sequence according to the fourth or fifth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1. In a preferred embodiment, a polynucleotide sequence according to the fourth or fifth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody light chain defined in SEQ ID NO: 2. In a preferred embodiment, a polynucleotide sequence according to the fourth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2. In a preferred embodiment, a polynucleotide sequence according to the fifth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2.

[0019] According to a sixth aspect of the present invention, there is provided a cell comprising a polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, the immunoglobulin chain comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2.

[0020] According to a seventh aspect of the present invention, there is provided a cell comprising a polynucleotide sequence encoding a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, the antibody comprising an immunoglobulin chain comprising at least one CDR of an antibody heavy chain defined in SEQ ID NO: 1 and an immunoglobulin chain comprising at least one CDR of an antibody light chain defined in SEQ ID NO: 2.

[0021] In a preferred embodiment, a polynucleotide sequence present in a cell according to the sixth or seventh aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1. In a preferred embodiment, a polynucleotide sequence present in a cell according to the sixth or seventh aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody light chain defined in SEQ ID NO: 2. In a preferred embodiment, a polynucleotide sequence present in a cell according to the sixth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2. In a preferred embodiment, a polynucleotide sequence present in a cell according to the seventh aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2.

[0022] According to an eighth aspect of the present invention, there is provided a method for producing a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2, the method comprising expressing a first polynucleotide sequence encoding an immunoglobulin chain of the humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1, and expressing a second polynucleotide sequence encoding an immunoglobulin chain of the humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof comprising at least one CDR of the antibody light chain defined in SEQ ID NO: 2.

[0023] In a preferred embodiment, the first polynucleotide sequence expressed in the method according to the eighth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain set forth in SEQ ID NO: 1. In a preferred embodiment, the second polynucleotide sequence expressed in the method according to the eighth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody light chain set forth in SEQ ID NO: 2. In a preferred embodiment, the first polynucleotide sequence expressed in the method according to the eighth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody heavy chain set forth in SEQ ID NO: 1 and the second polynucleotide sequence expressed in the method according to the eighth aspect of the invention encodes an immunoglobulin chain comprising all three CDRs of the antibody light chain set forth in SEQ ID NO: 2.

[0024] According to a ninth aspect of the present invention, there is provided a method for preventing or treating a disease or condition in a subject in need thereof, comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody or antigen-binding fragment thereof, wherein the antibody comprises at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. The antibody may be an anti-PSGL-1 antibody or antigen-binding fragment thereof according to any embodiment of the first aspect of the invention, or an anti-PSGL-1 antibody or antigen-binding fragment thereof according to the second aspect of the invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises all three CDRs of the antibody heavy chain set forth in SEQ ID NO: 1 and all three CDRs of the antibody light chain set forth in SEQ ID NO: 2.

[0025] According to a tenth aspect of the present invention, there is provided an anti-PSGL-1 antibody or antigen-binding fragment thereof for use as a medicament, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2. The antibody may be an anti-PSGL-1 antibody or antigen-binding fragment thereof according to any embodiment of the first aspect of the invention, or an anti-PSGL-1 antibody or antigen-binding fragment thereof according to the second aspect of the invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises all three CDRs of the antibody heavy chain defined in SEQ ID NO: 1 and all three CDRs of the antibody light chain defined in SEQ ID NO: 2.

[0026] The antibodies or antigen-binding fragments thereof of the invention according to the first or second aspect of the invention are suitable for use in the inventive method of treatment of the ninth aspect of the invention or in the inventive medical use of the tenth aspect of the invention. They may also be incorporated into the pharmaceutical composition of the third aspect of the invention.

[0027] The therapeutic method according to the ninth aspect of the invention, or the antibody or antigen-binding fragment thereof used according to the tenth aspect of the invention, can be used in the prevention or treatment of diseases or conditions associated with the binding of PSGL-1 to its ligand.

[0028] The antibody or antigen-binding fragment thereof used according to the tenth aspect of the invention may be used in the form of a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient, such as the composition of the third aspect of the invention.

[0029] In an eleventh aspect of the present invention there is provided a polynucleotide sequence encoding the heavy chain variable sequence set out in SEQ ID NO:26.

[0030] In a twelfth aspect of the invention there is provided a polynucleotide sequence encoding the light chain variable sequence set out in SEQ ID NO:29.

[0031] In a thirteenth aspect of the present invention there is provided a cell comprising a polynucleotide sequence encoding the heavy chain sequence set out in SEQ ID NO:1 and a polynucleotide sequence encoding the light chain sequence set out in SEQ ID NO:2.

[0032] For purposes of this disclosure, a therapeutically effective amount of an antibody or antigen-binding fragment thereof or pharmaceutical composition may be considered to refer to an amount or dose of an antibody or fragment or a pharmaceutical composition comprising an antibody or fragment sufficient to produce a desired therapeutic or beneficial effect in a patient or individual. A therapeutically effective amount may be an amount sufficient to reduce or delay the onset of a disease or condition, or an amount sufficient to reduce or delay the onset of one or more symptoms of a disease or condition. A therapeutically effective amount may be an amount sufficient to provide at least partial relief of a disease or condition, or an amount sufficient to provide at least partial relief of one or more symptoms of a disease or condition. A therapeutically effective amount may be an amount sufficient to completely prevent the onset of a disease or condition, or an amount sufficient to completely prevent the onset of one or more symptoms of a disease or condition. A therapeutically effective amount may be an amount sufficient to completely alleviate a disease or condition, or an amount sufficient to completely alleviate one or more symptoms of a disease or condition. Other specific considerations regarding therapeutically effective amounts of an antibody, antibody fragment, or pharmaceutical composition, taking into account the particular therapeutic outcome of a subject, are described elsewhere herein. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram of the SelK2 antibody, showing the arrangement and linkage of the antibody domains. [Figure 2] FIG. 1 shows the results of a kinetic assay of SelSP1 binding to captured parental antibody. [Figure 3] FIG. 1 shows the results of a kinetic assay of SelSP1 binding to captured SelK2 (high density surface). [Figure 4] FIG. 1 shows the results of a kinetic assay of SelSP1 binding to captured SelK2 (low density surface). [Figure 5] FIG. 1 shows the results of a study demonstrating the ability of SelK2 to inhibit neutrophil rolling. [Figure 6] 1 shows a parental antibody and a SelK2 antibody of the invention that inhibit the binding of the chemokine CCL27 and its interaction with PSGL-1. A complete assay cycle for CCL27. Injection "A" is surface blocking, injection "B" is CCL27, injection "C" is SDS, and injection "D" is NaOH. [Figure 7] Blocking of parental and SelK2 antibodies, zoomed in on injection "B" and re-normalized to before injection. Injection of CCL27 (990 nM). The solid curve represents the buffer-blocked surface, the dash-dotted curve represents the K2-blocked surface, and the dashed curve represents the SelK2-blocked surface. [Figure 8] FIG. 1 shows SelK2 inhibition of human neutrophil binding to P-selectin under flow. [Figure 9] FIG. 1 shows the change in mean serum concentration of SelK2 over time (PK) after two doses of 7.5 mg / kg administered 21 days apart. [Figure 10] FIG. 1 shows the change in mean % inhibition of Psel-Ig as a function of SelK2 serum concentration over time (PD) after two doses of 7.5 mg / kg, 21 days apart. [Figure 11] FIG. 1 shows the % decline in FEV1 (mean±SE) in placebo subjects during screening versus placebo subjects during treatment after allergen challenge. [Figure 12] FIG. 1 shows the % decline in FEV1 (mean±SE) at day 36 (after 5 weeks of treatment) following allergen challenge in asthmatic subjects receiving placebo or SelK2 treatment according to the invention. [Figure 13] FIG. 1 shows the mean (±SE) absolute delta eosinophil counts (10E6 / g) in sputum of asthmatic patients treated with placebo or SelK2 after allergen challenge. [Figure 14]FIG. 1 shows the mean (±SE) absolute eosinophil counts (10E9 / L) in the blood of asthmatic patients treated with placebo or SelK2 after allergen challenge. [Figure 15] FIG. 1 shows mean (±SE) differential eosinophil counts (%) in sputum of asthmatic patients treated with placebo or SelK2 after allergen challenge. [Figure 16] FIG. 1 shows the mean (±SE) differential eosinophil counts (%) in the blood of asthmatic patients treated with placebo or SelK2 after allergen challenge. [Figure 17] Figure 1 shows the mean (±SE) absolute delta eosinophil counts (10E6 / g) in sputum of COPD patients treated with placebo or SelK2. Day 15, p=0.0494, Day 22, p=0.0181. [Figure 18] FIG. 1 shows the mean (±SE) absolute differential eosinophil counts (10E6 / L) in the blood of COPD patients treated with placebo or SelK2. [Figure 19] Mean (±SE) absolute differential epithelial cell counts (10E6 / g) in sputum of COPD patients treated with placebo or SelK2. P=0.0167 [Figure 20] FIG. 1 shows a comparison of % decline in FEV1 after allergen challenge in patients with mild asthma treated with SelK2 versus tezepelumab. [Figure 21] FIG. 1 shows the mean (±SE) absolute number of eosinophils in sputum at baseline and day 22 in COPD patients treated with placebo or SelK2. [Figure 22] FIG. 1 shows the mean (±SE) absolute number of neutrophils in sputum at baseline and day 22 in COPD patients treated with placebo or SelK2. [Figure 23] FIG. 1 shows the mean (±SE) total number of cells per gram of sputum at baseline and day 22 in COPD patients treated with placebo or SelK2. DETAILED DESCRIPTION OF THE INVENTION

[0034] Embodiments of the present disclosure provide humanized antibodies and antigen-binding fragments thereof that bind to PSGL-1. Potential advantages of these antibodies include their ability to bind to PSGL-1 with high affinity and the potential for beneficial therapeutic activity. Advantages of the disclosed antibodies may include their ability to function as blocking antibodies, which may be capable of inhibiting the formation of a complex between bound PSGL-1 and P-selectin. Alternatively, or additionally, the disclosed antibodies may be capable of disrupting a pre-formed complex between PSGL-1 and P-selectin. It is readily apparent that these properties may confer therapeutic utility to the disclosed antibodies, leading to methods of treatment and medical uses using the antibodies (or antigen-binding fragments thereof) of the invention, as well as pharmaceutical compositions comprising the antibodies (or antigen-binding fragments thereof).

[0035] Antibody humanization is a well-established technique. However, the reduced immunogenicity of humanized antibodies often comes at the cost of reduced affinity compared to the parent antibody from which they are derived. Surprisingly, the antibodies of the present invention exhibit improved affinity compared to their parent antibodies, while also exhibiting the low immunogenicity required for therapeutic use.

[0036] Furthermore, clinical trials using the antibodies of the invention have demonstrated that they provide surprisingly effective treatments for respiratory conditions such as asthma and COPD.

[0037] Treatment with the antibodies of the present invention reduced the number of eosinophils in the sputum of asthma patients. Furthermore, the lung function of asthma patients was significantly improved by treatment with the antibodies disclosed herein. In fact, the therapeutic effect achieved was greater than that obtained with alternative drugs currently available in the clinical treatment of asthma, indicating that treatment with these new antibodies may be an improvement over currently available treatments.

[0038] In patients with COPD, treatment with the antibodies of the invention resulted in a significant reduction in the number of eosinophils, neutrophils and epithelial cells in sputum samples.

[0039] Moreover, the properties of the antibodies of the present invention clearly indicate that these benefits may be conferred with respect to many other pathologies characterized by the presence of unwanted inflammation.

[0040] Excessive or abnormal recruitment of leukocytes is at the heart of many disorders, particularly inflammatory diseases or conditions. Extravasation of white blood cells, such as eosinophils or neutrophils, leads to their accumulation in tissues, where their presence can have deleterious effects. Adhesion and rolling of white blood cells are necessary steps before extravasation, and PSGL-1 plays an important role in these processes. PSGL-1 on eosinophils may also bind to P-selectin on the surface of activated platelets, and this binding may initiate the upregulation of additional adhesion molecules on eosinophils, which also increases their binding to the blood vessel wall and extravasation.

[0041] Upregulation of P-selectin on endothelial cells and platelets in patients with sickle cell disease contributes to cell-cell interactions involved in the pathogenesis of vaso-occlusive and pain crises associated with this disease. White blood cell extravasation also plays a role in the excessive inflammation that can occur in SCD.

[0042] Without wishing to be bound by any hypothesis, the ability of the antibodies of the present invention, or antibodies suitable for use in the therapeutic methods or medical uses of the present invention, to inhibit or disrupt the binding of PSGL-1 to its ligand may facilitate the ability to reduce or reverse the adhesion and / or rolling activity of leukocytes, thereby preventing leukocyte extravasation. This impact on central processes within the inflammatory pathway indicates that these treatments may provide therapies for a wide range of diseases or conditions associated with leukocyte recruitment and inflammation.

[0043] Similarly, the ability of antibodies to inhibit or disrupt the binding of PSGL-1 to its ligand may enable these antibodies to reduce or reverse the cell-cell interactions involved in the vaso-occlusive and / or pain crises associated with SCD, and thus treatment with such antibodies may result in the effective prevention or alleviation of these symptoms of SCD.

[0044] The invention will now be further described with reference to the following terms and definitions.

[0045] Antibodies of the Invention A first aspect of the present invention provides a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2.

[0046] The antibody or antigen-binding fragment thereof of the present invention may comprise at least two CDRs of the antibody heavy chain set forth in SEQ ID NO: 1. The antibody or antigen-binding fragment thereof of the present invention may comprise three CDRs of the antibody heavy chain set forth in SEQ ID NO: 1.

[0047] The antibody or antigen-binding fragment thereof of the present invention may comprise at least two CDRs of the antibody light chain set forth in SEQ ID NO: 2. The antibody or antigen-binding fragment thereof of the present invention may comprise three CDRs of the antibody light chain set forth in SEQ ID NO: 2.

[0048] An antibody or antigen-binding fragment thereof of the invention may comprise the three CDRs of the antibody heavy chain set forth in SEQ ID NO:1 and the three CDRs of the antibody light chain set forth in SEQ ID NO:2.

[0049] These antibodies or antigen-binding fragments thereof of the invention can be used in the pharmaceutical compositions of the third aspect of the invention, and their immunoglobulin chains can also be encoded by polynucleotides of the fourth or fifth aspect of the invention.

[0050] The anti-PSGL-1 antibody or antigen-binding fragment thereof of the present invention can be defined as comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29. In another preferred example, the anti-PSGL-1 antibody or antigen-binding fragment thereof of the present invention can be defined as comprising a heavy chain sequence set forth in SEQ ID NO: 1 and a light chain sequence set forth in SEQ ID NO: 2.

[0051] An exemplary anti-PSGL-1 antibody or antigen-binding fragment thereof of the invention is defined by the heavy chain amino acid sequence set forth in SEQ ID NO:1 and the light chain amino acid sequence set forth in SEQ ID NO:2.

[0052] The heavy and light chain sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively, comprise the variable region sequences set forth in SEQ ID NO:26 and SEQ ID NO:29.

[0053] This results in the second aspect of the invention, comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, Anti-PSGL-1 antibodies or antigen-binding fragments thereof are provided.

[0054] The antibody or antigen-binding fragment thereof according to this second aspect of the invention may not comprise the constant regions set forth in SEQ ID NO:1 and SEQ ID NO:2, but may comprise a heavy chain variable sequence set forth in SEQ ID NO:26 and a light chain variable sequence set forth in SEQ ID NO:29.

[0055] An antibody or antigen-binding fragment thereof according to this second aspect of the invention may comprise the heavy chain amino acid sequence set out in SEQ ID NO: 1 and the light chain amino acid sequence set out in SEQ ID NO: 2. An antibody according to this aspect of the invention may consist of the heavy chain amino acid sequence set out in SEQ ID NO: 1 and the light chain amino acid sequence set out in SEQ ID NO: 2.

[0056] In a preferred embodiment, the antibody according to the second aspect of the invention is a humanized antibody.

[0057] Any antibody or antigen-binding fragment thereof according to either the first or second aspect of the present invention may be referred to as an "antibody of the present invention." For the avoidance of doubt, antibodies of the present invention should also be construed to encompass antigen-binding antibody fragments that meet specified requirements, such as affinity or ability to reduce binding of PSGL-1 to P-selectin or other ligands. Functional properties that suitable antigen-binding fragments of antibodies may possess are further discussed elsewhere herein. Unless the context requires otherwise, antigen-binding fragments of antibodies may be used as a suitable example of "antibody" in any of the aspects or embodiments of the present invention described herein, regardless of whether such use is explicitly mentioned.

[0058] Antibodies suitable for use in the therapeutic methods or medical uses of the present invention The ninth and tenth aspects of the present invention relate to methods of treatment using anti-PSGL-1 antibodies and medical uses of such anti-PSGL-1 antibodies, respectively. In each of these aspects of the present invention, the anti-PSGL-1 antibody is defined as comprising at least one CDR in the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR in the antibody light chain set forth in SEQ ID NO: 2.

[0059] It will be understood that the anti-PSGL-1 antibodies of the present invention represent suitable examples of anti-PSGL-1 antibodies that may be used in the therapeutic methods or medical uses of the present invention. Indeed, it will be understood that any of the anti-PSGL-1 antibodies of the present invention or antigen-binding fragments thereof described herein represent suitable examples of anti-PSGL-1 antibodies that may be used in the therapeutic methods or medical uses of the present invention.

[0060] Therefore, unless the context requires otherwise, all discussion in this disclosure regarding "antibody" or "antibodies" should be interpreted as relating to both the anti-PSGL-1 antibodies of the present invention and the anti-PSGL-1 antibodies that may be used in the therapeutic methods or medical uses of the present invention.

[0061] Antibodies that may be used in the therapeutic methods or medical uses of the present invention should be construed to include antibody fragments, particularly antigen-binding fragments, that meet the specified requirements, as discussed in more detail elsewhere herein.

[0062] Exemplary anti-PSGL-1 antibodies of the present invention comprising or consisting of a heavy chain amino acid sequence set forth in SEQ ID NO: 1 and a light chain amino acid sequence set forth in SEQ ID NO: 2 may be used in the therapeutic methods or medical uses of the present invention.

[0063] Anti-PSGL-1 antibody The antibodies of the present invention and antibodies suitable for use in the therapeutic methods or medical uses of the present invention are all antibodies that specifically bind to human PSGL-1, and therefore, all references to antibodies in the context of various aspects of the present disclosure should be construed as referring to anti-human PSGL-1 antibodies, unless the context requires otherwise.

[0064] In a preferred embodiment, the anti-PSGL-1 antibodies of the invention bind to the same epitope as the antibodies defined by SEQ ID NO:1 and SEQ ID NO:2.

[0065] In a preferred embodiment, an anti-PSGL-1 antibody suitable for use in the therapeutic methods or medical uses of the present invention binds to the same epitope as that bound by the antibodies defined by SEQ ID NO:1 and SEQ ID NO:2.

[0066] Exemplary anti-PSGL-1 antibodies of the invention suitable for use in the therapeutic methods and medical uses of the invention An exemplary anti-PSGL-1 antibody of the invention is designated by the inventors as SelK2. The amino acid heavy chain sequence of this antibody is set forth in SEQ ID NO: 1. The amino acid light chain sequence of this antibody is set forth in SEQ ID NO: 2.

[0067] Preferably, the anti-PSGL-1 antibody of the present invention may comprise an amino acid sequence comprising SEQ ID NO: 1. Preferably, the anti-PSGL-1 antibody of the present invention may comprise an amino acid sequence comprising SEQ ID NO: 2. Preferably, the anti-PSGL-1 antibody of the present invention may comprise both an amino acid sequence comprising SEQ ID NO: 1 and an amino acid sequence comprising SEQ ID NO: 2.

[0068] In a second aspect, the present invention provides a method for producing a composition comprising: comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, Anti-PSGL-1 antibodies are provided.

[0069] In a preferred embodiment, the antibody according to this second aspect of the invention consists of the heavy chain sequence set out in SEQ ID NO:1 and the light chain sequence set out in SEQ ID NO:2.

[0070] In a preferred embodiment of the third aspect of the present invention, comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, A pharmaceutical composition is provided that includes a humanized anti-PSGL-1 antibody and a pharmaceutically acceptable excipient.

[0071] In an eleventh aspect of the present invention, there is provided a polynucleotide sequence encoding the heavy chain variable sequence set forth in SEQ ID NO: 26. Suitably, such polynucleotide of the eleventh aspect of the present invention may encode the heavy chain sequence set forth in SEQ ID NO: 1. In a twelfth aspect of the present invention, there is provided a polynucleotide sequence encoding the light chain variable sequence set forth in SEQ ID NO: 29. Suitably, such polynucleotide of the twelfth aspect of the present invention may encode the light chain sequence set forth in SEQ ID NO: 2.

[0072] In a thirteenth aspect of the present invention there is provided a cell comprising a polynucleotide sequence encoding the heavy chain sequence set out in SEQ ID NO:1 and a polynucleotide sequence encoding the light chain sequence set out in SEQ ID NO:2.

[0073] In a preferred embodiment of the eighth aspect of the present invention, there is provided a method for producing a humanized anti-PSGL-1 antibody comprising a heavy chain sequence defined in SEQ ID NO: 1 and a light chain sequence defined in SEQ ID NO: 2, the method comprising expressing a polynucleotide sequence encoding the heavy chain sequence defined in SEQ ID NO: 1 and expressing a polynucleotide sequence encoding the light chain sequence defined in SEQ ID NO: 2.

[0074] In a preferred embodiment of the ninth aspect of the invention there is provided a method of preventing or treating a disease or condition in a subject in need thereof, comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody, wherein the antibody: comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, A method is provided.

[0075] In a preferred embodiment of the tenth aspect of the present invention, there is provided an anti-PSGL-1 antibody for use as a medicament, comprising: comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, Anti-PSGL-1 antibodies are provided.

[0076] Alternative anti-PSGL-1 antibodies of the invention suitable for use in the therapeutic methods and medical uses of the invention Other anti-PSGL-1 antibodies of the invention suitable for use in the therapeutic methods and medical uses of the invention can be defined by reference to their similarity to the reference heavy and light chain sequences set forth, respectively, in SEQ ID NO: 1 and SEQ ID NO: 2. These similarities can be defined by reference to exemplary heavy or light chain variable region sequences or by reference to full-length heavy or light chain sequences.

