WO2022061032A1 - Methods and compositions for treating viral infection - Google Patents

Abstract

The present disclosure provides methods for the treatment of viral infection, inhibiting virus -induced inflammation, and inhibiting virus-induced activation of mast cells and/or eosinophils. The present disclosure also provides articles of manufacture or kits comprising antibodies that bind to human Siglec-8 for the treatment of viral infection, inhibiting virus- induced inflammation, and inhibiting virus -induced activation of mast cells and/or eosinophils.

Description

METHODS AND COMPOSITIONS FOR TREATING VIRAL INFECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial Nos. 63/079,893, filed September 17, 2020, and 63/225,303, filed July 23, 2021, the disclosures of each of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 701712001640SEQLIST.TXT, date recorded: September 13, 2021, size: 107,975 bytes).

FIELD OF THE INVENTION

[0003] The present disclosure relates to methods for treating viral infection by administration of antibodies that bind to human Siglec-8 and compositions comprising said antibodies.

BACKGROUND

[0004] Siglec-8, a member of the CD33-related family of sialic acid-binding, immunoglobulin-like lectins (Siglecs), is a transmembrane cell surface protein with restricted tissue distribution, expressed selectively on the surface of eosinophils, mast cells and, at lower levels, on basophils. Siglec-8 contains 3 extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail containing 2 tyrosine-based signaling motifs including an immunoreceptor tyrosine-based inhibitory motif with inhibitory function. Engagement of Siglec-8 in mast cells can result in inhibition of mediator release, and in eosinophils can induce apoptosis (Bochner, B. (2009) Clin. Exp. Allergy 39:317-324).

[0005] Recent worldwide pandemics have arisen from various infectious viruses such as H1N1 influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), etc. In particular, the rapid spread of severe respiratory syndrome coronavirus 2 (SARS-Cov-2) and resulting coronavirus disease 2019 (COVID-19) poses an unprecedented health crisis. While the majority of cases get resolved with mild symptoms, some patients develop fatal complications, such as acute respiratory distress syndrome (ARDS) for which effective therapeutic strategies are urgently needed. In these severe cases, a hyper-inflammatory response or cytokine storm has been observed and is suspected to be one of the primary drivers of pathology. Indeed, transcriptomic profiling and histologic examination of the lungs or bronchoalveolar lavage (BAL) fluid of COVID-19 patients have revealed extensive immune cell infiltration and significantly elevated levels of cytokines, chemokines, and other pro- inflammatory mediators that correlate with disease severity. Evidence currently points to immune dysfunction as a potential driver of these hallmark syndromes of COVID- 19, however an understanding of the specific immune responses to SARS-CoV-2 remain extremely limited. [0006] As such, there remains a need for an effective treatment for viral infections and virus- induced inflammation, such as that observed with certain COVID-19 patients.

[0007] All references cited herein, including patent applications, patent publications, and scientific literature, are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

[0008] To meet this and other needs, the present disclosure relates, inter alia, to methods of treating viral infection, inhibiting virus-induced inflammation, and/or inhibiting virus-induced activation of mast cells and/or eosinophils by administration of antibodies that bind to human Siglec-8 and/or compositions comprising said antibodies. These are based at least in part on the findings demonstrated herein that elevated activation of mast cells and eosinophils is found in patients suffering from viral infection. Without wishing to be bound to theory, it is thought that viral infection can lead to innate immune stimulation independent of specific virus: viral receptor interactions, and that engagement of Siglec-8 with a monoclonal antibody could serve as a potential therapeutic option in viral infection, e.g., by reducing aberrant MC and eosinophil activation and hyperinflammation.

[0009] Accordingly, certain aspects of the present disclosure relate to methods for treating viral infection, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody that binds to human Siglec-8. Other aspects of the present disclosure relate to methods for inhibiting inflammation in an individual with a viral infection, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8. Other aspects of the present disclosure relate to methods for inhibiting mast cell and/or eosinophil activation in an individual with a viral infection, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8.

[0010] In some embodiments according to any of the embodiments described herein, the virus is an enveloped virus. In some embodiments, the virus is a Coronavirus. In some embodiments, the virus is selected from the group consisting of severe acute respiratory syndrome coronavirus 1 (SARS-Cov-1), severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), and Middle East respiratory syndrome-related coronavirus (MERS-Cov). In some embodiments, the virus is an Orthomyxovirus. In some embodiments, the virus is an influenza A virus. In some embodiments, the virus is an Orthopneumovirus. In some embodiments, the virus is respiratory syncytial virus (RSV).

[0011] In some embodiments according to any of the embodiments described herein, the individual has or has been diagnosed with acute respiratory distress syndrome (ARDS). In some embodiments, the individual has or has been diagnosed with post-acute COVID-19 syndrome. [0012] Certain aspects of the present disclosure relate to methods for treating post-acute COVID-19 syndrome, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody that binds to human Siglec-8. Other aspects of the present disclosure relate to methods for treating one or more symptoms of post-acute COVID-19 syndrome, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody that binds to human Siglec-8. Other aspects of the present disclosure relate to methods for inhibiting inflammation in an individual with postacute COVID- 19 syndrome, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8. Other aspects of the present disclosure relate to methods for inhibiting mast cell and/or eosinophil activation in an individual with post-acute COVID- 19 syndrome, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8. In some embodiments, the individual has or has been diagnosed with post-acute COVID-19 syndrome. In some embodiments, the individual has a prior COVID- 19 infection. In some embodiments, one or more symptom(s) of post-acute COVID-19 syndrome in the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g., prior to administration). In some embodiments, the one or more symptom(s) include loss of smell and/or taste, shortness of breath, fatigue, myalgia, dysautonomia, decreased exercise capacity, hypoxia, reduced diffusion capacity, restrictive pulmonary physiology, pulmonary fibrosis, diminished quality of life, muscular weakness, joint pain, dyspnea, cough, persistent oxygen requirement, anxiety, depression, sleep disturbances, post-traumatic stress disorder (PTSD), cognitive disturbance (e.g, “brain fog”), headache, palpitations, chest pain, increased cardiometabolic demand, myocardial fibrosis or scarring, arrhythmia, tachycardia, autonomic dysfunction, thromboembolism, chronic kidney disease, impaired renal function, new or worsening diabetes mellitus (type II diabetes), subacute thyroiditis, bone demineralization, multisystem inflammatory syndrome (MIS-C), and hair loss. Any of the antibodies or compositions described herein may find use in the methods of the present disclosure.

[0013] In some embodiments according to any of the embodiments described herein, prior to administration of the composition, the individual has increased mast cell activation, e.g., as compared to a reference or reference value. In some embodiments, the individual has increased mast cell activation in peripheral blood. In some embodiments, a peripheral blood sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present. In some embodiments, the individual has increased mast cell activation in lung tissue. In some embodiments, a lung-derived sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present. In some embodiments, prior to administration of the composition, the individual has increased eosinophil activation, e.g, as compared to a reference or reference value. In some embodiments, the individual has increased eosinophil activation in peripheral blood. In some embodiments, a peripheral blood sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present. In some embodiments, the individual has increased eosinophil activation in lung tissue. In some embodiments, a lung-derived sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present. In some embodiments, a serum sample obtained from the individual prior to administration of the composition has an elevated level of one or more pro-inflammatory cytokine(s) or growth factor(s), e.g., as compared to a reference or reference value. In some embodiments, the one or more pro- inflammatory cytokine(s) or growth factor(s) are selected from the group consisting of CCL2, IP- 10, IL-8, VEGF, histamine, leukotriene C4, leukotriene E4, prostaglandin D2, eotaxin, periostin, eosinophil peroxidase (EPX), IL-6, TNF, C-reactive protein (CRP), ferritin, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and IFN-y. In some embodiments, a serum sample obtained from the individual prior to administration of the composition has an elevated level of one or more polypeptide(s) expressed by mast cells, e.g, as compared to a reference or reference value. In some embodiments, the one or more polypeptide(s) expressed by mast cells are selected from the group consisting of chymase, -tryptase, and CPA3. In some embodiments, a lung-derived sample obtained from the individual prior to administration of the composition has an elevated level of one or more polypeptide(s) expressed by mast cells, pro-inflammatory cytokine(s), or chemokine(s), e.g., as compared to a reference or reference value; or an elevated level of one or more polynucleotides encoding one or more polypeptide(s) expressed by mast cells, pro-inflammatory cytokine(s), or chemokine(s), e.g., as compared to a reference or reference value. In some embodiments, the one or more polypeptide(s) expressed by mast cells and/or pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, CCL4, IL-8, IP-10, TPSB2, TPSAB1, and FCER1G. In some embodiments, the lung-derived sample is a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample. In some embodiments, prior to administration of the composition, the individual has eosinopenia in peripheral blood. In some embodiments, a serum sample obtained from the individual prior to administration of the composition has an elevated level of one or more polypeptide(s) expressed by eosinophils, e.g., as compared to a reference or reference value. In some embodiments, the one or more polypeptide(s) expressed by eosinophils comprises EDN. In some embodiments, a lung-derived sample obtained from the individual prior to administration of the composition has an elevated level of one or more polypeptide(s) expressed by eosinophils, e.g., as compared to a reference or reference value; or an elevated level of one or more polynucleotides encoding one or more polypeptide(s) expressed by eosinophils, e.g, as compared to a reference or reference value. In some embodiments, the one or more polypeptide(s) expressed by eosinophils are selected from the group consisting of EDN and Galectin-10. In some embodiments, the lung-derived sample is a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample. In some embodiments, one or more symptom(s) of viral infection in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition. In some embodiments, one or more symptom(s) of inflammation in the individual are reduced after administration of the composition e.g., as compared to a reference, reference value, or baseline level before administration of the composition. In some embodiments, one or more symptom(s) of ARDS in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition. In some embodiments, one or more symptom(s) of post-acute COVID-19 syndrome in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition. In some embodiments, level of one or more pro-inflammatory cytokine(s) or chemokine(s) in a serum sample obtained from the individual are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline level in a serum sample obtained before administration of the composition. In some embodiments, the one or more pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, IP- 10, IL-6, ferritin, C-reactive protein (CRP), and TNF. In some embodiments, level of one or more polypeptide(s) expressed by mast cells in a serum sample obtained from the individual are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline level in a serum sample obtained before administration of the composition. In some embodiments, the one or more polypeptide(s) expressed by mast cells comprises chymase. In some embodiments, level of one or more pro-inflammatory cytokine(s) or chemokine(s) or one or more polynucleotides encoding one or more pro-inflammatory cytokine(s) or chemokine(s)in a lung-derived sample obtained from the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline level in a lung-derived sample obtained before administration of the composition. In some embodiments, the one or more pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, IL-6, CXCL2, and IL-ip. In some embodiments, number(s) of eosinophils, monocytes, and/or neutrophils in a peripheral blood sample obtained from the individual are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline level in a peripheral blood sample obtained before administration of the composition. In some embodiments, number(s) of eosinophils, monocytes, and/or neutrophils in a lung-derived sample obtained from the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline level in a lung-derived sample obtained before administration of the composition. In some embodiments, level of one or more polypeptide(s) expressed by eosinophils in a serum sample obtained from the individual are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline level in a serum sample obtained before administration of the composition. In some embodiments, level of one or more polypeptide(s) expressed by eosinophils or one or more polynucleotides encoding one or more polypeptide(s) expressed by eosinophils in a lung-derived sample obtained from the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline level in a lung-derived sample obtained before administration of the composition. In some embodiments, the one or more polypeptide(s) expressed by eosinophils comprise eosinophil peroxidase (EPX). In some embodiments, eosinophil activation in the individual is reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline level before administration of the composition. In some embodiments, eosinophil activation in peripheral blood is reduced after administration of the composition. In some embodiments, eosinophil activation in a lung-derived sample is reduced after administration of the composition. In some embodiments, mast cell activation in the individual is reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline level before administration of the composition. In some embodiments, mast cell activation in peripheral blood is reduced after administration of the composition. In some embodiments, mast cell activation in a lung-derived sample is reduced after administration of the composition. In some embodiments, the lung-derived sample is a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample.

[0014] In some embodiments according to any of the embodiments described herein, the composition is administered by subcutaneous injection. In some embodiments, the composition is administered by intravenous infusion.

[0015] In some embodiments that may be combined with any other embodiments described herein, the antibody comprises a Fc region and N-gly coside-linked carbohydrate chains linked to the Fc region, wherein less than 50% of the N-gly coside-linked carbohydrate chains of the antibody in the composition contain a fucose residue. In some embodiments, substantially none of the N-gly coside-linked carbohydrate chains of the antibody in the composition contain a fucose residue. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence selected from SEQ ID NOs:67-70; and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and/or a light chain variable region comprising the amino acid sequence selected from SEQ ID NO: 16 or 21. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence selected from SEQ ID NOs: 11- 14; and/or a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs:23-24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence selected from SEQ ID NOs:2-14; and/or a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs: 16-24. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence selected from SEQ ID NOs:2-10; and/or a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs: 16-22. In some embodiments, the antibody comprises: (a) heavy chain variable region comprising: (1) an HC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:26-29; (2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61; (3) an HC-FR2 comprising the amino acid sequence selected from SEQ ID NOs:31-36; (4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62; (5) an HC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:38-43; (6) an HVR- H3 comprising the amino acid sequence of SEQ ID NO:63; and (7) an HC-FR4 comprising the amino acid sequence selected from SEQ ID NOs:45-46, and/or (b) a light chain variable region comprising: (1) an LC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:48- 49; (2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64; (3) an LC-FR2 comprising the amino acid sequence selected from SEQ ID NOs:51-53; (4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65; (5) an LC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:55-58; (6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 66; and (7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the antibody comprises: (a) heavy chain variable region comprising: (1) an HC-FR1 comprising the amino acid sequence of SEQ ID NO:26; (2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61; (3) an HC-FR2 comprising the amino acid sequence of SEQ ID NO:34; (4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62; (5) an HC-FR3 comprising the amino acid sequence of SEQ ID NO:38; (6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and (7) an HC-FR4 comprising the amino acid sequence of SEQ ID NOs:45; and/or (b) a light chain variable region comprising: (1) an LC-FR1 comprising the amino acid sequence of SEQ ID NO:48; (2) an HVR- L1 comprising the amino acid sequence of SEQ ID NO:64; (3) an LC-FR2 comprising the amino acid sequence of SEQ ID NO:51; (4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65; (5) an LC-FR3 comprising the amino acid sequence of SEQ ID NO:55; (6) an HVR- L3 comprising the amino acid sequence of SEQ ID NO:66; and (7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, the antibody comprises: (a) heavy chain variable region comprising: (1) an HC-FR1 comprising the amino acid sequence of SEQ ID NO:26; (2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61; (3) an HC-FR2 comprising the amino acid sequence of SEQ ID NO:34; (4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62; (5) an HC-FR3 comprising the amino acid sequence of SEQ ID NO:38; (6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and (7) an HC-FR4 comprising the amino acid sequence of SEQ ID NOs:45; and/or (b) a light chain variable region comprising: (1) an LC-FR1 comprising the amino acid sequence of SEQ ID NO:48; (2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64; (3) an LC-FR2 comprising the amino acid sequence of SEQ ID NO:51; (4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65; (5) an LC-FR3 comprising the amino acid sequence of SEQ ID NO:58; (6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66; and (7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, the antibody comprises: a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 88, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:94; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 103; a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104; or a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:96; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 99, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 106; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 109; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 107; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 110; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 108; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111. In some embodiments, the antibody binds to a human Siglec-8 and a non-human primate Siglec-8. In some embodiments, the non-human primate is a baboon. In some embodiments, the antibody binds to an epitope in Domain 1 of human Siglec-8, wherein Domain 1 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody binds to an epitope in Domain 3 of human Siglec-8, wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody binds to the same epitope as antibody 4F11. In some embodiments, the antibody binds to an epitope in Domain 2 or Domain 3 of human Siglec-8. In some embodiments, Domain 2 comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antibody binds to the same epitope as antibody 1C3. In some embodiments, Domain 3 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody binds to the same epitope as antibody 1H10. In some embodiments, the antibody binds to an epitope in Domain 1 of human Siglec-8 and competes with antibody 4F11 for binding to Siglec-8. In some embodiments, the antibody does not compete with antibody 2E2 for binding to Siglec-8. In some embodiments, the antibody is not antibody 2E2. In some embodiments, Domain 1 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises a heavy chain Fc region comprising a human IgG Fc region. In some embodiments, the human IgG Fc region comprises a human IgGl Fc region. In some embodiments, the human IgGl Fc region is non-fucosylated. In some embodiments, the human IgG Fc region comprises a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat. In some embodiments, the antibody depletes blood eosinophils and/or inhibits mast cell activation. In some embodiments, the antibody has been engineered to improve antibody-dependent cell- mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody comprises at least one amino acid substitution in the Fc region that improves ADCC activity. In some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated. In some embodiments, the antibody is produced in a cell line having a alphal,6-fucosyltransferase (Fut8) knockout. In some embodiments, the antibody is produced in a cell line overexpressing pi,4-N-acetylglucosminyltransferase III (GnT-III). In some embodiments, the cell line additionally overexpresses Golgi p-mannosidase II (Manll). In some embodiments, the cell line is a mammalian cell line, e.g., a Chinese hamster ovary (CHO) cell line. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:75; and/or a light chain comprising the amino acid sequence selected from SEQ ID NO:76 or 77. In some embodiments, the antibody is a monoclonal antibody.

[0016] In some embodiments according to any of the embodiments described herein, the composition is administered in combination with one or more additional therapeutic agent(s) for treating viral infection and/or inhibiting inflammation. In some embodiments, the one or more additional therapeutic agent(s) are selected from the group consisting of corticosteroids, hydroxychloroquine, azithromycin, colchicine, remdesivir, IL-6 antagonists, antigen-binding moieties that specifically bind viral spike protein, Ramatroban, convalescent plasma, and favipiravir. In some embodiments, the individual is a human. In some embodiments, the composition is a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.

[0017] Other aspects of the present disclosure relate to kits or articles of manufacture comprising a medicament comprising a composition comprising an antibody that binds to human Siglec-8 and a package insert comprising instructions for administration of the medicament in an individual in need thereof according to any one of the above embodiments.

[0018] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to one of skill in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1E show that pro-inflammatory cytokines and mast cell-specific proteases are significantly elevated in SARS-CoV-2 patient serum. (FIG. 1A) Cytokine and chemokine levels in serum from SARS-CoV-2 negative (left; n=9) or positive (right; n=10) patients determined by the Abbott RT-PCR nasal swab test. (FIG. IB) Levels of chymase, [3-tryptase, and CPA3 in SARS-CoV-2 patient serum compared to healthy controls. (FIG. 1C) Levels of mature tryptase in serum as determined by the Tosyl-Gly-Pro-Lys-pNA-based method. (FIG. ID) Spearman correlation matrix for MC proteases and cytokines in SARS-CoV-2 and healthy serum. (FIG. IE) Representative spearman correlations for active tryptase, IP- 10, and CCL2 in serum from healthy and SARS-CoV-2 patients. Data are plotted as individual donors +/- SD; ** p = <0.01; *** p = <0.001 as determined by Mann Whitney U test.

[0020] FIG. 2 shows that genes associated with mast cell and eosinophil mediators are significantly elevated in lungs from COVID-19 patients.

[0021] FIGS. 3A & 3B show that stimulation with poly (EC) and R848 directly induce TLR- mediated human mast cell activation and production of proteases and cytokines. FIG. 3A shows fold cytokine (IL-8, CCL3, and CCL4) induction from MCs (top) or fold cytokine (IL-8 and CCL4) induction from eosinophils stimulated with the synthetic analogs of ssRNA and dsRNA, R848 (middle) and poly (EC; right), as compared to control MCs or eosinophils (left). FIG. 3B shows fold chymase induction from MCs stimulated with R848 and poly (I:C), as compared to control MCs.

[0022] FIGS. 4A-4F show that Siglec-8 mAh treatment suppresses TLR-driven inflammation induced by poly (EC) administration. (FIG. 4A) Schematic of poly (LC)-mediated viral inflammation model, showing administration of Anti-S8 or isotype control. (FIGS. 4B & 4C) Total leukocytes, MHCII- monocytes, MHCII+ monocytes, and neutrophils in the BAL fluid or (FIG. 4D) monocytes and neutrophils in the blood of vehicle (left), ISO + poly (EC) (middle), or anti-S8 + poly (EC) (right) treated mice as determined by flow cytometry. Levels of (FIG. 4E) CCL2, IL-6, CXCL2, and IL-lb in BAL fluid or (FIG. 4F) CCL2, IP-10, IL-6, and TNF in the serum of vehicle (left), ISO + poly (EC) (middle), or anti-S8 + poly (EC) (right) treated mice. Data are plotted as mean +/- SEM (5-6 mice/group) and are representative of at least 2 experiments. * p = <0.05; ** p = <0.01; *** p = <0.001; **** p = <0.0001 by one-way ANOVA with Tukey’s multiple-comparisons test. BAL bronchoalveolar lavage, ISO isotype control, Anti-S8 anti-Siglec-8 antibody. [0023] FIGS. 5A-5D show that Poly (I:C)-driven inflammation is associated with aberrant MC and eosinophil activation that is suppressed with a Siglec-8 mAh. (FIG. 5A) Eosinophils in the BAL fluid and blood and (FIG. 5B) EPX levels in BAL fluid or serum from vehicle (left), ISO + poly (I:C) (middle), or anti-S8 + poly (I:C) (right) treated mice. (FIG. 5C) Levels of MCPT-4 from overnight ex vivo lung cultures and serum in vehicle (left), ISO + poly (I:C) (middle), or anti-S8 + poly (I:C) (right) treated mice. (FIG. 5D) Spearman correlations for serum MCPT4, CCL2, IP-10, and IL-6 in vehicle, ISO + poly (I:C), or anti-S8 + poly (EC) mice. Data are plotted as mean +/- SEM (5-6 mice/group) and are representative of at least 2 experiments. * p = <0.05; ** p = <0.01; *** p = <0.001; **** p = <0.0001 by one-way ANOVA with Tukey’s multiple-comparisons test. BAL bronchoalveolar lavage, ISO isotype control, MCPT4 mast cell protease-4, Anti-S8 anti-Siglec-8 antibody.

[0024] FIG. 6A shows expression level of the cell entry receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), on HEK293 cells, eosinophils, and purified human MCs, relative to Calu-3 cells.

[0025] FIG. 6B shows that R848, but not poly (EC), induced eosinophil activation as evidenced by increased expression of CD69 (surface expression on human blood eosinophils, as determined by flow cytometry). MFI, mean fluorescence intensity.

[0026] FIG. 6C shows levels of eosinophil-derived neurotoxin (EDN) in serum from SARS- CoV-2 negative (left; n=9) or positive (right; n=10) patients. Data are plotted as individual donors +/- SD; **** p = <0.0001 as determined by Mann Whitney U test.

[0027] FIG. 7A provides a diagram of a study testing the effect of anti-Siglec-8 monoclonal antibody (mAb) treatment in a mouse model of RSV infection in transgenic mice expressing Siglec-8. Mice were dosed with isotype control or anti-Siglec-8 antibody, then administered RSV-A2 (IxlO⁶ PFU) intranasally 4 hours later. On day 7, measurements of lung inflammation were obtained.

[0028] FIG. 7B shows measurements of body weight during RSV infection according to the study shown in FIG. 7A. Mice infected with RSV and treated with anti-Siglec-8 antibody (m2E2, triangles) displayed significantly less weight loss, as compared to mice infected with RSV and treated with isotype control antibody (squares). Data are plotted as mean body weights +/- SEM; n=4-5 mice/group; ** p = <0.01 as determined by Mann Whitney U test.

[0029] FIGS. 7C-7F show lung inflammation at day 7 after RSV infection, as determined by immune cell infiltration by monocytes (FIG. 7C), lymphocytes (FIG. 7D), neutrophils (FIG. 7E), and eosinophils (FIG. 7F). Mice infected with RSV and treated with anti-Siglec-8 antibody (right) displayed less immune cell infiltration in BAL fluid, as compared to mice infected with RSV and treated with isotype control antibody (middle). Data are plotted as mean cell number +/- SEM; n=4-5 mice/group; * p = <0.05 as determined by Mann Whitney U test. [0030] FIGS. 8A-8F show selective profiles of elevated inflammatory cytokines and mast cell-derived proteases in long covid patient sera. FIGS. 8A & 8B show levels of cytokines, chemokines, or mast cell-derived proteases in sera from symptomatic long covid patients (middle; n=13), asymptomatic short covid patients (right; n=13), or healthy controls (left; n=20). FIG. 8C shows Spearman correlations for sera levels of active tryptase and IL-6 and CXCL1 from patients and controls shown in panels of FIGS. 8A & 8B. FIGS. 8D-8F show the levels of cytokines and mast cell-derived proteases in sera from long covid patients (middle; n=13), actively infected SARS-CoV-2 positive patients (right; n=19), or healthy controls (left; n=20). Data are plotted as box and whisker plots (max, min, and median) with individual donors represented as dots; *P <0.05; **P <0.01; ***P <0.001; ****p <0.0001 as determined by oneway ANOVA with Holm-Sidak’s multiple comparisons test; ns = not significant.

[0031] FIGS. 9A-9D show inflammatory mediators not elevated in long covid patient sera. FIGS. 9A-9C show levels of cytokines, chemokines, or mast cell-derived proteases in sera from symptomatic long covid patients (middle; n=13), asymptomatic short covid patients (right; n=13), or healthy controls (left; n=20). FIG. 9D shows Spearman correlations for sera levels of CP A3 and IL-6 and CXCL1 from patients and controls shown in FIGS. 9A & 9B.

[0032] FIG. 9E shows ROC curves calculated for these selected individual parameters using PASC patients against PAAC patients and healthy controls. Data are plotted as box and whisker plots (max, min, and median) with individual donors represented as dots; *P <0.05; **P <0.01; *** > <0.001; ****p <0.0001 as determined by one-way ANOVA with Holm-Sidak’s multiple comparisons test.

[0033] FIG. 10 shows the levels of cytokines and chemokines in sera from symptomatic long covid patients or actively infected SARS-CoV-2 positive patients. Levels of cytokines, chemokines, and growth factors in sera from long co vid patients (middle; n=13), actively infected SARS-CoV-2 positive patients (right; n=19), or healthy controls (left; n=20). Data are plotted as box and whisker plots (max, min, and median) with individual donors represented as dots; *P <0.05; ** <0.01; *** <0.001; ****/> <0.0001 as determined by one-way ANOVA with Holm-Sidak’s multiple comparisons test. DETAILED DESCRIPTION

I. Definitions

[0034] It is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0035] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0036] It is understood that aspects and embodiments of the present disclosure include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0037] The term “antibody” includes polyclonal antibodies, monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with poly epitopic specificity, multispecific antibodies (e.g, bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

[0038] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and chains and four CH domains for p and s isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (Cnl). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0039] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 8, s, y and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in the present disclosure. Common allotypic variants in human populations are those designated by the letters a, f, n, z.

[0040] An “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly). In some embodiments, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody is prepared by at least one purification step.

[0041] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or posttranslation modifications (e.g, isomerizations, amidations) that may be present in minor amounts. In some embodiments, monoclonal antibodies have a C-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C- terminus of heavy chain and/or light chain. In some embodiments, the C-terminal cleavage removes a C-terminal lysine from the heavy chain. In some embodiments, monoclonal antibodies have an N-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of heavy chain and/or light chain. In some embodiments, monoclonal antibodies are highly specific, being directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific, being directed against multiple antigenic sites (such as a bispecific antibody or a multispecific antibody). The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage-display technologies, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0042] The term “naked antibody” refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0043] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g, human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions. [0044] An “antibody fragment” comprises a portion of an intact antibody, the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0045] Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (Cnl). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the Cnl domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0046] The Fc fragment comprises the carboxy -terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0047] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0048] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0049] “Functional fragments” of the antibodies of the present disclosure comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fv region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0050] The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibodies.”

[0051] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. In some embodiments, the number of these amino acid substitutions in the FR are no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, humanized antibodies are directed against a single antigenic site. In some embodiments, humanized antibodies are directed against multiple antigenic sites. An alternative humanization method is described in U.S. Pat. No. 7,981,843 and U.S. Patent Application Publication No. 2006/0134098.

[0052] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

[0053] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody-variable domain that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0054] A number of HVR delineations are in use and are encompassed herein. The HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia HVRs refer instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Loop Kabat Chothia Contact

LI L24-L34 L26-L34 L30-L36

L2 L50-L56 L50-L56 L46-L55

L3 L89-L97 L91-L96 L89-L96

Hl H31-H35B H26-H32 H30-H35B (Kabat Numbering)

Hl H31-H35 H26-H32 H30-H35 (Chothia Numbering)

H2 H50-H65 H53-H56 H47-H58

H3 H95-H102 H95-H102 H93-H101

[0055] Unless otherwise indicated, the variable-domain residues (HVR residues and framework region residues) are numbered according to Kabat et al., supra.

[0056] “Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.

[0057] The expression “variable-domain residue-numbering as in Kabat” or “amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0058] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0059] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0060] An antibody that “binds to”, “specifically binds to” or is “specific for” a particular a polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, binding of an anti-Siglec-8 antibody described herein (e.g., an antibody that binds to human Siglec-8) to an unrelated non-Siglec-8 polypeptide is less than about 10% of the antibody binding to Siglec-8 as measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)). In some embodiments, an antibody that binds to a Siglec-8 (e.g, an antibody that binds to human Siglec-8) has a dissociation constant (Kd) of < IpM, < 100 nM, < 10 nM, < 2 nM, < 1 nM, < 0.7 nM, <0 .6 nM, < 0.5 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'⁸ M or less, e.g. from 10'⁸M to 10'¹³ M, e.g., from 10'⁹ M to 10'¹³ M).

[0061] The term “anti-Siglec-8 antibody” or “an antibody that binds to human Siglec-8” refers to an antibody that binds to a polypeptide or an epitope of human Siglec-8 without substantially binding to any other polypeptide or epitope of an unrelated non-Siglec-8 polypeptide.

[0062] The term “Siglec-8” as used herein refers to a human Siglec-8 protein. The term also includes naturally occurring variants of Siglec-8, including splice variants or allelic variants. The amino acid sequence of an exemplary human Siglec-8 is shown in SEQ ID NO:72. The amino acid sequence of another exemplary human Siglec-8 is shown in SEQ ID NO:73. In some embodiments, a human Siglec-8 protein comprises the human Siglec-8 extracellular domain fused to an immunoglobulin Fc region. The amino acid sequence of an exemplary human Siglec- 8 extracellular domain fused to an immunoglobulin Fc region is shown in SEQ ID NO:74. The amino acid sequence underlined in SEQ ID NO:74 indicates the Fc region of the Siglec-8 Fc fusion protein amino acid sequence.

Human Siglec-8 Amino Acid Sequence

GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATN NPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLN YKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPG PTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDA TASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVH VRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFC IIFIIVRSCRKKSARPAAGVGDTGMEDAKAIRGSASQGPLTESWKDGNPLKKPPPAVAPS SGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACLRNHNPSSKEV RG (SEQ ID NO: 72)

Human Siglec-8 Amino Acid Sequence

GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATN NPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLN YKTKQLSVFVTALTHRPDILILGTLESGHPRNLTCSVPWACKQGTPPMISWIGASVSSPG PTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDA TASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVH VRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFC IIFIIVRSCRKKSARPAAGVGDTGMEDAKAIRGSASQGPLTESWKDGNPLKKPPPAVAPS SGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACLRNHNPSSKEV RG (SEQ ID NO: 73)

Siglec-8 Fc Fusion Protein Amino Acid Sequence

GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATN NPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLN YKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPG PTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDA TASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVH VRDEGEFTCRAONAOGSOHISLSLSLQNEGTGTSRPVSOVTLAAVGGIEGRSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGOPRE POVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSF

FLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74)

[0063] Antibodies that “induce apoptosis” or are “apoptotic” are those that induce programmed cell death as determined by standard apoptosis assays, such as binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). For example, the apoptotic activity of the anti-Siglec-8 antibodies (e.g, an antibody that binds to human Siglec-8) of the present disclosure can be shown by staining cells with annexin V.

[0064] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g, B cell receptors); and B cell activation.

[0065] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g, natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for killing of the target cell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457- 92 (1991). In some embodiments, an anti-Siglec-8 antibody (e.g, an antibody that binds to human Siglec-8) described herein enhances ADCC. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alter ADCC activity and other antibody properties include those disclosed by Ghetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al, Nature 332:563-564, 1988; Lund et al., J. Immunol 147:2657-2662, 1991; Lund et al, Mol Immunol 29:53-59, 1992; Alegre et al, Transplantation 57:1537-1543, 1994; Hutchins et al., Proc Natl. Acad Sci USA 92:11980-11984, 1995; Jefferis et al, Immunol Lett. 44:111-117, 1995; Lund et al., FASEB J9: 115-119, 1995; Jefferis et al, Immunol Lett 54:101-104, 1996; Lund et al, J Immunol 157:4963-4969, 1996; Armour et al., Eur J Immunol 29:2613-2624, 1999; Idusogie et al, J Immunol 164:4178-4184, 200; Reddy et al, J Immunol 164:1925-1933, 2000; Xu et al., Cell Immunol 200:16-26, 2000; Idusogie et al, J Immunol 166:2571-2575, 2001; Shields et al., J Biol Chem 276:6591-6604, 2001; Jefferis et al, Immunol Lett 82:57-65. 2002; Presta et al., Biochem Soc Trans 30:487-490, 2002; Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010, 2006; U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; 7,335,742; and 7,317,091.

[0066] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4. A single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) may be introduced to abolish the heterogeneity observed in recombinant IgG4 antibody. See Angal, S. et al. (1993) Mol Immunol 30, 105-108.

[0067] “Non-fucosylated” or “fucose-deficient” antibody refers to a glycosylation antibody variant comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, an antibody with reduced fucose or lacking fucose has improved ADCC function. Non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. In some embodiments, a non-fucosylated or fucose-deficient antibody composition contemplated herein is a composition wherein less than about 50% of the N-linked glycans attached to the Fc region of the antibodies in the composition comprise fucose.

[0068] The terms "fucosylation" or “fucosylated” refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody comprises a (l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the antibody Fc region, e.g. at position Asn 297 of the human IgGl Fc domain (EU numbering of Fc region residues). Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in immunoglobulins .

[0069] The "degree of fucosylation" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides identified by methods known in the art e.g., in an N-glycosidase F treated antibody composition assessed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a composition of a "fully fucosylated antibody" essentially all oligosaccharides comprise fucose residues, i.e. are fucosylated. In some embodiments, a composition of a fully fucosylated antibody has a degree of fucosylation of at least about 90%. Accordingly, an individual antibody in such a composition typically comprises fucose residues in each of the two N-linked oligosaccharides in the Fc region. Conversely, in a composition of a "fully non-fucosylated" antibody essentially none of the oligosaccharides are fucosylated, and an individual antibody in such a composition does not contain fucose residues in either of the two N-linked oligosaccharides in the Fc region. In some embodiments, a composition of a fully non- fucosylated antibody has a degree of fucosylation of less than about 10%. In a composition of a "partially fucosylated antibody" only part of the oligosaccharides comprise fucose. An individual antibody in such a composition can comprise fucose residues in none, one or both of the N- linked oligosaccharides in the Fc region, provided that the composition does not comprise essentially all individual antibodies that lack fucose residues in the N-linked oligosaccharides in the Fc region, nor essentially all individual antibodies that contain fucose residues in both of the N- linked oligosaccharides in the Fc region. In one embodiment, a composition of a partially fucosylated antibody has a degree of fucosylation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%).

[0070] “Binding affinity” as used herein refers to the strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g, an antigen). In some embodiments, the binding affinity of an antibody for a Siglec-8 (which may be a dimer, such as the Siglec-8-Fc fusion protein described herein) can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0071] “Binding avidity” as used herein refers to the binding strength of multiple binding sites of a molecule (e.g, an antibody) and its binding partner (e.g, an antigen).

[0072] An “isolated” nucleic acid molecule encoding the antibodies herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.

[0073] The term “pharmaceutical formulation” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to an individual to which the formulation would be administered. Such formulations are sterile.

[0074] “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0075] As used herein, the term “treatment” or “treating” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disease (e.g, viral infection) are mitigated or eliminated. For example, an individual is successfully “treated” if treatment results in increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required for treating the disease, reducing the frequency of recurrence of the disease, lessening severity of the disease, delaying the development or progression of the disease, and/or prolonging survival of individuals.

[0076] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0077] As used herein, the term “prevention” or “preventing” includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to a disease, susceptible to a disease, or at risk of developing a disease, but has not yet been diagnosed with the disease. In some embodiments, anti-Siglec-8 antibodies (e.g, an antibody that binds to human Siglec-8) described herein are used to delay development of a disease (e.g, viral infection, virus-induced inflammation, and/or virus-induced mast cell/ eosinophil activation).

[0078] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired or indicated effect, including a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in individuals prior to or at the earlier stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

[0079] “Chronic” administration refers to administration of the medicament(s) in a continuous as opposed to acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0080] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0081] As used herein, an “individual” or a “subject” is a mammal. A “mammal” for purposes of treatment includes humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

II. Methods

[0082] Provided herein are methods for treating and/or preventing viral infection, virus- induced inflammation, and/or virus-induced mast cell/eosinophil activation in an individual comprising administering to the individual an effective amount of an antibody described herein that binds to human Siglec-8 (e.g., an anti-Siglec-8 antibody) or compositions comprising said antibodies. In some embodiments, the antibody is in a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier. In some embodiments, the individual is a human.

[0083] Certain aspects of the present disclosure relate to viral infections, virus-induced inflammation, and/or virus-induced activation of mast cells and/or eosinophils. In some embodiments, the virus is an enveloped virus. Examples of enveloped viruses are well known in the art and include, without limitation, the virus families of Arenavirus, Arterivirus, Asfarvirus, Baculovirus, Bunyavirus, Coronavirus, Cystovirus, Deltavirus, Filovirus, Flavivirus, Fusellovirus, Hepadnavirus, Herpesvirus, Iridovirus, Lipothrixivirus, Orthomyxovirus, Paramyxovirus, Plasmavirus, Polydnavirus, Poxvirus, Retrovirus, Rhabdovirus, and Togavirus. In some embodiments, the virus is a Coronavirus, e.g., severe acute respiratory syndrome coronavirus 1 (SARS-Cov-1), severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), or Middle East respiratory syndrome-related coronavirus (MERS-Cov). In some embodiments, the virus is an Orthomyxovirus, e.g, influenza virus A, B, or C. In some embodiments, the virus is an Orthopneumovirus, e.g., respiratory syncytial virus (RSV). In some embodiments, the virus is associated with elevated activation of mast cells and/or eosinophils. Assays for detecting the presence of various viruses and viral infections are known in the art and include, e.g., nucleic acid tests (NATs), viral antigen tests, antibody tests, viral culturing, and so forth. Imagining of infected tissue (e.g., lung tissue), such as by computerized tomography (CT) scanning, may also be used in some cases to diagnose viral infection and/or inflammation. [0084] In some embodiments, an individual has or has been diagnosed with acute respiratory distress syndrome (ARDS), e.g., prior to treatment with an anti-Siglec-8 antibody. Methods for diagnosis of ARDS include, without limitation, chest X-ray, CT scanning, and/or measurement of oxygen levels.

[0085] In some embodiments, an individual (e.g., a “COVID long-hauler”) has or has been diagnosed with post-acute COVID-19 syndrome, e.g, prior to treatment with an anti-Siglec-8 antibody. Post-acute COVID-19 syndrome, also known as post-acute sequelae of COVID-19 (PASC), long COVID, and long-hauler syndrome, refers to persistent or new symptoms that arise at least 4-12 weeks after initial COVID-19 infection, or onset of acute COVID-19. See, e.g., Nalbandian, A. et al. (2021) Nat. Med. 27:601-615 and Sudre CH, et al. Nat Med 2021;27:626-631. In some embodiments, a serum sample obtained from the individual (e.g., prior to treatment with an anti-Siglec-8 antibody) has an elevated level of IL-6, CXCL1, active tryptase, and/or CP A3, e.g., compared to a reference, reference value, or baseline value.

[0086] In some embodiments, e.g., prior to treatment with an anti-Siglec-8 antibody, an individual has increased mast cell activation. In some embodiments, the individual has increased mast cell activation in peripheral blood (e.g., in a blood or plasma sample). In some embodiments, a peripheral blood sample (e.g., a blood or plasma sample) obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present. In some embodiments, the individual has increased mast cell activation in the gastrointestinal tract (e.g., in a Gl-derived sample, such as a GI biopsy specimen). In some embodiments, a Gl-derived sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present. In some embodiments, the individual has increased mast cell activation in lung tissue (e.g., in a lung-derived sample). In some embodiments, a lung-derived sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present. In some embodiments, the individual has increased mast cell activation in skin (e.g, in a skin-derived sample, such as a skin biopsy). In some embodiments, a skin-derived sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present. In some embodiments, mast cell activation and/or marker(s) indicative of mast cell activation are compared against a reference or reference value. Exemplary cytokines and other mast cell products are described and exemplified herein.

[0087] In some embodiments, e.g, prior to treatment with an anti-Siglec-8 antibody, an individual has increased eosinophil activation. In some embodiments, the individual has increased eosinophil activation in peripheral blood (e.g, in a blood or plasma sample). In some embodiments, a peripheral blood sample (e.g, a blood or plasma sample) obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present. In some embodiments, the individual has increased eosinophil activation in lung tissue (e.g, in a lung-derived sample). In some embodiments, a lung-derived sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present. In some embodiments, the individual has increased eosinophil activation in the gastrointestinal tract (e.g, in a Gl-derived sample, such as a GI biopsy specimen). In some embodiments, a Gl-derived sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present. In some embodiments, the individual has increased eosinophil activation in skin (e.g, in a skin- derived sample, such as a skin biopsy). In some embodiments, a skin-derived sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present. In some embodiments, eosinophil activation and/or marker(s) indicative of eosinophil activation are compared against a reference or reference value. Exemplary cytokines and other eosinophil products are described and exemplified herein.

[0088] In some embodiments, e.g, prior to treatment with an anti-Siglec-8 antibody, an individual has an elevated level of one or more pro-inflammatory cytokine(s), growth factor(s), or mast cell/eosinophil products, e.g., compared to a reference, reference value, or baseline value. In some embodiments, the level of the one or more pro-inflammatory cytokine(s), growth factor(s), or mast cell/eosinophil products are measured in a peripheral blood sample, e.g., a serum sample. Exemplary pro-inflammatory cytokine(s), growth factor(s), or mast cell/eosinophil products include, without limitation, CCL2, IP-10, IL-8, VEGF, histamine, leukotriene C4, leukotriene E4, prostaglandin D2, eotaxin, periostin, eosinophil peroxidase (EPX), IL-6, TNF, C-reactive protein (CRP), ferritin, major basic protein (MBP), eosinophil- derived neurotoxin (EDN), and IFN-y.

[0089] In some embodiments, e.g, prior to treatment with an anti-Siglec-8 antibody, an individual has an elevated level of one or more polypeptide(s) expressed by mast cells, e.g, compared to a reference, reference value, or baseline value. In some embodiments, the level of the one or more polypeptide(s) expressed by mast cells are measured in a peripheral blood sample, e.g., a serum sample. Exemplary polypeptide(s) expressed by mast cells include, without limitation, chymase, p-tryptase, and CP A3. In some embodiments, the levels of the one or more polypeptide(s) themselves are measured. In some embodiments, the levels of polynucleotide(s) encoding the one or more polypeptide(s) are measured.

[0090] In some embodiments, a sample obtained from the individual (e.g., prior to treatment with an anti-Siglec-8 antibody) has an elevated level of one or more polypeptide(s) expressed by mast cells, pro-inflammatory cytokine(s), or chemokine(s) or an elevated level of one or more polynucleotides encoding one or more polypeptide(s) expressed by mast cells, pro-inflammatory cytokine(s), or chemokine(s), e.g, compared to a reference, reference value, or baseline value. Exemplary polypeptides expressed by mast cells, pro-inflammatory cytokines, or chemokines include, without limitation, CCL2, CCL4, IL-8, IP- 10, TPSB2, TPSAB1, and FCER1G. In some embodiments, the sample is a lung-derived or serum sample.