[0077] In particular, further anti-PSGL-1 antibodies of the present invention suitable for use in the therapeutic methods and medical uses of the present invention can be defined by reference to the amino acid sequences of the reference heavy and light chain sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, or the degree of identity to the variable regions of these sequences (set forth in SEQ ID NO: 26 and SEQ ID NO: 29, respectively).

[0078] The percentage identity for a sequence is often referred to as "sequence identity" and is a measure used to quantify the degree of similarity between two biological sequences, such as DNA, RNA, or protein sequences. This measurement is expressed as a percentage and indicates how closely the sequences match at the same positions or residues. Those skilled in the art will recognize that there are several common methods that can be used to calculate the percentage identity. Suitable methods that can be used to calculate the percentage identity include aligning the sequences using various algorithms available for this purpose, such as BLAST (Basic Local Alignment Search Tool), ClustalW, or specialized protein alignment software such as MUSCLE or MAFFT, then counting the matching positions, determining the total number of aligned positions, and calculating the percentage identity using the following formula: percentage identity = (number of matching positions / total number of aligned positions) × 100.

[0079] Surrogate antibodies comprising modified versions of the variable regions present in SEQ ID NO: 1 and SEQ ID NO: 2 may share one or more of the functional properties defined herein for the antibodies defined by SEQ ID NO: 1 and SEQ ID NO: 2. For example, they may share the affinity of the antibodies defined by SEQ ID NO: 1 and SEQ ID NO: 2 and / or the ability to block PSGL-1 binding that these antibodies exhibit.

[0080] SEQ ID NO: 26 is the heavy chain variable region of the heavy chain sequence set forth in SEQ ID NO: 1. In a preferred embodiment, an antibody of the invention or an antibody suitable for use in the therapeutic methods or medical uses of the invention comprises a heavy chain variable region based on that set forth in SEQ ID NO: 26.

[0081] For example, such an antibody may comprise a heavy chain variable region having an amino acid sequence that contains no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO: 26. Such an antibody may comprise a heavy chain variable region having an amino acid sequence that contains no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid changes compared to the sequence set forth in SEQ ID NO: 26.

[0082] SEQ ID NO: 29 is the light chain variable region of the light chain sequence set forth in SEQ ID NO: 2. In a preferred embodiment, an antibody of the invention or an antibody suitable for use in the therapeutic methods or medical uses of the invention comprises a light chain variable region based on that set forth in SEQ ID NO: 29.

[0083] For example, such an antibody may comprise a light chain variable region having an amino acid sequence that contains no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO: 29. Such an antibody may comprise a light chain variable region having an amino acid sequence that contains no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid changes compared to the sequence set forth in SEQ ID NO: 29.

[0084] In preferred embodiments, an antibody of the invention, or an antibody suitable for use in the therapeutic methods or medical uses of the invention, comprises a heavy chain variable region that shares at least 90% identity with the heavy chain variable sequence set forth in SEQ ID NO: 26. In preferred embodiments, such an antibody comprises a heavy chain variable region that shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the heavy chain sequence set forth in SEQ ID NO: 26. In such embodiments, the light chain variable sequence of the antibody may comprise or consist of that set forth in SEQ ID NO: 29.

[0085] Alternatively, or additionally, a suitable antibody may comprise a light chain variable region that shares at least 90% identity with the light chain variable region sequence set forth in SEQ ID NO: 29. In preferred embodiments, such antibodies may comprise a light chain variable region that shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the light chain variable sequence set forth in SEQ ID NO: 29. Suitably, the heavy chain variable sequence of such antibodies may comprise or consist of that set forth in SEQ ID NO: 26.

[0086] In a preferred embodiment, an antibody of the invention, or an antibody suitable for use in a therapeutic method or medical use of the invention, comprises both a heavy chain variable region and a light chain variable region that share at least 90% identity with the heavy chain variable sequence set forth in SEQ ID NO: 26 and at least 90% identity with the light chain variable sequence set forth in SEQ ID NO: 29, respectively. In a preferred embodiment, such an antibody comprises a heavy chain variable region and a light chain variable region that share at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the heavy chain variable sequence set forth in SEQ ID NO: 26 and the light chain variable sequence set forth in SEQ ID NO: 29, respectively.

[0087] In the antibody embodiments defined in the preceding paragraph, the CDRs of the antibody may be identical to the CDRs of the anti-PSGL-1 antibodies defined by SEQ ID NO: 1 and SEQ ID NO: 2, and thus may be identical to the CDRs present in the variable regions of SEQ ID NO: 26 and SEQ ID NO: 29. In such cases, the non-identical residues or regions of the antibody may be limited to the non-CDR portions of the relevant variable regions.

[0088] Thus, an antibody of the invention, or an antibody suitable for use in the therapeutic methods or medical uses of the invention, may suitably comprise the CDRs of the antibody heavy chain sequence set forth in SEQ ID NO: 1 and the CDRs of the antibody light chain sequence set forth in SEQ ID NO: 2, as well as a heavy chain variable region sharing at least 90% identity with the heavy chain sequence set forth in SEQ ID NO: 26 and / or a light chain variable region sharing at least 90% identity with the light chain sequence set forth in SEQ ID NO: 29. In a preferred embodiment, such an antibody comprises the CDRs of SEQ ID NO:1 and SEQ ID NO:2, and a heavy chain variable region that shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of a heavy chain variable region set forth in SEQ ID NO:26 and / or a light chain variable region that shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of a light chain variable region set forth in SEQ ID NO:29.

[0089] Suitably, an antibody of the invention, or an antibody suitable for use in the therapeutic methods or medical uses of the invention, may suitably comprise the CDRs of the antibody heavy chain sequence set forth in SEQ ID NO: 1 and the CDRs of the antibody light chain sequence set forth in SEQ ID NO: 2, and comprises both a heavy chain variable region that shares at least 90% identity with the heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable region that shares at least 90% identity with the light chain variable sequence set forth in SEQ ID NO: 29. In a preferred embodiment, such an antibody comprises the CDRs of SEQ ID NO: 1 and SEQ ID NO: 2, and a heavy chain variable region and a light chain variable region that both share at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with either the heavy chain variable region sequence set forth in SEQ ID NO: 26 or the light chain variable region sequence set forth in SEQ ID NO: 29, respectively.

[0090] Specific examples of heavy chain sequences that may be incorporated into antibodies according to these embodiments include those comprising the heavy chain variable sequences set forth in SEQ ID NO: 27 and SEQ ID NO: 28. These heavy chain variable sequences are components of the surrogate antibodies of the invention generated by the inventors.

[0091] As noted above, SEQ ID NO:26 is the heavy chain variable region of the heavy chain sequence set forth in SEQ ID NO: 1. The serine residue at position 98 of SEQ ID NO:26 constitutes a back mutation compared to the lysine residue present in the acceptor sequence used to generate the humanized antibodies of the invention.

[0092] SEQ ID NO: 27 corresponds to SEQ ID NO: 26, except that an additional backmutation has been incorporated at position 75 of SEQ ID NO: 27. The backmutation at position 75 is a substitution of a serine residue with a proline residue.

[0093] SEQ ID NO:28 corresponds to SEQ ID NO:26 except that four additional backmutations have been incorporated at positions 16, 18, 42, and 75 of SEQ ID NO:28. The backmutation at position 16 is a substitution of an arginine residue with a glycine residue. The backmutation at position 18 is a substitution of a lysine residue with an arginine residue. The backmutation at position 42 is a substitution of a glycine residue with a glutamic acid residue. The backmutation at position 75 is a substitution of a serine residue with a proline residue.

[0094] SEQ ID NO:26 contains the CDRs of SEQ ID NO:1, and SEQ ID NO:27 and SEQ ID NO:28 both retain all of the CDRs of SEQ ID NO:1. SEQ ID NO:27 shares 99% identity with SEQ ID NO:26, and SEQ ID NO:28 shares 96% identity with SEQ ID NO:26.

[0095] SEQ ID NO:29 is the light chain variable region of the light chain sequence set forth in SEQ ID NO:2.

[0096] Examples of alternative light chain sequences that can be incorporated into antibodies according to these embodiments include the light chain sequence set forth in SEQ ID NO: 30, which corresponds to the sequence set forth in SEQ ID NO: 29, except that it incorporates two backmutations at positions 15 and 18 of SEQ ID NO: 30. The backmutation at position 15 is a substitution of a proline residue with a leucine residue. The backmutation at position 18 is a substitution of a proline residue with a glutamine residue.

[0097] SEQ ID NO:30 retains all of the CDRs of SEQ ID NO:2 and shares 98% identity with SEQ ID NO:29.

[0098] Antibodies incorporating either the heavy chain variable sequence of SEQ ID NO: 27, the heavy chain variable sequence of SEQ ID NO: 28 or the light chain variable sequence of SEQ ID NO: 30 may be used in the therapeutic methods or medical uses of the invention.

[0099] It will be understood that the anti-PSGL-1 antibodies of the present invention suitable for use in the therapeutic methods and medical uses of the present invention can also be defined by reference to their similarity to the full-length heavy and light chain sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0100] In a preferred embodiment, an antibody of the invention, or an antibody suitable for use in a therapeutic method or medical use of the invention, comprises a heavy chain having an amino acid sequence which contains no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO: 1. Such an antibody may comprise a heavy chain having an amino acid sequence which contains no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid changes compared to the sequence set forth in SEQ ID NO: 1.

[0101] In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises a light chain having an amino acid sequence which contains no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO: 2. Such an antibody may comprise a light chain having an amino acid sequence which contains no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid changes compared to the sequence set forth in SEQ ID NO: 2.

[0102] In preferred embodiments, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises a heavy chain that shares at least 90% identity with the heavy chain sequence set forth in SEQ ID NO: 1. In preferred embodiments, such an antibody comprises a heavy chain that shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the heavy chain sequence set forth in SEQ ID NO: 1. In such embodiments, the light chain sequence of the antibody may comprise or consist of that set forth in SEQ ID NO:2.

[0103] Alternatively or additionally, a suitable antibody may comprise a light chain that shares at least 90% identity with the light chain sequence set forth in SEQ ID NO: 2. In preferred embodiments, such an antibody may comprise a light chain that shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the light chain sequence set forth in SEQ ID NO: 2. Suitably, the heavy chain sequence of such an antibody may comprise or consist of that set forth in SEQ ID NO: 1.

[0104] In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention shares at least 90% identity with the heavy chain sequence set forth in SEQ ID NO: 1 and at least 90% identity with the light chain sequence set forth in SEQ ID NO: 2. In a preferred embodiment, such an antibody shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with both the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2.

[0105] In the antibody embodiments defined in the preceding paragraph, the CDRs of the antibody may be identical to the CDRs of the anti-PSGL-1 antibody defined by SEQ ID NO: 1 and SEQ ID NO: 2, and the non-identical regions may be limited to the non-CDR portions of the antibody.

[0106] Thus, an antibody of the invention, or an antibody suitable for use in the therapeutic methods or medical uses of the invention, may suitably comprise the CDRs of the antibody heavy chain sequence set forth in SEQ ID NO: 1 and the CDRs of the antibody light chain sequence set forth in SEQ ID NO: 2, and a heavy chain sharing at least 90% identity with the heavy chain sequence set forth in SEQ ID NO: 1 and / or a light chain sharing at least 90% identity with the light chain sequence set forth in SEQ ID NO: 2. In preferred embodiments, such an antibody comprises the CDRs of SEQ ID NO: 1 and SEQ ID NO: 2, and a heavy chain sharing at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the heavy chain sequence set forth in SEQ ID NO: 1 and / or a light chain sharing at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the light chain sequence set forth in SEQ ID NO:2.

[0107] In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention may suitably comprise the CDRs of the antibody heavy chain sequence set forth in SEQ ID NO: 1 and the antibody light chain sequence set forth in SEQ ID NO: 2, and share at least 90% identity with both the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2. In a preferred embodiment, such an antibody comprises the CDRs of SEQ ID NO: 1 and SEQ ID NO: 2, and shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with both the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2.

[0108] CDRs of the antibodies of the present invention or antibodies used in the therapeutic method or medical use The anti-PSGL-1 antibodies of the invention and that may be used in the therapeutic methods or medical uses of the invention are defined by reference to the CDRs they contain. In particular, these antibodies are defined by reference to comprising at least one CDR from the reference antibody heavy chain sequence set forth in SEQ ID NO: 1 and at least one CDR from the reference antibody light chain sequence set forth in SEQ ID NO: 2.

[0109] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises a heavy chain CDR H1 as present in SEQ ID NO: 1. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises a heavy chain CDR H2 as present in SEQ ID NO: 1. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises a heavy chain CDR H3 as present in SEQ ID NO: 1.

[0110] Preferably, the antibody of the invention or the antibody used in the therapeutic methods and medical uses of the invention comprises each of the CDR H1 present in SEQ ID NO: 1, the CDR H2 present in SEQ ID NO: 1 and the CDR H3 present in SEQ ID NO: 1.

[0111] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the light chain CDR L1 present in SEQ ID NO: 2. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the light chain CDR L2 present in SEQ ID NO: 2. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the light chain CDR L3 present in SEQ ID NO: 2.

[0112] Preferably, the antibody of the present invention or the antibody used in the therapeutic methods and medical uses of the present invention comprises each of the CDR L1 present in SEQ ID NO:2, the CDR L2 present in SEQ ID NO:2 and the CDR L3 present in SEQ ID NO:2.

[0113] Preferably, the antibody of the invention or the antibody used in the therapeutic methods and medical uses of the invention comprises each of CDR H1 present in SEQ ID NO: 1, CDR H2 present in SEQ ID NO: 1, CDR H3 present in SEQ ID NO: 1, CDR L1 present in SEQ ID NO: 2, CDR L2 present in SEQ ID NO: 2 and CDR L3 present in SEQ ID NO: 2.

[0114] It will be appreciated that there are many different schemes and conventions by which the amino acid residues that make up a CDR within an antibody heavy or light chain may be identified, including, by way of example only, the Kabat numbering scheme, the Chothia numbering scheme, and the IMGT numbering scheme.

[0115] As a guide to those skilled in the art seeking to practice the invention, Table 15 lists the CDRs of exemplary antibody SelK2: A combination of the Kabat and Chothia numbering schemes, Kabat numbering scheme, IMGT numbering scheme, and Chothia numbering scheme, The sequences are defined in SEQ ID NO: 1 and SEQ ID NO: 2 as identified by the following:

[0116] CDRs that may be contained in the antibodies of the present invention or antibodies suitable for use in the therapeutic methods and medical uses of the present invention may preferably be selected from the sequences of SelK2 listed in Table 15.

[0117] Thus, CDR H1 of an antibody of the invention or an antibody suitable for use in the therapeutic methods and medical uses of the invention may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 and SEQ ID NO: 44. CDR H2 of an antibody of the invention or an antibody suitable for use in the therapeutic methods and medical uses of the invention may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16 and SEQ ID NO: 45. CDR H3 of an antibody of the invention or an antibody suitable for use in the therapeutic methods and medical uses of the invention may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 46.

[0118] CDR L1 of the antibody of the invention or of an antibody suitable for use in the therapeutic methods and medical uses of the invention may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 47. CDR L2 of the antibody of the invention or of an antibody suitable for use in the therapeutic methods and medical uses of the invention may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 and SEQ ID NO: 48. CDR L3 of the antibody of the invention or of an antibody suitable for use in the therapeutic methods and medical uses of the invention may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20 and SEQ ID NO: 49.

[0119] Preferably, the CDRs comprised in an antibody of the invention or an antibody suitable for use in the therapeutic methods and medical uses of the invention can each be selected with reference to the same numbering scheme. Alternatively, each of the CDRs of an antibody of the invention or an antibody suitable for use in the therapeutic methods and medical uses of the invention can be independently selected from those set out in Table 15, such that the antibody comprises CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3.

[0120] Preferably, the antibodies of the invention or antibodies used in the therapeutic methods and medical uses of the invention comprise one or more CDRs from SEQ ID NO: 1 and one or more CDRs from SEQ ID NO: 2, as determined by reference to a combination of the Kabat numbering scheme and the Chothia numbering scheme.

[0121] Preferably, the antibody of the invention according to this embodiment or the antibody for use in the methods of treatment and medical uses of the invention comprises a Kabat / Chothia-defined heavy chain CDR H1 of SEQ ID NO: 3. Preferably, the antibody of the invention according to this embodiment or the antibody for use in the methods of treatment and medical uses of the invention comprises a Kabat / Chothia-defined heavy chain CDR H2 of SEQ ID NO: 4. Preferably, the antibody of the invention according to this embodiment or the antibody for use in the methods of treatment and medical uses of the invention comprises a Kabat / Chothia-defined heavy chain CDR H3 of SEQ ID NO: 5.

[0122] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the Kabat / Chothia-defined CDR H1 of SEQ ID NO: 3, the Kabat / Chothia-defined CDR H2 of SEQ ID NO: 4 and the Kabat / Chothia-defined CDR H3 of SEQ ID NO: 5.

[0123] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Kabat / Chothia-defined light chain CDR L1 of SEQ ID NO: 6. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Kabat / Chothia-defined light chain CDR L2 of SEQ ID NO: 7. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Kabat / Chothia-defined light chain CDR L3 of SEQ ID NO: 8.

[0124] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the Kabat / Chothia-defined CDR L1 of SEQ ID NO: 6, the Kabat / Chothia-defined CDR L2 of SEQ ID NO: 7 and the Kabat / Chothia-defined CDR L3 of SEQ ID NO: 8.

[0125] Suitably, the antibodies of the invention or antibodies for use in the methods of treatment and medical uses of the invention comprise each of the Kabat / Chothia-defined CDR H1 of SEQ ID NO: 3, the Kabat / Chothia-defined CDR H2 of SEQ ID NO: 4, the Kabat / Chothia-defined CDR H3 of SEQ ID NO: 5, the Kabat / Chothia-defined CDR L1 of SEQ ID NO: 6, the Kabat / Chothia-defined CDR L2 of SEQ ID NO: 7 and the Kabat / Chothia-defined CDR L3 of SEQ ID NO: 8.

[0126] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise one or more CDRs from SEQ ID NO: 1 and one or more CDRs from SEQ ID NO: 2, as determined with reference to the Kabat numbering scheme.

[0127] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises a Kabat-defined heavy chain CDR H1 of SEQ ID NO: 9. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises a Kabat-defined heavy chain CDR H2 of SEQ ID NO: 10. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises a Kabat-defined heavy chain CDR H3 of SEQ ID NO: 11.

[0128] Suitably, the antibodies of the invention or used in the methods of treatment and medical uses of the invention comprise each of the Kabat-defined CDR H1 of SEQ ID NO:9, CDR H2 of SEQ ID NO:10 and CDR H3 of SEQ ID NO:11.

[0129] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Kabat-defined light chain CDR L1 of SEQ ID NO: 12. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Kabat-defined light chain CDR L2 of SEQ ID NO: 13. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Kabat-defined light chain CDR L3 of SEQ ID NO: 14.

[0130] Preferably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the Kabat-defined CDR L1 of SEQ ID NO: 12, the Kabat-defined CDR L2 of SEQ ID NO: 13 and the Kabat-defined CDR L3 of SEQ ID NO: 14.

[0131] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the Kabat-defined CDR H1 of SEQ ID NO: 9, the Kabat-defined CDR H2 of SEQ ID NO: 10, the Kabat-defined CDR H3 of SEQ ID NO: 11, the Kabat-defined CDR L1 of SEQ ID NO: 12, the Kabat-defined CDR L2 of SEQ ID NO: 13 and the Kabat-defined CDR L3 of SEQ ID NO: 14.

[0132] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise one or more CDRs from SEQ ID NO: 1 and one or more CDRs from SEQ ID NO: 2 as determined with reference to the IMGT numbering scheme.

[0133] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises an IMGT-defined heavy chain CDR H1 of SEQ ID NO: 15. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises an IMGT-defined heavy chain CDR H2 of SEQ ID NO: 16. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises an IMGT-defined heavy chain CDR H3 of SEQ ID NO: 17.

[0134] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the IMGT-defined CDR H1 of SEQ ID NO: 15, the IMGT-defined CDR H2 of SEQ ID NO: 16 and the IMGT-defined CDR H3 of SEQ ID NO: 17.

[0135] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the IMGT-defined light chain CDR L1 of SEQ ID NO: 18. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the IMGT-defined light chain CDR L2 of SEQ ID NO: 19. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the IMGT-defined light chain CDR L3 of SEQ ID NO: 20.

[0136] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the IMGT-defined CDR L1 of SEQ ID NO: 18, the IMGT-defined CDR L2 of SEQ ID NO: 19 and the IMGT-defined CDR L3 of SEQ ID NO: 20.

[0137] Suitably, the antibodies of the invention or antibodies for use in the methods of treatment and medical uses of the invention comprise each of the IMGT-defined CDR H1 of SEQ ID NO: 15, the IMGT-defined CDR H2 of SEQ ID NO: 16, the IMGT-defined CDR H3 of SEQ ID NO: 17, the IMGT-defined CDR L1 of SEQ ID NO: 18, the IMGT-defined CDR L2 of SEQ ID NO: 19 and the IMGT-defined CDR L3 of SEQ ID NO: 20.

[0138] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise one or more CDRs from SEQ ID NO: 1 and one or more CDRs from SEQ ID NO: 2 as determined with reference to the Chothia numbering scheme.

[0139] Preferably, the antibody of the invention according to this embodiment or the antibody for use in the methods of treatment and medical uses of the invention comprises the Chothia-defined heavy chain CDR H1 of SEQ ID NO: 44. Preferably, the antibody of the invention according to this embodiment or the antibody for use in the methods of treatment and medical uses of the invention comprises the Chothia-defined heavy chain CDR H2 of SEQ ID NO: 45. Preferably, the antibody of the invention according to this embodiment or the antibody for use in the methods of treatment and medical uses of the invention comprises the Chothia-defined heavy chain CDR H3 of SEQ ID NO: 46.

[0140] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the Chothia-defined CDR H1 of SEQ ID NO: 44, the Chothia-defined CDR H2 of SEQ ID NO: 45 and the Chothia-defined CDR H3 of SEQ ID NO: 46.

[0141] Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Chothia-defined light chain CDR L1 of SEQ ID NO: 47. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Chothia-defined light chain CDR L2 of SEQ ID NO: 48. Preferably, the antibody of the invention or the antibody for use in the methods of treatment and medical uses of the invention comprises the Chothia-defined light chain CDR L3 of SEQ ID NO: 49.