[0091] In some embodiments, a sample obtained from the individual (e.g., prior to treatment with an anti-Siglec-8 antibody) has an elevated level of one or more polypeptide(s) expressed by eosinophils or an elevated level of one or more polynucleotides encoding one or more polypeptide(s) expressed by eosinophils, e.g., compared to a reference, reference value, or baseline value. Exemplary polypeptides expressed by eosinophils include, without limitation, EDN and Galectin-10. In some embodiments, the sample is a lung-derived or serum sample. [0092] In some embodiments, e.g, prior to treatment with an anti-Siglec-8 antibody, an individual has eosinopenia in peripheral blood. [0093] In some embodiments, one or more symptom(s) of viral infection in the individual are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline value (e.g., prior to administration). In some embodiments, one or more symptom(s) of inflammation in the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, one or more symptom(s) of ARDS in the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration).

[0094] In some embodiments, one or more symptom(s) of post-acute COVID-19 syndrome in the individual are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). Symptoms of postacute COVID-19 syndrome can vary, but may include, e.g, one or more of: loss of smell and/or taste, shortness of breath, fatigue, myalgia, dysautonomia, decreased exercise capacity, hypoxia, reduced diffusion capacity, restrictive pulmonary physiology, pulmonary fibrosis, diminished quality of life, muscular weakness joint pain, dyspnea, cough, persistent oxygen requirement, anxiety, depression, sleep disturbances, post-traumatic stress disorder (PTSD), cognitive disturbance (e.g, “brain fog”), headache, palpitations, chest pain, increased cardiometabolic demand, myocardial fibrosis or scarring, arrhythmia, tachycardia, autonomic dysfunction, thromboembolism, chronic kidney disease, impaired renal function, new or worsening diabetes mellitus (type II diabetes), subacute thyroiditis, bone demineralization, multisystem inflammatory syndrome (MIS-C), and hair loss.

[0095] In some embodiments, level of one or more pro-inflammatory cytokine(s) or chemokine(s) in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or serum sample. For example, in some embodiments, the one or more pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, IP- 10, IL-6, ferritin, C-reactive protein (CRP), and TNF.

[0096] In some embodiments, level of one or more polypeptide(s) or other products expressed by mast cells in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, level of one or more polynucleotide(s) encoding one or more polypeptide(s) or other products expressed by mast cells in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or serum sample. For example, the one or more polypeptide(s) or other products expressed by mast cells can include chymase.

[0097] In some embodiments, level of one or more polypeptide(s) or other products expressed by eosinophils in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, level of one or more polynucleotide(s) encoding one or more polypeptide(s) or other products expressed by eosinophils in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or serum sample. For example, the one or more polypeptide(s) or other products expressed by eosinophils can include eosinophil peroxidase (EPX).

[0098] In some embodiments, level of pro-inflammatory cytokine(s) or chemokine(s) in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or serum sample. For example, the pro-inflammatory cytokine(s) or chemokine(s) can include CCL2, IL-6, CXCL2, and/or IL-ip.

[0099] In some embodiments, number(s) of eosinophils, monocytes, and/or neutrophils in the individual, or in a sample obtained from the individual, are reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or serum sample.

[0100] In some embodiments, eosinophil activation in the individual, or in a sample obtained from the individual, is reduced after administration of the composition, e.g, as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or peripheral blood (e.g, serum) sample. Exemplary markers of eosinophil activation are described and exemplified herein. [0101] In some embodiments, mast cell activation in the individual, or in a sample obtained from the individual, is reduced after administration of the composition, e.g., as compared to a reference, reference value, or baseline value (e.g, prior to administration). In some embodiments, the sample is a lung-derived or peripheral blood (e.g., serum) sample. Exemplary markers of mast cell activation are described and exemplified herein.

[0102] The terms “baseline” or “baseline value” used interchangeably herein can refer to a measurement or characterization of a symptom before the administration of the therapy (e.g, an anti-Siglec-8 antibody) or at the beginning of administration of the therapy. The baseline value can be compared to a reference value in order to determine the reduction or improvement of a symptom of viral infection/inflammation contemplated herein. A reference value and/or baseline value can be obtained from one individual, from two different individuals or from a group of individuals (e.g., a group of two, three, four, five or more individuals).

[0103] The terms “reference” or “reference value” used interchangeably herein can refer to a measurement or characterization of a value or symptom in an individual without viral infection (or in a group of such individuals). A “reference value” can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value; a mean value; or a value as compared to a baseline value. Similarly, a “baseline value” can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value; a mean value; or a value as compared to a reference value. A reference value can be obtained from one individual, from two different individuals or from a group of individuals (e.g, a group of two, three, four, five or more individuals). In some embodiments, a reference value refers to a standard or benchmark value in the field. In some embodiments, a reference value refers to a value calculated de novo from one or more individuals (e.g., without viral infection).

[0104] In some embodiments, the level of a biomarker or value described herein is measured in a sample, e.g., obtained from an individual of the present disclosure. In some embodiments, the sample is a peripheral blood sample (e.g., a whole blood or serum sample). In some embodiments, the sample is a lung-derived sample (e.g., a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample). In some embodiments, the sample is a skin-derived sample (e.g., from a skin biopsy). In some embodiments, the sample is from the gastrointestinal tract (e.g., from a biopsy of the GI tract, such as an endoscopic, esophageal, gastric, duodenal, jejunal, ileal, or colonic biopsy). [0105] In some embodiments, the level of a biomarker or value described herein is measured based on polypeptide levels. Exemplary assays for measuring polypeptides are known in the art and include, without limitation, ELISA, Western blotting, mass spectrometry, enzymatic assays, etc. In some embodiments, the level of a biomarker or value described herein is measured based on polynucleotide levels. Exemplary assays for measuring polynucleotides are known in the art and include, without limitation, Northern or Southern blotting, direct sequencing (e.g., NGS sequencing), microarray analysis, in situ hybridization, polymerase chain reaction (PCR) and real-time or quantitative PCR (RT-PCR or q-PCR), etc.

Administration

[0106] For the prevention or treatment of disease, the appropriate dosage of an active agent, will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the individual's clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the individual at one time or over a series of treatments. In some embodiments, an interval between administrations of an anti- Siglec-8 antibody (e.g., an antibody that binds to human Siglec-8) described herein is about one month or longer. In some embodiments, the interval between administrations is about 1 month, about two months, about three months, about four months, about five months, about six months or longer. As used herein, an interval between administrations refers to the time period between one administration of the antibody and the next administration of the antibody. As used herein, an interval of about one month includes four weeks. Accordingly, in some embodiments, the interval between administrations is about four weeks, about five weeks, about six weeks, about seven weeks, about eight weeks, about nine weeks, about ten weeks, about eleven weeks, about twelve weeks, about sixteen weeks, about twenty weeks, about twenty four weeks, or longer. In some embodiments, the treatment includes multiple administrations of the antibody, wherein the interval between administrations may vary. For example, the interval between the first administration and the second administration is about one month, and the intervals between the subsequent administrations are about three months. In some embodiments, the interval between the first administration and the second administration is about one month, the interval between the second administration and the third administration is about two months, and the intervals between the subsequent administrations are about three months. In some embodiments, an anti- Siglec-8 antibody described herein (e.g, an antibody that binds to human Siglec-8) is administered at a flat dose. In some embodiments, an anti-Siglec-8 antibody described herein (e.g, an antibody that binds to human Siglec-8) is administered to an individual at a dosage from about 0.1 mg to about 1800 mg per dose. In some embodiments, the anti-Siglec-8 antibody (e.g., an antibody that binds to human Siglec-8) is administered to an individual at a dosage of about any of 0.1 mg, 0.5 mg, 1 mg, 5 mg , 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, and 1800 mg per dose. In some embodiments, an anti-Siglec-8 antibody described herein (e.g., an antibody that binds to human Siglec-8) is administered to an individual at a dosage from about 150 mg to about 450 mg per dose. In some embodiments, the anti-Siglec-8 antibody (e.g., an antibody that binds to human Siglec-8) is administered to an individual at a dosage of about any of 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, and 450 mg per dose. In some embodiments, an anti-Siglec-8 antibody described herein (e.g, an antibody that binds to human Siglec-8) is administered to an individual at a dosage from about 0.1 mg/kg to about 20 mg/kg per dose. In some embodiments, an anti- Siglec-8 antibody described herein (e.g, an antibody that binds to human Siglec-8) is administered to an individual at a dosage from about 0.01 mg/kg to about 10 mg/kg per dose. In some embodiments, an anti-Siglec-8 antibody described herein (e.g., an antibody that binds to human Siglec-8) is administered to an individual at a dosage from about 0.1 mg/kg to about 10 mg/kg, about 1.0 mg/kg to about 10 mg/kg, or about 0.3mg/kg to about 1.0 mg/kg. In some embodiments, an anti-Siglec-8 antibody described herein is administered to an individual at a dosage of about any of 0.1 mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5 mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg. Any of the dosing frequency described above may be used. Any dosing frequency described above may be used in the methods or uses of the compositions described herein. Efficacy of treatment with an antibody described herein (e.g, an antibody that binds to human Siglec-8) can be assessed using any of the methodologies or assays described herein at intervals ranging between every week and every three months. In some embodiments, efficacy of treatment (e.g., reduction or improvement of one or more symptoms) is assessed about every one month, about every two months, about every three months, about every four months, about every five months, about every six months or longer after administration of an antibody that binds to human Siglec-8. In some embodiments, efficacy of treatment (e.g., reduction or improvement of one or more symptoms) is assessed about every one week, about every two weeks, about every three weeks, about every four weeks, about every five weeks, about every six weeks, about every seven weeks, about every eight weeks, about every nine weeks, about every ten weeks, about every eleven weeks, about every twelve weeks, about every sixteen weeks, about every twenty weeks, about every twenty four weeks, or longer. [0107] In some embodiments, an anti-Siglec-8 antibody described herein (e.g, an antibody that binds to human Siglec-8) is administered to an individual (e.g, by intravenous infusion) at one or more doses comprising between about 0.1 mg/kg and about 4.0 mg/kg of the antibody. In some embodiments, the antibody is administered to an individual by intravenous infusion at one or more doses comprising between about 0.3 mg/kg and about 3.0 mg/kg of the antibody, e.g, at about 0.3 mg/kg antibody, about 0.5 mg/kg antibody, about 1.0 mg/kg antibody, about 1.5 mg/kg antibody, about 2.0 mg/kg antibody, about 2.5 mg/kg antibody, or about 3.0 mg/kg antibody. In some embodiments, the antibody is administered to the individual (e.g, by intravenous infusion) in two or more doses (e.g, comprising between about 0.3 mg/kg and about 3.0 mg/kg of the antibody) at an interval of about 28 days. In some embodiments, the antibody is administered to the individual (e.g, by intravenous infusion) monthly in two or more doses (e.g, comprising between about 0.3 mg/kg and about 3.0 mg/kg of the antibody). In some embodiments, the antibody is administered to the individual (e.g, by intravenous infusion) in two or more doses (e.g, comprising between about 0.3 mg/kg and about 3.0 mg/kg of the antibody) at an interval of about 4 weeks. In some embodiments, the antibody is administered to the individual (e.g, by intravenous infusion) according to the following schedule: Day 1, Day 29, Day 57, Day 85, Day 113, and Day 141. In some embodiments, the antibody is administered to the individual by intravenous infusion at a first dose comprising about 0.3 mg/kg of the antibody, a second dose comprising about 1.0 mg/kg of the antibody, a third dose comprising about 1.0 mg/kg of the antibody, a fourth dose comprising about 1.0 mg/kg to about 3.0 mg/kg of the antibody, a fifth dose comprising about 1.0 mg/kg to about 3.0 mg/kg of the antibody, and a sixth dose comprising about 1.0 mg/kg to about 3.0 mg/kg of the antibody. In some embodiments, the antibody is administered to the individual by intravenous infusion at a first dose comprising about 0.3 mg/kg of the antibody, a second dose comprising about 1.0 mg/kg of the antibody, a third dose comprising about 1.0 mg/kg of the antibody, a fourth dose comprising about 1.0 mg/kg or about 3.0 mg/kg of the antibody, a fifth dose comprising about 1.0 mg/kg or about 3.0 mg/kg of the antibody, and a sixth dose comprising about 1.0 mg/kg or about 3.0 mg/kg of the antibody. In some embodiments, the antibody is administered to the individual by intravenous infusion at a first dose comprising about 0.3 mg/kg of the antibody, a second dose comprising about 1.0 mg/kg of the antibody, a third dose comprising about 1.0 mg/kg of the antibody, a fourth dose comprising about 1.0 mg/kg of the antibody, a fifth dose comprising about 1.0 mg/kg of the antibody, and a sixth dose comprising about 1.0 mg/kg of the antibody. In some embodiments, the antibody is administered to the individual by intravenous infusion according to the following schedule: about 0.3 mg/kg of the antibody on Day 1, about 1.0 mg/kg of the antibody on Day 29, about 1.0 mg/kg of the antibody on Day 57, about 1.0 mg/kg or about 3.0 mg/kg of the antibody on Day 85, about 1.0 mg/kg or about 3.0 mg/kg of the antibody on Day 113, and about 1.0 mg/kg or about 3.0 mg/kg of the antibody on Day 141.

[0108] In some embodiments, the methods of the present disclosure comprise administering an anti-Siglec-8 antibody or composition of the present disclosure in one or more doses. In some embodiments, a corticosteroid is administered to the individual at least 6 hours prior to administration of the first dose of the composition (i.e., comprising an antibody that binds to human Siglec-8). In some embodiments, the methods comprise administering a corticosteroid to the individual, then administering to the individual a first dose of the composition (i.e., comprising an antibody that binds to human Siglec-8) at least 6 hours after administering the corticosteroid. In some embodiments, the corticosteroid is administered to the individual at least 12 hours prior to administration of the composition. In some embodiments, the corticosteroid is administered to the individual within 24 hours prior to administration of the composition, e.g., 6- 24 hours prior, or 12-24 hours prior.

[0109] In some embodiments, the first dose of the composition is administered to the individual by intravenous infusion over a period of about 4 hours. In some embodiments, a corticosteroid is administered to the individual at least 6 hours prior to administration of the first dose of the composition (i.e., comprising an antibody that binds to human Siglec-8). In some embodiments, the methods comprise administering a corticosteroid to the individual, then administering to the individual a first dose of the composition (i.e., comprising an antibody that binds to human Siglec-8) at least 6 hours after administering the corticosteroid, wherein the first dose of the composition is administered to the individual by intravenous infusion over a period of about 4 hours. [0110] In some embodiments, the corticosteroid is prednisone. In some embodiments, the corticosteroid is methylprednisolone, hydrocortisone, or dexamethasone. In some embodiments, the corticosteroid is prednisone, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or prednisolone. In some embodiments, the corticosteroid is self-administered by the individual being treated with the anti-Siglec-8 antibody. In some embodiments, the corticosteroid is administered orally.

[oni] In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of greater than 0.5mg/kg, or about Img/kg. In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of 80mg. For example, in some embodiments, the methods comprise administering to the individual greater than 0.5mg/kg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12- 24 hours prior to administration of the first dose of the anti-Siglec-8 antibody. In some embodiments, the methods comprise administering to the individual 0.5mg/kg to Img/kg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the first dose of the anti- Siglec-8 antibody. In some embodiments, the methods comprise administering to the individual Img/kg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the first dose of the anti-Siglec-8 antibody. In some embodiments, the methods comprise administering to the individual 60mg or 80mg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the first dose of the anti-Siglec-8 antibody.

[0112] In some embodiments, the first dose of the composition is administered to the individual by intravenous infusion over a period of about 4 hours. In some embodiments, less than 50% of total volume of the first dose is administered to the individual in the first 2 hours of the infusion. In some embodiments, less than 30% of total volume of the first dose is administered to the individual in the first 2 hours of the infusion. In some embodiments, the first dose is administered to the individual by intravenous infusion according to the following schedule, in chronological order: 1 mL/hour for 15 minutes, 5 mL/hour for 15 minutes, 10 mL/hour for 30 minutes, 15 mL/hour for 30 minutes, 25 mL/hour for 30 minutes, 30 mL/hour for 30 minutes, 35 mL/hour for 30 minutes, and 40 mL/hour for 62 minutes. In some embodiments, the first dose is administered to the individual by intravenous infusion according to the schedule shown in Table A.

Table A. Infusion rate schedule for 4-hour infusion.

[0113] In some embodiments, administration of the first dose of the composition by intravenous infusion over a period of about 4 hours reduces the risk of infusion-related reaction (IRR) in the individual, as compared to administration of the first dose by intravenous infusion over a period that is less than about 4 hours. In some embodiments, administration of the first dose of the composition by intravenous infusion over a period of about 4 hours reduces the severity of infusion-related reaction (IRR) in the individual, as compared to administration of the first dose by intravenous infusion over a period that is less than about 4 hours.

[0114] In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between O.lmg/kg and lOmg/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between Img/kg and lOmg/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between 0. Img/kg and 3mg/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between O.lmg/kg and Img/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between about Img/kg and about 3mg/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at 3mg/kg in the first dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at about any of 0.1 mg/kg, 0.5 mg/kg, 1,0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg in the first dose.

[0115] In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual in the first dose via the intravenous or subcutaneous route.

[0116] In some embodiments, the methods further comprise administering a corticosteroid to the individual 1-2 hours prior to administration of the first dose. That is, the methods can comprise administering a corticosteroid at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the first dose, as well as administering a corticosteroid within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the corticosteroid is prednisone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, the corticosteroid is hydrocortisone or dexamethasone. In some embodiments, the corticosteroid is administered orally. In some embodiments, the corticosteroid is administered intravenously. In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of greater than 0.5mg/kg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of about 1 mg/kg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of 80mg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the corticosteroid (e.g, methylprednisolone) is administered at a dose of lOOmg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose.

[0117] In some embodiments, the methods further comprise administering an antihistamine to the individual 1-2 hours prior to administration of the first dose. In some embodiments, the methods can comprise administering an antihistamine within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the antihistamine is cetirizine. In some embodiments, the antihistamine is administered orally. In some embodiments, the antihistamine (e.g, cetirizine) is administered at a dose of lOmg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the antihistamine (e.g, cetirizine) is administered at a dose of lOmg 40 minutes to 180 minutes prior to administration of the first dose.

[0118] In some embodiments, the methods further comprise administering an antipyretic or non-steroidal anti-inflammatory drug (NSAID) to the individual 1-2 hours prior to administration of the first dose. In some embodiments, the methods can comprise administering an antipyretic or NSAID within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose. In some embodiments, the antipyretic or NSAID is acetaminophen. In some embodiments, the antipyretic or NSAID is administered orally. In some embodiments, the antipyretic or NSAID (e.g., acetaminophen) is administered at a dose of 975-1000mg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose.

[0119] In some embodiments according to any of the embodiments described herein, the methods further comprise administering to the individual a second dose of a composition comprising an antibody that binds to human Siglec-8. For example, the second dose can be administered about 28 days, about 4 weeks, or about 1 month after administration of the first dose. In some embodiments, the second dose is administered to the individual without administration of a corticosteroid to the individual at least 6 hours, at least 12 hours, 6-24 hours, or 12-24 hours prior to administration of the second dose. That is, in some embodiments, corticosteroid is administered to the individual at least 6 hours, at least 12 hours, 6-24 hours, or 12-24 hours prior to administration of only the first dose, but not subsequent doses, of the anti- Siglec-8 antibody.

[0120] In some embodiments, the second dose of the composition is administered to the individual by intravenous infusion over a period of about 4 hours. In some embodiments, less than 50% of total volume of the second dose is administered to the individual in the first 2 hours of the infusion. In some embodiments, less than 30% of total volume of the second dose is administered to the individual in the first 2 hours of the infusion. In some embodiments, the second dose is administered to the individual by intravenous infusion according to the following schedule, in chronological order: 1 mL/hour for 15 minutes, 5 mL/hour for 15 minutes, 10 mL/hour for 30 minutes, 15 mL/hour for 30 minutes, 25 mL/hour for 30 minutes, 30 mL/hour for 30 minutes, 35 mL/hour for 30 minutes, and 40 mL/hour for 62 minutes. In some embodiments, the second dose is administered to the individual by intravenous infusion according to the schedule shown in Table A. In some embodiments, the first and the second doses are administered to the individual by intravenous infusion according to the schedule shown in Table A.

[0121] In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between O. lmg/kg and lOmg/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between Img/kg and lOmg/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between O. lmg/kg and 3mg/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between O. lmg/kg and Img/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between about Img/kg and about 3mg/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at 3mg/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at about any of 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg in the second dose (and optionally any subsequent doses). In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between about O. lmg/kg and about Img/kg in the first dose and between about 3mg/kg and about lOmg/kg in the second dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between about Img/kg and about 3mg/kg in the first dose and between about Img/kg and about 3mg/kg in the second dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the first dose and at 3mg/kg in the second dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at 3mg/kg in the first dose and at lOmg/kg in the second dose.