[0142] Suitably, the antibodies of the invention or antibodies used in the methods of treatment and medical uses of the invention comprise each of the Chothia-defined CDR L1 of SEQ ID NO: 47, the Chothia-defined CDR L2 of SEQ ID NO: 48 and the Chothia-defined CDR L3 of SEQ ID NO: 49.

[0143] Suitably, the antibodies of the invention or antibodies for use in the methods of treatment and medical uses of the invention comprise each of the Chothia-defined CDR H1 of SEQ ID NO: 44, the Chothia-defined CDR H2 of SEQ ID NO: 45, the Chothia-defined CDR H3 of SEQ ID NO: 46, the Chothia-defined CDR L1 of SEQ ID NO: 47, the Chothia-defined CDR L2 of SEQ ID NO: 48 and the Chothia-defined CDR L3 of SEQ ID NO: 49.

[0144] Preferably, the anti-PSGL-1 antibodies of the invention or suitable for use in the therapeutic methods and medical uses of the invention are At least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and at least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, or At least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and at least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or At least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and at least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, or at least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, and at least one, two or three CDRs having a sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49; The antibody comprises:

[0145] Humanized antibodies of the present invention The antibody of the present invention may be a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof. The antibody used in the therapeutic method or medical use of the present invention may also be a humanized anti-PSGL-1 antibody.

[0146] In a preferred embodiment, the humanized antibody comprises the CDRs set forth in the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 2, and human germline acceptor sequences.

[0147] In a preferred embodiment, the heavy chain of a humanized antibody of the invention comprises the CDRs set forth in SEQ ID NO: 1 in the human germline acceptor sequence VH3 1-3 3-30.5 (SEQ ID NO: 23). Suitably, the framework 4 region of the heavy chain of such an antibody may be of the human germline heavy joining sequence JH4a.

[0148] In a preferred embodiment, the light chain of the humanized antibody of the invention comprises the human germline acceptor sequence IGKV2-40 * 01 (SEQ ID NO: 24) comprises the CDRs set forth in SEQ ID NO: 2. Suitably, the framework 4 region of the light chain of such an antibody may be of the human germline kappa joining sequence JK5.

[0149] For the avoidance of doubt, the murine parent antibody does not constitute a humanized antibody according to the present invention.

[0150] Framework Regions of Antibodies of the Invention The antibodies of the present invention may comprise human framework acceptor sequences. In a preferred embodiment, one, two, three, or four framework sequences of the heavy chain variable region may be human framework sequences. In a preferred embodiment, one, two, three, or four framework sequences of the light chain variable region may be human framework sequences.

[0151] The antibodies of the present invention may comprise a substantially human framework acceptor incorporating one or more backmutations. Preferably, the antibodies of the present invention may comprise a substantially human framework acceptor incorporating a single backmutation. An example of such a framework region is found in SEQ ID NO: 1, where the serine residue at position 117 of SEQ ID NO: 1 constitutes a backmutation replacing the lysine residue found in the naturally occurring human sequence.

[0152] In a preferred embodiment, an antibody of the present invention comprises a heavy chain framework region FR H1 of SEQ ID NO: 32. Preferably, an antibody of the present invention comprises a heavy chain framework region FR H2 of SEQ ID NO: 33. Preferably, an antibody of the present invention comprises a heavy chain framework region FR H3 of SEQ ID NO: 34. Preferably, an antibody of the present invention comprises a heavy chain framework region FR H4 of SEQ ID NO: 35.

[0153] Preferably, the antibody of the present invention comprises FR H1 of SEQ ID NO:32, FR H2 of SEQ ID NO:33, FR H3 of SEQ ID NO:34 and FR H4 of SEQ ID NO:35.

[0154] In a preferred embodiment, an antibody of the present invention comprises a light chain framework region FR L1 of SEQ ID NO: 36. Preferably, an antibody of the present invention comprises a light chain framework region FR L2 of SEQ ID NO: 37. Preferably, an antibody of the present invention comprises a light chain framework region FR L3 of SEQ ID NO: 38. Preferably, an antibody of the present invention comprises a light chain framework region FR L4 of SEQ ID NO: 39.

[0155] Preferably, the antibody of the present invention comprises FR L1 of SEQ ID NO:36, FR L2 of SEQ ID NO:37, FR L3 of SEQ ID NO:38 and FR L4 of SEQ ID NO:39.

[0156] In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises FR H1 of SEQ ID NO: 32, FR H2 of SEQ ID NO: 33, FR H3 of SEQ ID NO: 34, FR H4 of SEQ ID NO: 35, FR L1 of SEQ ID NO: 36, FR L2 of SEQ ID NO: 37, FR L3 of SEQ ID NO: 38 and FR L4 of SEQ ID NO: 39.

[0157] The exemplary sequences of these framework regions listed in the Sequence Information Table were calculated using a combination of the Kabat and Chothia numbering systems. It will be understood that the definitions of framework regions may vary slightly based on alternative numbering systems. Such variations correspond to changes in the definitions of CDRs determined by these alternative numbering systems (as listed in Table 15), and therefore it may be preferable to use a consistent numbering system to determine both CDRs and framework regions.

[0158] Preferably, a humanized antibody of the invention or for use in the therapeutic methods and medical uses of the invention comprises a heavy chain incorporating no more than five back mutations. For example, such a humanized antibody may incorporate no more than four, no more than three, no more than two, or no more than two back mutations in the heavy chain. Preferably, a humanized antibody of the invention or for use in the therapeutic methods and medical uses of the invention comprises a single back mutation in the heavy chain.

[0159] In a preferred embodiment, the humanized antibodies of the invention or used in the therapeutic methods and medical uses of the invention comprise a back mutation in the heavy chain to introduce a serine residue adjacent to CDR H3.

[0160] The inventors have found that back mutations introduced into the light chain of an antibody of the invention have a greater effect on affinity than back mutations introduced into the heavy chain. Accordingly, a preferred humanized antibody of the invention or for use in the therapeutic methods and medical uses of the invention may comprise a light chain incorporating five or fewer back mutations. For example, such a humanized antibody may incorporate four or fewer, three or fewer, two or fewer, or one or fewer back mutations in the light chain. Preferably, a humanized antibody of the invention or for use in the therapeutic methods and medical uses of the invention does not comprise a back mutation in the light chain.

[0161] Variable regions of the antibodies of the present invention SEQ ID NO: 26 defines the variable region of the heavy chain of SEQ ID NO: 1. An antibody of the invention or an antibody suitable for use in the therapeutic methods or medical uses of the invention may comprise a heavy chain comprising the variable sequence defined in SEQ ID NO: 26. As mentioned above, the serine residue at position 98 of SEQ ID NO: 26 constitutes a back mutation.

[0162] In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises a heavy chain variable region that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises a heavy chain variable region of SEQ ID NO: 26. Such an antibody may retain the serine residue at position 98 of SEQ ID NO: 26.

[0163] SEQ ID NO: 29 defines the variable region of the light chain of SEQ ID NO: 2. An antibody of the invention or an antibody suitable for use in the therapeutic methods or medical uses of the invention may comprise a light chain comprising a variable sequence defined in SEQ ID NO: 29.

[0164] In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises a light chain variable region that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 29. In a preferred embodiment, an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention comprises a light chain variable region of SEQ ID NO: 26.

[0165] In a preferred embodiment, an antibody of the invention or suitable for use in the therapeutic methods or medical uses of the invention comprises a heavy chain variable region of SEQ ID NO:26 and a light chain variable region of SEQ ID NO:29.

[0166] Further details of suitable antibodies incorporating specific modifications in the variable region sequences are provided elsewhere herein.

[0167] Constant regions of the antibodies of the present invention The antibodies of the present invention or antibodies suitable for use in the methods or uses of the present invention may comprise any desired constant region, and those skilled in the art can readily select a suitable constant region taking into account the requirements of the application for which the antibody will be used.

[0168] By way of example only, a suitable antibody may comprise the human IgG2 heavy chain constant region of SEQ ID NO:40.

[0169] Incorporation of a human IgG2 heavy chain constant region into antibodies of the invention may be advantageous for a number of reasons. For example, the constant region of IgG2 exhibits low binding to Fc receptors and low binding to complement component 1q (C1q), making antibodies incorporating this constant region less likely to induce antibody effector functions. The human IgG2 heavy chain constant region of SEQ ID NO: 40 is particularly less likely to induce such functions due to the incorporation of a modification that further reduces C1q binding (an alanine residue at position 201 of SEQ ID NO: 40, replacing the lysine residue found at this position in naturally occurring human IgG2).

[0170] Preferably, the antibody of the present invention comprises a human kappa light chain constant region. For example, such an antibody may comprise the human kappa light chain constant region of SEQ ID NO:41.

[0171] Post-translational modifications Preferably, the anti-PSGL-1 antibody of the present invention or an anti-PSGL-1 antibody suitable for use in the therapeutic method or medical use of the present invention may comprise one or more post-translational modifications, which may be selected from the group consisting of glycosylation, N-terminal glutaminyl cyclization, and C-terminal lysine clipping.

[0172] In a preferred embodiment, the anti-PSGL-1 antibody of the invention or an anti-PSGL-1 antibody suitable for use in the therapeutic methods or medical uses of the invention has a heavy chain glycosylated at Asn-289 with G0F as the predominant N-glycan structure.

[0173] antibody fragments The advantages provided by the antibodies of the invention and antibodies suitable for use in the therapeutic methods or medical uses of the invention generally may also be construed as being conferred by antigen-binding fragments of such antibodies, and therefore the invention should also be construed as disclosing and relating to antigen-binding fragments of the antibodies described herein.

[0174] Suitable antigen-binding fragments of the antibodies disclosed herein, such as the antibodies of the invention or antibodies used in the therapeutic methods or medical uses of the invention, may comprise the variable regions of the antibody. Indeed, such fragments may consist essentially of the variable regions of the antibody.

[0175] For the avoidance of doubt, the present invention provides an antigen-binding fragment of a humanized antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. Such an antigen-binding fragment of a humanized antibody may be used in the pharmaceutical composition of the third aspect of the invention.

[0176] Similarly, the present invention provides polynucleotide sequences encoding antigen-binding fragments of immunoglobulin chains of humanized antibodies comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. The present invention also provides polynucleotide sequences encoding antigen-binding fragments of immunoglobulin chains comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2.

[0177] Furthermore, the invention provides cells comprising a polynucleotide according to these embodiments of the invention that encodes an antigen-binding antibody fragment. Such cells may be used in the production method of the eighth aspect of the invention to produce an antigen-binding fragment of an antibody according to the invention.

[0178] The method of treatment according to the ninth aspect of the invention can be carried out using a suitable antigen-binding fragment of an antibody. An antigen-binding fragment of an antibody comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2 can be used as a medicament according to the tenth aspect of the invention.

[0179] Indeed, unless the context requires otherwise, references in this disclosure to "antibody(s) of the invention" should be construed to also encompass antigen-binding fragments of such antibodies.

[0180] By way of example only, suitable antigen-binding fragments of antibodies of the invention may be selected from the group consisting of antigen-binding fragments (Fab), single-domain antibody fragments (sdAb), nanobodies, single-chain variable fragments (scFv), antibody variable fragments (Fv), Fd fragments and autonomous VH domains.

[0181] Those skilled in the art will be well aware of the many ways in which one can determine whether a fragment of an antibody of the invention retains the ability to bind to a PSGL-1 antibody and constitutes an antigen-binding fragment of an antibody of the invention. By way of example only, the ability of an antibody fragment to bind to PSGL-1 can be determined by the methods used in the Examples to determine antibody affinity or the functional attributes of an antibody.

[0182] Functional properties of the antibodies of the invention or antibodies or antibody fragments suitable for use in the therapeutic methods or medical uses of the invention In addition to the primarily sequence-based and structural definitions above, antibodies of the invention and antibodies suitable for use in the therapeutic methods and medical uses of the invention can be usefully characterized with respect to their functional properties, which can be assessed in vitro or in vivo, as appropriate.

[0183] The inventors have found that the antibodies of the present invention have functional properties that provide significant advantages in terms of their therapeutic use.

[0184] affinity The antibodies of the present invention bind to PSGL-1 with high affinity.

[0185] Humanized antibodies often exhibit reduced affinity for their targets compared to the parent antibody from which they are derived, particularly when germline sequences are used as framework acceptors. In contrast, the present inventors have demonstrated that the humanized anti-PSGL-1 antibodies of the present invention have higher affinity for PSGL-1 than their parent antibodies. While the parent antibody from which the humanized anti-PSGL-1 antibodies of the present invention are derived has an affinity constant of 16.6 nM for PSGL-1, the exemplary antibody SelK2 (comprising SEQ ID NO: 1 and SEQ ID NO: 2) of the present invention has an affinity constant of 4.29 nM for PSGL-1. This represents an almost four-fold increase in affinity compared to the parent antibody, which is highly unusual for a humanized antibody using germline framework acceptor sequences.

[0186] The improved affinity of the antibodies of the invention is a surprising and beneficial discovery. Without wishing to be bound by any hypothesis, the inventors believe that this high affinity supports the unexpectedly effective results that may be achieved by the therapeutic use of the antibodies of the invention in the treatment methods or medical uses of the invention.

[0187] In preferred embodiments, the high affinity of the antibodies of the invention and antibodies suitable for use in the therapeutic methods and medical uses of the invention described herein means that these antibodies are not only able to inhibit binding of PSGL-1 to its ligand, but also to disrupt such binding that has already occurred. As explained further below, this opens up a new therapeutic window in which treatments may achieve benefit even when response-driven binding of PSGL-1 to its ligand has already begun.

[0188] In a preferred embodiment, an antibody of the present invention or an antibody used in the therapeutic methods and medical uses of the present invention may have an affinity constant for PSGL-1 of less than 16 nM. For example, an antibody of the present invention may have an affinity constant for PSGL-1 of less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, or less than 5 nM. Preferably, an antibody of the present invention may have an affinity constant for PSGL-1 of about 4 nM, for example, 4.29 nM.

[0189] In preferred embodiments, the antibodies of the invention or antibodies used in the therapeutic methods and medical uses of the invention may have even greater affinity for PSGL-1, for example an affinity constant of 4 nM or less, 2 nM or less, or 1 nM or less.

[0190] The high affinity of the antibodies of the present invention makes them particularly suitable for carrying out the therapeutic methods or medical uses of the present invention, although it will be appreciated that in alternative embodiments, it may also be possible to carry out these methods or uses using anti-PSGL-1 antibodies with lower affinity.

[0191] The affinity of antibodies of the invention can be determined by any suitable method known to those of skill in the art. By way of example only, affinities such as the affinity values ​​described in the previous paragraph can be derived by surface plasmon resonance (SPR) using a series of increasing concentrations up to saturation. Details of suitable techniques and the results obtained using them with antibodies of the invention are described in the Examples.

[0192] Suitable antigen-binding fragments of antibodies can have affinities according to any of the above parameters.

[0193] Inhibition or disruption of the binding of PSGL-1 to its ligands As demonstrated in the Examples, the antibodies of the present invention are capable of inhibiting and / or disrupting the binding of PSGL-1 to its ligands.

[0194] In the context of the present invention, "inhibition" of binding between PSGL-1 and a ligand can be considered to refer to the prevention of the formation of new interactions between PSGL-1 and the ligand in question.

[0195] On the other hand, "disruption" of binding between PSGL-1 and a ligand can be considered to refer to the disruption of a previously formed interaction between PSGL-1 and the ligand in question.

[0196] As further discussed elsewhere in this disclosure, these capabilities of the antibodies of the present invention and the antibodies used in the therapeutic methods or medical uses of the present invention are suitable for both the prevention of diseases or conditions caused by the harmful binding of PSGL-1 to its ligand, and the treatment of diseases or conditions caused by the harmful binding of PSGL-1 to its ligand.

[0197] Generally, an antibody of the invention or an antibody suitable for use in a method of treatment or medical use of the invention will inhibit binding of PSGL-1 to a ligand by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, It may be possible to inhibit or destroy by at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0198] Those skilled in the art will be able to easily identify suitable assays for determining the extent of inhibition or disruption of binding achieved. Those skilled in the art will recognize that there are several common techniques that can be used to determine the extent of inhibition or disruption of binding achieved. Suitable techniques that can be used to determine the extent of inhibition or disruption of binding achieved include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, Western blotting, immunoprecipitation (IP), and competitive binding assays. These can be selected with reference to a particular ligand of interest (e.g., binding of PSGL-1 to P-selectin or a chemokine) or with reference to a particular binding-related activity of interest (e.g., cell rolling or extravasation).

[0199] Suitable antigen-binding fragments of antibodies may have the ability to inhibit or disrupt binding of PSGL-1 to its ligand according to any of the above parameters.

[0200] Inhibition or disruption of binding to selectins PSGL-1 is known to bind to selectins, including those selected from the group consisting of P-selectin, E-selectin, and L-selectin.

[0201] As described in the Examples, the inventors have demonstrated that the antibodies of the invention can inhibit or disrupt binding of PSGL-1 to certain selectin ligands, particularly P-selectin and L-selectin, and such inhibition or disruption of binding prevents or reduces selectin-mediated biological activity.

[0202] Suitably, the antibody of the invention or an antibody suitable for use in the therapeutic methods or medical uses of the invention inhibits the binding of PSGL-1 to a selectin (such as P-selectin or L-selectin) by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, or at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51% , at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least It may be possible to inhibit or destroy at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0203] The ability of an antibody to inhibit or disrupt binding of PSGL-1 to a selectin ligand (such as P-selectin or L-selectin) can be determined by any suitable means, for example, by cell-based assays such as those discussed in Example 4 or Example 6.

[0204] Suitable antigen-binding fragments of antibodies may have the ability to inhibit or disrupt binding of PSGL-1 to a selectin according to any of the above parameters.

[0205] Inhibition or disruption of binding to chemokines PSGL-1 is known to bind to chemokines including those selected from the group consisting of CCL27, CCL19 and CCL21.

[0206] As described in the Examples, the inventors have demonstrated that the antibodies of the invention can inhibit or disrupt the binding of PSGL-1 to chemokine ligands, exemplified by CCL27, such that inhibition or disruption of binding prevents or reduces chemokine-mediated biological activity.

[0207] Preferably, the antibody of the invention or an antibody suitable for use in the therapeutic method or medical use of the invention inhibits the binding of PSGL-1 to a chemokine (such as CCL27) by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least At least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77% %, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% inhibited or destroyed.

[0208] The ability of an antibody to inhibit or disrupt binding of PSGL-1 to a chemokine ligand (such as CCL27) can be determined by any suitable means, for example, it can be assessed by an SPR-based assay such as that discussed in Example 5.

[0209] Suitable antigen-binding fragments of antibodies may have the ability to inhibit or disrupt binding between PSGL-1 and a chemokine according to any of the above parameters.

[0210] Inhibition or disruption of cell adhesion The interaction of PSGL-1 with its ligands contributes to increased cell adhesion through a number of different mechanisms. The attachment of PSGL-1 on white blood cells to its ligands on the vascular wall is known to be an essential step in cell adhesion to substrates such as the circulatory lining or platelets. The interaction of PSGL-1 on cells such as eosinophils with P-selectin on the surface of activated platelets also increases the expression of adhesion molecules by leukocytes, increasing their adhesive propensity. In either case, PSGL-1-mediated adhesion of leukocytes such as eosinophils may be followed by other activities related to cell migration or extravasation.

[0211] As described in the Examples, the present inventors have demonstrated that the antibodies of the present invention can inhibit or disrupt the binding between PSGL-1 and its ligands, which is involved in cell adhesion. Inhibition or disruption of such binding prevents or reduces cell adhesion, thereby preventing or reducing the biological activity resulting from such cell adhesion.

[0212] The cell may be a leukocyte. The cell may be an epithelial cell. The cell may be selected from the group consisting of an eosinophil, a neutrophil, and an epithelial cell. The biological activity that is prevented or reduced by inhibiting or disrupting cell adhesion may be determined with reference to the cell type in question.

[0213] Suitably, the antibody of the invention or an antibody suitable for use in the method of treatment or medical use of the invention inhibits PSGL-1 mediated cell adhesion by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, It may be possible to inhibit or destroy at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0214] The ability of an antibody to inhibit or disrupt cell adhesion mediated by PSGL-1 binding can be determined by any suitable means, for example, it can be assessed by assays such as those discussed in Example 4 or Example 6.

[0215] Suitable antigen-binding fragments of antibodies may have the ability to inhibit or disrupt cell adhesion according to any of the above parameters.

[0216] Inhibition or disruption of cell rolling The interaction of PSGL-1 with its ligands is known to contribute to the process of cell rolling, which is an early stage of cell adhesion to substrates such as the endothelium of the circulation, and which may be associated with activation of the rolling cells and / or their subsequent migration or extravasation.

[0217] As described in the Examples, the inventors have demonstrated that the antibodies of the present invention can inhibit or disrupt cell rolling mediated by binding of PSGL-1 to its ligand, which inhibits or disrupts "downstream" activities such as cell extravasation.

[0218] As mentioned above, the cell rolling inhibited or disrupted may be a leukocyte. The cell may be a non-leukocyte cell, such as a metastatic cancer cell. The cell may be selected from the group consisting of a neutrophil, an eosinophil, and a metastatic cell. The biological activity that is prevented or reduced by inhibiting or disrupting cell rolling may be determined with reference to the cell type in question.

[0219] Suitably, the antibody of the invention, or an antibody suitable for use in the method of treatment or medical use of the invention, inhibits PSGL-1 mediated cell rolling by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, It may be possible to inhibit or destroy by at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0220] The ability of an antibody to inhibit or disrupt cell adhesion mediated by PSGL-1 binding can be determined by any suitable means, for example, it can be assessed by an assay such as the neutrophil rolling assay described in Example 4.

[0221] Suitable antigen-binding fragments of antibodies may have the ability to inhibit or disrupt cell rolling according to any of the above parameters.

[0222] Inhibition or disruption of cell extravasation Binding of cells to the endothelium of the circulatory system through the interaction of PSGL-1 with its ligand is a necessary step in the process of cell extravasation. Extravasated cells can contribute to inflammatory responses within tissues, potentially causing detrimental effects in the tissues in question.

[0223] As described in the Examples, the present inventors have demonstrated that the antibodies of the present invention can inhibit or disrupt cell extravasation mediated by the binding of PSGL-1 to its ligand, and thus, such inhibition or disruption of cell extravasation can prevent or reduce inflammation mediated by extravasated cells.

[0224] The extravasation inhibited or disrupted may be that of leukocytes or other cells, such as metastatic cancer cells. The cells may be selected from the group consisting of eosinophils, neutrophils, and metastatic cancer cells. The biological activity prevented or reduced by inhibiting or disrupting cell rolling can be determined with reference to the cell type in question. In the case of leukocytes, this typically results in reduced inflammation, and in the case of metastatic cells, it may result in reduced cancer metastasis.

[0225] Suitably, the antibody of the invention, or an antibody suitable for use in a method of treatment or medical use of the invention, reduces cell extravasation by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least It may be possible to inhibit or destroy at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0226] The ability of an antibody to inhibit or disrupt cell extravasation mediated by PSGL-1 binding can be determined by any suitable means, for example, it can be assessed by an assay such as that described in Example 8.

[0227] Importantly, our results demonstrate that the reduction in leukocyte (e.g., eosinophil or neutrophil) extravasation upon treatment with the antibodies of the invention is not associated with a reduction in the number of leukocytes present in the circulation, indicating that the reduction in extravasation is not simply a function of a reduction in the number of circulating leukocytes, but is specifically associated with a reduction in the number of leukocytes (e.g., eosinophils or neutrophils) that extravasate from the bloodstream into underlying tissues during an inflammatory response.

[0228] Maintaining circulating white blood cell counts is important because these cells play a critical role in mounting an appropriate, as opposed to an aberrant, immune response. By way of example only, circulating eosinophils play a key role in the body's response to parasites, and neutrophils play a major role in the innate immune response.

[0229] Thus, the antibodies, methods of treatment or medical uses of the invention can be used to therapeutically reduce the number of extravasated leukocytes (such as eosinophils or neutrophils) without adversely affecting the recipient's immunity.

[0230] Suitable antigen-binding fragments of antibodies may have the ability to inhibit or disrupt cell extravasation according to any of the above parameters.

[0231] Decreased cell count in sputum As described below, the antibodies of the invention and the therapeutic methods and medical uses of the invention are of particular interest in the treatment of respiratory conditions or diseases such as asthma or COPD.

[0232] Respiratory conditions or diseases may be associated with the accumulation of cells in sputum. This accumulation may occur as a result of cell extravasation (e.g., in the case of white blood cells) or cell shedding (e.g., in the case of epithelial cells lining the respiratory tract). Thus, the ability of an antibody to cause a reduction in the number of cells in sputum can provide an indication that the antibody alleviates the respiratory condition in a manner suitable for achieving a therapeutic effect. The reduction may be observed for any suitable cell of interest, such as cells selected from the group consisting of eosinophils, neutrophils, and epithelial cells.

[0233] The inventors have found that the antibodies of the invention can reduce the number of cells that accumulate in sputum, which can be a useful surrogate for assessing the severity or progression of the disease.

[0234] Suitably, the antibody of the invention or an antibody suitable for use in the method of treatment or medical use of the invention reduces the number of cells in sputum by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at 8%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, or less At least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, It may be possible to achieve a reduction of at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0235] The ability of an antibody to inhibit or disrupt cell extravasation mediated by PSGL-1 binding can be determined by any suitable means, for example, it can be assessed by assays such as the absolute or ratio differential eosinophil count studies described in Example 8 or the absolute or ratio differential eosinophil count studies described in Example 11.

[0236] Suitable antigen-binding fragments of antibodies may be capable of causing a reduction in the number of cells in sputum according to any of the above parameters.

[0237] Improvement in pulmonary function tests Perhaps the most significant finding from our studies conducted in human subjects as part of clinical trials is that the antibodies of the invention can result in improved lung function as demonstrated by improvements in pulmonary function tests.

[0238] In particular, the inventors have demonstrated that asthma patients receiving treatment with the antibodies of the invention achieve dramatic and statistically significant improvements in forced expiratory volume (FEV1). FEV1 provides a measure of the maximum volume of air an individual can forcibly exhale in one second. Higher FEV1 values ​​indicate better lung function, while lower FEV1 values ​​indicate more impaired lung function.

[0239] A subject's or patient's FEV1 can be measured at baseline (before treatment with the experimental drug or placebo) and after treatment. These values ​​can then be compared to calculate the percentage change in FEV1 (e.g., the maximum decrease in FEV1), or the area under the curve (AUC) can be calculated for the resulting FEV1 values. Either of these statistics can be useful in determining the effect of the antibody on improving lung function.

[0240] As described in Example 8, asthma patients challenged with an appropriate allergen exhibit a decline in FEV1 associated with airway obstruction. Treatment with the antibody of the invention (SelK2), embodying a method of treatment or medical use according to the invention, reduces the decline in FEV1 during the delayed allergen response (LAR; 3 to 8 hours after allergen challenge). This indicates an improvement in pulmonary function in asthma patients receiving treatment with the antibody of the invention compared to control patients receiving placebo. This is observed in terms of both the area under the curve for the % decline in FEV1 and the maximum % decline in FEV1. The change in maximum % decline indicates a statistically significant improvement in pulmonary function in subjects receiving treatment with the antibody of the invention.

[0241] The antibodies of the invention, or antibodies suitable for use in the methods of treatment or medical uses of the invention, may improve lung function by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53% %, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% improvement may be possible.

[0242] In the Examples, it has been demonstrated that antibodies of the invention, or preferred embodiments of antibodies for use in the methods of treatment or medical uses of the invention, can improve lung function in asthma patients by at least 35% compared to placebo. Indeed, such antibodies can improve lung function in asthma patients by at least 50% compared to placebo. These changes are greater than can be achieved using currently available therapies, and therefore demonstrate the improved treatments that the inventors' developments will make available to patients with lung disease.

[0243] Preferably, the improvement in lung function can be demonstrated in terms of an improvement in maximum % decline in FEV1 compared to a placebo control in asthmatic patients undergoing allergen challenge. Preferably, such improvement can occur during the LAR.

[0244] Suitable antigen-binding fragments of antibodies may have the ability to produce an improvement in a pulmonary function test according to any of the above parameters.

[0245] Low immunogenicity of the antibodies of the invention or antibodies suitable for use in the therapeutic methods or medical uses of the invention It is generally recognized that therapeutic antibodies can generate immune responses in their recipients, which is particularly problematic for therapeutic antibodies that must be administered over a long period of time, such as for the treatment of chronic conditions or diseases.

[0246] Even humanized antibodies can elicit such immune responses. The immune response to humanized therapeutic antibodies results in the production of human anti-human antibodies (HAHA). HAHA can bind to the therapeutic antibody and interfere with its ability to bind to its corresponding antigen (e.g., PSGL-1). As a result, the presence of HAHA can significantly reduce the therapeutic efficacy of the antibody in some cases.

[0247] It would therefore be advantageous if therapeutic antibodies exhibited low immunogenicity, thereby reducing the risk of HAHA formation and loss of therapeutic efficacy.

[0248] The antibodies of the present invention, exemplified by SelK2, showed low immunogenicity in recipients. As described in Example 10 and shown in Table 13, immunogenicity analysis conducted as part of clinical trials using SelK2 showed low-level responses, some of which were not repeated at later time points, and no neutralizing activity of the antibodies of the present invention was observed. Furthermore, some patients who received a placebo were also found to show low-level responses according to the assay used.

[0249] It will therefore be appreciated that the antibodies of the present invention exhibit low immunogenicity, making them well suited for practical therapeutic use, particularly for use in the treatment of chronic conditions or diseases.

[0250] A suitable antigen-binding fragment of an antibody can have low immunogenicity according to any of the above parameters.

[0251] Treatment methods and medical uses As noted above, the antibodies of the invention have properties that make them suitable for therapeutic use in many contexts. These properties lead to methods of treatment using the antibodies of the invention and medical uses of the antibodies of the invention, which are presented in more detail below.

[0252] Given these beneficial properties of the antibodies of the present invention and considering the relationship between the amino acid sequence, structure, and function of antibodies, it will be understood that antibodies sharing sequence characteristics with the antibodies of the present invention can also be used in corresponding therapeutic methods and medical uses. In particular, antibodies that contain sequences in common with the sequences of the antibodies of the present invention and therefore share binding characteristics with the antibodies of the present invention can be used in such therapeutic methods and medical uses. Thus, the methods or medical uses of the present invention can use not only the antibodies of the present invention, but also antibodies comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. Specific embodiments of such antibodies have been discussed in detail above.

[0253] Treatment / Medical Use Protection According to a ninth aspect of the present invention, there is provided a method for preventing or treating a disease or condition in a subject in need thereof, the method comprising providing a therapeutically effective amount of an anti-PSGL-1 antibody to the subject, wherein the antibody comprises at least one CDR of an antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of an antibody light chain defined in SEQ ID NO: 2.

[0254] The antibody provided in the method of the ninth aspect of the invention may be an anti-PSGL-1 antibody according to any embodiment of the first aspect of the invention or an anti-PSGL-1 antibody according to the second aspect of the invention.

[0255] The disease or condition prevented or treated by the method of the ninth aspect of the invention may be a disease or condition associated with the binding of PSGL-1 to its ligand. Examples of such conditions or diseases are discussed below.

[0256] According to a tenth aspect of the present invention, there is provided an anti-PSGL-1 antibody for use as a medicament, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2. The antibody may be an anti-PSGL-1 antibody according to any embodiment of the first aspect of the invention or an anti-PSGL-1 antibody according to the second aspect of the invention.

[0257] The antibody for use according to the tenth aspect of the invention may be used in the prevention or treatment of a disease or condition associated with the binding of PSGL-1 to its ligand.

[0258] The antibody for use according to the tenth aspect of the invention may be used in the form of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable excipient, and such a composition may also be used to provide the antibody required for the treatment method of the ninth aspect of the invention.

[0259] "Prevention or treatment" of diseases or conditions associated with the binding of PSGL-1 to its ligand Those skilled in the art will recognize a wide range of diseases or conditions associated with the binding of PSGL-1 to its ligands. Such diseases or conditions may be caused by the binding of PSGL-1 to one or more of its ligands. Examples of such diseases or conditions are discussed in more detail in the following pages. The antibodies of the present invention and the therapeutic methods and medical uses of the present invention can be used to prevent and / or treat such diseases or conditions.

[0260] In the context of the present disclosure, reference to "prevention" of a disease or condition associated with binding of PSGL-1 to its ligand may be interpreted as referring to prophylactic medical intervention intended to prevent the occurrence or further development or progression of one or more of the diseases or conditions associated with binding of PSGL-1 to its ligand. Antibodies can be used to prevent diseases or conditions by inhibiting the formation of new bonds between PSGL-1 and one or more of its ligands.

[0261] References in this disclosure to "prevention" of a disease or condition, either generally or specifically, may be construed to encompass delaying the symptoms or onset of the relevant disease or condition. Furthermore, references in this disclosure to "prevention" of a disease or condition, either generally or specifically, should also be construed to encompass incomplete or partial prevention of the relevant disease or condition. Thus, prevention may result in complete avoidance of the symptoms of the disease or condition (e.g., such that asthma symptoms do not occur upon exposure to an allergen) or partial avoidance of the symptoms of the disease or condition (e.g., such that symptoms experienced upon exposure to an allergen are reduced or delayed).

[0262] In the case of treatment of a respiratory disease or condition, such as asthma or COPD, prevention of the disease or condition can be demonstrated by improved pulmonary function compared to the function achieved in the absence of treatment. As discussed in more detail elsewhere herein, pulmonary function can be assessed with reference to the change in FEV1 in response to allergen challenge achieved with or without treatment. Pulmonary function can be improved by at least 20%, at least 30%, at least 40%, at least 50% or more compared to the function occurring in the absence of treatment.

[0263] Alternatively or additionally, in the prevention of respiratory diseases or conditions such as asthma or COPD, disease prevention can be demonstrated by a reduction in the number of localized inflammatory cells (such as eosinophils or neutrophils) present compared to the number of such cells present in the absence of treatment. The number of localized inflammatory cells present in the lungs of patients with respiratory diseases or conditions such as asthma or COPD has been reported to correlate with the severity of the disease. The amount of localized inflammatory cells present in the lungs can be assessed with reference to the number of such cells present in sputum, as discussed elsewhere herein. The number of localized inflammatory cells present in the lungs can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more compared to the number of such cells present in sputum in the absence of treatment.

[0264] In therapeutic methods or medical uses according to such embodiments of the present invention, a concentration of antibody can be maintained in the circulation sufficient to achieve at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition of binding between PSGL-1 and its ligand. This approach can thus prevent steps in leukocyte adhesion, rolling, and extravasation (particularly of eosinophils) that would otherwise contribute to disease or pathology (e.g., asthma attacks). Suitable routes of administration and dosing regimens can be selected with this goal in mind.

[0265] References to "treatment" of a disease or condition associated with the binding of PSGL-1 to its ligand may be interpreted as referring to a medical intervention intended to alleviate one or more pre-existing symptoms of a disease or condition associated with the binding of PSGL-1 to its ligand (as opposed to the prophylactic, preventative use described above). "Treatment" in this context may include complete treatment, in which all symptoms are completely alleviated, or partial treatment, in which symptoms are incompletely alleviated. Partial treatment, in which symptoms are alleviated by at least 20%, at least 40%, at least 60%, or at least 80% compared to symptoms that would occur in the absence of treatment, may be sufficient to provide beneficial relief to a subject.

[0266] Without wishing to be bound by any hypothesis, the antibodies can be used to treat diseases or conditions by disrupting the existing binding between PSGL-1 and one or more of its ligands.

[0267] For example, in therapeutic methods or medical uses according to these embodiments of the present invention, a concentration of antibody can be established in the circulation sufficient to disrupt the binding between PSGL-1 and its ligand. This approach can thus disrupt existing binding associated with an ongoing disease or condition (such as an ongoing asthma attack). This disruption of binding can reverse ongoing leukocyte adhesion and rolling (e.g., for eosinophils) and prevent further extravasation associated with the condition or disease. Suitable routes of administration and dosing regimens can be selected with this goal in mind.

[0268] The antibodies, methods of treatment or medical uses of the invention may be useful in the prevention or treatment of diseases or conditions that require the inhibition or disruption of PSGL-1 mediated binding between leukocytes (such as eosinophils) and endothelial cells.

[0269] The antibodies, methods of treatment or medical uses of the invention may be useful in the prevention or treatment of diseases or conditions that require the inhibition or disruption of PSGL-1 mediated binding between leukocytes (such as eosinophils) and platelets.

[0270] The antibodies, methods of treatment or medical uses of the invention may be useful in the prevention or treatment of diseases or conditions that require the inhibition or disruption of PSGL-1 mediated binding between endothelial cells and sickle red blood cells.

[0271] Subject or patient The therapeutic methods of the present invention are carried out on a subject in need of prevention or treatment of a disease or condition. The terms "subject" and "patient" may be used interchangeably in the context of this disclosure.

[0272] Preferably, the subject or patient may be in need of prophylaxis or treatment for a disease or condition associated with binding of PSGL-1 to its ligand.

[0273] The subject or patient may have symptoms of a disease or condition in need of treatment or may be identified as being at increased risk of developing a disease in need of prevention.

[0274] In a preferred embodiment, the subject or patient may be suffering from an acute disease or condition, in which case the methods or medical uses of the invention may be used to treat the disease, thereby alleviating the symptoms of the acute disease or condition, and such treatment may take advantage of the ability of a suitable antibody to disrupt pre-existing binding between PSGL-1 and its ligand.

[0275] In a preferred embodiment, the subject or patient may be suffering from a chronic disease. In such an embodiment, the therapeutic method or medical use of the present invention may be used to prevent the worsening of the chronic disease or the onset of an acute disease. Such therapeutic prevention may take advantage of the ability of a suitable antibody to inhibit de novo binding between PSGL-1 and its ligand.

[0276] Conditions being treated Binding of PSGL-1 to its ligands, e.g., P-selectin and L-selectin, is involved in many biological processes. Therefore, the ability to inhibit or disrupt such binding allows for the modulation of these processes for clinical purposes. In the Examples described herein, the inventors demonstrate the ability of antibodies of the invention to inhibit or disrupt PSGL-1 binding to selectins (e.g., P-selectin) or chemokines (e.g., CCL27).

[0277] These ligands are known to be involved in the homing of inflammatory cells to sites of inflammation. The ability to inhibit or disrupt the binding of PSGL-1 to these ligands can help inhibit the pathophysiological recruitment and emigration of leukocytes (including eosinophils, neutrophils, and lymphocytes) to lung tissues and airways, such as occurs in asthma and COPD. Thus, the results obtained to date support the scientific rationale for using the antibodies, therapeutic methods, or medical uses of the present invention to treat asthma or COPD.

[0278] The results also demonstrate the surprising efficacy of the antibodies, methods and uses in such treatments.

[0279] Given this surprising clinical efficacy, and the general role that binding of PSGL-1 to its ligands plays in the initiation or propagation of deleterious biological activity, particularly activity associated with inflammatory cell extravasation and influx, it will also be understood by those skilled in the art that the antibodies, methods of treatment and medical uses of the present invention can be expected to have clinical applications beyond those directly demonstrated herein.

[0280] Anti-inflammatory uses The data presented in the Examples demonstrate that the antibodies of the invention are suitable for use as anti-inflammatory agents. Similarly, the therapeutic methods and medical uses of the invention can be used to prevent or treat inflammation or inflammation-related diseases or conditions.

[0281] The antibodies, medical uses or methods of treatment of the invention may be useful for the prevention or treatment of an inflammatory condition or disease selected from the group consisting of asthma, chronic obstructive pulmonary disease, allergic reactions, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), transplant rejection, graft versus host disease, psoriasis, dermatitis, nephritis, lupus erythematosus, scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis, atherosclerosis and thyroiditis.

[0282] The antibodies, methods of treatment and medical uses of the present invention are particularly useful for the prevention or treatment of inflammatory diseases or conditions associated with the extravasation and accumulation of inflammatory cell types selected from the group consisting of eosinophils and neutrophils.

[0283] The antibodies of the present invention can be used as immunosuppressants. Those skilled in the art will readily be able to identify clinical situations in which it may be desirable to therapeutically utilize the immunosuppressive activity of an antibody of the present invention, or an antibody suitable for use in a method or use of the present invention.

[0284] Eosinophilic conditions and diseases Eosinophils are a type of white blood cell that contributes to the initiation and regulation of inflammation. Eosinophilic pathologies occur when large numbers of eosinophils are recruited to a particular site in the body. This can occur particularly as a result of extravasation, where eosinophils cross blood vessel walls and enter inflamed tissue. Increased numbers of eosinophils in tissues can result in tissue damage.

[0285] As demonstrated in the examples, the antibodies of the invention and the methods and medical uses of the invention have proven surprisingly clinically effective in alleviating eosinophilic conditions. The use of the antibodies of the invention and / or the methods and medical uses of the invention for the prevention or treatment of eosinophilic conditions therefore constitutes a particularly preferred embodiment of these various aspects of the invention.

[0286] Preferably, the eosinophilic condition or disease to be prevented or treated is eosinophilic asthma, allergies, eosinophilic esophagitis, eosinophilic dermatitis, contact dermatitis, acute myeloid leukemia (AML), ascariasis, atopic dermatitis (eczema), bullous pemphigoid, cancer (such as Hodgkin's lymphoma, leukemia and certain myeloproliferative neoplasms), Churg-Strauss syndrome, drug allergies, eosinophilic cardiomyopathy, eosinophilic cellulitis (Wells' syndrome), eosinophilic colitis, eosinophilic enteritis, eosinophilic fasciitis, The disease may be selected from the group consisting of eosinophilic gastrointestinal disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic leukemia, eosinophilic myocarditis, hay fever (allergic rhinitis), Hodgkin's lymphoma (Hodgkin's disease), hypereosinophilic syndrome, idiopathic hypereosinophilic syndrome (HES), IgG4-related disease, inflammatory bowel disease (Crohn's disease, ulcerative colitis), lymphatic filariasis, neuromyelitis optica (NMO), ovarian cancer, parasitic infection, primary biliary cirrhosis, primary immunodeficiency, or trichinellosis.

[0287] asthma Asthma is a disease involving long-term inflammation of the lung airways. During an asthma attack, airway narrowing and bronchospasm cause shortness of breath, chest tightness, and other symptoms such as coughing and wheezing. Attacks can be triggered by activity or various environmental conditions, and asthma may have an allergic component. Asthma is believed to have caused approximately 461,000 deaths worldwide in 2019.

[0288] As shown in the Examples, the inventors have demonstrated that the antibody of the present invention and the therapeutic method and medical use of the present invention can be used very effectively for the prevention or treatment of asthma. The antibody and therapeutic method or medical use of the present invention can be effective for the prevention or treatment of moderate or severe asthma. In particular, they can be useful for the prevention or treatment of eosinophilic asthma.

[0289] As clearly demonstrated by the Examples, treatment of asthma with the antibodies of the present invention (in one example of the therapeutic methods or medical uses of the present invention) can result in a statistically significant improvement in lung function. Therefore, the therapeutic methods and medical uses of the present invention are effective asthma therapies. Indeed, these results demonstrate that the improvements brought about by the agents and treatments of the present invention are greater than those achieved with the primary treatments known from the prior art, and therefore represent an important advance in the therapies available to asthma patients.

[0290] The ability of the antibodies identified by the inventors to inhibit or disrupt the binding of PSGL-1 to its ligands indicates that these agents may be advantageously used in the management and therapy of asthma to prevent the occurrence of asthma attacks or to alleviate symptoms associated with ongoing attacks.

[0291] In particular, the therapeutic methods or medical uses of the present invention can be used to prevent or treat asthma by attenuating allergen-induced bronchoconstriction. The data disclosed in the Examples show that the treatments of the present invention are effective in reducing such constriction.

[0292] Suitably, the asthma to be prevented or treated may be selected from the group consisting of eosinophilic asthma, allergic asthma and severe neutrophilic asthma.

[0293] The antibodies of the invention and / or the therapeutic methods and medical uses of the invention may be particularly useful in the prevention or treatment of eosinophilic asthma, as they may result in a reduction in the extravasation of eosinophils that drive eosinophilic asthma.

[0294] A preferred embodiment of the ninth aspect of the present invention provides a method for preventing or treating asthma in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody of the second aspect of the present invention.