[0122] In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual in the second dose (and optionally any subsequent doses) via the intravenous or subcutaneous route. [0123] In some embodiments, the second dose is administered without administration of a corticosteroid at least 6 hours, at least 12 hours, within 24 hours, 6-24 hours, or 12-24 hours prior to administration of the second dose. In other embodiments, a corticosteroid is administered to the individual at least 6 hours, at least 12 hours, within 24 hours, 6-24 hours, or 12-24 hours prior to administration of the second dose. In some embodiments, the corticosteroid is prednisone. In some embodiments, the corticosteroid is methylprednisolone, hydrocortisone, or dexamethasone. In some embodiments, the corticosteroid is self-administered by the individual being treated with the anti-Siglec-8 antibody. In some embodiments, the corticosteroid is administered orally. In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of greater than 0.5mg/kg, or about Img/kg. In some embodiments, the corticosteroid (e.g, prednisone) is administered at a dose of 80mg. For example, in some embodiments, the methods comprise administering to the individual greater than 0.5mg/kg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the second dose of the anti-Siglec-8 antibody. In some embodiments, the methods comprise administering to the individual Img/kg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the second dose of the anti-Siglec-8 antibody. In some embodiments, the methods comprise administering to the individual 80mg prednisone at least 6 hours (and optionally within 24 hours), at least 12 hours (and optionally within 24 hours), 6-24 hours, or 12-24 hours prior to administration of the second dose of the anti-Siglec-8 antibody. In some embodiments, administration of the corticosteroid at least 6 hours prior to administration of the second dose reduces the risk of infusion-related reaction (IRR) in the individual, as compared to administration of the second dose without administration of the corticosteroid at least 6 hours prior. In some embodiments, administration of the corticosteroid at least 12 hours prior to administration of the second dose reduces the risk of infusion-related reaction (IRR) in the individual, as compared to administration of the second dose without administration of the corticosteroid at least 12 hours prior. In some embodiments, administration of the corticosteroid 12-24 hours prior to administration of the second dose reduces the risk of infusion-related reaction (IRR) in the individual, as compared to administration of the second dose without administration of the corticosteroid 12-24 hours prior. In some embodiments, administration of the corticosteroid at least 6 hours prior to administration of the second dose reduces the severity of infusion-related reaction (IRR) in the individual, as compared to administration of the second dose without administration of the corticosteroid at least 6 hours prior. In some embodiments, administration of the corticosteroid at least 12 hours prior to administration of the second dose reduces the severity of infusion-related reaction (IRR) in the individual, as compared to administration of the second dose without administration of the corticosteroid at least 12 hours prior. In some embodiments, administration of the corticosteroid 12-24 hours prior to administration of the second dose reduces the severity of infusion-related reaction (IRR) in the individual, as compared to administration of the second dose without administration of the corticosteroid 12-24 hours prior. In some embodiments, a corticosteroid is administered to the individual at least 6 hours, at least 12 hours, within 24 hours, 6-24 hours, or 12-24 hours prior to administration of the first and second doses. In some embodiments, a corticosteroid is administered to the individual at least 6 hours, at least 12 hours, within 24 hours, 6-24 hours, or 12-24 hours prior to administration of the first and second doses, but not prior to any subsequent doses of the anti-Siglec-8 antibody.

[0124] In some embodiments, the methods further comprise administering a corticosteroid to the individual 1-2 hours prior to administration of the second dose (and optionally any subsequent doses). In some embodiments, the corticosteroid administered to the individual 1-2 hours prior to administration of the first dose is methylprednisolone. In some embodiments, lOOmg methylprednisolone is administered to the individual within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the first dose (e.g, intravenously). [0125] In some embodiments, the methods further comprise administering an antihistamine to the individual 1-2 hours prior to administration of the second dose (and optionally any subsequent doses). In some embodiments, the methods can comprise administering an antihistamine within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the second dose (and optionally any subsequent doses). In some embodiments, the antihistamine is cetirizine. In some embodiments, the antihistamine is administered orally. In some embodiments, the antihistamine (e.g, cetirizine) is administered at a dose of lOmg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the second dose (and optionally any subsequent doses).

[0126] In some embodiments, the methods further comprise administering an antipyretic or non-steroidal anti-inflammatory drug (NSAID) to the individual 1-2 hours prior to administration of the second dose (and optionally any subsequent doses). In some embodiments, the methods can comprise administering an antipyretic or NSAID within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the second dose (and optionally any subsequent doses). In some embodiments, the antipyretic or NSAID is acetaminophen. In some embodiments, the antipyretic or NSAID is administered orally. In some embodiments, the antipyretic or NSAID e.g., acetaminophen) is administered at a dose of 975-1000mg within 1 hour, about 1 hour, within 2 hours, about 2 hours, or 1-2 hours prior to administration of the second dose (and optionally any subsequent doses).

[0127] In some embodiments according to any of the embodiments described herein, the methods further comprise administering to the individual a third dose of a composition comprising an antibody that binds to human Siglec-8 (e.g, subsequent to administration of a second dose as described herein). For example, the third dose can be administered about 28 days, about 4 weeks, or about 1 month after administration of the second dose and/or about 56 days, about 8 weeks, or about 2 months after administration of the first dose. In some embodiments, the third dose is administered to the individual without administration of a corticosteroid to the individual at least 6 hours, at least 12 hours, 6-24 hours, or 12-24 hours prior to administration of the third dose.

[0128] In some embodiments, the third dose of the composition is administered to the individual by intravenous infusion over a period of about 2 hours to about 4 hours. In some embodiments, the third dose of the composition is administered to the individual by intravenous infusion over a period of about 1 hour to about 4 hours. In some embodiments, the third dose of the composition is administered to the individual by intravenous infusion over a period of about 2 hours. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the following schedule, in chronological order: 10 mL/hour for 30 minutes, 25 mL/hour for 15 minutes, 40 mL/hour for 15 minutes, 55 mL/hour for 15 minutes, 70 mL/hour for 15 minutes, 85 mL/hour for 15 minutes, and 100 mL/hour for 16 minutes. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the schedule shown in Table B. In some embodiments, the third dose of the composition is administered to the individual by intravenous infusion over a period of about 3 hours. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the following schedule, in chronological order: 2 mL/hour for 30 minutes, 10 mL/hour for 30 minutes, 20 mL/hour for 30 minutes, 40 mL/hour for 30 minutes, and 60 mL/hour for 64 minutes. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the schedule shown in Table C. In some embodiments, the third dose of the composition is administered to the individual by intravenous infusion over a period of about

4 hours. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the following schedule, in chronological order: 1 mL/hour for 15 minutes,

5 mL/hour for 15 minutes, 10 mL/hour for 30 minutes, 15 mL/hour for 30 minutes, 25 mL/hour for 30 minutes, 30 mL/hour for 30 minutes, 35 mL/hour for 30 minutes, and 40 mL/hour for 62 minutes. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the schedule shown in Table A. In some embodiments, the third dose of the composition is administered to the individual by intravenous infusion over a period of about 1 hour. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the following schedule, in chronological order: 24 mL/hour for 15 minutes, and 125.3 mL/hour for 45 minutes. In some embodiments, the third dose is administered to the individual by intravenous infusion according to the schedule shown in Table D.

Table B. Infusion rate schedule for 2-hour infusion.

Table C. Infusion rate schedule for 3-hour infusion.

Table D. Infusion rate schedule for 1-hour infusion.

[0129] In some embodiments, the first and the second doses of the composition are administered to the individual by intravenous infusion over a period of about 4 hours, and the third dose of the composition is administered to the individual by intravenous infusion over a period of about 1 hour to about 4 hours. For example, in some embodiments, the first, second, and third doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g, according to Table A. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g., according to Table A, and the third dose is administered to the individual by intravenous infusion over a period of about 3 hours, e.g, according to Table C. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g., according to Table A, and the third dose is administered to the individual by intravenous infusion over a period of about 2 hours, e.g., according to Table B. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g, according to Table A, and the third dose is administered to the individual by intravenous infusion over a period of about 1 hour, e.g., according to Table D. For example, the third dose can be administered to the individual over a shorter infusion time, e.g., according to physician’s judgement, if no or mild infusion-related reaction(s) occur after administration of the first and/or second doses.

[0130] In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between 0. Img/kg and lOmg/kg in the third dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between Img/kg and lOmg/kg in the third dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at 3mg/kg in the third dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the third dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the first dose, followed by 3mg/kg in the second and third doses. [0131] In some embodiments according to any of the embodiments described herein, the methods further comprise administering to the individual a fourth dose of a composition comprising an antibody that binds to human Siglec-8 (e.g, subsequent to administration of a third dose as described herein). For example, the fourth dose can be administered about 28 days, about 4 weeks, or about 1 month after administration of the third dose; about 56 days, about 8 weeks, or about 2 months after administration of the second dose; and/or about 84 days, about 12 weeks, or about 3 months after administration of the first dose. In some embodiments, the fourth dose is administered to the individual without administration of a corticosteroid to the individual at least 6 hours, at least 12 hours, 6-24 hours, or 12-24 hours prior to administration of the third dose. In some embodiments, six doses or more of a composition comprising an antibody that binds to human Siglec-8 are administered to the individual (e.g, administered every 28 days, every 4 weeks, or every month).

[0132] In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion over a period of about 2 hours to about 4 hours. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion over a period of about 1 hour to about 4 hours. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion over a period of about 2 hours. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the following schedule, in chronological order: 10 mL/hour for 30 minutes, 25 mL/hour for 15 minutes, 40 mL/hour for 15 minutes, 55 mL/hour for 15 minutes, 70 mL/hour for 15 minutes, 85 mL/hour for 15 minutes, and 100 mL/hour for 16 minutes. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the schedule shown in Table B. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion over a period of about 3 hours. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the following schedule, in chronological order: 2 mL/hour for 30 minutes, 10 mL/hour for 30 minutes, 20 mL/hour for 30 minutes, 40 mL/hour for 30 minutes, and 60 mL/hour for 64 minutes. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the schedule shown in Table C. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion over a period of about 4 hours. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the following schedule, in chronological order: 1 mL/hour for 15 minutes, 5 mL/hour for 15 minutes, 10 mL/hour for 30 minutes, 15 mL/hour for 30 minutes, 25 mL/hour for 30 minutes, 30 mL/hour for 30 minutes, 35 mL/hour for 30 minutes, and 40 mL/hour for 62 minutes. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the schedule shown in Table A. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion over a period of about 1 hour. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the following schedule, in chronological order: 24 mL/hour for 15 minutes, and 125.3 mL/hour for 45 minutes. In some embodiments, the fourth and/or subsequent dose(s) of the composition are administered to the individual by intravenous infusion according to the schedule shown in Table D.

[0133] In some embodiments, the first and the second doses of the composition are administered to the individual by intravenous infusion over a period of about 4 hours, and the third and fourth doses of the composition are administered to the individual by intravenous infusion over a period of about 1 hour to about 4 hours. For example, in some embodiments, the first, second, third, and fourth doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g, according to Table A. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g., according to Table A, and the third and/or fourth doses are administered to the individual by intravenous infusion over a period of about 3 hours, e.g., according to Table C. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g, according to Table A, and the third and/or fourth doses are administered to the individual by intravenous infusion over a period of about 2 hours, e.g., according to Table B. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours, e.g., according to Table A, and the third and/or fourth doses are administered to the individual by intravenous infusion over a period of about 1 hour, e.g, according to Table D. In some embodiments, the first and second doses are administered to the individual by intravenous infusion over a period of about 4 hours (e.g., according to Table A), followed by 4 subsequent doses (e.g, administered every 28 days, every 4 weeks, or every month) administered to the individual by intravenous infusion over a period of about 1 hour to about 4 hours (e.g, according to Table B, C, or D). For example, the third, fourth, and/or subsequent dose(s) can be administered to the individual over a shorter infusion time, e.g, according to physician’s judgement, if no or mild infusion-related reaction(s) occur after administration of the first and/or second third doses.

[0134] In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between O.lmg/kg and lOmg/kg in the fourth dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at between Img/kg and lOmg/kg in the fourth dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at 3mg/kg in the fourth dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the fourth dose. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the first dose, followed by 3mg/kg in the second, third, and fourth doses. In some embodiments, the antibody that binds to human Siglec-8 is administered to the individual at Img/kg in the first dose, followed by 3mg/kg for 5 subsequent doses (e.g, administered every 28 days, every 4 weeks, or every month).

[0135] Antibodies described herein that bind to human Siglec-8 can be used either alone or in combination with other agents in the methods described herein.

[0136] In some embodiments, a composition of the present disclosure comprising an anti- Siglec-8 antibody is administered in combination with one or more additional therapeutic agent(s). In some embodiments, the additional therapeutic agent(s) are for treating viral infection and/or inhibiting inflammation. Examples of additional therapeutic agents include, without limitation, corticosteroids, hydroxychloroquine, azithromycin, colchicine, remdesivir, IL-6 antagonists, antigen-binding moieties (e.g, monoclonal antibodies, antigen-binding fragments, single chain antibodies, etc.) that specifically bind a viral spike protein or other viral surface antigens, Ramatroban, convalescent plasma, and favipiravir.

[0137] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the present disclosure can occur prior to, simultaneously, and/or following, administration of the one or more additional therapeutic agents. In some embodiments, administration of an anti-Siglec-8 antibody described herein and administration of one or more additional therapeutic agents occur within about one month, about two months, about three months, about four months, about five months or about six months of each other. In some embodiments, administration of an anti-Siglec-8 antibody described herein and administration of one or more additional therapeutic agents occur within about one week, about two weeks or about three weeks of each other. In some embodiments, administration of an anti-Siglec-8 antibody described herein and administration of one or more additional therapeutic agents occur within about one day, about two days, about three days, about four days, about five days, or about six days of each other.

[0138] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the present disclosure can occur prior to, simultaneously, and/or following, administration of the one or more additional therapeutic agents. In some embodiments, administration of an anti-Siglec-8 antibody described herein and administration of one or more additional therapeutic agents occur within about one month, about two months, about three months, about four months, about five months or about six months of each other. In some embodiments, administration of an anti-Siglec-8 antibody described herein and administration of one or more additional therapeutic agents occur within about one week, about two weeks or about three weeks of each other. In some embodiments, administration of an anti-Siglec-8 antibody described herein and administration of one or more additional therapeutic agents occur within about one day, about two days, about three days, about four days, about five days, or about six days of each other.

[0139] Anti-Siglec8 antibodies and/or one or more additional therapeutic agents may be administered via any suitable route of administration known in the art, including, without limitation, by oral administration, sublingual administration, buccal administration, topical administration, rectal administration, via inhalation, transdermal administration, subcutaneous injection, intradermal injection, intravenous (IV) injection, intra-arterial injection, intramuscular injection, intracardiac injection, intraosseous injection, intraperitoneal injection, transmucosal administration, vaginal administration, intravitreal administration, intra-articular administration, peri-articular administration, local administration, epicutaneous administration, or any combinations thereof.

Antibodies [0140] Certain aspects of the present disclosure provide isolated antibodies that bind to a human Siglec-8 (e.g., an agonist antibody that binds to human Siglec-8). In some embodiments, an anti-Siglec-8 antibody described herein has one or more of the following characteristics: (1) binds a human Siglec-8; (2) binds to an extracellular domain of a human Siglec-8; (3) binds a human Siglec-8 with a higher affinity than mouse antibody 2E2 and/or mouse antibody 2C4; (4) binds a human Siglec-8 with a higher avidity than mouse antibody 2E2 and/or mouse antibody 2C4; (5) has a T_m of about 70°C-72°C or higher in a thermal shift assay; (6) has a reduced degree of fucosylation or is non-fucosylated; (7) binds a human Siglec-8 expressed on eosinophils and induces apoptosis of eosinophils; (8) binds a human Siglec-8 expressed on mast cells and depletes or reduces the number of mast cells; (9) binds a human Siglec-8 expressed on mast cells and inhibits FceRI-dependent activities of mast cells (e.g, histamine release, PGD2 release, Ca²⁺ flux, and/or P-hexosaminidase release, etc.); (10) has been engineered to improve ADCC activity; (11) binds a human Siglec-8 expressed on mast cells and kills mast cells by ADCC activity (in vitro, and/or in vivo); (12) binds to Siglec-8 of a human and a non-human primate; (13) binds to Domain 1, Domain 2, and/or Domain 3 of human Siglec-8, or binds a Siglec-8 polypeptide comprising Domain 1, Domain 2, and/or Domain 3 of human Siglec-8 (e.g., fusion proteins described herein); and (14) depletes activated eosinophils with an EC50 less than the EC50 of mouse antibody 2E2 or 2C4. Any of the antibodies described in U.S. Pat. No. 9,546,215 and/or W02015089117 may find use in the methods, compositions, and kits provided herein. [0141] In one aspect, the present disclosure provides antibodies that bind to a human Siglec-8. In some embodiments, the human Siglec-8 comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, the human Siglec-8 comprises an amino acid sequence of SEQ ID NO:73. In some embodiments, an antibody described herein binds to a human Siglec-8 expressed on mast cells and depletes or reduces the number of mast cells. In some embodiments, an antibody described herein binds to a human Siglec-8 expressed on mast cells and inhibits mast cell-mediated activity.

[0142] In one aspect, the invention provides antibodies that bind to a human Siglec-8. In some embodiments, the human Siglec-8 comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, the human Siglec-8 comprises an amino acid sequence of SEQ ID NO:73. In some embodiments, the antibody described herein binds to an epitope in Domain 1 of human Siglec-8, wherein Domain 1 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody described herein binds to an epitope in Domain 2 of human Siglec-8, wherein Domain 2 comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antibody described herein binds to an epitope in Domain 3 of human Siglec-8, wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody described herein binds to a fusion protein comprising the amino acid of SEQ ID NO: 116 but not to a fusion protein comprising the amino acid of SEQ ID NO: 115. In some embodiments, the antibody described herein binds to a fusion protein comprising the amino acid of SEQ ID NO: 117 but not to a fusion protein comprising the amino acid of SEQ ID NO: 115. In some embodiments, the antibody described herein binds to a fusion protein comprising the amino acid of SEQ ID NO: 117 but not to a fusion protein comprising the amino acid of SEQ ID NO:116. In some embodiments, the antibody described herein binds to a linear epitope in the extracellular domain of human Siglec-8. In some embodiments, the antibody described herein binds to a conformational epitope in the extracellular domain of human Siglec- 8. In some embodiments, an antibody described herein binds to a human Siglec-8 expressed on eosinophils and induces apoptosis of eosinophils. In some embodiments, an antibody described herein binds to a human Siglec-8 expressed on mast cells and depletes mast cells. In some embodiments, an antibody described herein binds to a human Siglec-8 expressed on mast cells and inhibits mast cell-mediated activity. In some embodiments, an antibody described herein binds to a human Siglec-8 expressed on mast cells and kills mast cells by ADCC activity. In some embodiments, an antibody described herein depletes mast cells and inhibits mast cell activation. In some embodiments, an antibody herein depletes activated eosinophils and inhibits mast cell activation. In some embodiments, an antibody herein (e.g., a non-fucosylated anti- Siglec-8 antibody) depletes blood eosinophils and inhibits mast cell activation. In some embodiments, an antibody herein (e.g., a non-fucosylated anti-Siglec-8 antibody) depletes eosinophils from the peripheral blood and inhibits mast cell activation.

[0143] Provided herein is an isolated anti-Siglec-8 antibody that binds to human Siglec-8 and non-human primate Siglec-8. Identification of antibodies with primate cross-reactivity would be useful for preclinical testing of anti-Siglec-8 antibodies in non-human primates. In one aspect, the invention provides antibodies that bind to a non-human primate Siglec-8. In one aspect, the invention provides antibodies that bind to a human Siglec-8 and a non-human primate Siglec-8. In some embodiments, the non-human primate Siglec-8 comprises an amino acid sequence of SEQ ID NO: 118 or a portion thereof. In some embodiments, the non-human primate Siglec-8 comprises an amino acid sequence of SEQ ID NO: 119 or a portion thereof. In some embodiments, the non-human primate is a baboon (e.g., Papio Anubis). In some embodiments, the antibody that binds to a human Siglec-8 and a non-human primate Siglec-8, binds to an epitope in Domain 1 of human Siglec-8. In a further embodiment, Domain 1 of human Siglec-8 comprises the amino acid sequence of SEQ ID NO:112. In some embodiments, the antibody that binds to a human Siglec-8 and a non-human primate Siglec-8, binds to an epitope in Domain 3 of human Siglec-8. In a further embodiment, Domain 3 of human Siglec-8 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody that binds to a human Siglec-8 and a non-human primate Siglec-8 is a humanized antibody, a chimeric antibody, or a human antibody. In some embodiments, the antibody that binds to a human Siglec-8 and anon-human primate Siglec-8 is a murine antibody. In some embodiments, the antibody that binds to a human Siglec-8 and a non-human primate Siglec-8 is a human IgGl antibody.

[0144] In one aspect, an anti-Siglec-8 antibody described herein is a monoclonal antibody. In one aspect, an anti-Siglec-8 antibody described herein is an antibody fragment (including antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In one aspect, an anti-Siglec-8 antibody described herein comprises an antibody fragment (including antigenbinding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In one aspect, an anti- Siglec-8 antibody described herein is a chimeric, humanized, or human antibody. In one aspect, any of the anti-Siglec-8 antibodies described herein are purified.

[0145] In one aspect, anti-Siglec-8 antibodies that compete with murine 2E2 antibody and murine 2C4 antibody binding to Siglec-8 are provided. Anti-Siglec-8 antibodies that bind to the same epitope as murine 2E2 antibody and murine 2C4 antibody are also provided. Murine antibodies to Siglec-8, 2E2 and 2C4 antibody are described in U.S. Pat. No. 8,207,305; U.S. Pat. No. 8,197,811, U.S. Pat. No. 7,871,612, and U.S. Pat. No. 7,557,191.

[0146] In one aspect, anti-Siglec-8 antibodies that compete with any anti-Siglec-8 antibody described herein (e.g., HEKA, HEKF, 1C3, 1H10, 4F11, 2C4, 2E2) for binding to Siglec-8 are provided. Anti-Siglec-8 antibodies that bind to the same epitope as any anti-Siglec-8 antibody described herein (e.g., HEKA, HEKF, 1C3, 1H10, 4F11, 2C4, 2E2) are also provided.

[0147] In one aspect of the present disclosure, polynucleotides encoding anti-Siglec-8 antibodies are provided. In certain embodiments, vectors comprising polynucleotides encoding anti-Siglec-8 antibodies are provided. In certain embodiments, host cells comprising such vectors are provided. In another aspect of the present disclosure, compositions comprising anti- Siglec-8 antibodies or polynucleotides encoding anti-Siglec-8 antibodies are provided. In certain embodiments, a composition of the present disclosure is a pharmaceutical formulation for the treatment of viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation. In certain embodiments, a composition of the present disclosure is a pharmaceutical formulation for the prevention of virus-induced inflammation and/or virus- induced mast cell/eosinophil activation (e.g., in an individual suffering from viral infection).

[0148] In one aspect, provided herein is an anti-Siglec-8 antibody comprising 1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 2C4. In one aspect, provided herein is an anti- Siglec-8 antibody comprising 1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 2E2. In some embodiments, the HVR is a Kabat CDR or a Chothia CDR.

[0149] In one aspect, provided herein is an anti-Siglec-8 antibody comprising 1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 1C3. In one aspect, provided herein is an anti- Siglec-8 antibody comprising 1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 4F11. In one aspect, provided herein is an anti-Siglec-8 antibody comprising 1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 1H10. In some embodiments, the HVR is a Kabat CDR or a Chothia CDR.