[0295] A preferred embodiment of the tenth aspect of the invention provides an anti-PSGL-1 antibody of the second aspect of the invention for use in the prevention or treatment of asthma.

[0296] Neutrophilic Diseases and Disorders Neutrophils are highly abundant white blood cells that contribute to the initiation and regulation of inflammation. Neutrophilic pathologies occur when large numbers of neutrophils are recruited to a particular site in the body. This can occur particularly as a result of extravasation, where neutrophils cross blood vessel walls and enter inflamed tissue. Increased neutrophils in tissues can result in tissue damage.

[0297] As demonstrated in the Examples, the antibodies of the invention and the therapeutic methods and medical uses of the invention have proven surprisingly clinically effective in reducing neutrophil extravasation and accumulation. Thus, the antibodies of the invention and the therapeutic methods and medical uses show promise in alleviating neutrophilic conditions. Therefore, the use of the antibodies of the invention and / or the therapeutic methods and medical uses of the invention for the prevention or treatment of neutrophilic conditions constitutes a particularly preferred embodiment of these various aspects of the invention.

[0298] Suitably, the neutrophilic condition or disease to be prevented or treated is COPD, neutrophilic dermatoses (e.g., Sweet's syndrome, amicrobial pustulosis of the folds, erythema elevatum perstans, sterile pustulosis of the scalp / legs, sterile subcorneal pustulosis, and pyoderma gangrenosum), neutrophilic asthma, rheumatoid arthritis (RA), cystic fibrosis (CF), neutrophilic vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis), gout, IBD (e.g., Crohn's disease and ulcerative colitis), neutrophilic meningitis, Behcet's disease, PAPA syndrome. syndrome, hidradenitis suppurativa, EGFRI-induced acne, PASH syndrome, PAPASH syndrome, neutrophilic panniculitis, sterile abscess syndrome, systemic inflammatory response syndrome (SIRS), severe sepsis, cryopyrin-associated periodic fever syndrome (CAPS), neutrophilic otitis externa, allergy, chronic neutrophilic leukemia (CNL), hay fever (allergic rhinitis), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), SARS-CoV, SARS-CoV-2, and necrotizing colitis (NEC).

[0299] Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by coughing and difficulty breathing. Forms of COPD can include emphysema and chronic bronchitis. Prolonged inflammation in the lungs, generally mediated by eosinophils, neutrophils, or macrophage inflammatory cells, can cause airway remodeling and narrowing, contributing to decreased lung function. Currently, there is no cure available for COPD, but the disease can be prevented or managed with appropriate treatment. In 2019, COPD caused approximately 3.2 million deaths worldwide.

[0300] Extravasation of eosinophils into lung tissue contributes to the establishment of elevated local eosinophil counts in certain populations of COPD subjects. The number of inflammatory cells present in the lung is known to correlate well with the severity of the disease.

[0301] In the results of a clinical trial reported in Example 8, the inventors demonstrated that treatment with an antibody of the invention (exemplified by SelK2) reduces the number of extravasated eosinophils in the lungs of patients with COPD. The same results also showed that treatment of COPD patients with an antibody of the invention reduces the number of neutrophils present.

[0302] Those skilled in the art will appreciate that these results, including a reduction in the numbers of two types of white blood cells that are thought to play an important role in driving the inflammation that underlies COPD, indicate that the antibodies of the invention (and thus the therapeutic methods and medical uses of the invention) may provide an effective therapy for the treatment of COPD.

[0303] The reduction in neutrophil numbers may result from reduced adhesion, rolling, and extravasation of these cells due to inhibition or disruption of PSGL-1-mediated binding, which is consistent with the reduced neutrophil adhesion and rolling observed in Examples 4 and 6.

[0304] In view of the above, it will be appreciated that the use of the antibodies of the invention or the methods of treatment or medical uses of the invention in the prevention or treatment of COPD represents a particularly preferred embodiment of these various aspects of the invention.

[0305] The inventors have also observed that COPD patients receiving treatment according to the present invention exhibit a reduced number of epithelial cells present in their sputum (when compared to controls).

[0306] A preferred embodiment of the ninth aspect of the present invention provides a method for preventing or treating COPD in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody of the second aspect of the present invention.

[0307] A preferred embodiment of the tenth aspect of the invention provides an anti-PSGL-1 antibody of the second aspect of the invention for use in the prevention or treatment of COPD.

[0308] Respiratory disease or condition Given the success observed with the antibodies and therapeutic methods of the present invention in both asthma and COPD, it will be appreciated that the present invention is well suited for use in the context of preventing or treating respiratory diseases or conditions. In particular, the antibodies or therapeutic methods or medical uses of the present invention can be used to prevent or treat respiratory diseases or conditions associated with inflammation. The antibodies or therapeutic methods or medical uses of the present invention can be used to prevent or treat respiratory diseases or conditions associated with eosinophil or neutrophil activity.

[0309] By way of example, the antibodies of the invention or the methods of treatment or medical uses of the invention may be used to prevent or treat a respiratory disease or condition selected from the group consisting of eosinophilic pneumonia (whether chronic or acute), simple pulmonary eosinophilia (Löffler's syndrome), allergic bronchopulmonary aspergillosis, eosinophilic granulomatosis with polyangiitis, and iatrogenic diseases such as eosinophilia caused by exposure to sulfonamides.

[0310] Sickle cell disease (SCD) SCD is an inherited blood disorder characterized by the presence of an abnormal hemoglobin known as hemoglobin S (HbS), which causes red blood cells to assume a characteristic sickle or crescent shape and leads to a variety of health complications, including pain crises, anemia, and organ damage.

[0311] Key pathological features of SCD are vaso-occlusion, in which sickle red blood cells aggregate and block small blood vessels, and chronic inflammation associated with increased eosinophil numbers or activation. These processes lead to the symptoms of SCD, such as pain crises and tissue damage.

[0312] The formation of a complex between PSGL-1 and P-selectin plays a major role in both of these processes. As discussed above, the binding of PSGL-1 to its ligand plays an important role in the extravasation and influx of inflammatory cells associated with inflammation. Furthermore, upregulation of P-selectin in endothelial cells and platelets contributes to the cell-cell interactions involved in the pathogenesis of vaso-occlusion and pain crises in SCD. Given that the antibodies of the present invention have demonstrated the ability to inhibit the formation of a complex between PSGL-1 and P-selectin and to disrupt such complexes that have already formed, it will be appreciated that the present invention is well suited for use in the context of preventing or treating SCD or its symptoms. In particular, the antibodies, therapeutic methods, or medical uses of the present invention can be used to prevent or treat SCD or its symptoms.

[0313] A preferred embodiment of the ninth aspect of the present invention provides a method for preventing or treating SCD in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody of the second aspect of the present invention.

[0314] A preferred embodiment of the tenth aspect of the invention provides an anti-PSGL-1 antibody of the second aspect of the invention for use in the prevention or treatment of SCD.

[0315] In the context of this disclosure, particularly with reference to the ninth or tenth aspects of the present invention, prevention or treatment of SCD should be interpreted as including treating or preventing any of the processes, such as vascular occlusion or chronic inflammation, that result in the deleterious effects of SCD. Preferably, prevention or treatment of SCD should be interpreted as including preventing or treating symptoms associated with SCD. In particular, prevention or treatment of SCD should be interpreted as including preventing or treating pain crises associated with SCD.

[0316] The ability of the antibodies of the invention (or antibodies suitable for use in the methods or uses of the invention) to inhibit the formation of a complex between PSGL-1 and P-selectin indicates their usefulness in preventing SCD or a symptom of SCD, such as a pain crisis, before the disease or symptom has occurred. The ability of the antibodies of the invention (or antibodies suitable for use in the methods or uses of the invention) to disrupt an already formed complex between PSGL-1 and P-selectin indicates their usefulness in treating SCD or a symptom of SCD, such as a pain crisis, after the disease or symptom has already progressed. Given the suffering inflicted on patients by an SCD pain crisis, it will be appreciated that agents that can treat patients suffering from such a crisis and thereby provide pain relief would be highly desirable.

[0317] Treatment regimen In the method of treatment or medical use according to the present invention, the nature of the condition or disease; the characteristics of the antibody used, whether prophylaxis or treatment is required; Treatment regimens can be used that are selected taking into consideration factors including:

[0318] Preferably, the treatment regimen will also take into account factors such as the age or weight of the subject to be treated and the severity of the subject's disease.

[0319] The pharmacokinetic properties of antibodies used in the therapeutic methods or medical uses of the invention can inform treatment regimens. As a guide, Example 7 demonstrates that an exemplary antibody of the invention, SelK2, has a half-life and pharmacodynamic profile that indicates that effective concentrations of the antibody in serum can be maintained by monthly administration.

[0320] As discussed elsewhere herein, in applications where it is desired to prevent a pathological condition or disease, it may be necessary to establish and maintain a serum antibody concentration sufficient to inhibit the binding of PSGL-1 to its ligand, and in applications where it is desired to treat a pathological condition or disease, it may be necessary to establish and maintain a serum antibody concentration sufficient to disrupt the binding of PSGL-1 to its ligand.

[0321] By way of example only, treatment incidences may be repeated multiple times a day or daily, treatment incidences may be repeated weekly, every two weeks, every three weeks, every four weeks, every month, every five weeks, every six weeks, or at longer intervals.

[0322] The Examples demonstrate the efficacy of a treatment regimen in which an antibody of the invention (e.g., at a dose of 7.5 mg / kg body weight) is administered at 3-week intervals, which may be effective in treating patients with respiratory disorders such as asthma or COPD.

[0323] A preferred embodiment of a dosing regimen that may be used in the therapeutic methods or medical uses of the present invention involves a loading dose approach: the first two treatments are given two weeks apart, and further treatments may be given approximately once every four weeks.

[0324] Therapeutically effective amount of antibody Therapeutic methods of the invention include providing a therapeutically effective amount of an antibody of the invention (or an antibody suitable for use in a method or use of the invention). Such a therapeutically effective amount of the required antibody may be provided in a single treatment or may be accumulated over several treatments.

[0325] A therapeutically effective amount for an antibody of the invention or an antibody suitable for use in a method of treatment or medical use of the invention can be defined in terms of the amount of antibody administered per kg of total body weight of the patient receiving the antibody.

[0326] The Examples demonstrate the efficacy of a treatment regimen in which an antibody of the invention is administered at a dose of 7.5 mg / kg body weight (e.g., with three weeks between doses), which may be effective in treating patients with respiratory disorders such as asthma or COPD.

[0327] For example, the antibody can be administered to a patient in a therapeutically effective amount of about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, or about 30 mg / kg.

[0328] In a preferred embodiment, the therapeutically effective amount of the antibody is about 10 mg to about 5000 mg. For example, in a preferred embodiment, the therapeutically effective amount of the antibody is about 100 mg to about 1500 mg, for example, about 200 mg to about 1000 mg or about 450 mg to about 750 mg. Preferably, the therapeutically effective amount of the antibody of the present invention is about 600 mg.

[0329] Route of administration When practicing the therapeutic methods or medical uses of the invention, an antibody, such as an antibody of the invention, can be provided to a subject by any suitable route of administration.

[0330] It will be appreciated that because antibodies may be useful in preventing the extravasation of leukocytes from the bloodstream, in preferred embodiments of the invention, the antibodies may be administered intravenously, which may be by intravenous injection or infusion, as further described in the Examples.

[0331] Infusion according to this embodiment of the invention can be carried out for any suitable period of time. By way of example only, the infusion can be for 30 minutes (or about 30 minutes).

[0332] Preferably, the antibody can be provided by intravenous infusion, a route of administration that has been shown to be effective in the prevention or treatment of asthma and COPD.

[0333] Injection is a well-established route for administration of therapeutic antibodies and is therefore a preferred embodiment for delivery in practicing the therapeutic methods or medical uses of the present invention.

[0334] Preferably, the antibody is provided to a subject in need of prevention or treatment by injection. Preferably, the antibody is provided to a subject in need of prevention or treatment by intravenous injection. Preferably, the antibody is provided to a subject in need of prevention or treatment by intramuscular injection. Preferably, the antibody is provided to a subject in need of prevention or treatment by subcutaneous injection.

[0335] Pharmaceutical Composition Pharmaceutical compositions of the invention comprise an antibody of the invention or an antibody suitable for use in a therapeutic method or medical use of the invention. In addition to the antibody, pharmaceutical compositions of the invention comprise a suitable pharmaceutically acceptable excipient.

[0336] Pharmaceutical compositions can be formulated with reference to their intended route of administration. After selecting a chosen route of administration, those skilled in the art can readily identify examples of suitable formulations.

[0337] Preferably, the pharmaceutical compositions of the present invention are suitable for administration by intravenous infusion. An exemplary pharmaceutical composition of the present invention suitable for administration by intravenous infusion is described in the Examples, and this composition has been shown to be effective in the prevention or treatment of asthma and COPD.

[0338] Preferably, the pharmaceutical composition of the present invention is suitable for injection. Preferably, the pharmaceutical composition of the present invention is suitable for intravenous injection. Preferably, the pharmaceutical composition of the present invention is suitable for intramuscular injection. Preferably, the pharmaceutical composition of the present invention is suitable for subcutaneous injection.

[0339] In the case of a pharmaceutical composition of the present invention for administration by injection, suitable excipients may include diluents.

[0340] The pharmaceutical composition of the present invention may suitably comprise an antibody of the present invention or an antibody suitable for use in the therapeutic method or medical use of the present invention, and a phosphate buffered saline solution. In a preferred embodiment of such a composition, the phosphate buffered saline comprises sodium phosphate at a concentration of about 25 mM and sodium chloride at a concentration of about 190 mM.

[0341] In a preferred embodiment, the pharmaceutical compositions of the present invention include an excipient such as polysorbate 80. By way of example, the pharmaceutical compositions of the present invention may include polysorbate 80 at a concentration of about 0.02% (w / w). Examples of other excipients that may be used in the pharmaceutical compositions of the present invention are described elsewhere in this disclosure.

[0342] The pharmaceutical composition of the present invention may have a substantially neutral pH. In such an embodiment, the pharmaceutical composition of the present invention may have a pH of 7.0±0.5.

[0343] Preferably, the pharmaceutical composition of the present invention may contain the antibody of the present invention or an antibody suitable for use in the therapeutic method or medical use of the present invention at a concentration of about 1 mg / mL to about 1000 mg / mL. For example, the pharmaceutical composition of the present invention may contain the antibody of the present invention or an antibody suitable for use in the therapeutic method or medical use of the present invention at a concentration of about 2 mg / mL to about 90 mg / mL, about 3 mg / mL to about 80 mg / mL, about 4 mg / mL to about 70 mg / mL, about 5 mg / mL to about 60 mg / mL, about 6 mg / mL to about 50 mg / mL, about 7 mg / mL to about 40 mg / mL, about 8 mg / mL to about 30 mg / mL, or about 9 mg / mL to about 20 mg / mL.

[0344] Preferably, the pharmaceutical composition of the present invention may contain the antibody at a concentration of about 10 mg / mL.

[0345] Preferably, the pharmaceutical composition of the present invention can be provided in the form of a dosage unit of about 1 mL to about 100 mL. For example, the pharmaceutical composition of the present invention can be provided in the form of a dosage unit of about 2 mL to about 90 mL, about 3 mL to about 80 mL, about 4 mL to about 70 mL, about 5 mL to about 60 mL, about 6 mL to about 50 mL, about 7 mL to about 40 mL, about 8 mL to about 30 mL, or about 9 mL to about 20 mL.

[0346] Preferably, the pharmaceutical composition of the present invention can be provided in the form of a dosage unit of about 10 mL.

[0347] Preferably, the pharmaceutical composition of the present invention is suitable for administration by intravenous infusion. The pharmaceutical composition of the present invention administered by intravenous infusion has been shown to be effective in the prevention or treatment of asthma and COPD.

[0348] Preferably, the pharmaceutical composition of the present invention is suitable for injection. Preferably, the pharmaceutical composition of the present invention is suitable for intravenous injection. Preferably, the pharmaceutical composition of the present invention is suitable for intramuscular injection. Preferably, the pharmaceutical composition of the present invention is suitable for subcutaneous injection.

[0349] Polynucleotide sequences, expression vectors and cells of the invention The invention provides polynucleotide sequences (sometimes simply referred to as "polynucleotides") that encode the antibodies of the invention, or antibodies suitable for use in the therapeutic methods or medical uses of the invention, or that encode antigen-binding fragments of such antibodies. The antibodies may be according to any of the aspects or embodiments described herein.

[0350] The polynucleotide sequences of the present invention can encode the immunoglobulin chains of a suitable antibody. Preferably, the polynucleotide sequences encode the immunoglobulin chains of an antibody of the present invention. Reference to an immunoglobulin chain encoded by a polynucleotide of the present invention should be interpreted to encompass the variable region of the appropriate antibody, a full-length immunoglobulin chain of the appropriate antibody, or an antigen-binding fragment of the appropriate antibody (e.g., an scFv fragment).

[0351] According to a fourth aspect of the present invention, there is provided a polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody, the polynucleotide sequence comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2.

[0352] According to a fifth aspect of the present invention, there is provided a polynucleotide sequence encoding a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, comprising an immunoglobulin chain comprising at least one CDR of an antibody heavy chain defined in SEQ ID NO: 1 and an immunoglobulin chain comprising at least one CDR of an antibody light chain defined in SEQ ID NO: 2.

[0353] By way of example, a polynucleotide according to any of these aspects of the invention may encode an immunoglobulin heavy chain of a humanized anti-PSGL-1 antibody that comprises at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1. Such a polynucleotide may encode an immunoglobulin heavy chain of a humanized anti-PSGL-1 antibody that comprises at least two CDRs of the antibody heavy chain set forth in SEQ ID NO: 1. Such a polynucleotide may encode an immunoglobulin heavy chain of a humanized anti-PSGL-1 antibody that comprises all three CDRs of the antibody heavy chain set forth in SEQ ID NO: 1.

[0354] Alternatively or additionally, polynucleotides according to these aspects of the invention may encode an immunoglobulin light chain of a humanized anti-PSGL-1 antibody that comprises at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. Such polynucleotides may encode an immunoglobulin light chain of a humanized anti-PSGL-1 antibody that comprises at least two CDRs of the antibody light chain set forth in SEQ ID NO: 2. Such polynucleotides may encode an immunoglobulin light chain of a humanized anti-PSGL-1 antibody that comprises all three CDRs of the antibody light chain set forth in SEQ ID NO: 2.

[0355] A suitable polynucleotide of the invention may encode both a heavy chain comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and a light chain comprising at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. A suitable polynucleotide of the invention may encode both a heavy chain comprising at least two CDRs of the antibody heavy chain set forth in SEQ ID NO: 1 and a light chain comprising at least two CDRs of the antibody light chain set forth in SEQ ID NO: 2. A suitable polynucleotide of the invention may encode both a heavy chain comprising all three CDRs of the antibody heavy chain set forth in SEQ ID NO: 1 and a light chain comprising all three CDRs of the antibody light chain set forth in SEQ ID NO: 2.

[0356] Preferred polynucleotide sequences of the present invention can be further defined with reference to the antibody variable sequences that they encode.

[0357] For example, the eleventh aspect of the invention provides a polynucleotide sequence encoding the heavy chain variable sequence set out in SEQ ID NO: 26. Suitably, such a polynucleotide of the eleventh aspect of the invention may encode the heavy chain sequence set out in SEQ ID NO: 1.

[0358] Furthermore, the twelfth aspect of the invention provides a polynucleotide sequence encoding the light chain variable sequence set out in SEQ ID NO: 29. Suitably, such a polynucleotide of the twelfth aspect of the invention may encode the light chain sequence set out in SEQ ID NO: 2.

[0359] As an example of a polynucleotide in accordance with the present invention, there is provided a polynucleotide set forth in SEQ ID NO: 42, which encodes a polypeptide comprising the heavy chain sequence of SEQ ID NO: 1 and an N-terminal 19 amino acid leader peptide. The present invention also provides a polynucleotide set forth in SEQ ID NO: 43, which encodes a polypeptide comprising the light chain sequence of SEQ ID NO: 2 and an N-terminal 19 amino acid leader peptide.

[0360] In a preferred embodiment, a nucleic acid according to this aspect of the invention comprises the sequence set forth in SEQ ID NO: 42 encoding an immunoglobulin heavy chain and the sequence set forth in SEQ ID NO: 43 encoding an immunoglobulin light chain. In a preferred embodiment, a polynucleotide of the invention shares at least 90% identity with the sequence set forth in SEQ ID NO: 42 or the sequence set forth in SEQ ID NO: 43. In a preferred embodiment, a polynucleotide of the invention shares at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the light chain sequence set forth in SEQ ID NO: 15 and the heavy chain sequence set forth in SEQ ID NO: 16. Such identity calculations may not take into account the portion of the polynucleotide that encodes the leader peptide.

[0361] Suitable polynucleotides may include one or more of polymers of deoxyribonucleic acid (DNA), polymers of ribonucleic acid (RNA), analogs of these DNA or RNA generated using nucleotide analogs, and derivatives, fragments, and homologs thereof.

[0362] Polynucleotides according to the invention may be optimized for expression in mammalian cells. Suitable polynucleotides may be optimized for expression in mammalian cell lines, such as Chinese hamster ovary (CHO) cells.

[0363] Polynucleotides of the invention can be provided as part of a larger nucleic acid molecule. By way of example only, polynucleotides of the invention can be provided as part of an expression construct comprising a first polynucleotide sequence of the invention encoding a first immunoglobulin chain comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1, and a second polynucleotide sequence of the invention encoding a second immunoglobulin chain comprising at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. Such expression constructs may further comprise a promoter and one or more internal ribosome entry sites located between the first and second polynucleotide sequences.

[0364] The invention provides expression vectors comprising polynucleotides of the invention and host cells comprising the expression vectors of the invention. Host cells comprising the expression vectors of the invention can be used in methods for producing antibodies of the invention, for example, by recombinant expression.

[0365] The term "expression vector" as used herein refers to a nucleic acid molecule that comprises a polynucleotide of the present invention, is linear or circular, and comprises one or more expression units. In a preferred embodiment, the expression vector comprising a polynucleotide according to the present invention may be a DHFR expression vector.

[0366] The present invention also provides a cell comprising a polynucleotide sequence according to the invention. Such a cell may comprise a polynucleotide sequence according to the fourth, fifth, eleventh or twelfth aspect of the invention.

[0367] For example, a sixth aspect of the present invention provides a cell comprising a polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, the immunoglobulin chain comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2.

[0368] A seventh aspect of the present invention provides a cell comprising a polynucleotide sequence encoding a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, the antibody comprising an immunoglobulin chain comprising at least one CDR of an antibody heavy chain defined in SEQ ID NO: 1 and an immunoglobulin chain comprising at least one CDR of an antibody light chain defined in SEQ ID NO: 2.

[0369] Furthermore, as noted above, the thirteenth aspect of the present invention provides a cell comprising a polynucleotide sequence encoding the heavy chain sequence set forth in SEQ ID NO:1 and a polynucleotide sequence encoding the light chain sequence set forth in SEQ ID NO:2.

[0370] In any of these aspects of the invention, the polynucleotide sequence(s) may be provided in the form of an expression vector.

[0371] A cell of the present invention may be a "host cell" capable of supporting the replication or expression of an expression vector. A cell of the present invention, such as a host cell, may be a prokaryotic cell, such as E. coli, or a eukaryotic cell. Preferably, a cell of the present invention, such as a host cell, is a cell of a mammalian cell line. Preferably, a cell of the present invention, such as a host cell, is a CHO cell.

[0372] A polynucleotide according to the present invention may be an isolated polynucleotide.

[0373] Manufacturing method An eighth aspect of the present invention provides a method for producing a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2. The method comprises: expressing a first polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1; expressing a second polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody light chain set forth in SEQ ID NO:2; Includes:

[0374] Preferably, the expression of the first polynucleotide and the second polynucleotide can occur in the same cell. Alternatively, the expression of the first polynucleotide and the second polynucleotide can occur in a first cell population and a second cell population. The cells used in accordance with this aspect of the invention can be those discussed for the cells of the invention and host cells described above.

[0375] The first and second polynucleotide sequences used in the methods of the present invention can be introduced into cells where they are expressed. Such a step of introducing the first and second polynucleotide sequences can be a feature of such a method. The first and second polynucleotide sequences can be incorporated into one or more expression vectors.

[0376] The method according to this aspect of the invention may further comprise the step of purifying the antibodies produced. [Example]

[0377] Example 1. Humanization of SelK2 Humanization of the heavy and light chains was performed by CDR grafting, using germline framework acceptor regions to reduce the potential immunogenicity of the antibodies. Engineered IgG2 constant regions were used to reduce intrinsic antibody effector functions such as Fc receptor binding and complement activation.

[0378] Parent antibody The amino acid sequence of the parent murine antibody was determined by LC-MS-MS. The amino acid sequence of the heavy chain variable region of the parent murine antibody is set forth in SEQ ID NO: 21, and the amino acid sequence of the light chain variable region of the parent murine antibody is set forth in SEQ ID NO: 22. For the avoidance of doubt, the parent antibody does not constitute a humanized anti-PSGL-1 antibody of the present invention.

[0379] Design, production and testing of several humanized versions To minimize potential immunogenicity, human germline sequences were chosen as acceptor sequences. For the heavy chain, the human germline sequence VH3 1-3 3-30.5 (SEQ ID NO: 23) was chosen, and the framework 4 sequence (highlighted in green) was derived from the human germline heavy binding sequence JH4a. For the light chain, the human germline sequence IGKV2-40 * 01 (SEQ ID NO: 24) was chosen, and the framework 4 sequence (FGQGTRLEIKR) was derived from the human germline kappa binding sequence JK5.

[0380] Humanized versions of the heavy chain (SEQ ID NO: 25) and light chain (SEQ ID NO: 29) were designed by grafting the CDRs of the parent antibody onto human acceptor framework sequences without further modification or backmutation (linear grafted version).

[0381] The humanized versions were also designed with one, two, or five backmutations (B1, B2, or B5) in the heavy chain (SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively) and two or four backmutations (B2 or B4) in the light chain (SEQ ID NO: 30 and SEQ ID NO: 31, respectively). The backmutations were made to the original amino acid residues of the parent antibody as listed in Table 1.

[0382] [Table 1]

[0383] Humanized versions were engineered as human IgG1 / κ antibodies and transiently expressed by the OMRF Antibody Expression Group. This was accomplished by subcloning the light chain variable region into an expression vector containing the human κ constant region coding sequence and the heavy chain variable region into an expression vector containing the human IgG1 constant region coding sequence. Both the light and heavy chain expression vectors were driven by a CMV promoter. The heavy and light chain expression vectors for each antibody construct were then cotransfected into HEK 293A cells and transiently expressed. The resulting antibodies were purified from the culture medium using protein A chromatography. A full description of the methodology used is presented (Smith, K., et al., 2009).

[0384] Affinity testing of humanized versions of the parent antibody was performed using surface plasmon resonance (SPR) by SensiQ Technologies (Oklahoma City, Oklahoma). Testing was performed on a streptavidin-coated SPR chip loaded with biotinylated GSP-6, a glycosulfopeptide matching the N-terminal region of human PSGL-1. An initial screen of the humanized versions was performed to identify the best candidates for further development.

[0385] The light and heavy chain combinations tested were LG-HG (SEQ ID NO:25 + SEQ ID NO:29), LB2-HG (SEQ ID NO:25 + SEQ ID NO:30), LG-HB1 (SEQ ID NO:26 + SEQ ID NO:29), LG-HB2 (SEQ ID NO:27 + SEQ ID NO:29) and LG-HB5 (SEQ ID NO:28 + SEQ ID NO:29).

[0386] Because the backmutations present in the light chain disrupted binding affinity, a linear graft version (LG) of the light chain, in which the CDRs of the parent antibody were placed into the human acceptor framework sequences without further modification or backmutation, minimized disruption of antibody binding and functionality. Surprisingly, backmutations in the heavy chain improved binding affinity.

[0387] Therefore, based on the affinity test results, the HB1 version of the heavy chain (i.e., containing one back mutation) (SEQ ID NO: 26) and the LG graft version of the light chain (i.e., containing no back mutations) (SEQ ID NO: 29) were selected for the SelK2 antibody to improve the binding affinity and functionality of the antibody and reduce any potential immunogenicity. Then, a DHFR-based expression vector encoding SelK2 was developed to generate a CHO-based production cell line expressing SelK2.

[0388] For the final SelK2 antibody, the heavy chain version (B1) was re-engineered into a human IgG2 constant region to eliminate potential antibody effector functions. The IgG2 isotype has been shown to have minimal binding capacity for Fc receptors and the complement component C1q. To further eliminate C1q binding (SEQ ID NO: 1), a single amino acid change was introduced into the constant region, changing the K residue at position 333 of SEQ ID NO: 1 to an A residue. This design was enabled by DNA synthesis using GenScript (4), subcloned by the OMRF Antibody Expression Group, and sequence verified by both suppliers. The light chain version (graft) was maintained as a human kappa construct (SEQ ID NO: 2). The SelK2 antibody was transiently expressed and purified by the OMRF Antibody Expression Group.

[0389] Example 2. Characterization of SelK2 As shown in Table 2, the monosaccharide composition of SelK2 is as follows:

[0390] [Table 2]

[0391] Sialic Acid Analysis The sialic acid content of both N-glycolneuraminic acid (NGNA) and N-acetylneuraminic acid (NANA) was determined after liberation of sialic acid by mild acid hydrolysis. After reaction with o-phenylenediamine, the derivatized sialic acid samples were separated by reversed-phase HPLC with fluorescence detection. Quantitation was performed against an external calibration curve. This method allows for the separation and quantitation of both NGNA and NANA. NGNA (N-glycolylneuraminic acid) was not detected. The detected level of NANA (N-acetylneuraminic acid) is below the LOQ of the assay.

[0392] From these results, it can be concluded that the stoichiometry of each reference standard is below the LOQ of 0.1 moles of sialic acid per mole of protein. This work was performed by Charles River Laboratories.

[0393] Example 3. Parental antibodies and affinity of SelK2 SelK2 and parental antibodies were immobilized and their interaction with SelSP1 (PSGL-1 sulfopeptide) was characterized using SensiQ Pioneer.

[0394] Materials and Methods All SPR measurements were performed at a controlled temperature of 25°C using a SensiQ Pioneer (S / N 13110154). SelK2 (Lot No. 14-2189) was dispensed into three 1 mL aliquots in a biosafety cabinet. Protein G was purchased from BioVision (Cat. No. 6510). The running buffer for the Pioneer contained 10 mM HEPES (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween-20. All other reagents were purchased from Sigma-Aldrich.

[0395] A new COOH2 biosensor was installed and conditioned according to the CH0012 protocol. Protein G was immobilized by amine coupling. A mixture containing 0.1 M EDC and 0.025 M NHS was injected into flow channel 1 (FC1) and FC2 for 4 min. 50 μg / mL Protein G in 10 mM sodium acetate buffer (pH 4.5) was injected into FC1 and FC2 for 15 min. 1 M ethanolamine HCl solution (pH 8.0) was injected into FC1 and FC2 for 5 min. Approximately 1145 response units (RU) of Protein G were coupled to FC1 and FC2 of the sensor chip. 50 nM SelK2 in system buffer was injected, capturing approximately 2000 RU of SelK2 in FC1 and 1000 RU in FC2. A dilution series of SelSP1 at 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM was prepared in system buffer. Each sample was injected for 3 minutes at a flow rate of 40 μL / min. Dissociation was monitored for 4 minutes after each injection at the same flow rate. Samples were tested in duplicate, ranging from low to high concentrations.

[0396] Kinetic model fitting was performed using Qdat analysis software (SensiQ Technologies, Inc., version 2.5.1.56). A simple 1:1 kinetic model was fitted to the data to determine best-fit values ​​for the association and dissociation rate constants, k and k. To account for incomplete dissociation of SelSP1 after some analyte injections, a local Rmax was fitted to each curve. Global analysis of the binding responses revealed good agreement in kinetics and affinity between the 2000 (high) and 1000 (low) RU SelK2 surfaces. Table 2 shows the kinetic rate and affinity constants for this lot of SelK2.

[0397] result

[0398] [Table 3]

[0399] As shown in Figure 2 and Table 3, the parent antibody binds to SelSP1 with an equilibrium dissociation constant (KD) of 16.4 nM. As shown in Figures 3, 4, and Table 4, the SelK2 antibody binds to SelSP1 with an equilibrium dissociation constant (KD) of 4.28 nM. This result was consistent when two surface densities of antibody were tested and when two replicates were used for every SelSP1 concentration. Thus, surprisingly, the SelK2 antibody has improved binding affinity to SelSP1 compared to the parent antibody.

[0400] Example 4. SelK2 can functionally block neutrophil rolling on P-selectin In vitro rolling assay The ability of SelK2 to functionally block PSGL-1 was tested by assessing its ability to block neutrophil rolling on P-selectin. In this assay, human neutrophils roll onto and bind to P-selectin coated on plates at densities similar to those found on activated endothelial cells under flow conditions simulating shear stress within blood vessels.

[0401] Materials and Methods P-selectin (mP-selectin) purified from platelet membranes was prepared as described (Ushiyama et al., 1993). The parent antibody was purchased from Millipore (catalog number MAB4092). Human neutrophils were isolated from healthy donors as described (Zimmerman et al., 1985). 30 μl of human mP-selectin (1 μg / ml) was added dropwise to a defined area on a 35 mm culture plate (Corning) and incubated overnight at 4°C. The area was washed twice with HBSS and then blocked with HBSS containing 1% human serum albumin for 2 hours at room temperature. Human neutrophils (4.5 × 10 in HBSS containing 0.5% human serum albumin) were cultured in a 5% CO2-free medium. 5The cells (cells / ml) were incubated with buffer alone (control) or SelK2 at concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.06 μg / mL, 0.03 μg / mL, or 0.015 μg / mL for 5 minutes before starting the rolling experiment. Then, the cells were incubated at 1.0 dyn / cm 2 Cells were perfused over adsorbed mP-selectin in a parallel-plate flow chamber under a shear stress of 100 Hz (Ramachandran et al., 1999). After 5 min, dynamic images of several fields were captured and digitized using a Hamamatsu Photonics ORCA-Flash 2.8 CCD digital camera. The cumulative numbers of bound and slow-rolling neutrophils (SelK2 concentrations) from each run were measured in five or six different fields using image analysis software Element (Nikon Solutions Corporation), and the mean cell number and standard deviation were calculated.

[0402] [Table 4]

[0403] As shown in Figure 5 and Table 4, SelK2 inhibited neutrophil rolling in this assay in a dose-dependent manner over the concentration range of 0.125 μg / mL to 1 μg / mL, achieving near-complete inhibition at 0.5 μg / mL. These results demonstrate the ability of the SelK2 antibody to block PSGL-1 and inhibit neutrophil binding to P-selectin and rolling under shear stress.

[0404] Example 5. SelK2 can functionally block and inhibit the binding and interaction of the chemokine CCL27 with PSGL-1 The ability of SelK2 to block and inhibit the binding and interaction of the chemokine CCL27 with PSGL-1 was assessed using an SPR-based assay that uses a sulfopeptide (SelSP1) modeled on the N-terminus of human PSGL-1, which interacts with CCL27 and is bound by K2 and SelK2.

[0405] Materials and Methods All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. SensiQ Pioneer was used for all SPR experiments. CCL27 (376-CT-025 / CF) was supplied by Selexys, an R&D Systems company. SelSP1, K2, and SelK2 were also supplied by Selexys. The assay buffer contained 10 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl2, and 0.01% (w / w) Tween-20. The buffer was sterile filtered before use.

[0406] Sensor preparation: A new COOH2 sensor was installed and conditioned with two 10-second injections of each of the following: 10 mM HCl, 50 mM NaOH, and 0.1% SDS. The device was primed with assay buffer. A solution containing 75 μg / mL streptavidin, 4 mM EDC, and 1 mM NHS in 10 mM acetate buffer (pH 4.5) was injected into all three flow channels (FC) until approximately 3,000 RU of protein was captured. To cap unreacted NHS esters, 1 M ethanolamine (pH 8.0) was injected for 4 minutes to inactivate the sensor. A solution containing 500 nM SelSP1 in assay buffer was injected into FC3 and FC2 for 3 minutes. Approximately 300 RU were captured in FC2 and 250 RU in FC3. FC1 was blocked by injecting 13 μM biotin-PEO4-NH2 for 2 minutes. The system was primed again with assay buffer.

[0407] CCL27 Binding Assay: The CCL27 binding assay consisted of four injections: the first injection was either 40 μg / mL K2 or 40 μg / mL SelK2 in buffer; the second injection was 990 nM CCL27 in assay buffer; and the third and fourth injections were 0.1% SDS and 20 mM NaOH. Injection 1 was 20 min long, injection 2 was 2 min long, and injections 3 and 4 were 1 min long each. Injections were run through all three FCs. Each assay cycle was repeated three times in sequence.

[0408] Data analysis: Data were processed and analyzed using Qdat (BioLogic Software & SensiQ Technologies, Inc.). Assay curves were overlaid on the X-axis, and the Y-axis was normalized to the time point immediately before CCL27 injection. To remove nonspecific artifacts, the reference channel signal was subtracted from the SelSP1 channel signal. The identity of the blocking injection sample was used to distinguish assay cycles and compare CCL27 binding. Blocking injection samples were alternated, and each was repeated in triplicate. Because an overall decrease in CCL27 binding was observed for each of the three blocking samples, each replicate was compared together. Therefore, for each replicate set, the Ab-blocked CCL27 injection was referenced to the buffer-blocked cycle. Percent inhibition was calculated using the CCL27 injection after buffer injection as a control for each Ab-blocked replicate.

[0409] result Figure 6 shows a complete assay cycle for each blocking sample, with the parental and SelK2 cycles (dash-dotted and dashed curves, respectively) showing a large antibody binding response in injection "A," while the solid curve has negligible binding in this injection. Figure 7 shows the same data expanded to injection "B" and renormalized to just before this injection. This figure shows that CCL27 binds to SelSP1 with weak affinity (KD 10 μM, fitting not shown) in the absence of antibody blocking (buffer control). This binding is reduced when SelSP1 is blocked with either antibody.

[0410] [Table 5]

[0411] Table 5 shows the percent inhibition of the parental and SelK2 antibodies for each replicate and two SelSP1 reaction FCs. SelK2 was found to inhibit CCL27 binding by 62±6%. The parental antibody was found to inhibit CCL27 binding by 50±5%. The slightly increased inhibition of SelK2 over K2 is most likely due to its greater ability to bind SelSP1 than K2. These results demonstrate the ability of the SelK2 antibody to block chemokine binding through PSGL-1 inhibition.

[0412] We believe that this binding property of SelK2 may be related to either a binding conformation that allows better packing density of the antibody or to a higher affinity for the SelSP1 target.

[0413] Thus, this study demonstrates that these two antibodies that bind to SelSP1 can specifically block CCL27 binding to SelSP1, indicating that CCL27 recognizes a site on SelSP1 that overlaps with the antibody-binding site. The affinity of both antibodies for SelSP1 is several orders of magnitude stronger than that of CCL27, and therefore it is highly likely that the antibodies can displace CCL27 in vivo.

[0414] Taken together, the data in Examples 4 and 5 demonstrate the PSGL-1 binding affinity, specificity, and functionality of the SelK2 antibody. These results also demonstrate the dual function of SelK2. The N-terminal domain of PSGL-1 contains a selectin-binding domain that overlaps with a chemokine-binding domain. SelK2 binds to this domain and effectively blocks both selectin and chemokine binding.

[0415] Example 6. SelK2 effectively inhibits human neutrophil binding to P-selectin under flow To determine the efficacy of SelK2 in preventing adhesive interactions between PSGL-1 and its major ligand, P-selectin, human neutrophils were incubated with SelK2 before perfusion of the cells over P-selectin-coated plates under physiological flow.

[0416] Human neutrophils were incubated with buffer alone (control) or SelK2 at concentrations of 0.5 μg / mL, 0.25 μg / mL, or 0.125 μg / mL for 5 minutes. Then, a 1.0 dyn / cm 2 Cells were perfused over a surface coated with human P-selectin in a parallel-plate flow chamber under a shear stress of 100 s. Dynamic images of representative fields were recorded using a digital camera. Elongated streaks indicate cells that initially rolled on the plate-bound P-selectin surface, while rounded cells represent cells that transiently adhered to P-selectin.

[0417] As shown in Figure 8, SelK2 inhibited neutrophil rolling on P-selectin in a dose-dependent manner over the concentration range of 0.125 μg / mL to 0.5 μg / mL, achieving nearly complete inhibition at 0.5 μg / mL.

[0418] Example 7. Pharmacokinetic analysis PK ELISA assay A PK ELISA assay was used to measure SelK2 concentrations, as determined by ligand-binding antigen ELISA (LBA-ELISA). Subjects were administered 7.5 mg / kg of Selk2 at Visit 4 (Dose 1) on Day 1 and Visit 8 (Dose 2) on Day 22. Serum samples for SelK2 analysis were then collected at 15 minutes, 30 minutes, 1 hour, 24 hours, 8 days, 15 days, 29 days, 36 days, 43 days, 50 days, and 57 days after administration, along with a pre-dose collection.

[0419] Biotinylated SP-1 peptide was loaded onto a neutravidin-coated 96-well ELISA plate, followed by the loading of diluted serum samples to capture SelK2. SelK2 was then detected with HRP-conjugated goat anti-human IgG. A colorimetric signal can then be generated by developing with TMB substrate. The concentration of SelK2 in the serum sample was determined by measuring the sample signal against the signal of a known standard curve containing multiple concentrations of SelK2.

[0420] The mean and median half-lives were 322 hours and 268 hours, respectively, as shown in Figure 9 and Table 6. In addition, the mean and median times to maximum observed serum concentrations were 1.97 hours and 0.9 hours, respectively, for Dose 1 and 4.84 hours and 1 hour, respectively, for Dose 2.

[0421] [Table 6]

[0422] PD assay A PD assay was used to measure the relative inhibition of Psel-Ig binding to PSGL-1 (GSP6) by SelK2 in serum samples using surface plasmon response technology.

[0423] First, biotinylated GSP6 was loaded onto a streptavidin-coated gold sensor chip, and diluted serum samples were injected to allow active SelK2 to bind to GSP6. Finally, Psel-Ig was injected to bind any free GSP6. Signal was assessed by the change in response units (RU) from before Psel-Ig injection to 25 seconds after injection, and nonspecific signal was subtracted using a control in-line reference channel consisting of streptavidin without GSP6. Relative inhibition was determined by comparing the Psel-Ig signal of the serum sample with that of a negative control (specific "pre-dose").

[0424] As shown in Figure 10, Psel-Ig is simultaneously blocked with the observed increase in SelK2 serum concentration. When the mean serum concentration is between 35 μg / mL and 50 μg / mL, the inhibition rate of Psel-Ig is greater than 80%. In this assay, a concentration of 80% or greater is considered complete blockade. Notably, 58.6% inhibition of Psel-Ig is achieved even at the lowest serum concentration of 27.3 μg / mL on day 57 (±3 days) after administration 2.

[0425] Example 8. Study assessing the safety and efficacy of SelK2 on airway responses following allergen challenge in subjects with asthma and COPD (ClinicalTrials.gov Identifier: NCT04540042) SelK2 was administered to humans in a recent two-part, randomized, double-blind, placebo-controlled, parallel-group phase II study. The primary objective of Part 1 of the study was to determine how safe and effective two doses of SelK2 were in participants with mild asthma. Lung function and inflammatory cell counts were measured in response to allergen challenge in the lungs in the presence or absence of SelK2. Part 2 of the study examined how safe and effective a single dose of SelK2 was in participants with chronic obstructive pulmonary disease (COPD). Lung function and inflammatory cell counts were measured in the presence or absence of SelK2 in COPD patients.

[0426] Key inclusion criteria for Part 1: Male or female, aged 18-65 (inclusive). 18.0 kg / m 2 More than 35.0kg / m 2 Body mass index (BMI) below. Documented physician-diagnosed asthma for at least 4 months prior to screening. Pre-bronchodilator FEV1 ≥ 70% predicted at screening. Documented allergy to at least one common allergen confirmed by skin prick testing. Dual responders to inhaled bronchial allergen challenge as evidenced by positive allergen-induced early (EAR) and late airway bronchoconstriction (LAR) at screening.