[0150] In some embodiments, the antibody described herein binds to an epitope in Domain 1 of human Siglec-8, wherein Domain 1 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody described herein binds to an epitope in Domain 2 of human Siglec-8, wherein Domain 2 comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antibody described herein binds to an epitope in Domain 3 of human Siglec-8, wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114.

[0151] In some embodiments, the antibody described herein binds to a fusion protein comprising the amino acid of SEQ ID NO: 116 but not to a fusion protein comprising the amino acid of SEQ ID NO:115. In some embodiments, the antibody described herein binds to a fusion protein comprising the amino acid of SEQ ID NO: 117 but not to a fusion protein comprising the amino acid of SEQ ID NO: 115. In some embodiments, the antibody described herein binds to a fusion protein comprising the amino acid of SEQ ID NO: 117 but not to a fusion protein comprising the amino acid of SEQ ID NO: 116.

[0152] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:94; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the antibody described herein binds to an epitope in Domain 2 of human Siglec-8, wherein Domain 2 comprises the amino acid sequence of SEQ ID NO: 113. [0153] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antibody described herein binds to an epitope in Domain 3 of human Siglec-8, wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody described herein binds to human Siglec-8 and non-human primate Siglec-8.

[0154] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:96; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody described herein binds to an epitope in Domain 1 of human Siglec-8, wherein Domain 1 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody described herein binds to human Siglec-8 and non-human primate Siglec-8.

[0155] In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

[0156] In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence selected from SEQ ID NOs:67-70; and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

[0157] In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71.

[0158] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence selected from SEQ ID NOs:67-70; and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71.

[0159] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:94; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:103.

[0160] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104.

[0161] In another aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:96; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:105.

[0162] An anti-Siglec-8 antibody described herein may comprise any suitable framework variable domain sequence, provided that the antibody retains the ability to bind human Siglec-8. As used herein, heavy chain framework regions are designated "HC-FR1-FR4," and light chain framework regions are designated "LC-FR1-FR4." In some embodiments, the anti-Siglec-8 antibody comprises a heavy chain variable domain framework sequence of SEQ ID NO:26, 34, 38, and 45 (HC-FR1, HC-FR2, HC-FR3, and HC-FR4, respectively). In some embodiments, the anti-Siglec-8 antibody comprises a light chain variable domain framework sequence of SEQ ID NO:48, 51, 55, and 60 (LC-FR1, LC-FR2, LC-FR3, and LC-FR4, respectively). In some embodiments, the anti-Siglec-8 antibody comprises a light chain variable domain framework sequence of SEQ ID NO:48, 51, 58, and 60 (LC-FR1, LC-FR2, LC-FR3, and LC-FR4, respectively). [0163] In one embodiment, an anti-Siglec-8 antibody comprises a heavy chain variable domain comprising a framework sequence and hypervariable regions, wherein the framework sequence comprises the HC-FR1-HC-FR4 sequences SEQ ID NOs:26-29 (HC-FR1), SEQ ID NOs:31-36 (HC-FR2), SEQ ID NOs:38-43 (HC-FR3), and SEQ ID NOs:45 or 46 (HC-FR4), respectively; the HVR-H1 comprises the amino acid sequence of SEQ ID NO:61; the HVR-H2 comprises the amino acid sequence of SEQ ID NO:62; and the HVR-H3 comprises an amino acid sequence of SEQ ID NO:63. In one embodiment, an anti-Siglec-8 antibody comprises a heavy chain variable domain comprising a framework sequence and hypervariable regions, wherein the framework sequence comprises the HC-FR1-HC-FR4 sequences SEQ ID NOs:26- 29 (HC-FR1), SEQ ID NOs:31-36 (HC-FR2), SEQ ID NOs:38-43 (HC-FR3), and SEQ ID NOs:45 or 46 (HC-FR4), respectively; the HVR-H1 comprises the amino acid sequence of SEQ ID NO:61; the HVR-H2 comprises the amino acid sequence of SEQ ID NO:62; and the HVR- H3 comprises an amino acid sequence selected from SEQ ID NOs:67-70. In one embodiment, an anti-Siglec-8 antibody comprises a light chain variable domain comprising a framework sequence and hypervariable regions, wherein the framework sequence comprises the LC-FR1- LC-FR4 sequences SEQ ID NOs:48 or 49 (LC-FR1), SEQ ID NOs:51-53 (LC-FR2), SEQ ID NOs:55-58 (LC-FR3), and SEQ ID NO:60 (LC-FR4), respectively; the HVR-L1 comprises the amino acid sequence of SEQ ID NO:64; the HVR-L2 comprises the amino acid sequence of SEQ ID NO:65; and the HVR-L3 comprises an amino acid sequence of SEQ ID NO:66. In one embodiment, an anti-Siglec-8 antibody comprises a light chain variable domain comprising a framework sequence and hypervariable regions, wherein the framework sequence comprises the LC-FR1-LC-FR4 sequences SEQ ID NOs:48 or 49 (LC-FR1), SEQ ID NOs:51-53 (LC-FR2), SEQ ID NOs:55-58 (LC-FR3), and SEQ ID NO:60 (LC-FR4), respectively; the HVR-L1 comprises the amino acid sequence of SEQ ID NO:64; the HVR-L2 comprises the amino acid sequence of SEQ ID NO:65; and the HVR-L3 comprises an amino acid sequence of SEQ ID NO:71. In one embodiment of these antibodies, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs:2-10 and the light chain variable domain comprises and amino acid sequence selected from SEQ ID NOs: 16-22. In one embodiment of these antibodies, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs:2-10 and the light chain variable domain comprises and amino acid sequence selected from SEQ ID NOs: 23 or 24. In one embodiment of these antibodies, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 11-14 and the light chain variable domain comprises and amino acid sequence selected from SEQ ID NOs:16- 22. In one embodiment of these antibodies, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 11-14 and the light chain variable domain comprises and amino acid sequence selected from SEQ ID NOs:23 or 24. In one embodiment of these antibodies, the heavy chain variable domain comprises an amino acid sequence of SEQ ID NO:6 and the light chain variable domain comprises and amino acid sequence of SEQ ID NO: 16. In one embodiment of these antibodies, the heavy chain variable domain comprises an amino acid sequence of SEQ ID NO:6 and the light chain variable domain comprises and amino acid sequence of SEQ ID NO:21.

[0164] In some embodiments, the heavy chain HVR sequences comprise the following: a) HVR-H1 (IYGAH (SEQ ID NO: 61)); b) HVR-H2 (VIWAGGSTNYNSALMS (SEQ ID NO:62)); and c) HVR-H3 (DGSSPYYYSMEY (SEQ ID NO:63); DGSSPYYYGMEY (SEQ ID

NO:67); DGSSPYYYSMDY (SEQ ID NO:68); DGSSPYYYSMEV (SEQ ID NO:69); or DGSSPYYYGMDV (SEQ ID NO:70)).

[0165] In some embodiments, the heavy chain HVR sequences comprise the following: a) HVR-H1 (SYAMS (SEQ ID NO:88); DYYMY (SEQ ID NO:89); or SSWMN (SEQ ID NO:90)); b) HVR-H2 (IISSGGSYTYYSDSVKG (SEQ ID NO:91); RIAPEDGDTEYAPKFQG (SEQ ID NO:92); or QIYPGDDYTNYNGKFKG (SEQ ID NO:93)); and c) HVR-H3 (HETAQAAWFAY (SEQ ID NO:94); EGNYYGSSILDY (SEQ ID NO:95); or LGPYGPFAD (SEQ ID NO:96)).

[0166] In some embodiments, the heavy chain FR sequences comprise the following: a) HC-FR1 (EVQLVESGGGLVQPGGSLRLSCAASGFSLT (SEQ ID NO:26); EVQLVESGGGLVQPGGSLRLSCAVSGFSLT (SEQ ID NO:27); QVQLQESGPGLVKPSETLSLTCTVSGGSIS (SEQ ID NO:28); or QVQLQESGPGLVKPSETLSLTCTVSGFSLT (SEQ ID NO:29)); b) HC-FR2 (WVRQAPGKGLEWVS (SEQ ID NO:31); WVRQAPGKGLEWLG (SEQ ID NO:32); WVRQAPGKGLEWLS (SEQ ID NO: 33); WVRQAPGKGLEWVG (SEQ ID NO:34); WIRQPPGKGLEWIG (SEQ ID NO:35); or WVRQPPGKGLEWLG (SEQ ID NO:36)); c) HC-FR3 (RFTISKDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO:38);

RLSISKDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO:39); RLTISKDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO:40); RFSISKDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID N0:41); RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO:42); or RLSISKDNSKNQVSLKLSSVTAADTAVYYCAR (SEQ ID NO:43)); and d) HC-FR4 (WGQGTTVTVSS (SEQ ID NO:45); or WGQGTLVTVSS (SEQ ID NO:46)).

[0167] In some embodiments, the light chain HVR sequences comprise the following: a) HVR-L1 (SATSSVSYMH (SEQ ID NO:64)); b) HVR-L2 (STSNLAS (SEQ ID NO:65)); and c) HVR-L3 (QQRSSYPFT (SEQ ID NO:66); or QQRSSYPYT (SEQ ID NO:71)).

[0168] In some embodiments, the light chain HVR sequences comprise the following: a) HVR-L1 (SASSSVSYMH (SEQ ID NO:97); RASQDITNYLN (SEQ ID NO:98); or SASSSVSYMY (SEQ ID NO:99)); b) HVR-L2 (DTSKLAY (SEQ ID NO: 100); FTSRLHS (SEQ ID NO: 101); or DTSSLAS (SEQ ID NO: 102)); and c) HVR-L3 (QQWSSNPPT (SEQ ID NO: 103); QQGNTLPWT (SEQ ID NO: 104); or QQWNSDPYT (SEQ ID NO: 105)).

[0169] In some embodiments, the antibody comprises: a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:94; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 103; a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104; or a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:96; and/or a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 105.

[0170] In some embodiments, the light chain FR sequences comprise the following: a) LC-FR1 (EIVLTQSPATLSLSPGERATLSC (SEQ ID NO:48); or EIILTQSPATLSLSPGERATLSC (SEQ ID NO:49)); b) LC-FR2 (WFQQKPGQAPRLLIY (SEQ ID NO:51); WFQQKPGQAPRLWIY (SEQ ID NO:52); or WYQQKPGQAPRLLIY (SEQ ID NO: 53)); c) LC-FR3 (GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO:55);

GVPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO:56);

GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO:57); or GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO:58)); and d) LC-FR4 (FGPGTKLDIK (SEQ ID NO:60)).

[0171] In some embodiments, provided herein is an anti-Siglec-8 antibody (e.g, a humanized anti-Siglec-8) antibody that binds to human Siglec-8, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises:

(a) heavy chain variable domain comprising:

(1) an HC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:26-29;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61;

(3) an HC-FR2 comprising the amino acid sequence selected from SEQ ID NOs:31-36;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62;

(5) an HC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:38-43;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and

(7) an HC-FR4 comprising the amino acid sequence selected from SEQ ID NOs:45-46, and/or

(b) a light chain variable domain comprising:

(1) an LC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:48-49;

(2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64;

(3) an LC-FR2 comprising the amino acid sequence selected from SEQ ID NOs:51-53; (4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65;

(5) an LC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:55-58;

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 66; and

(7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60.

[0172] In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain selected from SEQ ID NOs:2-10 and/or comprising a light chain variable domain selected from SEQ ID NOs: 16-22. In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain selected from SEQ ID NOs:2-14 and/or comprising a light chain variable domain selected from SEQ ID NOs: 16-24. In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain selected from SEQ ID NOs:2-10 and/or comprising a light chain variable domain selected from SEQ ID NO:23 or 24. In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain selected from SEQ ID NOs: 11-14 and/or comprising a light chain variable domain selected from SEQ ID NOs: 16-22. In one aspect, provided herein is an anti- Siglec-8 antibody comprising a heavy chain variable domain selected from SEQ ID NOs: 11-14 and/or comprising a light chain variable domain selected from SEQ ID NO:23 or 24. In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain of SEQ ID NO:6 and/or comprising a light chain variable domain selected from SEQ ID NO: 16 or 21.

[0173] In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain selected from SEQ ID NOs: 106-108 and/or comprising a light chain variable domain selected from SEQ ID NOs: 109-111. In one aspect, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain of SEQ ID NO: 106 and/or comprising a light chain variable domain of SEQ ID NO: 109. In one aspect, provided herein is an anti-Siglec- 8 antibody comprising a heavy chain variable domain of SEQ ID NO: 107 and/or comprising a light chain variable domain of SEQ ID NO: 110. In one aspect, provided herein is an anti-Siglec- 8 antibody comprising a heavy chain variable domain of SEQ ID NO: 108 and/or comprising a light chain variable domain of SEQ ID NO: 111.

[0174] In some embodiments, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:2-14. In some embodiments, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 106-108. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody comprising that amino acid sequence retains the ability to bind to human Siglec-8. In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the HVRs (i.e. , in the FRs). In some embodiments, an anti-Siglec-8 antibody comprises a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO:6. In some embodiments, an anti-Siglec-8 antibody comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 106- 108.

[0175] In some embodiments, provided herein is an anti-Siglec-8 antibody comprising a light chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 16-24. In some embodiments, provided herein is an anti-Siglec-8 antibody comprising a light chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 109-111. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody comprising that amino acid sequence retains the ability to bind to human Siglec-8. In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the HVRs (i.e., in the FRs). In some embodiments, an anti-Siglec-8 antibody comprises a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 16 or 21. In some embodiments, an anti-Siglec-8 antibody comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 109-111.

[0176] In one aspect, the present disclosure provides an anti-Siglec-8 antibody comprising (a) one, two, or three VH HVRs selected from those shown in Table 1 and/or (b) one, two, or three VL HVRs selected from those shown in Table 1.

[0177] In one aspect, the present disclosure provides an anti-Siglec-8 antibody comprising (a) one, two, or three VH HVRs selected from those shown in Table 2 and/or (b) one, two, or three VL HVRs selected from those shown in Table 2. [0178] In one aspect, the present disclosure provides an anti-Siglec-8 antibody comprising (a) one, two, three or four VH FRs selected from those shown in Table 3 and/or (b) one, two, three or four VL FRs selected from those shown in Table 3.

[0179] In some embodiments, provided herein is an anti-Siglec-8 antibody comprising a heavy chain variable domain and/or a light chain variable domain of an antibody shown in Table 4, for example, HAKA antibody, HAKB antibody, HAKC antibody, etc.

Table 1. Amino acid sequences of HVRs of antibodies

Table 2. Amino acid sequences of HVRs from murine 1C3, 1H10, and 4F11 antibodies

Table 3. Amino acid sequences of FRs of antibodies

Table 4. Amino acid sequences of variable regions of antibodies

[0180] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 8, s, y and p, respectively. The y and a classes are further divided into subclasses e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a,f,n,z or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In further embodiments, the human IgG Fc region comprises a human IgGl or IgG4. In some embodiments, the antibody is an IgGl antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the human IgG4 comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat. In some embodiments, the human IgGl comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the human IgG4 comprises the amino acid sequence of SEQ ID NO:79.

[0181] In some embodiments, provided herein is an anti-Siglec-8 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:75; and/or a light chain comprising the amino acid sequence selected from SEQ ID NOs:76 or 77. In some embodiments, the antibody may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 87; and/or a light chain comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-Siglec-8 antibody induces apoptosis of activated eosinophils. In some embodiments, the anti-Siglec-8 antibody induces apoptosis of resting eosinophils. In some embodiments, the anti-Siglec-8 antibody depletes activated eosinophils and inhibits mast cell activation. In some embodiments, the anti-Siglec-8 antibody depletes or reduces mast cells and inhibits mast cell activation. In some embodiments, the anti-Siglec-8 antibody depleted or reduces the number of mast cells. In some embodiments, the anti-Siglec-8 antibody kills mast cells by ADCC activity. In some embodiments, the antibody depletes or reduces mast cells expressing Siglec-8 in a tissue. In some embodiments, the antibody depletes or reduces mast cells expressing Siglec-8 in a biological fluid.

1. Antibody Affinity

[0182] In some aspects, an anti-Siglec-8 antibody described herein binds to human Siglec-8 with about the same or higher affinity and/or higher avidity as compared to mouse antibody 2E2 and/or mouse antibody 2C4. In certain embodiments, an anti-Siglec-8 antibody provided herein has a dissociation constant (Kd) of < IpM, < 150 nM, < 100 nM, < 50 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10-8 M or less, e.g. from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M). In some embodiments, an anti-Siglec-8 antibody described herein binds to human Siglec-8 at about 1.5-fold, about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold higher affinity than mouse antibody 2E2 and/or mouse antibody 2C4. In some embodiments, the anti-Siglec-8 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; and/or a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs:16 or 21.

[0183] In one embodiment, the binding affinity of the anti-Siglec-8 antibody can be determined by a surface plasmon resonance assay. For example, the Kd or Kd value can be measured by using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore® Inc.) are activated with N-ethyl- N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) according to the supplier's instructions. Capture antibodies (e.g., anti-human-Fc) are diluted with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 30 pl/minute and further immobilized with an anti-Siglec-8 antibody. For kinetics measurements, two-fold serial dilutions of dimeric Siglec-8 are injected in PBS with 0.05% Tween 20 (PBST) at 25° C at a flow rate of approximately 25 pl/min. Association rates (k_on) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881.

[0184] In another embodiment, biolayer interferometry may be used to determine the affinity of anti-Siglec-8 antibodies against Siglec-8. In an exemplary assay, Siglec-8-Fc tagged protein is immobilized onto anti-human capture sensors, and incubated with increasing concentrations of mouse, chimeric, or humanized anti-Siglec-8 Fab fragments to obtain affinity measurements using an instrument such as, for example, the Octet Red 384 System (ForteBio).

[0185] The binding affinity of the anti-Siglec-8 antibody can, for example, also be determined by the Scatchard analysis described in Munson et al., Anal. Biochem, 107:220 (1980) using standard techniques well known in the relevant art. See also Scatchard, G., Ann. N.Y. Acad. Sci. 51:660 (1947).

2. Antibody Avidity

[0186] In some embodiments, the binding avidity of the anti-Siglec-8 antibody can be determined by a surface plasmon resonance assay. For example, the Kd or Kd value can be measured by using a BIAcore T100. Capture antibodies (e.g., goat-anti -human-Fc and goat-anti- mouse-Fc) are immobilized on a CM5 chip. Flow-cells can be immobilized with anti -human or with anti-mouse antibodies. The assay is conducted at a certain temperature and flow rate, for example, at 25oC at a flow rate of 30pl/min. Dimeric Siglec-8 is diluted in assay buffer at various concentrations, for example, at a concentration ranging from 15nM to

1.88pM. Antibodies are captured and high performance injections are conducted, followed by dissociations. Flow cells are regenerated with a buffer, for example, 50mM glycine pH 1.5. Results are blanked with an empty reference cell and multiple assay buffer injections, and analyzed with 1:1 global fit parameters.

3. Competition Assays

[0187] Competition-assays can be used to determine whether two antibodies bind the same epitope by recognizing identical or sterically overlapping epitopes or one antibody competitively inhibits binding of another antibody to the antigen. These assays are known in the art. Typically, antigen or antigen expressing cells is immobilized on a multi-well plate and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured. Common labels for such competition assays are radioactive labels or enzyme labels. In some embodiments, an anti- Siglec-8 antibody described herein competes with a 2E2 antibody described herein, for binding to the epitope present on the cell surface of a cell (e.g., a mast cell). In some embodiments, an anti-Siglec-8 antibody described herein competes with an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, for binding to the epitope present on the cell surface of a cell (e.g., a mast cell). In some embodiments, an anti- Siglec-8 antibody described herein competes with a 2C4 antibody described herein, for binding to the epitope present on the cell surface of a cell (e.g, a mast cell). In some embodiments, an anti-Siglec-8 antibody described herein competes with an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:2 (as found in U.S. Pat. No. 8,207,305), and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 (as found in U.S. Pat. No. 8,207,305), for binding to the epitope present on the cell surface of a cell (e.g, a mast cell).

4. Thermal Stability

[0188] In some aspects, an anti-Siglec-8 described herein has a melting temperature (Tm) of at least about 70°C, at least about 71°C, or at least about 72°C in a thermal shift assay. In an exemplary thermal shift assay, samples comprising a humanized anti-Siglec-8 antibody are incubated with a fluorescent dye (Sypro Orange) for 71 cycles with 1°C increase per cycle in a qPCR thermal cycler to determine the Tm. In some embodiments, the anti-Siglec-8 antibody has a similar or higher Tm as compared to mouse 2E2 antibody and/or mouse 2C4 antibody. In some embodiments, the anti-Siglec-8 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and/or a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs:16 or 21. In some embodiments, the anti-Siglec-8 antibody has the same or higher Tm as compared to a chimeric 2C4 antibody. In some embodiments, the anti-Siglec-8 antibody has the same or higher Tm as compared to an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

5. Biological Activity Assays

[0189] In some embodiments, an anti-Siglec-8 antibody described herein depletes eosinophils and inhibits mast cells. Assays for assessing apoptosis of cells are well known in the art, for example staining with Annexin V and the TUNNEL assay.

[0190] In some embodiments, an anti-Siglec-8 antibody described herein induces ADCC activity. In some embodiments, an anti-Siglec-8 antibody described herein kills eosinophils expressing Siglec-8 by ADCC activity. In some embodiments, a composition comprises non- fucosylated (i.e., afucosylated) anti-Siglec-8 antibodies. In some embodiments, a composition comprising non-fucosylated anti-Siglec-8 antibodies described herein enhances ADCC activity against Siglec-8 expressing eosinophils as compared to a composition comprising partially fucosylated anti-Siglec-8 antibodies. Assays for assessing ADCC activity are well known in the art and described herein. In an exemplary assay, to measure ADCC activity, effector cells and target cells are used. Examples of effector cells include natural killer (NK) cells, large granular lymphocytes (LGL), lymphokine-activated killer (LAK) cells and PBMC comprising NK and LGL, or leukocytes having Fc receptors on the cell surfaces, such as neutrophils, eosinophils and macrophages. Effector cells can be isolated from any source including individuals with a disease of interest (e.g., viral infection). The target cell is any cell which expresses on the cell surface antigens that antibodies to be evaluated can recognize. An example of such a target cell is an eosinophil which expresses Siglec-8 on the cell surface. Another example of such a target cell is a cell line (e.g., Ramos cell line) which expresses Siglec-8 on the cell surface (e.g., Ramos 2C10)). Target cells can be labeled with a reagent that enables detection of cytolysis. Examples of reagents for labeling include a radio-active substance such as sodium chromate (Na2 ⁵¹CrC>4). See, e.g., Immunology, 14, 181 (1968); J. Immunol. Methods., 172, 227 (1994); and J. Immunol. Methods., 184, 29 (1995).

[0191] In an exemplary assay to assess ADCC and apoptotic activity of anti-Siglec-8 antibodies on mast cells, human mast cells are isolated from human tissues or biological fluids according to published protocols (Guhl et al., Biosci. Biotechnol. Biochem., 2011, 75:382-384; Kulka et al., In Current Protocols in Immunology, 2001, (John Wiley & Sons, Inc.)) or differentiated from human hematopoietic stem cells, for example as described by Yokoi et al., J Allergy Clin Immunol., 2008, 121:499-505. Purified mast cells are resuspended in Complete RPMI medium in a sterile 96-well U-bottom plate and incubated in the presence or absence of anti-Siglec-8 antibodies for 30 minutes at concentrations ranging between 0.0001 ng/ml and 10 pg/ml. Samples are incubated for a further 4 to 48 hours with and without purified natural killer (NK) cells or fresh PBL to induce ADCC. Cell-killing by apoptosis or ADCC is analyzed by flow cytometry using fluorescent conjugated antibodies to detect mast cells (CD117 and FcsRl) and Annexin-V and 7AAD to discriminate live and dead or dying cells. Annexin-V and 7AAD staining are performed according to manufacturer’s instructions.

[0192] In some aspects, an anti-Siglec-8 antibody described herein inhibits mast cell-mediated activities. Mast cell tryptase has been used as a biomarker for total mast cell number and activation. For example, total and active tryptase as well as histamine, N-methyl histamine, and 11 -beta-prostaglandin F2 can be measured in blood or urine to assess the reduction in mast cells. See, e.g, U.S. Patent Application Publication No. US 20110293631 for an exemplary mast cell activity assay.

E. Antibody Preparation

[0193] The antibody described herein (e.g., an antibody that binds to human Siglec-8) is prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.

1. Antibody Fragments

[0194] The present disclosure encompasses antibody fragments. Antibody fragments may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

[0195] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from A’. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above.

Alternatively, Fab'-SH fragments can be directly recovered from A’. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.

2. Humanized Antibodies

[0196] The present disclosure encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0197] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called “best- fit” method, the sequence of the variable domain of a rodent (e.g., mouse) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) Immunol., 151:2623.

[0198] It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those, skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

3. Human Antibodies

[0199] Human anti-Siglec-8 antibodies of the present disclosure can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal anti-Siglec-8 antibodies of the present disclosure can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).

[0200] It is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy -chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.

USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

[0201] Gene shuffling can also be used to derive human antibodies from non-human (e.g. , rodent) antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting”, either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Sel ection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, i.e., the epitope governs the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non- human origin.

4. Bispecific Antibodies

[0202] Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for Siglec-8 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of Siglec-8. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Siglec-8. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2bispecific antibodies).

[0203] Methods for making bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305: 537 (1983), WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J., 10: 3655 (1991). For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

5. Single-Domain Antibodies

[0204] In some embodiments, an antibody of the present disclosure is a single-domain antibody. A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl). In one embodiment, a single-domain antibody consists of all or a portion of the heavy chain variable domain of an antibody.

6. Antibody Variants

[0205] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.

[0206] A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

[0207] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody. [0208] In some embodiments, monoclonal antibodies have a C-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C- terminus of heavy chain and/or light chain. In some embodiments, the C-terminal cleavage removes a C-terminal lysine from the heavy chain. In some embodiments, monoclonal antibodies have an N-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of heavy chain and/or light chain. In some embodiments, truncated forms of monoclonal antibodies can be made by recombinant techniques.

[0209] In certain embodiments, an antibody of the present disclosure is altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0210] Addition or deletion of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) is created or removed. The alteration may also be made by the addition, deletion, or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0211] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in US Pat Appl No US 2003/0157108 (Presta, L.). See also US 2004/0093621 (KyowaHakko Kogyo Co., Ltd). Antibodies with a bisecting N- acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003/011878, Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody are reported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US 2005/0123546 (Umana et al.) on antigen-binding molecules with modified glycosylation.

[0212] In certain embodiments, a glycosylation variant comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues). Examples of publications related to “defucosylated” or “fucose-deficient” antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; Okazaki et al. J. Mol. Biol. 336:1239- 1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane- Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and cells overexpressing [31,4-N- acetylglycosminyltransferase III (GnT-III) and Golgi p-mannosidase II (Manll).

[0213] Antibodies are contemplated herein that have reduced fucose relative to the amount of fucose on the same antibody produced in a wild-type CHO cell. For example, the antibody has a lower amount of fucose than it would otherwise have if produced by native CHO cells (e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In certain embodiments, an anti-Siglec-8 antibody provided herein is one wherein less than about 50%, 40%, 30%, 20%, 10%, 5% or 1% of the N-linked glycans thereon comprise fucose. In certain embodiments, an anti-Siglec-8 antibody provided herein is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely without fucose, or has no fucose or is non-fucosylated or is afucosylated. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. In some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated.

[0214] In one embodiment, the antibody is altered to improve its serum half-life. To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (US 2003/0190311, U.S. Pat. No. 6,821,505; U.S. Pat. No. 6,165,745; U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,648,260; U.S. Pat. No. 6,165,745; U.S. Pat. No. 5,834,597).

[0215] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 5 under the heading of “preferred substitutions.” If such substitutions result in a desirable change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table 5, or as further described below in reference to amino acid classes, may be introduced and the products screened.

Table 5.

[0216] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or c) the bulk of the side chain. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

[0217] Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0218] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites.

[0219] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have modified (e.g., improved) biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least part of a phage coat protein (e.g., the gene III product of M13) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). In order to identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigenantibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to techniques known in the art, including those elaborated herein. Once such variants are generated, the panel of variants is subjected to screening using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0220] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody. [0221] It may be desirable to introduce one or more amino acid modifications in an Fc region of antibodies of the present disclosure, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions including that of a hinge cysteine. In some embodiments, the Fc region variant comprises a human IgG4 Fc region. In a further embodiment, the human IgG4 Fc region comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat.

[0222] In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody of the present disclosure may comprise one or more alterations as compared to the wild type counterpart antibody, e.g. in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (i.e. , either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351 concerning other examples ofFc region variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or diminished binding to FcRs. The content of these patent publications are specifically incorporated herein by reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591- 6604 (2001). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in U.S. Pat. No. 6,194,551B1, WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

7. Vectors. Host Cells, and Recombinant Methods

[0223] For recombinant production of an antibody of the present disclosure, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

Generating Antibodies Using Prokaryotic Host Cells: a) Vector Construction

[0224] Polynucleotide sequences encoding polypeptide components of the antibody of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

[0225] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genesencoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.

[0226] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as ZGEM.TM.- I I may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392. [0227] The expression vector of the present disclosure may comprise two or more promoter- cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.

[0228] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present disclosure. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

[0229] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the [3- galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.

[0230] In one aspect of the present disclosure, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of the present disclosure should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the present disclosure, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0231] In another aspect, the production of the immunoglobulins according to the present disclosure can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coli trxB-strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0232] Antibodies of the present disclosure can also be produced by using an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the present disclosure. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.

[0233] One technique for modulating translational strength is disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which can alter the amino acid sequence. In certain embodiments, changes in the nucleotide sequence are silent. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgamo sequences, along with alterations in the signal sequence. One method for generating mutant signal sequences is the generation of a “codon bank” at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i. e. , the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0234] In one embodiment, a set of vectors is generated with a range of TIR strengths for each cistron therein. This limited set provides a comparison of expression levels of each chain as well as the yield of the desired antibody products under various TIR strength combinations. TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the present disclosure. [0235] Prokaryotic host cells suitable for expressing antibodies of the present disclosure include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the present disclosure. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli/. 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture. b) Antibody Production [0236] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0237] Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs polyethylene glycol/DMSO. Yet another technique used is electroporation. [0238] Prokaryotic cells used to produce the polypeptides of the present disclosure are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.

[0239] Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thiogly collate, dithioerythritol and dithiothreitol.

[0240] The prokaryotic host cells are cultured at suitable temperatures. In certain embodiments, for E. coli growth, growth temperatures range from about 20° C. to about 39° C.; from about 25° C. to about 37° C.; or about 30° C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. In certain embodiments, for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

[0241] If an inducible promoter is used in the expression vector of the present disclosure, protein expression is induced under conditions suitable for the activation of the promoter. In one aspect of the present disclosure, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. In certain embodiments, the phosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety of other inducers may be used, according to the vector construct employed, as is known in the art. [0242] In one embodiment, the expressed polypeptides of the present disclosure are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0243] In one aspect of the present disclosure, antibody production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, and in certain embodiments, about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose. Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.

[0244] In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.

[0245] To improve the production yield and quality of the polypeptides of the present disclosure, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

[0246] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present disclosure. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0247] In one embodiment, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the present disclosure. c) Antibody Purification

[0248] In one embodiment, the antibody protein produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cationexchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0249] In one aspect, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody products of the present disclosure. Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein A is immobilized can be a column comprising a glass or silica surface, or a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent, such as glycerol, to possibly prevent nonspecific adherence of contaminants.

[0250] As the first step of purification, a preparation derived from the cell culture as described above can be applied onto a Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase would then be washed to remove contaminants non-specifically bound to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

[0251] A vector for use in a eukaryotic host cell generally includes one or more of the following non-limiting components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. a) Signal Sequence Component

[0252] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such a precursor region is ligated in reading frame to DNA encoding the antibody. b) Origin of Replication

[0253] Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter. c) Selection Gene Component

[0254] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media. [0255] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0256] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0257] For example, in some embodiments, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, an appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0258] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199. Host cells may include NSO, CHOK1, CHOK1SV or derivatives, including cell lines deficient in glutamine synthetase (GS). Methods for the use of GS as a selectable marker for mammalian cells are described in U.S. Pat. No. 5,122,464 and U.S. Pat. No. 5,891,693. d) Promoter Component

[0259] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding a polypeptide of interest (e.g., an antibody). Promoter sequences are known for eukaryotes. For example, virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into eukaryotic expression vectors.

[0260] Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heatshock promoters, provided such promoters are compatible with the host cell systems.

[0261] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describing expression of human [3-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. e) Enhancer Element Component

[0262] Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) describing enhancer elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide-encoding sequence, but is generally located at a site 5' from the promoter. f) Transcription Termination Component

[0263] Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein. g) Selection and Transformation of Host Cells

[0264] Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; CHOK1 cells, CHOK1SV cells or derivatives and a human hepatoma line (Hep G2). [0265] Host cells are transformed with the above-described-expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. h) Culturing the Host Cells

[0266] The host cells used to produce an antibody of the present disclosure may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. i) Purification of Antibody

[0267] When using recombinant techniques, the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems may be first concentrated using a commercially available protein concentration filter, for example, an Ami con or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants. [0268] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human yl, y2, or y4 heavy chains (Lindmark et al., J. Immunol. Methods 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a poly aspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0269] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to further purification, for example, by low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, performed at low salt concentrations (e.g., from about 0-0.25M salt).

[0270] In general, various methodologies for preparing antibodies for use in research, testing, and clinical use are well-established in the art, consistent with the above-described methodologies and/or as deemed appropriate by one skilled in the art for a particular antibody of interest.

Production of non-fucosylated antibodies

[0271] Provided herein are methods for preparing antibodies with a reduced degree of fucosy lati on. For example, methods contemplated herein include, but are not limited to, use of cell lines deficient in protein fucosylation (e.g., Lecl3 CHO cells, alpha- 1,6-fucosyltransferase gene knockout CHO cells, cells overexpressing pi,4-N-acetylglycosminyltransferase III and further overexpressing Golgi p-mannosidase II, etc.), and addition of a fucose analog(s) in a cell culture medium used for the production of the antibodies. See Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; WO 2004/056312 Al; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); and US Pat. No. 8,574,907. Additional techniques for reducing the fucose content of antibodies include Glymaxx technology described in U.S. Patent Application Publication No. 2012/0214975. Additional techniques for reducing the fucose content of antibodies also include the addition of one or more glycosidase inhibitors in a cell culture medium used for the production of the antibodies. Glycosidase inhibitors include a-glucosidase I, a-glucosidase II, and a-mannosidase I. In some embodiments, the glycosidase inhibitor is an inhibitor of a-mannosidase I (e.g., kifunensine). [0272] As used herein, “core fucosylation” refers to addition of fucose (“fucosylation”) to N- acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan. Also provided are antibodies produced by such methods and compositions thereof.

[0273] In some embodiments, fucosylation of complex N-gly coside-linked sugar chains bound to the Fc region (or domain) is reduced. As used herein, a “complex N-gly coside-linked sugar chain” is typically bound to asparagine 297 (according to the number of Kabat), although a complex N-gly coside linked sugar chain can also be linked to other asparagine residues. A “complex N-gly coside-linked sugar chain” excludes a high mannose type of sugar chain, in which only mannose is incorporated at the non-reducing terminal of the core structure, but includes 1) a complex type, in which the non-reducing terminal side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally has a sialic acid, bisecting N- acetylglucosamine or the like; or 2) a hybrid type, in which the non-reducing terminal side of the core structure has both branches of the high mannose N-gly coside-linked sugar chain and complex N-gly coside-linked sugar chain.

[0274] In some embodiments, the “complex N-gly coside-linked sugar chain” includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the nonreducing terminal side of Gal-GlcNAc optionally further has a structure such as a sialic acid, bisecting N-acetylglucosamine or the like.

[0275] According to the present methods, typically only a minor amount of fucose is incorporated into the complex N-gly coside-linked sugar chain(s). For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the antibody has core fucosylation by fucose in a composition. In some embodiments, substantially none (i.e., less than about 0.5%) of the antibody has core fucosylation by fucose in a composition. In some embodiments, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% of the antibody is nonfucosylated in a composition.

[0276] In some embodiments, provided herein is an antibody wherein substantially none (i.e., less than about 0.5%) of the N-gly coside-linked carbohydrate chains contain a fucose residue. In some embodiments, provided herein is an antibody wherein at least one or two of the heavy chains of the antibody is non-fucosylated.

[0277] As described above, a variety of mammalian host-expression vector systems can be utilized to express an antibody. In some embodiments, the culture media is not supplemented with fucose. In some embodiments, an effective amount of a fucose analog is added to the culture media. In this context, an “effective amount” refers to an amount of the analog that is sufficient to decrease fucose incorporation into a complex N-gly coside-linked sugar chain of an antibody by at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%. In some embodiments, antibodies produced by the instant methods comprise at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% non-core fucosy lated protein (e.g., lacking core fucosylation), as compared with antibodies produced from the host cells cultured in the absence of a fucose analog.

[0278] The content (e.g., the ratio) of sugar chains in which fucose is not bound to N- acetylglucosamine in the reducing end of the sugar chain versus sugar chains in which fucose is bound to N-acetylglucosamine in the reducing end of the sugar chain can be determined, for example, as described in the Examples. Other methods include hydrazinolysis or enzyme digestion (see, e.g., Biochemical Experimentation Methods 23: Method for Studying Glycoprotein Sugar Chain (Japan Scientific Societies Press), edited by Reiko Takahashi (1989)), fluorescence labeling or radioisotope labeling of the released sugar chain and then separating the labeled sugar chain by chromatography. Also, the compositions of the released sugar chains can be determined by analyzing the chains by the HPAEC-PAD method (see, e.g., J. Liq Chromatogr. 6:1557 (1983)). (See generally U.S. Patent Application Publication No. 2004/0110282.). III. Compositions

[0279] In some aspects, also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the anti-Siglec-8 antibodies described herein (e.g., an antibody that binds to Siglec-8). In some aspects, provided herein is a composition comprising an anti- Siglec-8 antibody described herein, wherein the antibody comprises a Fc region and N- gly coside-linked carbohydrate chains linked to the Fc region, wherein less than about 50% of the N-gly coside-linked carbohydrate chains contain a fucose residue. In some embodiments, the antibody comprises a Fc region and N-gly coside-linked carbohydrate chains linked to the Fc region, wherein less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or about 15% of the N-gly coside-linked carbohydrate chains contain a fucose residue. In some aspects, provided herein is a composition comprising an anti-Siglec-8 antibody described herein, wherein the antibody comprises a Fc region and N-gly coside-linked carbohydrate chains linked to the Fc region, wherein substantially none of the N-gly coside-linked carbohydrate chains contain a fucose residue.

[0280] Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.

[0281] Buffers can be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers can be present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may be comprised of histidine and trimethylamine salts such as Tris.

[0282] Preservatives can be added to prevent microbial growth, and are typically present in a range from about 0.2%-1.0% (w/v). Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0283] Tonicity agents, sometimes known as “stabilizers” can be present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intramolecular interactions. Tonicity agents can be present in any amount between about 0.1% to about 25% by weight or between about 1 to about 5% by weight, taking into account the relative amounts of the other ingredients. In some embodiments, tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0284] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0285] Non-ionic surfactants or detergents (also known as “wetting agents”) can be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml or about 0.07 mg/ml to about 0.2 mg/ml. In some embodiments, non-ionic surfactants are present in a range of about 0.001% to about 0.1% w/v or about 0.01% to about 0.1% w/v or about 0.01% to about 0.025% w/v.

[0286] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poly oxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0287] In order for the formulations to be used for in vivo administration, they must be sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0288] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means. In some embodiments, a composition or anti-Siglec-8 antibody of the present disclosure is administered by intravenous infusion once a month for 3 or more months. In some embodiments, a composition or anti-Siglec-8 antibody of the present disclosure is administered by intravenous infusion once per cycle (e.g., on Day 1) for 1, 2, 3, 4, 5, or 6 cycles, wherein each cycle is 1 month, 4 weeks, or 28 days.

[0289] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active compounds are suitably present in combination in amounts that are effective for the purpose intended.

IV. Articles of Manufacture or Kits

[0290] In another aspect, an article of manufacture or kit is provided which comprises an anti- Siglec-8 antibody described herein (e.g., an antibody that binds human Siglec-8). The article of manufacture or kit may further comprise instructions for use of the antibody in the methods of the present disclosure. Thus, in certain embodiments, the article of manufacture or kit comprises instructions for the use of an anti-Siglec-8 antibody that binds to human Siglec-8 in methods for treating and/or preventing viral infection, virus-induced inflammation, and/or virus-induced mast cell/ eosinophil activation in an individual comprising administering to the individual an effective amount of an anti-Siglec-8 antibody that binds to human Siglec-8. In certain embodiments, the article of manufacture comprises a medicament comprising an antibody that binds to human Siglec-8 and a package insert comprising instructions for administration of the medicament in an individual in need thereof to treat and/or prevent viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation. In some embodiments, the package insert further indicates that the treatment is effective in reducing one or more symptoms in the individual with viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation as compared to a baseline level before administration of the medicament. In some embodiments, the individual is diagnosed with viral infection, virus- induced inflammation, and/or virus-induced mast cell/eosinophil activation before administration of the medicament comprising the antibody. In certain embodiments, the individual is a human.

[0291] The article of manufacture or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as single or dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation.

[0292] The article of manufacture or kit may further comprise a label or a package insert, which is on or associated with the container, may indicate directions for reconstitution and/or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other modes of administration for treating and/or preventing viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation in an individual. The container holding the formulation may be a single-use vial or a multi-use vial, which allows for repeat administrations of the reconstituted formulation. The article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. [0293] In a specific embodiment, the present disclosure provides kits for a single doseadministration unit. Such kits comprise a container of an aqueous formulation of therapeutic antibody, including both single or multi-chambered pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0294] In another embodiment, provided herein is an article of manufacture or kit comprising the formulations described herein for administration in an auto-injector device. An auto-injector can be described as an injection device that upon activation, will deliver its contents without additional necessary action from the patient or administrator. They are particularly suited for self-medication of therapeutic formulations when the delivery rate must be constant and the time of delivery is greater than a few moments.

[0295] In another aspect, an article of manufacture or kit is provided which comprises an anti- Siglec-8 antibody described herein (e.g., an antibody that binds human Siglec-8). The article of manufacture or kit may further comprise instructions for use of the antibody in the methods of the present disclosure. Thus, in certain embodiments, the article of manufacture or kit comprises instructions for the use of an anti-Siglec-8 antibody that binds to human Siglec-8 in methods for treating or preventing viral infection, virus-induced inflammation, and/or virus-induced mast cell/ eosinophil activation in an individual comprising administering to the individual an effective amount of an anti-Siglec-8 antibody that binds to human Siglec-8. In certain embodiments, the article of manufacture or kit comprises a medicament comprising an antibody that binds to human Siglec-8 and a package insert comprising instructions for administration of the medicament in an individual in need thereof to treat and/or prevent viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation.

[0296] The present disclosure also provides an article of manufacture or kit which comprises an anti-Siglec-8 antibody described herein (e.g., an antibody that binds human Siglec-8) in combination with one or more additional medicament (e.g., a second medicament) for treating or preventing viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation in an individual. The article of manufacture or kit may further comprise instructions for use of the antibody in combination with one or more additional medicament in the methods of the present disclosure. For example, the article of manufacture or kit herein optionally further comprises a container comprising a second medicament, wherein the anti-Siglec-8 antibody is a first medicament, and which article or kit further comprises instructions on the label or package insert for treating the individual with the second medicament, in an effective amount. Thus in certain embodiments, the article of manufacture or kit comprises instructions for the use of an anti-Siglec-8 antibody that binds to human Siglec-8 in combination with one or more additional medicament in methods for treating or preventing viral infection, virus-induced inflammation, and/or virus-induced mast cell/eosinophil activation in an individual. In certain embodiments, the article of manufacture or kit comprises a medicament comprising an antibody that binds to human Siglec-8 (e.g., a first medicament), one or more additional medicament and a package insert comprising instructions for administration of the first medicament in combination with the one or more additional medicament (e.g., a second medicament). Exemplary additional medicaments contemplated for use with an antibody that binds to human Siglec-8 are provided supra.