[0427] Key exclusion criteria for Part 1: Pulmonary disease other than stable mild asthma; for example, any exacerbation of asthma in the past 4 weeks that required a change in asthma therapy or that the investigator considered clinically significant. Current or recent (within 8 weeks prior to screening or randomization) diagnosis of bacterial, protozoan, viral, or parasitic infection; suspected of having or being at high risk for a parasitic infection, or history of 2 or more episodes of herpes zoster infection. Have a history of life-threatening asthma, defined as an asthmatic episode requiring intubation and / or involving hypercapnia, respiratory arrest, and / or hypoxic attacks. Have been hospitalized or taken to the emergency room for asthma in the 12 months prior to screening or randomization. History of tuberculosis (latent or active) or systemic fungal disease.

[0428] Key inclusion criteria for Part 2: Male or female, aged 40 to 75 years (inclusive) at the time of informed consent. Physician-confirmed diagnosis of COPD with symptoms compatible with COPD for at least 1 year prior to screening. 18.0 kg / m 2 More than 35.0kg / m 2 BMI at screening as follows: Able to tolerate sputum induction and produce an adequate sputum sample with a neutrophil differential count of >55% at screening. At screening, post-bronchodilator FEV1 was 30% to 80% of the predicted normal value, and post-bronchodilator FEV1 / FVC was less than 0.7. Current or former tobacco smokers with a smoking history of at least 10 pack-years (10 pack-years defined as 20 cigarettes per day for 10 years or 10 cigarettes per day for 20 years). Have a negative blood test result for tuberculosis (TB) at screening.

[0429] Key exclusion criteria for Part 2: COPD exacerbation requiring oral steroids and / or antibiotics within 8 weeks prior to screening or randomization. Positive sputum culture at screening indicating ongoing infection. Other Respiratory Disorders: Subjects with a currently diagnosed asthma, active tuberculosis, lung cancer, bronchiectasis, sarcoidosis, pulmonary fibrosis, interstitial lung disease, known alpha-1 antitrypsin deficiency, or other active lung disease other than COPD. History of life-threatening COPD, including intensive care unit admission and / or need for intubation within the past 5 years. Two or more hospitalizations for COPD in the past year before screening. Previous lung resection, lung reduction surgery, or lung transplant. Occasionally requires supplemental oxygen. Any infection requiring hospitalization or intravenous antibiotics within 6 months prior to screening or randomization. Current or recent (within 8 weeks prior to screening or randomization) diagnosis of bacterial, protozoan, viral, or parasitic infection; suspected of having or being at high risk for a parasitic infection, or history of 2 or more episodes of herpes zoster infection. Active participation in a pulmonary rehabilitation program. History of tuberculosis (latent or active) or systemic fungal disease.

[0430] Bronchial allergen challenge was performed according to standard procedures using the method described by Taylor et al. (Taylor, Harris, and O'Connor, 2000; Comparison of incremental and bolus dose inhaled allergen challenge in asthmatic patients; Clinical and Experimental Allergy; 30: 56-63), and the presence of early and late responses was confirmed at the screening visit. The cumulative dose of allergen required to achieve a response during the screening phase was administered as a bolus during the treatment phase. Only subjects who experienced a significant symptomatic decline in FEV1 (exceeding 20% ​​and 15% expected values, respectively) during the EAR and / or LAR phases at screening were randomized at the investigator's discretion. Alternatively, if deemed safe by the investigator, subjects could undergo a repeat bronchial allergen challenge at screening, using a lower cumulative dose of the same allergen or a different allergen. If a subject met both the EAR and LAR and underwent a repeat allergen challenge, the repeat challenge was to be delayed by at least 21 days.

[0431] FEV1 time points for allergen challenge are listed below: Screening: Pre-dilution, Post-dilution, +5 min (multiple times depending on allergen titration), +10 min (multiple times depending on allergen titration), +15 min (multiple times depending on allergen titration), +20 min (multiple times depending on allergen titration), +30 min (multiple times depending on allergen titration), +45 min, 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 4.5 hr, 5 hr, 5.5 hr, 6 hr, 6.5 hr, 7 hr, 7.5 hr, 8 hr and 24 hr.

[0432] Treatment phases: pre-dilution, post-dilution, +5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 4.5 hr, 5 hr, 5.5 hr, 6 hr, 6.5 hr, 7 hr, 7.5 hr, 8 hr and 24 hr.

[0433] Failure to achieve both the EAR and LAR during the allergen challenge administered at screening indicated that the subject was not responsive to the allergen used in the challenge. Because asthmatic subjects tend to be atopic to more than one airborne allergen, repeated allergen challenge with a different allergen than that used in the initial challenge may be performed if deemed safe by the investigator. If a repeat allergen challenge is required, it should be performed at least 7 days later.

[0434] Spirometry (FEV1, FVC, FEV1% predicted, FVC% predicted, FEV1 / FVC (reported as %), FEF 25%-75%, and FEF 25%-75% predicted%) was measured using a spirometer in accordance with the American Thoracic Society (ATS) / European Respiratory Society (ERS) recommendations. Spirometry was performed according to the ATS / ERS 2019 criteria. Predicted normal values ​​used were based on the Global Lung Function Initiative (GLI) (2012) (Quanjer, et al., 2012; Multi-ethnic reference values ​​for spirometry for the 3-95-year age range: the global lung function 2012 equations; Eur Respir J; 40: 1324-1343).

[0435] [Table 7]

[0436] As outlined in Table 7, the Phase II Part 1 study involved a 42-day screening period prior to treatment with SelK2 or placebo administered intravenously on days 1 and 22, followed by a subsequent allergen challenge on day 36. As outlined in Tables 8 and 9, the study consisted of 9 patients treated with SelK2 antibody and 15 patients treated with placebo. Primary and secondary endpoints were assessed at day 36.

[0437] As shown in Tables 9 and 10, subject breakdown and demographics were fairly evenly distributed between SelK2-treated and placebo-treated patients.

[0438] [Table 8]

[0439] [Table 9]

[0440] Figure 11 shows the allergen challenge response curves (EAR and LAR) of placebo patients during screening and treatment. If the allergen challenge model is properly controlled, the two curves should overlap, as shown in the figure. Table 10 and Figure 12 show that treatment with SelK2 antibody reduced the area under the curve (AUC) of the rate of decline in FEV1 from 3 to 8 hours after allergen challenge compared to the placebo control, resulting in a mean improvement in lung function of 45.7%. Furthermore, as shown in Table 11 and Figure 12, treatment with SelK2 antibody reduced the mean maximum decline (%) from 3 to 8 hours after allergen challenge compared to the placebo control, resulting in a mean improvement in lung function of 52.4%.

[0441] [Table 10]

[0442] [Table 11]

[0443] As shown in Figures 13 and 15, SelK2-treated asthma patients showed significant decreases in absolute and percentage differential eosinophil counts at 8 and 24 hours post-challenge compared to placebo-treated patients. Figure 13 shows that the mean absolute differential eosinophil count (10E6 / g) decreased from 1.15 to 0.44 at 8 hours post-challenge and from 1.36 to 0.47 at 24 hours post-challenge in response to SelK2 treatment.

[0444] Figure 15 shows that the percent differential eosinophil counts decreased from 17.2% to 8.6% at 8 hours post-challenge and from 18.7% to 8.3% at 24 hours post-challenge in response to SelK2 treatment. In contrast, Figures 14 and 16 show that no decrease in eosinophils was observed in the blood of asthmatic patients at 8 and 24 hours post-challenge. Thus, the decrease in lung eosinophils with SelK2 treatment is not due to depletion of eosinophils in the bloodstream, as is characteristic of other approved therapies. Rather, the decrease in lung eosinophils with SelK2 treatment is due to a decrease in extravasation of these cells through the endothelium into the lung.

[0445] This is supported by Figure 17, which shows that COPD patients treated with SelK2 showed a significant decrease in sputum absolute differential eosinophil counts compared to placebo-treated patients from days 8 to 28 of the study. Figure 17 shows that on day 15, the mean absolute differential eosinophil count (10E6 / g) decreased significantly from 0.198 to 0.045, on day 22, the mean absolute differential eosinophil count (10E6 / g) decreased significantly from 0.229 to 0.016, and on day 28, the mean absolute differential eosinophil count (10E6 / g) decreased significantly from 0.187 to 0.048.

[0446] Furthermore, Figure 18 shows that no decrease in eosinophils was observed in the blood of COPD patients treated with SelK2.

[0447] Finally, Figure 19 shows that COPD patients treated with SelK2 showed a significant decrease in absolute epithelial count difference in sputum compared to placebo-treated patients starting on day 22 of the study. On day 22, the mean absolute epithelial count difference (10E6 / g) significantly decreased from 0.350 to 0.038, on day 29, the mean absolute epithelial count difference (10E6 / g) significantly decreased from 0.286 to 0.128, and on day 43, the mean absolute epithelial count difference (10E6 / g) significantly decreased from 0.332 to 0.128.

[0448] Thus, this phase II study confirms the role of SelK2 antibodies in reducing eosinophil migration to the lung and improving lung function after allergen challenge in asthmatics. Importantly, blood eosinophil levels were unchanged, indicating no systemic immunosuppression of these cells.

[0449] Example 9. Comparative analysis of airway responses following allergen challenge in patients with stable allergic asthma treated with SelK2 and tezepelumab antibodies Figure 20 is a comparative analysis showing the rate of decline in FEV1 in patients with stable allergic asthma treated with a SelK2 antibody or the recently approved tezepelumab antibody. Patients in both groups were subjected to allergen challenge on day 36 for SelK2 and day 42 for tezepelumab. The tezepelumab graph is taken from a previously published study showing FEV1 response rates in 16 patients with mild asthma treated with a tezepelumab antibody or placebo control and subjected to allergen challenge (Gauvreau, et al., 2014, Effects of an Anti-TSLP Antibody on Allergen-Induced Asthmatic Responses; The New England Journal of Medicine; 370: 2102-2110).

[0450] As shown in Table 12 and Figure 20, treatment with tezepelumab antibody resulted in a 34% improvement in maximum FEV1 decline, a 33% improvement in FEV1AUC, and a 4% improvement in mean LAR decline (%). In contrast, SelK2 treatment resulted in a 55% improvement in maximum FEV1 decline, a 51% improvement in FEV1AUC, and a 15% improvement in mean LAR decline (%). All three parameters of lung function after allergen challenge were superior with SelK2 treatment compared with tezepelumab treatment. In addition, both drugs resulted in a significant reduction in pulmonary eosinophils.

[0451] [Table 12]

[0452] Example 10. Immunogenicity analysis The immune response to the antibodies of the invention was investigated in recipients who received SelK2 or a placebo as part of a clinical trial.

[0453] Each patient's serum, collected at the designated time points, was tested in triplicate using the AlphaLISA format. Diluted serum was mixed with donor and acceptor beads previously coated with SelK2 antibodies. If anti-SelK2 antibodies are present in the serum, the anti-antibodies cross-link the two bead types, resulting in an increase in fluorescence. The combined test results of the pre-dose serum were used to establish the positive / negative cutoff. Triplicate measurements of pre-dose serum (collected before the first loading dose) were used to calculate the mean and standard deviation of the measurements. The number of data points used to calculate the standard deviation was three times the number of pre-dose specimens collected. The 95% positive / negative cutoff was determined using the following formula: 95% upper cutoff = mean signal + (1.96 x standard deviation of all signals)

[0454] result The results of this analysis are shown in Table 13. As can be seen, the incidence of immune response was 27.8% in SelK2-treated subjects and 16.7% in placebo-treated subjects. Furthermore, the immune response was low-level, some of which were not repeated at later time points, and no neutralizing activity of the antibody of the present invention was observed.

[0455] [Table 13]

[0456] Example 11. Study evaluating the effect of SelK2 on absolute immune cell counts in sputum of COPD patients A recent randomized, double-blind, placebo-controlled, parallel-group study investigated the effect of SelK2 on sputum inflammatory cells compared with placebo in subjects diagnosed with COPD. The primary objective of this study was to investigate the hypothesis that SelK2 inhibits or disrupts inflammatory cell extravasation into the pulmonary airways in subjects with COPD and to evaluate the safety and efficacy of intravenously administered SelK2 compared with placebo. Approximately 24 subjects diagnosed with COPD and with a sputum neutrophil count of greater than 55% at screening were planned to be recruited. Subjects were randomized in a 2:1 ratio to receive either 7.5 mg / kg SelK2 or placebo.

[0457] All subjects received a single dose of study medication on Day 1. The appropriate unit dose of SelK2 was prepared (by unblinded pharmacy personnel or designee) as a SelK2-saline mixture to deliver the target dose to subjects who received 100 mL of infusion.

[0458] [Table 14]

[0459] As shown in Figures 21, 22, and 23 and Table 14, SelK2-treated subjects experienced statistically significant decreases from baseline in absolute eosinophil and neutrophil cell counts and total cells per gram of sputum at Day 22 compared to placebo-treated subjects (p=0.0181, 0.0413, and 0.0257, respectively, based on adjusted least squares (LS) means).

[0460] Specifically, the reduction in absolute neutrophil counts in the SelK2-treated group was 4.1 ± 3.1 × 10 cells at baseline. 6 / g to 1.7±1.4×10 on the 22nd day 6 / g, whereas the mean cell count in the placebo group was 5.8 ± 8.4 × 10 6 / g and 5.8±7.5×10 on day 22 6 / g (p=0.0413 based on adjusted LS means).

[0461] Next, looking at the reduction in absolute eosinophil counts in the SelK2 treatment group, the cell count was an average of 0.10 ± 0.09 × 10 cells at baseline. 6 / g to 0.02±0.02×10 on the 22nd day 6 / g, whereas the mean eosinophil count in the placebo group was 0.17 ± 0.22 × 10 6 / g to 0.23±0.35×10 on the 22nd day 6 / g (p=0.0181 based on adjusted LS means).

[0462] Similarly, the total number of cells per gram of sputum in the SelK2-treated group averaged 4.8 ± 0.8 × 10 at baseline. 6 / g to 2.0±1.5×10 on the 22nd day 6 / g, whereas the number in the placebo group was a mean of 7.1 ± 3.5 × 10 at baseline. 6 / g to 7.4±9.7×10 on day 22 6 / g (p=0.0257 based on adjusted LS means).

[0463] Because both neutrophils and eosinophils are involved in the pathogenesis of COPD, the ability to achieve a targeted reduction of these cell types in the lung with a single dose of SelK2 is promising. The chronic nature of COPD means that it is unexpectedly positive that this type of reduction can be achieved within the relatively short treatment period evaluated. It can be hypothesized that long-term treatment with SelK2 will have further beneficial effects.

[0464] Although the number of neutrophils and eosinophils present in the sputum of COPD patients was reduced, no corresponding changes in the number of neutrophils and eosinophils were observed in the blood of subjects treated with SelK2. Without being bound by theory, this suggests that the reduction in neutrophils and eosinophils may be due to inhibition of their extravasation to the lungs rather than a reduction in the number of these cells in the circulation. Of note, previously published therapies rely on depletion of these inflammatory cells in the bloodstream to achieve the desired depletion in the lungs. Because SelK2 specifically inhibits the extravasation of these cells to the lungs while leaving the number of cells in the blood intact, it is expected that SelK2 treatment will not adversely affect other beneficial functions of these cells in the blood.

[0465] Sequence information

[0466] [Table 15]

[0467] [Table 16] TIFF2025531889000018.tif253170TIFF2025531889000019.tif253170TIFF2025531889000020.tif239170

[0468] Aspects and Embodiments of the Invention The following paragraphs define aspects and embodiments of the invention that do not form part of the scope of claims, but for which protection may be sought. 1. A humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1 and at least one CDR of the antibody light chain set forth in SEQ ID NO:2. 2. The humanized anti-PSGL-1 antibody of paragraph 1, wherein CDR H1 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:44. 3. The humanized anti-PSGL-1 antibody of paragraph 1 or 2, wherein CDR H2 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16 and SEQ ID NO:45. 4. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 3, wherein the CDR H3 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17 and SEQ ID NO:46. 5. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 4, wherein CDR L1 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18 and SEQ ID NO:47. 6. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 5, wherein CDR L2 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19 and SEQ ID NO:48. 7. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 6, wherein CDR L3 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:49. 8. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 7, comprising each of the Kabat / Chothia-defined CDR H1 of SEQ ID NO: 3, the Kabat / Chothia-defined CDR H2 of SEQ ID NO: 4, the Kabat / Chothia-defined CDR H3 of SEQ ID NO: 5, the Kabat / Chothia-defined CDR L1 of SEQ ID NO: 6, the Kabat / Chothia-defined CDR L2 of SEQ ID NO: 7, and the Kabat / Chothia-defined CDR L3 of SEQ ID NO: 8. 9. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 7, comprising each of a Kabat-defined CDR H1 of SEQ ID NO: 9, a Kabat-defined CDR H2 of SEQ ID NO: 10, a Kabat-defined CDR H3 of SEQ ID NO: 11, a Kabat-defined CDR L1 of SEQ ID NO: 12, a Kabat-defined CDR L2 of SEQ ID NO: 13, and a Kabat-defined CDR L3 of SEQ ID NO: 14. 10. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 7, comprising each of an IMGT-defined CDR H1 of SEQ ID NO: 15, an IMGT-defined CDR H2 of SEQ ID NO: 16, an IMGT-defined CDR H3 of SEQ ID NO: 17, an IMGT-defined CDR L1 of SEQ ID NO: 18, an IMGT-defined CDR L2 of SEQ ID NO: 19, and an IMGT-defined CDR L3 of SEQ ID NO: 20. 11. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 7, comprising each of a Chothia-defined CDR H1 of SEQ ID NO: 44, a Chothia-defined CDR H2 of SEQ ID NO: 45, a Chothia-defined CDR H3 of SEQ ID NO: 46, a Chothia-defined CDR L1 of SEQ ID NO: 47, a Chothia-defined CDR L2 of SEQ ID NO: 48, and a Chothia-defined CDR L3 of SEQ ID NO: 49. 12. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 11, comprising a heavy chain variable region having an amino acid sequence that contains no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO:26. 13. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 12, comprising a heavy chain variable region that shares at least 95% identity with the heavy chain variable region amino acid sequence set forth in SEQ ID NO:26. 14. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 13, comprising a heavy chain comprising the variable sequence set forth in SEQ ID NO:26. 15. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 14, comprising a light chain variable region having an amino acid sequence that contains no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO:29. 16. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 15, comprising a light chain variable region that shares at least 95% identity with the light chain variable region amino acid sequence set forth in SEQ ID NO:29. 17. The humanized anti-PSGL-1 antibody of any of paragraphs 1 to 16, comprising a light chain comprising the variable sequence set forth in SEQ ID NO:29. 18. comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, Anti-PSGL-1 antibody. 19. The anti-PSGL-1 antibody of any of paragraphs 1 to 18, having an affinity for PSGL-1 of less than 10 nM. 20. The anti-PSGL-1 antibody of paragraph 19, having an affinity for PSGL-1 of less than 20.5 nM. 21. The anti-PSGL-1 antibody of paragraph 20, having an affinity for PSGL-1 of about 4.29 nM. 22. The anti-PSGL-1 antibody of any of paragraphs 1 to 21, which is capable of inhibiting and disrupting the binding of PSGL-1 to its ligand. 23. The anti-PSGL-1 antibody of any of paragraphs 1 to 22, which is capable of improving lung function in asthma patients by at least 35% compared to placebo. 24. The anti-PSGL-1 antibody of paragraph 23, which is capable of improving lung function in asthma patients by about 50% compared to placebo. 25. A pharmaceutical composition comprising a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2, and a pharmaceutically acceptable excipient. 26. The pharmaceutical composition of paragraph 25, wherein the antibody is described in any of paragraphs 1 to 24. 27. A method for preventing or treating a disease or condition in a subject in need thereof, comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody, wherein the antibody comprises at least one CDR of an antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of an antibody light chain set forth in SEQ ID NO: 2. 28. A method of preventing or treating a disease or condition of Paragraph 27, wherein the antibody is described in any of Paragraphs 1 to 24. 29. A method of preventing or treating a disease or condition of paragraph 27 or 2, wherein said antibody is provided in a pharmaceutical composition of paragraph 25 or 26. 30. A method for preventing or treating the disease or condition of any of paragraphs 27 to 29 for the prevention or treatment of an inflammatory condition or disease selected from the group consisting of asthma, chronic obstructive pulmonary disease, allergic reactions, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), graft rejection, graft-versus-host disease, psoriasis, dermatitis, nephritis, lupus erythematosus, scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis, atherosclerosis, and thyroiditis. 31. The method of any of paragraphs 27 to 30 for the prevention or treatment of a respiratory disease or condition selected from the group consisting of asthma and COPD. 32. A method of treatment as set forth in paragraph 31 for the prevention or treatment of asthma. 33. The method of treatment of paragraph 31 for the prevention or treatment of COPD. 34. Eosinophilic asthma, allergies, eosinophilic esophagitis, eosinophilic dermatitis, acute myeloid leukemia (AML), ascariasis, atopic dermatitis (eczema), bullous pemphigoid, cancer (Hodgkin's lymphoma, leukemia, and certain myeloproliferative neoplasms), Churg-Strauss syndrome, drug allergy, eosinophilic cardiomyopathy, eosinophilic cellulitis (Wells syndrome), eosinophilic colitis, eosinophilic enteritis, eosinophilic fasciitis, eosinophilic gastrointestinal disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic 30. The method of any of paragraphs 27 to 29 for the prevention or treatment of an eosinophilic condition or disease selected from the group consisting of leukemia, eosinophilic myocarditis, hay fever (allergic rhinitis), Hodgkin's lymphoma (Hodgkin's disease), hypereosinophilic syndrome, idiopathic hypereosinophilic syndrome (HES), IgG4-related disease, inflammatory bowel disease (Crohn's disease, ulcerative colitis), lymphatic filariasis, neuromyelitis optica (NMO), ovarian cancer, parasitic infection, primary biliary cirrhosis, primary immunodeficiency, or trichinellosis. 35. The method of treatment of paragraph 34 for the prevention or treatment of an eosinophilic condition or disease selected from the group consisting of eosinophilic asthma, eosinophilic esophagitis, and eosinophilic dermatitis. 36. An anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2, for use as a medicament. 37. An anti-PSGL-1 antibody for use according to paragraph 36, wherein the antibody is according to any of paragraphs 1 to 24. 38. An anti-PSGL-1 antibody for use according to paragraph 36 or 37, wherein the antibody is provided in a pharmaceutical composition according to paragraph 25 or 26. 39. The anti-PSGL-1 antibody for use according to any of paragraphs 36 to 38, wherein the antibody is used for the prevention or treatment of an inflammatory condition or disease selected from the group consisting of asthma, chronic obstructive pulmonary disease, allergic reactions, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), graft rejection, graft-versus-host disease, psoriasis, dermatitis, nephritis, lupus erythematosus, scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis, atherosclerosis, and thyroiditis. 40. An anti-PSGL-1 antibody for use according to any of paragraphs 36 to 39, for use in the prevention or treatment of a respiratory disease or condition selected from the group consisting of asthma and COPD. 41. An anti-PSGL-1 antibody for use according to paragraph 40, for use in the prevention or treatment of asthma. 42. An anti-PSGL-1 antibody for use according to paragraph 40, for use in the prevention or treatment of COPD. 43. Eosinophilic asthma, allergies, eosinophilic esophagitis, eosinophilic dermatitis, acute myeloid leukemia (AML), ascariasis, atopic dermatitis (eczema), bullous pemphigoid, cancer (Hodgkin's lymphoma, leukemia, and certain myeloproliferative neoplasms), Churg-Strauss syndrome, drug allergy, eosinophilic cardiomyopathy, eosinophilic cellulitis (Wells syndrome), eosinophilic colitis, eosinophilic enteritis, eosinophilic fasciitis, eosinophilic gastrointestinal disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic leukemia, eosinophilic 39. The anti-PSGL-1 antibody for use according to any of paragraphs 36 to 38, for use in the prevention or treatment of an eosinophilic condition or disease selected from the group consisting of myocarditis, hay fever (allergic rhinitis), Hodgkin's lymphoma (Hodgkin's disease), hypereosinophilic syndrome, idiopathic hypereosinophilic syndrome (HES), IgG4-related disease, inflammatory bowel disease (Crohn's disease, ulcerative colitis), lymphatic filariasis, neuromyelitis optica (NMO), ovarian cancer, parasitic infection, primary biliary cirrhosis, primary immunodeficiency, or trichinellosis. 44. The anti-PSGL-1 antibody for use according to paragraph 43, for use in the prevention or treatment of an eosinophilic condition or disease selected from the group consisting of eosinophilic asthma, eosinophilic esophagitis and eosinophilic dermatitis. 45. A polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody, comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1 and at least one CDR of the antibody light chain set forth in SEQ ID NO:2. 46. ​​The polynucleotide sequence of Paragraph 45, encoding an immunoglobulin chain of the humanized anti-PSGL-1 antibody of any of Paragraphs 1 to 23. 47. The polynucleotide sequence of paragraph 45 or 46, comprising SEQ ID NO:42. 48. The polynucleotide sequence of any of paragraphs 45 to 47, comprising SEQ ID NO: 43. 49. A cell comprising a polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody, the immunoglobulin chain comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2. 50. The cell of Paragraph 49, wherein the polynucleotide sequence is as set forth in any of Paragraphs 46 to 48. 51. A method for producing a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2, comprising: expressing a first polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1; expressing a second polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody light chain set forth in SEQ ID NO:2; A method comprising: 52. The method of paragraph 51, wherein the first polynucleotide sequence and / or the second polynucleotide sequence is as described in any of paragraphs 46 to 48.

Claims

1. A humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1 and at least one CDR of the antibody light chain set forth in SEQ ID NO:

2.

2. The humanized anti-PSGL-1 antibody of claim 1, comprising a heavy chain variable region defined as SEQ ID NO:26 and / or a light chain variable region defined as SEQ ID NO:

29.

3. 3. The humanized anti-PSGL-1 antibody of claim 1 or 2, wherein the CDR H1 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15 and SEQ ID NO:

44.

4. 4. The humanized anti-PSGL-1 antibody of any one of claims 1 to 3, wherein the CDR H2 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16 and SEQ ID NO:

45.

5. 5. The humanized anti-PSGL-1 antibody of any one of claims 1 to 4, wherein the CDR H3 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17 and SEQ ID NO:

46.

6. 6. The humanized anti-PSGL-1 antibody of any one of claims 1 to 5, wherein CDR L1 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18 and SEQ ID NO:

47.

7. 7. The humanized anti-PSGL-1 antibody of any one of claims 1 to 6, wherein CDR L2 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19 and SEQ ID NO:

48.

8. 8. The humanized anti-PSGL-1 antibody of any one of claims 1 to 7, wherein the CDR L3 of the antibody comprises or consists of a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:

49.

9. 9. The humanized anti-PSGL-1 antibody of any one of claims 1 to 8, comprising each of a Kabat / Chothia-defined CDR H1 of SEQ ID NO: 3, a Kabat / Chothia-defined CDR H2 of SEQ ID NO: 4, a Kabat / Chothia-defined CDR H3 of SEQ ID NO: 5, a Kabat / Chothia-defined CDR L1 of SEQ ID NO: 6, a Kabat / Chothia-defined CDR L2 of SEQ ID NO: 7, and a Kabat / Chothia-defined CDR L3 of SEQ ID NO:

8.

10. The humanized anti-PSGL-1 antibody of any one of claims 1 to 8, comprising each of a Kabat-defined CDR H1 of SEQ ID NO: 9, a Kabat-defined CDR H2 of SEQ ID NO: 10, a Kabat-defined CDR H3 of SEQ ID NO: 11, a Kabat-defined CDR L1 of SEQ ID NO: 12, a Kabat-defined CDR L2 of SEQ ID NO: 13, and a Kabat-defined CDR L3 of SEQ ID NO:

14.

11. 9. The humanized anti-PSGL-1 antibody of any one of claims 1 to 8, comprising each of an IMGT-defined CDR H1 of SEQ ID NO: 15, an IMGT-defined CDR H2 of SEQ ID NO: 16, an IMGT-defined CDR H3 of SEQ ID NO: 17, an IMGT-defined CDR L1 of SEQ ID NO: 18, an IMGT-defined CDR L2 of SEQ ID NO: 19, and an IMGT-defined CDR L3 of SEQ ID NO:

20.

12. 9. The humanized anti-PSGL-1 antibody of any one of claims 1 to 8, comprising each of a Chothia-defined CDR H1 of SEQ ID NO: 44, a Chothia-defined CDR H2 of SEQ ID NO: 45, a Chothia-defined CDR H3 of SEQ ID NO: 46, a Chothia-defined CDR L1 of SEQ ID NO: 47, a Chothia-defined CDR L2 of SEQ ID NO: 48, and a Chothia-defined CDR L3 of SEQ ID NO:

49.

13. The humanized anti-PSGL-1 antibody of any one of claims 1 to 12, comprising a heavy chain variable region having an amino acid sequence containing no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO:

26.

14. The humanized anti-PSGL-1 antibody of any one of claims 1 to 13, comprising a heavy chain variable region that shares at least 95% identity with the heavy chain variable region amino acid sequence set forth in SEQ ID NO:

26.

15. The humanized anti-PSGL-1 antibody of any one of claims 1 to 14, comprising a heavy chain comprising the variable sequence set forth in SEQ ID NO:

26.

16. The humanized anti-PSGL-1 antibody of any one of claims 1 to 15, comprising a light chain variable region having an amino acid sequence containing no more than 10 amino acid changes compared to the sequence set forth in SEQ ID NO:

29.

17. The humanized anti-PSGL-1 antibody of any one of claims 1 to 16, comprising a light chain variable region that shares at least 95% identity with the light chain variable region amino acid sequence set forth in SEQ ID NO:

29.

18. The humanized anti-PSGL-1 antibody of any one of claims 1 to 17, comprising a light chain comprising the variable sequence set forth in SEQ ID NO:

29.

19. comprising a heavy chain variable sequence set forth in SEQ ID NO: 26 and a light chain variable sequence set forth in SEQ ID NO: 29; or comprising a heavy chain sequence as set forth in SEQ ID NO: 1 and a light chain sequence as set forth in SEQ ID NO: 2, Anti-PSGL-1 antibody.

20. The anti-PSGL-1 antibody of any one of claims 1 to 19, having an affinity for PSGL-1 of less than 10 nM.

21. 21. The anti-PSGL-1 antibody of claim 20, having an affinity for PSGL-1 of less than 5 nM.

22. The anti-PSGL-1 antibody of claim 21, having an affinity for PSGL-1 of about 4.29 nM.

23. The anti-PSGL-1 antibody of any one of claims 1 to 22, which is capable of inhibiting and disrupting the binding of PSGL-1 to its ligand.

24. The anti-PSGL-1 antibody of any one of claims 1 to 23, which is capable of improving lung function in asthma patients by at least 35% compared to placebo.

25. The anti-PSGL-1 antibody of claim 24, which is capable of improving lung function in asthma patients by about 50% compared to placebo.

26. An antigen-binding fragment of the anti-PSGL-1 antibody of any one of claims 1 to 25.

27. A pharmaceutical composition comprising a humanized anti-PSGL-1 antibody, or an antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO: 2, and a pharmaceutically acceptable excipient.

28. The pharmaceutical composition of claim 27, wherein the antibody is as defined in any one of claims 1 to 25.

29. 28. The pharmaceutical composition of claim 27, wherein the antibody fragment is as described in claim 26.

30. 1. A method for preventing or treating a disease or condition in a subject in need thereof, comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody, wherein the antibody comprises at least one CDR of an antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of an antibody light chain set forth in SEQ ID NO: 2, or an antigen-binding fragment thereof.

31. 31. A method for preventing or treating a disease or condition according to claim 30, wherein the antibody is according to any one of claims 1 to 25.

32. 31. A method for preventing or treating a disease or condition according to claim 30, wherein the antigen-binding antibody fragment is as described in claim 26.

33. 33. A method for preventing or treating a disease or condition according to any one of claims 30 to 32, wherein said antibody or antigen-binding fragment thereof is provided in a pharmaceutical composition according to any one of claims 27 to 29.

34. 34. The method for preventing or treating a disease or condition according to any one of claims 30 to 33, wherein the disease is an inflammatory condition or disease selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), allergic reactions, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), transplant rejection, graft-versus-host disease, psoriasis, dermatitis, nephritis, lupus erythematosus, scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis, atherosclerosis, and thyroiditis.

35. A method for preventing or treating a disease or condition according to any one of claims 30 to 34, for the prevention or treatment of a respiratory disease or condition selected from the group consisting of asthma and COPD.

36. 36. A method for preventing or treating a disease or condition according to claim 35 for the prevention or treatment of asthma.

37. 36. A method for preventing or treating a disease or condition according to claim 35 for the prevention or treatment of COPD.

38. The disease or condition may be eosinophilic asthma, allergies, eosinophilic esophagitis, eosinophilic dermatitis, acute myeloid leukemia (AML), ascariasis, atopic dermatitis (eczema), bullous pemphigoid, cancer (such as Hodgkin's lymphoma, leukemia, and certain myeloproliferative neoplasms), Churg-Strauss syndrome, drug allergy, eosinophilic cardiomyopathy, eosinophilic cellulitis (Wells' syndrome), eosinophilic colitis, eosinophilic enteritis, eosinophilic fasciitis, eosinophilic gastrointestinal disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic leukemia, 34. The method for preventing or treating the disease or condition according to any one of claims 30 to 33, wherein the disease or condition is an eosinophilic condition or disease selected from the group consisting of eosinophilic myocarditis, hay fever (allergic rhinitis), Hodgkin's lymphoma (Hodgkin's disease), hypereosinophilic syndrome, idiopathic hypereosinophilic syndrome (HES), IgG4-related disease, inflammatory bowel disease (Crohn's disease, ulcerative colitis), lymphatic filariasis, neuromyelitis optica (NMO), ovarian cancer, parasitic infection, primary biliary cirrhosis, primary immunodeficiency syndrome, and trichinellosis.

39. 39. The method of preventing or treating a disease or condition according to claim 38, wherein the eosinophilic condition or disease is eosinophilic asthma, eosinophilic esophagitis or eosinophilic dermatitis.

40. The disease or condition is selected from the group consisting of COPD, neutrophilic dermatoses (e.g., Sweet's syndrome, sterile pustulosis of the folds, erythema elevatum perstans, sterile pustulosis of the scalp / legs, sterile subcorneal pustulosis, and pyoderma gangrenosum), neutrophilic asthma, rheumatoid arthritis (RA), cystic fibrosis (CF), neutrophilic vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis), gout, IBD (e.g., Crohn's disease and ulcerative colitis), neutrophilic meningitis, Behcet's disease, PAPA syndrome, hidradenitis suppurativa, EGFR-associated acne, PASH syndrome, and PAPASH syndrome. , neutrophilic panniculitis, sterile abscess syndrome, systemic inflammatory response syndrome (SIRS), severe sepsis, cryopyrin-associated periodic fever syndrome (CAPS), neutrophilic otitis externa, allergy, chronic neutrophilic leukemia (CNL), hay fever (allergic rhinitis), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), SARS-CoV, SARS-CoV-2, and necrotizing colitis (NEC).

41. 34. A method for preventing or treating a disease or condition according to any one of claims 30 to 33, wherein the disease or condition is sickle cell disease (SCD).

42. 42. A method of preventing or treating the disease or condition of claim 41, comprising preventing or treating a symptom of SCD selected from the group consisting of pain crisis, vascular occlusion, and chronic inflammation.

43. An anti-PSGL-1 antibody, or an antigen-binding fragment thereof, for use as a medicament, comprising at least one CDR of the antibody heavy chain defined in SEQ ID NO: 1 and at least one CDR of the antibody light chain defined in SEQ ID NO:

2.

44. The anti-PSGL-1 antibody for use according to claim 43, wherein the antibody is described in any one of claims 1 to 25.

45. The antigen-binding fragment of an anti-PSGL-1 antibody for use according to claim 43, wherein the antigen-binding antibody fragment is as described in claim 26.

46. The anti-PSGL-1 antibody for use according to any one of claims 43 to 45, wherein the antibody or antigen-binding fragment thereof is provided in a pharmaceutical composition according to any one of claims 27 to 29.

47. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to any one of claims 43 to 46, for use in the prevention or treatment of an inflammatory condition or disease selected from the group consisting of asthma, COPD, allergic reactions, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), transplant rejection, graft-versus-host disease, psoriasis, dermatitis, nephritis, lupus erythematosus, scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis, atherosclerosis, and thyroiditis.

48. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to any one of claims 43 to 47, for use in the prevention or treatment of a respiratory disease or condition selected from the group consisting of asthma and COPD.

49. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to claim 48, for use in the prevention or treatment of asthma.

50. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to claim 48, for use in the prevention or treatment of COPD.

51. Eosinophilic asthma, allergies, eosinophilic esophagitis, eosinophilic dermatitis, acute myeloid leukemia (AML), ascariasis, atopic dermatitis (eczema), bullous pemphigoid, cancer (such as Hodgkin's lymphoma, leukemia, and certain myeloproliferative neoplasms), Churg-Strauss syndrome, drug allergies, eosinophilic cardiomyopathy, eosinophilic cellulitis (Wells syndrome), eosinophilic colitis, eosinophilic enteritis, eosinophilic fasciitis, eosinophilic gastrointestinal disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic leukemia, eosinophilic myocarditis, hay fever (allergic 47. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to any one of claims 43 to 46, for use in the prevention or treatment of an eosinophilic condition or disease selected from the group consisting of eosinophilic syndrome, idiopathic hypereosinophilic rhinitis, Hodgkin's lymphoma (Hodgkin's disease), hypereosinophilic syndrome, idiopathic hypereosinophilic syndrome (HES), IgG4-related disease, inflammatory bowel disease (Crohn's disease, ulcerative colitis), lymphatic filariasis, neuromyelitis optica (NMO), ovarian cancer, parasitic infection, primary biliary cirrhosis, primary immunodeficiency, or trichinellosis.

52. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to claim 51, for use in the prevention or treatment of an eosinophilic condition or disease selected from the group consisting of eosinophilic asthma, eosinophilic esophagitis, and eosinophilic dermatitis.

53. COPD, neutrophilic dermatoses (e.g., Sweet's syndrome, sterile pustulosis of the folds, erythema elevatum, sterile pustulosis of the scalp / legs, sterile subcorneal pustulosis, and pyoderma gangrenosum), neutrophilic asthma, rheumatoid arthritis (RA), cystic fibrosis (CF), neutrophilic vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis), gout, IBD (e.g., Crohn's disease and ulcerative colitis), neutrophilic meningitis, Behcet's disease, PAPA syndrome, hidradenitis suppurativa, EGFR-associated acne, PASH syndrome, PAPASH syndrome, neutrophilic panniculitis, sterile abscess 47. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to any one of claims 43 to 46, for use in the prevention or treatment of a neutrophilic condition or disease selected from the group consisting of pulmonary embolism syndrome, systemic inflammatory response syndrome (SIRS), severe sepsis, cryopyrin-associated periodic fever syndrome (CAPS), neutrophilic otitis externa, allergy, chronic neutrophilic leukemia (CNL), hay fever (allergic rhinitis), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), SARS-CoV, SARS-CoV-2, and necrotizing colitis (NEC).

54. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to any one of claims 43 to 46, for use in the prevention or treatment of sickle cell disease (SCD).

55. The anti-PSGL-1 antibody or antigen-binding fragment thereof for use according to claim 54, for use in the prevention or treatment of a symptom of SCD selected from the group consisting of pain crisis, vascular occlusion, and chronic inflammation.

56. A polynucleotide sequence encoding the immunoglobulin chains of a humanized anti-PSGL-1 antibody or antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1 and at least one CDR of the antibody light chain set forth in SEQ ID NO:

2.

57. A polynucleotide sequence encoding a humanized anti-PSGL-1 antibody or antigen-binding fragment thereof, comprising an immunoglobulin chain comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1 and an immunoglobulin chain comprising at least one CDR of the antibody light chain set forth in SEQ ID NO:

2.

58. A polynucleotide sequence encoding the heavy chain variable sequence set forth in SEQ ID NO:

26.

59. A polynucleotide sequence encoding the light chain variable sequence set forth in SEQ ID NO:

29.

60. A polynucleotide sequence according to any one of claims 56 to 59, encoding an immunoglobulin chain of the humanized anti-PSGL-1 antibody according to any one of claims 1 to 25.

61. A polynucleotide according to any one of claims 56 to 60, encoding an antibody according to any one of claims 1 to 25.

62. A polynucleotide encoding the antibody of any one of claims 1 to 25.

63. 63. The polynucleotide sequence of any one of claims 56 to 62, comprising SEQ ID NO:

42.

64. 64. The polynucleotide sequence of any one of claims 56 to 63, comprising SEQ ID NO:

43.

65. A cell comprising a polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody or antigen-binding fragment thereof, comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO:

2.

66. A cell comprising a polynucleotide sequence encoding a humanized anti-PSGL-1 antibody or an antigen-binding fragment thereof, comprising an immunoglobulin chain comprising at least one CDR of an antibody heavy chain defined in SEQ ID NO: 1 and an immunoglobulin chain comprising at least one CDR of an antibody light chain defined in SEQ ID NO:

2.

67. A cell comprising a polynucleotide sequence encoding the heavy chain sequence set forth in SEQ ID NO:1 and a polynucleotide sequence encoding the light chain sequence set forth in SEQ ID NO:

2.

68. 68. The cell of any one of claims 65 to 67, wherein the polynucleotide sequence is as defined in any one of claims 56 to 64.

69. 1. A method for producing a humanized anti-PSGL-1 antibody comprising at least one CDR of an antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of an antibody light chain set forth in SEQ ID NO: 2, comprising: expressing a first polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO:1; expressing a second polynucleotide sequence encoding an immunoglobulin chain of a humanized anti-PSGL-1 antibody comprising at least one CDR of the antibody light chain set forth in SEQ ID NO:2; A method comprising:

70. 70. The method of claim 69, wherein the first polynucleotide sequence and / or the second polynucleotide sequence is as defined in any one of claims 56 to 64.

71. 1. A method for reducing neutrophil counts in sputum of a subject in need thereof, comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody or antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises at least one CDR of an antibody heavy chain set forth in SEQ ID NO:1 and at least one CDR of an antibody light chain set forth in SEQ ID NO:

2.

72. 72. The method of claim 71, wherein the antibody is described in any one of claims 1 to 25.

73. 72. The method of claim 71, wherein the antibody fragment is as described in claim 26.

74. 74. The method of any one of claims 71 to 73, wherein the therapeutically effective amount is an amount effective to inhibit neutrophil extravasation.

75. 75. The method of any one of claims 71 to 74, wherein the number of neutrophils in the blood of the subject is not reduced.

76. 1. A method for reducing eosinophil count in sputum of a subject in need thereof, comprising providing to the subject a therapeutically effective amount of an anti-PSGL-1 antibody or antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises at least one CDR of an antibody heavy chain set forth in SEQ ID NO:1 and at least one CDR of an antibody light chain set forth in SEQ ID NO:

2.

77. 77. The method of claim 76, wherein the antibody is described in any one of claims 1 to 25.

78. 77. The method of claim 76, wherein the antibody fragment is as described in claim 26.

79. 79. The method of any one of claims 76 to 78, wherein the therapeutically effective amount is an amount effective to inhibit eosinophil extravasation.

80. 80. The method of any one of claims 76 to 79, wherein the number of eosinophils in the blood of the subject is not reduced.

81. 81. The method of any one of claims 71 to 80, wherein the subject has an inflammatory condition or disease selected from the group consisting of chronic obstructive pulmonary disease, asthma, an allergic reaction, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), transplant rejection, graft versus host disease, psoriasis, dermatitis, nephritis, lupus erythematosus, scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis, atherosclerosis, and thyroiditis.

82. An antibody or antigen-binding fragment thereof comprising at least one CDR of the antibody heavy chain set forth in SEQ ID NO: 1 and at least one CDR of the antibody light chain set forth in SEQ ID NO: 2, for use in treating pulmonary inflammatory conditions by inhibiting neutrophil and / or eosinophil extravasation, thereby reducing the accumulation of neutrophils and / or eosinophils in sputum.

83. The anti-PSGL-1 antibody for use according to claim 82, wherein the antibody is described in any one of claims 1 to 25.

84. The antigen-binding fragment of an anti-PSGL-1 antibody for use according to claim 82, wherein the antigen-binding antibody fragment is as described in claim 26.

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