[0297] It is understood that the aspects and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLES

[0298] The present disclosure will be more fully understood by reference to the following examples. The examples should not, however, be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: Aberrant mast cell activation is associated with COVID-19 and TLR-mediated viral inflammation

[0299] Innate immune sensing serves as the first line of antiviral defense and is initiated by the recognition of conserved pathogen-associated molecular patterns by pattern recognition receptors (PRRs). In the case of RNA viruses such as SARS-CoV-2, the Toll-like receptors (TLRs) 3, 7, and 8 and cytosolic PPRs MDA-5 and RIG-1, specifically recognize singlestranded RNA (ssRNA) and double-stranded RNA (dsRNA). Upon PPR activation by viruses, downstream signaling cascades trigger the secretion of pro-inflammatory cytokines and type I/III interferons (IFNs), which can either limit viral infection or contribute to pathological inflammation. Indeed, activation of immune cells via PPRs has been postulated to drive the release of pro-inflammatory cytokines seen in severe COVID-19 patients. Mast cells (MCs) are tissue resident immune cells that constitute a major sensory arm of the innate immune system. They are crucially located at sites that interface with the external environment, such as the skin, lungs, and gastrointestinal tract, allowing them to be among the first cells to respond during pathogen invasion. MCs are equipped with TLRs and receptors for inflammatory mediators, such as cytokines and damage associated molecular patterns (DAMPs), allowing them to act as sentinels for tissue damage and pathogens. Upon activation, MCs release pre-formed granules containing inflammatory mediators, vasoactive molecules, and MC-specific proteases, including P-tryptase, chymase, and carboxypeptidase (CPA)-3. In addition, MC activation leads to de novo produced cytokines and lipid mediators, many of which are associated with the cytokine storm seen in COVID-19. Consequently, these MC products lead to tissue edema, vascular leakage, barrier disruption, and recruitment of immune cells, such as neutrophils and monocytes. [0300] MC responses to viral pathogens have not been extensively studied, however recent evidence suggests activation of MCs during viral infection can be detrimental, leading to excessive inflammation and pathology. Many viruses have been shown to directly induce MC degranulation, protease release, and cytokine production, including dengue (DENV), Japanese encephalitis (JEV), Zika, and influenza. For example, tryptase and chymase are elevated in plasma from patients with severe DENV infection and these MC proteases were shown to induce significant vascular leakage in peripheral tissues in response to the infection. In addition, JEV induces chymase release from MCs and increases brain endothelial barrier permeability via cleavage of tight junction molecules, leading to increased central nervous system infection and worsened morbidity and mortality that was shown to be dependent on MC-derived mediators. While the exact mechanism of viral pathogen-mediated MC activation has not been elucidated, viral detection by TLR-3/7/8 and RIG-I/MAVS appear to be central for production of MC mediators following viral infection.

[0301] Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on MCs and eosinophils that inhibits MC activation and induces eosinophil depletion when engaged with a monoclonal antibody (mAb). Siglec-8 mAbs have been shown to

-Il l- suppress immune cell infiltration, local and systemic inflammation, protease production, fibrosis, and anaphylaxis in both IgE-dependent and independent disease models.

[0302] Given the pathogenic role of MCs and eosinophils in many allergic and non-allergic inflammatory diseases and the putative role for them in COVID- 19 pathogenesis, MC and eosinophil activation was evaluated in SARS-CoV-2 patients, and the relevance of TLR- stimulation and Siglec-8 mAb-inhibition in COVID- 19 inflammation determined. This Example shows that MC-derived proteases and eosinophil-associated proteins are significantly elevated in SARS-CoV-2 patient serum and COVID- 19 autopsy lungs and correlate with pro-inflammatory cytokines associated with hyperinflammation. TLR stimulation of human MCs and eosinophils with the synthetic viral RNA mimetics, poly (EC) and R848 induced activation that resembled the mediator profiles seen in COVID-19 patients. Moreover, intratracheal poly (EC) administration reproduced many features of SARS-CoV-2 inflammation, including cytokine elevation, immune cell airway infiltration, and induction of MC proteases and eosinophil granule proteins. Treatment with a Siglec-8 mAh significantly suppressed poly (EC)-induced local and systemic inflammation and levels of MC and eosinophil -derived proteins.

[0303] These data provide evidence that MC and eosinophil activation are components of SARS-CoV-2 inflammation and demonstrate that targeting Siglec-8 with a monoclonal antibody (mAb) suppresses TLR-mediated inflammation, supporting further clinical investigation of Siglec-8 antibodies as a therapeutic approach for attenuating excessive inflammation during viral infections.

Materials and Methods

Mice

[0304] Siglec-8 transgenic (tg) mice were generated as previously described (Youngblood, B.A. et al. (2019) JCI Insight 4: 126219) and were used at 6-8 weeks of age. All animal experiments were done under an lACUC-approved protocol. For all studies, animals were formally randomized, technicians were blinded to treatment groups, and quantitative measurements were done without the opportunity for bias.

Flow cytometry

[0305] Approximately 1-5* 10⁶ cells were preincubated with CD 16/32 antibody to block nonspecific binding. Cells were then incubated at 4°C for 10 minutes with staining antibody panels, washed, and fixed in 2% paraformaldehyde. Data acquisition was performed using a NovoCyte flow cytometer (Acea Biosciences) and FlowJo (San Diego, CA) was used for data analysis. The following antibodies were purchased from eBioscience or Biolegend (clone indicated in brackets): CD3-BV650 (**), CD8-APC (53-6.7), CDllb-BV605 (MI/70), CD19- BV421 (), CD45-BV785 (30-F11), CD117-SB436 (2B8), CD206 PECy7 (**), F4/80-FITC (BM8), F4/80-PECy7 (BM8), Ly6G-BV510 (1A8), Ly6C-APCCy7 (HK1.4), MHCII-(IA/E)- AF488, SiglecF-PE (S17007L), FcERl-FITC (MAR-1), and Siglec-8 AF647 (1H10 clone, Allakos, Inc.). Gating strategy for cells from mouse tissues was as follows: mast cells: 7AAD' CD45⁺ IgER⁺CD117⁺; Eosinophils: 7AAD CD45⁺ SSC^Hi Siglec-F⁺; Neutrophils: 7AAD CD45⁺ Ly6C" Ly6G⁺; Monocytes: 7AAD CD45⁺ Ly6G'Ly6C^hl, interstitial Macrophages 7AAD CD45⁺ F4/80^hiSiglecF'CDl lb⁺MHCII⁺.

Intratracheal poly (I: C) model of inflammation

[0306] Siglec-8 tg mice were injected with an anti-Siglec-8 mlgGl mAB (2E2 clone) or Isotype-matched control mlgGl mAh (biolegend) intraperitoneally at 5mg/kg three hours before poly(EC) challenge (Invivogen, San Diego, CA, USA). On days 0 and 1, the mice were anesthetized by isoflurane inhalation and 50ul of PBS or poly(EC) (Img/ml) was administered intratracheally. On day 2, serum, bronchoalveolar lavage (BAL) fluid, lungs and peripheral blood were isolated and assessed for immune phenotyping, cytokine and mast cell protease quantification.

Measurement of inflammatory mediators

[0307] Cytokines in BALF and serum of Poly (EC) treated mice were measured using the multiplex Meso Scale Diagnostics (MSD) assay. Analytes included in the analysis measured were CCL2, IL-6, MIP2, IL-10.

MCPT-4 quantification

[0308] mMCPT4 levels were quantified in the lungs of Poly (I: C) challenged or sham mice by harvesting the lungs on day 2, followed by overnight culture in RPMI and collection of supernatants. Serum was also collected on day 2. Supernatants and serum were analyzed for mMCPT4 (LSBio, LS-F55860-1) by ELISA and according to the manufacturer’s instructions. Human peripheral blood derived MCs

[0309] Peripheral blood cells were isolated from residual cells in the leukocyte reduction chamber (TrimaAccel). Cells were eluted by gravity, and RBCs were lysed using IX lysis buffer (BioLegend). CD34+ progenitor cells were isolated using the CD34 MicroBead Kit UltraPure human kit (Miltenyi Biotec) and cultured as previously described (Saito, H et al. 2006). After 7 weeks in culture, cells were maintained IMDM supplemented with 5% FBS, 55 pM P- Mercaptoethanol, lOOng/mL SCF, and 50ng/mL IL-6.

In Vitro stimulation of Human peripheral blood derived MCs and cytokine/chemokine analysis

[0310] MCs were cultured in IMDM supplemented with 5% FBS and stimulated with lOug/ml of PolyLC or R848 overnight. Levels of cytokines and chemokines in supernatant were measured by meso-scale discovery (MSD).

Human Sera Samples

[0311] Sera from healthy controls (n=9) and SARS-CoV-2 patients (n=10) was obtained from Discovery Life Sciences Biobank (San Luis Obispo, CA, USA). The SARS-CoV-2 status of these donors was determined using the Abbott RT-PCR nasopharyngeal swab test. Serum cytokine levels were quantified using multiplex analysis (MSD). The cytokines measured were CCL2, IP- 10, IL-8, and VEGF.

[0312] Mast cell activation was quantified using serum surrogate markers such as tryptase activity (IMM001, Sigma- Aldrich), carboxypeptidase A3 levels (CP A3, LS-F7363, LS Bio), P- tryptase (ELH-TPSB2-1, Ray Biotech), or chymase levels (50-149-8059, Biomatik).

Statistical analysis

[0313] To determine statistical significance, nonparametric Mann-Whitney U test, unpaired 2- tailed t test, 2-tailed t test with Holm-Sidak’s posttest, or 1-way ANOVA with Tukey’s posttest for multiple comparisons was performed using Prism (GraphPad Software). A -value of 0.05 or less was considered significant. RNA-sequencing data processing and analysis

[0314] Raw RNA-seq data was obtained from three studies through the NCBI Sequence Read Archive: Blanco-Melo (GSE147507), GSE151803, and GSE150316. Reads were aligned to GRCh38 with STAR v2.6.1a (Dobin et al. https://doi.org/10.1093/bioinformatics/bts635) and multiple runs from the same sample were merged. PCR & optical duplicates were marked using Picard Tools v2.20.4 (Broad Institute). Gene counts were summarized using featureCounts from the subread package vl.6.3 (Liao et al. https://doi.org/10.1093/bioinformatics/btt656) and batch effects resulting from study were removed using ComBat-Seq. DESeq2 vl.22.2 was utilized to obtain differentially expressed genes (p.adj < 0.1) and a variance-stabilized matrix for visualization of individual samples. Heatmaps were generated using ComplexHeatmap v2.5.4 and data were restricted to a maximum absolute log2fold change of 5 for visualization.

Results

MC-specific proteases are significantly elevated in SARS-CoV-2 patient serum [0315] Previous studies have demonstrated that activation of MCs and subsequent protease release contributes to virus-induced inflammation and pathology, including vascular leak, excessive airway inflammation, barrier disruption, and fibrosis. As such, many reports have implicated MCs as putative effector cells in COVID- 19 pathogenesis.

[0316] To evaluate if systemic MC activation was associated with SARS-CoV-2 inflammation, serum of SARS-CoV-2 positive patients and healthy controls was tested for pro- inflammatory mediators and the MC-specific proteases, chymase, |3-tryptase, and CPA3. Consistent with previous reports, serum from SARS-CoV-2 patients had significantly higher levels of pro-inflammatory mediators compared to healthy controls, including CCL2, IP-10, IL-8 and VEGF (FIG. 1A). Significantly elevated levels of chymase, |3-tryptase, and CP A3 were also found in SARS-CoV-2 patient serum, strongly suggesting systemic and aberrant MC activation (FIG. IB). Consistent with increased MC activation, SARS-CoV-2 patient serum also had significantly increased levels catalytically active, mature tryptase compared to serum from healthy donors (FIG. 1C).

[0317] To gain additional insight into the association between inflammation and MC activation in SARS-CoV-2 patients, pro-inflammatory cytokines and chemokines with MC- derived proteases were correlated. Protease levels significantly correlated with many pro- inflammatory cytokines associated with COVID-19 disease severity, including IP-10, CCL2, and IL-8 (FIGS. 1D-1E). These data demonstrate that MC-specific proteases are elevated in SARS- CoV-2 patients and suggest MC activation could contribute to COVID-19 pathogenesis.

MC protease and eosinophil granule genes are increased in CO VID- 19 patient lungs [0318] Following the characterization of MC-specific proteases in SARS-CoV-2 patient serum, these mediators were next evaluated in lungs obtained from post-mortem COVID-19 patients compared to healthy controls using publicly available bulk RNA-seq datasets. Due to the small number of patients within each published study, three different bulk RNA-seq datasets were leveraged to generate a combined dataset with 10 COVID-19 patient lungs and 3 healthy control lungs to increase confidence in the differential gene expression analyses.

[0319] As expected, many inflammatory cytokines and chemokines were increased in lungs COVID-19 patients, including CCL2, CCL4, IL-8, and IP-10. Consistent with the serum findings from SARS-CoV-2 patients, the MC protease genes TPSB2 and TPSAB1 were significantly elevated in lungs from COVID-19 patients compared to healthy control lungs (FIG. 2). In addition, FCER1G, the gamma chain of the IgE receptor and other Fc receptors was significantly increased. In contrast, other MC-specific genes, such as HDC and KIT were not increased in COVID- 19 lung tissue, suggesting COVID- 19 inflammation is associated with increased activation of MCs and not elevated cell number. These data corroborate the findings in SARS-CoV-2 patient serum and further implicate aberrant MC activation in COVID-19 disease pathogenesis.

[0320] Eosinopenia has been a common finding in the peripheral blood of many COVID-19 patients, however very few studies have examined eosinophil numbers or associated mediators in diseased tissue. Strikingly, several eosinophil-associated granule genes were found to be significantly upregulated in COVID-19 patient lung tissue, including CLC (Galectin-10) and RNASE2 (EDN) (FIG. 2). To evaluate if eosinophil granule mediators were also elevated in serum, EDN was measured in SARS-CoV-2 patient and healthy donor serum. Consistent with the RNA-seq data, EDN was significantly elevated in SARS-CoV-2 patient serum (FIG. 6C). Collectively, these data show that MC-derived proteases and eosinophil-associated mediators are increased in COVID- 19 lungs and suggest elevated eosinophil activity is a component of COVID-19 inflammation. TLR stimulation ofMCs and eosinophil with synthetic viral RNA induces activation that resembles SARS-CoV-2 inflammation

[0321] The above data strongly suggested systemic and local MC activation and to a lesser extent, eosinophil activation, were associated with COVID- 19 inflammation. Potential mechanisms of MC and eosinophil activation during SARS-CoV-2 inflammation were investigated.

[0322] Purified human MCs and eosinophils had minimal expression of the cell entry receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), relative to Calu-3 cells, suggesting ACE2-independent activation (FIG. 6A). To determine if the engagement of TLRs by viral pathogens could mediated MC and eosinophil activation, these cells were stimulated with the synthetic analogs of ssRNA and dsRNA, R848 and poly (EC), respectively. Both R848 and poly (IC) induced significant MC activation as evidenced by increased cytokine production, including IL-8, CCL3, and CCL4 (FIGS. 3A & 3B). R848 also induced significant induction of IL-8 and CCL4 in eosinophils (FIG. 3A). In addition, R848 and poly (IC) induced production of chymase in the absence of MC degranulation (FIG. 3B). In contrast to MCs, R848, but not poly (EC), induced eosinophil activation as evidenced by increased expression of CD69 and cytokine production (FIGS. 3A & 6B).

[0323] These data demonstrate that TLR stimulation with synthetic analogs of viral RNA induce MC and eosinophil activation that resembles the cell-specific mediator profiles seen in SARS-CoV-2 serum and COVID- 19 lung samples. Moreover, these data suggest a potential mechanism of innate immune stimulation by SARS-CoV-2 independent of ACE2.

Siglec-8 mAb treatment suppresses TLR-driven inflammation induced by poly (I: C) [0324] Given that MC and eosinophil activation were observed in COVID- 19 patients and reproduced in vitro via TLR stimulation, the role of MCs and eosinophils was examined in a TLR-mediated viral inflammation model where the anti-inflammatory activity of a Siglec-8 mAh (anti-S8) could be evaluated. To this end, poly (EC) was intratracheally instilled to stimulate TLR3 and RIG-I-MDA5 pathways in vivo using Siglec-8 transgenic mice which express functional Siglec-8 on mouse MCs and eosinophils (FIG. 4A). Administration of poly (EC) in vivo has been shown to reproduce many features of SARS-CoV-2 inflammation and mimic acute respiratory exacerbations triggered by viral infections. [0325] Intratracheal administration of poly (LC) induced robust and significant airway inflammation as evidenced by infiltration of immune cells into the BAL fluid, including total leukocytes, neutrophils, monocytes, and macrophages compared to vehicle control (Figure 4B- C). In addition, intratracheal poly (I:C) increased blood monocytes and neutrophils (Figure 4D). One dose of anti-S8 significantly suppressed TLR-mediated infiltration of immune cells into the airway and periphery (Figure 4B-D).

[0326] Consistent with the robust immune cell infiltration into the airway, poly (I:C) administration increased cytokines and chemokines in the BAL fluid, including CCL2, IP- 10, MIP-2, and IL-lb (Figure 4E). Likewise, CCL2, IP-10, IL-6, and TNF were significantly increased in the serum of mice administered poly (LC) (Figure 4F). Treatment with anti-S8 significantly abrogated local and systemic cytokine and chemokine elevation induced by poly (LC) (Figure 4E and F).

[0327] These data suggest that intratracheal poly (LC) administration reproduces features of SARS-CoV-2 inflammation, including dysregulated secretion of pro-inflammatory cytokines and immune cell infiltration that is suppressed with a Siglec-8 mAh.

Poly (LC)-driven inflammation is associated with aberrant MC and eosinophil activation that is suppressed with a Siglec-8 mAb

[0328] Next, it was assessed whether the decrease in poly (EC)-mediated inflammation in mice treated with anti-S8 was associated with reduced MC and eosinophil activity. Interestingly, poly (I:C) administration significantly reduced eosinophil numbers in the BAL fluid and peripheral blood compared to vehicle control mice as determined by flow cytometry (Figure 5 A). In contrast, eosinophil peroxidase (EPX) levels in the BAL fluid and serum were significantly increased in mice that received poly (LC), suggesting TLR-driven inflammation induces activation and subsequent cytolysis of eosinophils (Figure 5B). Consistent with the known eosinophil depleting activity, anti-S8-treated mice had significantly reduced BAL fluid and blood eosinophils and decreased levels of EPX compared to isotype control mAb-treated mice administered poly (LC) (Figure 5A and B).

[0329] To evaluate MC activation, levels of the MC-specific mediator, mast cell protease-4 (MCPT4), were quantified in ex vivo cultured lung tissue and serum in poly (LC)-driven inflammation. Among the murine chymases, MCPT4 is most likely the functional counterpart of human chymase because it has similar substrate specificity and proteoglycan-binding properties. Intratracheal poly (I: C) administration significantly increased MCPT4 levels in the lung and serum, indicative of MC activation (Figure 5C).

[0330] Anti-Siglec-8 treatment significantly reduced lung and serum MCPT4 levels compared to isotype control mAb-treated mice, consistent with MC inhibition (Figure 5C). Lastly, to determine if MC protease levels associated with pro-inflammatory cytokines in our poly (I:C) inflammation model, serum MCPT4 levels were correlated with CCL2, IP- 10, and IL-6. As was seen in human serum, MC protease levels significantly correlated with many pro-inflammatory cytokines associated with poly (I:C) inflammation (Figure 5D).

[0331] These data demonstrate that TLR-mediated inflammation driven by synthetic viral RNA induces MC and eosinophil activation that resembles COVID- 19 inflammation. Moreover, anti-Siglec-8 treatment significantly reduced aberrant MC and eosinophil activation and hyperinflammation, suggesting that engagement of Siglec-8 with a mAh could serve as a potential therapeutic option in severe COVID- 19 patients.

Example 2: Anti-Siglec-8 treatment in a mouse model of RSV infection

[0332] This Example describes testing the effect of anti-Siglec-8 antibody treatment in a mouse model of RSV infection.

[0333] A diagram of the study is provided in FIG. 7A. Siglec-8 transgenic mice (as described in Example 1) were dosed with murine anti-Siglec-8 antibody m2E2 (as described in, e.g., W02015089117) or isotype control, then administered RSV-A2 (IxlO⁶ PFU) intranasally 4 hours after administration of the antibody. On day 7, mice were sacrificed for measurements of lung inflammation.

[0334] Body weight was measured during RSV infection. As shown in FIG. 7B, mice infected with RSV and treated with anti-Siglec-8 mAh displayed significantly less weight loss compared to isotype control mice infected with RSV.

[0335] Lung inflammation was also measured at day 7 of RSV infection. As shown in FIGS. 7C-7F, mice infected with RSV and treated with anti-Siglec-8 mAh displayed less immune cell infiltration in the BAL fluid compared to isotype control mice infected with RSV.

[0336] These results demonstrate that anti-Siglec-8 antibody treatment was able to reduce weight loss and lung inflammation (as assessed by immune cell infiltration) in RSV-infected mice. Example 3: Mast cell activation is associated with post-acute COVID- 19 syndrome

[0337] Up to a quarter of COVID-19 patients experience persistent morbidity including chronic fatigue, brain fog, body aches, and loss of smell, often lasting for months following acute infection. The etiology of this post-acute sequelae of COVID- 19 syndrome (PASC) remains poorly understood.

[0338] As of July 2021, over 30 million Americans have recovered from documented COVID- 19 infection, and prevalence studies suggest up to twice as many may be undocumented (Demonbreun AR, et al. JCI Insight 2021;6). For survivors of COVID-19, chronic morbid symptoms are common, and include fatigue, brain fog, body aches, and loss of smell (Sudre CH, et al. Nat Med 2021;27:626-631). These persistent symptoms have been referred to as post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, long COVID and Tong-hauler syndrome’. Among patients previously hospitalized for COVID- 19, up to 2/3 had fatigue or muscle weakness after 6 months, while over 10-30% of non-hospitalized patients experience brain fog and fatigue affecting cognition and quality of life compared to controls (Graham EL, et al. Ann Clin Transl Neurol 2021;8:1073-1085; Frontera JA, et al. J Neurol Sci

2021;426: 117486; Blomberg B, et al. Nat Med 2021). Health care costs and utilization attributed to PASC have not been fully described, but lost productivity from work is likely. The etiopathogenesis of PASC remains unclear, but inflammation may play a role (Doykov I, et al. FlOOORes 2020;9:1349), along with metabolic disturbances (Holmes E, et al. J Proteome Res 2021;20:3315-3329) and autoantibodies (Wallukat G, et al. J Transl Autoimmun

2021;4:100100). Notably, there are no known effective treatments for PASC, and with emerging cases and anticipated outbreaks due to the new Delta variant of SARS-CoV-2 likely leading to more patients suffering from PASC, studies to determine mechanisms with potential treatment targets are vital.

[0339] Recently, evidence of mast cell (MC) activation in sera and lung tissue was identified in patients with acute COVID- 19 infection (Gebremeskel S, et al. Front Immunol 2021;12:650331). Sofria-Castro similarly found serum carboxypeptidase A3 (CPA3) levels correlate with C-reactive protein in COVID- 19 patients with lung disease (Soria-Castro R, et al. J Leukoc Biol 2021). Moreover, post-mortem lung biopsies from COVID-19 patients showed increased numbers of mast cells (MCs) (Motta Junior JDS, et al. Front Immunol.

2020;! 1:574862. 2020 ), suggesting MCs are both activated and elevated in acute COVID-19. Whether this activation is persistent and contributes to morbidity associated with PASC is unknown. Notably, many of the common symptoms of PASC occur in mast cell activation syndrome (MCAS, US ICD-110 code D89.42) and indolent systemic mastocytosis (ISM, US ICD-110 code D47.02). Therefore it was hypothesized that PASC is an inflammatory condition associated with MC activation.

[0340] As noted above, evidence of mast cell (MC) activation in patients with acute COVID- 19 was previously found, and it was hypothesized that MCs may contribute to PASC. In this Example, inflammatory mediators and MC-derived proteases were assessed in sera from adults with symptomatic PASC, post-acute asymptomatic COVID-19 (PAAC), acute COVID-19, and healthy controls. Sera from PASC patients displayed significantly elevated levels of IL-6 and CXCL1 compared to PAAC and healthy controls. PASC patient sera also showed evidence of MC activation that differed from PAAC and acute COVID-19 patients, suggesting patients with PASC have a distinct MC activation profile. These data suggest PASC is associated with immune dysregulation and systemic MC activation.

Materials and Methods

Human serum samples

[0341] Sera from PASC (n=13) and PAAC patients (n=13) were obtained from Reprocell (Beltsville, MD, USA). The initial SARS-CoV-2 status of these patients was determined using the Abbott RT-PCR nasopharyngeal swab test. Upon serum collection these patients were negative on SARS-CoV-2 rapid antigen test and self-reported symptoms were collected. Healthy control sera were obtained from Stanford Blood Center (Palo Alto, CA, USA). Acute COVID-19 patient sera were collected as previously described (Gebremeskel S, et al. Front Immunol 2021;12:650331). Serum cytokine and chemokine levels were quantified using multiplex analysis Meso Scale Discovery (MSD). Mast cell activation was quantified by ELISA in serum using MC-derived proteases: carboxypeptidase A3 (CP A3, LS-F7363, LS Bio) or chymase (50- 149-8059, Biomatik). Mast cell tryptase activity (active tryptase) was determined by the Tosyl- Gly-Pro-Lys-pNA-based method (IMM001, Sigma- Aldrich) according to the manufacturer’s instructions.

Results

[0342] To evaluate systemic inflammation and MC activation in PASC, serum was obtained from 13 adults (median age: 29, IQR: 29, 58, 12/13 female) with symptomatic PASC with a history of positive RT-PCR for SARS-COV-2 between 39-301 days prior (median: 60, IQR: 54, 77). Among the PASC patients, 84% reported fatigue, while 46% reported body aches or loss of taste/smell, and 25% reported brain fog. To control for SARS-CoV-2 infection, serum was obtained from aged-matched post-acute asymptomatic COVID-19 (PAAC) patients with a history of positive RT-PCR as well as healthy controls. In addition, serum samples from an acute COVID- 19 patient cohort used in a prior study was re-assessed (Gebremeskel S, et al. Front Immunol 2021;12:650331).

[0343] Initially, specific cytokines and chemokines associated with inflammation in sera from patients with PASC and PAAC, as well as healthy controls, were profiled. Among these mediators, solely IL-6 and CXCL1 were significantly increased in PASC, but not PAAC patients, compared to controls (FIG. 8A). The following mediators showed no difference between PASC, PAAC, and control: IL-8, TNF, CCL2, CCL3, IL-17A, and VEGF (FIGS. 9A & 9B)

[0344] Next, levels of MC-derived proteases were assessed to evaluate MC activation. Active tryptase and CPA3 levels were significantly elevated in sera from PASC, but not PAAC patients, compared to healthy controls, highly suggestive of systemic MC activation (FIG. 8B). In contrast, chymase levels were not significantly different between these populations (FIG. 9C). To determine if MC activation was associated with elevated IL-6 and CXCL1, correlation analyses were performed using sera levels of MC proteases. Active tryptase significantly correlated with both IL-6 and CXCL1 levels, whereas CP A3 levels demonstrated modest association (FIGS. 8C & 9D). Collectively, these data suggest PASC is associated with systemic inflammation and MC activation that is not seen in PAAC or healthy controls.

[0345] To further characterize the inflammatory profile identified in PASC patient sera, cytokine, chemokine, and MC protease levels were compared against sera from a previously published cohort of acute COVID-19 patients (Gebremeskel S, et al. Front Immunol 2021;12:650331). Sera from symptomatic PASC patients displayed significantly reduced inflammatory cytokines and chemokines compared to sera from acute COVID-19 patients (FIGS. 8D & 10). Levels of active tryptase and chymase were also significantly lower in PASC patients compared to acute COVID- 19 patients (FIG. 8E). Notably, CP A3 levels in the serum were not significantly different between PASC and acute COVID- 19 patients (FIG. 8F), suggesting CPA3 levels may remain similarly elevated post-acute infection. Taken together, these findings support a potential role for immune dysfunction associated with MC activation in PASC.

[0346] PASC or “COVID-19 long hauler syndrome” has become increasingly recognized as common following COVID- 19 infection, even in patients with mild acute illness (Sudre CH, Murray B, Varsavsky T, et al. Nat Med 2021;27:626-631, Graham EL, Clark JR, Orban ZS, et al. Ann Clin Transl Neurol 2021;8:1073-1085, Frontera JA, Yang D, Lewis A, et al, J Neurol Sci 2021;426: 117486, Blomberg B, Mohn KG, Brokstad KA, et al. Nat Med 2021). The natural history is poorly understood, but PASC can persist beyond 6 months, as is evident in this cohort. A predominant feature of PASC is fatigue, which was reported by over 80% of this cohort. In addition, other notable symptoms were body aches, loss of taste/smell, and brain fog. To date, no mechanism has been described for PASC although studies have examined potential immune and metabolic derangement (Doykov I, Hallqvist J, Gilmour KC, et al. FlOOORes 2020;9:1349, Holmes E, Wist J, Masuda R, et al. J Proteome Res 2021;20:3315-3329, Wallukat G, Hohberger B, Wenzel K, et al. J Transl Autoimmun 2021;4:100100). As many of the symptoms of PASC are shared with MCAS and ISM, it was hypothesized that MC activation may be a critical mechanism of PASC. Using small cohorts of predominantly female PASC and PAAC patients with ahistory of PCR-positive SARS-CoV-2 infection and healthy controls, evidence of systemic MC activation and elevated inflammatory mediators was found in PASC patients. To date, this is the first immune mechanistic description of PASC.

[0347] MCs are tissue resident granulocytic cells that constitute a major sensory arm of the innate immune system. Upon activation, MCs release preformed granules containing inflammatory mediators, vasoactive autocoids, catalytically active MC-specific proteases, and de novo production of cytokines and lipid mediators, such as TNF, IL-6, and prostaglandin D2 (Wemersson S, Pejler G. Nat Rev Immunol. 2014;14(7):478-94). IL-6 is a pleiotropic cytokine that has been shown to promote MC development and enhance degranulation and mediator production (Brockow K, Akin C, Huber M, Metcalfe DD. Clin Sci (Lond) 2012;122:143-59). Moreover, IL-6 levels associate with the severity of multiple MC-associated diseases, including systemic mastocytosis, urticaria, and asthma (Brockow K, Akin C, Huber M, Metcalfe DD. Clin Sci (Lond) 2012;122:143-59; Fujii K, Konishi K, Kanno Y, Ohgou N. J.

Dermatol. 2001;28:248-50; Morjaria JB, Babu KS, Vijayanand P, Chauhan AJ, Davies DE, Holgate ST, Thorax. 2011 ;66: 537). IL-6 was found to be elevated in sera of symptomatic PASC patients, but not in PAAC patient sera. Indeed, IL-6 has been hypothesized to contribute to COVID-19-related long-term symptoms, including fatigue, sleeping difficulties, depression, and anxiety by regulating neuro-immune crosstalk (Kappelmann N, Dantzer R, Khandaker GM. Psychoneuroendocrinology. 2021; 131:105295). In acute COVID-19, therapeutic blockade of IL-6 has led to lower death rates across different disease severities (Rubin EJ, Longo DL, Baden LR. N Engl J Med. 2021), suggesting it could potentially be useful in PASC.

[0348] Significant evidence of MC activation in patients with PASC was found that differed from PAAC and acute COVID- 19. In humans, MCs are classified according to their protease content and tissue distribution, with the MCT subclass expressing only tryptase (P-tryptase) and being primarily found in mucosal tissues, and the MCTC subclass expressing tryptase, chymase and CPA-3 and located mainly in the skin (Wemersson S, Pejler G. Nat Rev Immunol. 2014;14(7):478-94). These data suggest that acute COVID-19 infection and PASC have differential MC activation profiles based on sera levels of active tryptase and chymase. Chymase levels were significantly elevated in acute COVID, but not PASC, while CP A3 levels were similar between these two populations. These data suggest that MC dysfunction may contribute to inflammation and symptoms seen in PASC. More importantly, these findings highlight MCs as potential therapeutic targets for patients with PASC.

[0349] Key strengths of this study were the breadth and duration of symptoms captured in PASC patients and having an aged-matched PAAC cohort as a comparator population. In addition, nearly all patients had detectable SARS-CoV-2 IgG antibodies. While nearly all patients in the cohort were women, this is commonly found in prevalence studies of PASC and interestingly aligns with MCAS demographics.

[0350] In summary, evidence of systemic inflammation and MC activation was found in patients with PASC.

SEQUENCES

All polypeptide sequences are presented N-terminal to C-terminal unless otherwise noted.

All nucleic acid sequences are presented 5’ to 3’ unless otherwise noted.

Amino acid sequence of mouse 2E2 heavy chain variable domain

QVQLKESGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNY NSALMSRLSISKDNSKSQVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVT VSS (SEQ ID NO:1)

Amino acid sequence of 2E2 RHA heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTN YNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTT VTVSS (SEQ ID NO:2)

Amino acid sequence of 2E2 RHB heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAVSGFSLTIYGAHWVRQAPGKGLEWLGVIWAGGSTN YNSALMSRLSISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTT VTVSS (SEQ ID NO:3)

Amino acid sequence of 2E2 RHC heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAVSGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTN YNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTT VTVSS (SEQ ID NO:4)

Amino acid sequence of 2E2 RHP heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWLSVIWAGGSTN YNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTT VTVSS (SEQ ID NO:5) Amino acid sequence of 2E2 RHE heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGT TVTVSS (SEQ ID NO:6)

Amino acid sequence of 2E2 RHF heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTN

YNSALMSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTT VTVSS (SEQ ID NO:7)

Amino acid sequence of 2E2 RHG heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTN

YNSALMSRFSISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTT VTVSS (SEQ ID NO: 8)

Amino acid sequence of 2E2 RHA2 heavy chain variable domain

QVQLQESGPGLVKPSETLSLTCTVSGGSISIYGAHWIRQPPGKGLEWIGVIWAGGSTNYN SALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGSSPYYYSMEYWGQGTLVTV SS (SEQ ID NOV)

Amino acid sequence of 2E2 RHB2 heavy chain variable domain

QVQLQESGPGLVKPSETLSLTCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTN YNSALMSRLSISKDNSKNQVSLKLSSVTAADTAVYYCARDGSSPYYYSMEYWGQGTL VTVSS (SEQ ID NOTO)

Amino acid sequence of 2E2 RHE S-G mutant heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST

NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYGMEYWGQGT TVTVSS (SEQ ID NO:11) Amino acid sequence of 2E2 RHE E-D heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMDYWGQGT TVTVSS (SEQ ID NO: 12)

Amino acid sequence of 2E2 RHE Y-V heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEVWGQGT TVTVSS (SEQ ID NO:13)

Amino acid sequence of 2E2 RHE triple mutant heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYGMDVWGQG TTVTVSS (SEQ ID NO: 14)

Amino acid sequence of mouse 2E2 light chain variable domain

QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRF SGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIK (SEQ ID NO:15)

Amino acid sequence of 2E2 RKA light chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO: 16)

Amino acid sequence of 2E2 RKB light chain variable domain

EIILTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLWIYSTSNLASGVPARF

SGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO: 17)

Amino acid sequence of 2E2 RKC light chain variable domain

EIILTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFS

GSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO: 18) Amino acid sequence of 2E2 RKD light chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLWIYSTSNLASGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO: 19)

Amino acid sequence of 2E2 RKE li ht chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGVPAR

FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO:20)

Amino acid sequence of 2E2 RKF light chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARF

SGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO:21)

Amino acid sequence of 2E2 RKG light chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGQAPRLLIYSTSNLASGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK (SEQ ID NO:22)

Amino acid sequence of 2E2 RKA F-Y mutant light chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPYTFGPGTKLDIK (SEQ ID NO:23)

Amino acid sequence of 2E2 RKF F-Y mutant light chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARF

SGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPYTFGPGTKLDIK (SEQ ID NO:24)

Amino acid sequence of HEKA heavy chain and HEKF heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST

NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGT

TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:75)

Amino acid sequence of HEKA light chain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:76)

Amino acid sequence of HEKF light chain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARF SGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:77)

Amino acid sequence of IgGl heavy chain constant region

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:78)

Amino acid sequence of IgG4 heavy chain constant region

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 79) Amino acid sequence of Ig kappa light chain constant region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 80)

Amino acid sequence of murine 2C4 and 2E2 IgGl heavy chain

QVQLKRASGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTN

YNSALMSRLSISKDNSKSQVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSV

TVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPA

VLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSS

VFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNST

FRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMA

KDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSN

WEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ ID NO:81)

Amino acid sequence of murine 2C4 kappa light chain

EIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRF

SGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKADAAPTVSIFPPSSEQ

LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT

KDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:82)

Amino acid sequence of murine 2E2 kappa light chain

QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRF

SGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKADAAPTVSIFPPSSEQ

LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT

KDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:83)

Amino acid sequence of chimeric 2C4 and 2E2 IgGl heavy chain

QVQLKRASGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTN

YNSALMSRLSISKDNSKSQVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSV

TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE

LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 84)

Amino acid sequence of chimeric 2C4 kappa light chain

EIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRF SGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)

Amino acid sequence of chimeric 2E2 kappa light chain

QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRF SGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 86)

Amino acid sequence of HEKA IgG4 heavy chain (IgG4 contains a S228P mutation)

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGST

NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGT TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL

TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:87)

Amino acid sequence of mouse 1C3 heavy chain variable domain (underlined residues comprise CDRs Hl and H2 according to Chothia numbering)

EVQVVESGGDLVKSGGSLKLSCAASGFPFSSYAMSWVRQTPDKRLEWVAIISSGGSYTY YSDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHETAQAAWFAYWGQGTLV TVSA (SEQ ID NO: 106) Amino acid sequence of mouse 1H10 heavy chain variable domainfunderlined residues comprise CDRs Hl and H2 according to Chothia numbering)

EVOLOQSGAELVRPGASVKLSCTASGFNIKDYYMYWVKORPEOGLEWIGRIAPEDGDT

EYAPKFQGKATVTADTSSNTAYLHLSSLTSEDTAVYYCTTEGNYYGSSILDYWGQGTT LTVSS (SEQ ID NO: 107)

Amino acid sequence of mouse 4F11 heavy chain variable domain (underlined residues comprise CDRs Hl and H2 according to Chothia numbering)

OVOLOQSGAELVKPGASVKISCKASGYAFRSSWMNWVKORPGKGLEWIGOIYPGDDY

TNYNGKFKGKVTLTADRSSSTAYMQLSSLTSEDSAVYFCARLGPYGPFADWGQGTLVT VS A (SEQ ID NO: 108)

Amino acid sequence of mouse 1C3 light chain variable domain

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLAYGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK (SEQ ID NO: 109)

Amino acid sequence of mouse 1H10 light chain variable domain

DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYFTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 110)

Amino acid sequence of mouse 4F11 light chain variable domain

QIVLTQSPAIVSASPGEKVTMTCSASSSVSYMYWYQQRPGSSPRLLIYDTSSLASGVPVR

FSGSGSGTSYSLTISRIESEDAANYYCQQWNSDPYTFGGGTKLEIK (SEQ ID NO: 111)

Amino acid sequence of human Siglec-8 Domain 1

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDR PYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGS MKWSYKSQLNYKTKQLSVFVTALTHRP (SEQ ID NO: 112)

Amino acid sequence of human Siglec-8 Domain 2

DILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDH

GTSLTCQVTLPGTGVTTTSTVRLDVS (SEQ ID NO: 113) Amino acid sequence of human Siglec-8 Domain 3

YPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTL CPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLA AVGG (SEQ ID NO: 114)

Amino acid sequence of human Siglec-8 Domain 1 Fusion Protein

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDR PYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGS MKWSYKSQLNYKTKQLSVFVTALTHRPIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115)

Amino acid sequence of human Siglec-8 Domains 1 and 2 Fusion Protein

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDR PYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGS MKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMI SWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSIEGRSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116)

Amino acid sequence of human Siglec-8 Domains 1, 2. and 3 Fusion Protein

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDR PYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGS MKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMI SWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWN LTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSS NPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGIE

GRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ

ID NO: 117

Claims

CLAIMS What is claimed is:

1. A method for treating viral infection, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody that binds to human Siglec-8.

2. A method for inhibiting inflammation in an individual with a viral infection, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8.

3. A method for inhibiting mast cell and/or eosinophil activation in an individual with a viral infection, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8.

4. The method of any one of claims 1-3, wherein the virus is an enveloped virus.

5. The method of claim 4, wherein the virus is a Coronavirus.

6. The method of claim 5, wherein the virus is selected from the group consisting of severe acute respiratory syndrome coronavirus 1 (SARS-Cov-1), severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), and Middle East respiratory syndrome-related coronavirus (MERS-Cov).

7. The method of claim 4, wherein the virus is an Orthomyxovirus.

8. The method of claim 7, wherein the virus is an influenza A virus.

9. The method of claim 4, wherein the virus is an Orthopneumovirus.

10. The method of claim 9, wherein the virus is respiratory syncytial virus (RSV).

11. The method of any one of claims 1-10, wherein the individual has or has been diagnosed with acute respiratory distress syndrome (ARDS).

12. A method for treating post-acute COVID-19 syndrome, comprising administering to an individual in need thereof an effective amount of a composition comprising an antibody that binds to human Siglec-8.

13. A method for inhibiting inflammation in an individual with post-acute COVID- 19 syndrome, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8.

14. A method for inhibiting mast cell and/or eosinophil activation in an individual with postacute COVID- 19 syndrome, comprising administering to the individual an effective amount of a composition comprising an antibody that binds to human Siglec-8.

15. The method of any one of claims 12-14, wherein, prior to administration of the composition, the individual has been diagnosed with post-acute COVID-19 syndrome.

16. The method of any one of claims 12-15, wherein, prior to administration of the composition, the individual has been diagnosed with COVID-19 infection.

17. The method of any one of claims 12-16, wherein one or more symptom(s) of post-acute COVID- 19 syndrome in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition.

18. The method of claim 17, wherein the one or more symptom(s) include one or more of: loss of smell and/or taste, shortness of breath, fatigue, myalgia, dysautonomia, decreased exercise capacity, hypoxia, reduced diffusion capacity, restrictive pulmonary physiology, pulmonary fibrosis, diminished quality of life, muscular weakness, joint pain, dyspnea, cough, persistent oxygen requirement, anxiety, depression, sleep disturbances, post-traumatic stress disorder (PTSD), cognitive disturbance, headache, palpitations, chest pain, increased cardiometabolic demand, myocardial fibrosis or scarring, arrhythmia, tachycardia, autonomic dysfunction, thromboembolism, chronic kidney disease, impaired renal function, new or worsening type II diabetes, subacute thyroiditis, bone demineralization, multisystem inflammatory syndrome (MIS-C), and hair loss.

19. The method of any one of claims 1-18, wherein, prior to administration of the composition, the individual has increased mast cell activation, as compared to a reference or reference value.

20. The method of claim 19, wherein a peripheral blood sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present.

21. The method of claim 19 or claim 20, wherein a lung-derived sample obtained from the individual prior to administration of the composition has one or more markers of mast cell activation present.

22. The method of any one of claims 1-21, wherein, prior to administration of the composition, the individual has increased eosinophil activation, as compared to a reference or reference value.

23. The method of claim 22, wherein a peripheral blood sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present.

24. The method of claim 22 or claim 23, wherein a lung-derived sample obtained from the individual prior to administration of the composition has one or more markers of eosinophil activation present.

25. The method of any one of claims 1-24, wherein a serum sample obtained from the individual prior to administration of the composition has an elevated level of one or more pro- inflammatory cytokine(s) or growth factor(s), as compared to a reference or reference value.

26. The method of claim 25, wherein the one or more pro-inflammatory cytokine(s) or growth factor(s) are selected from the group consisting of CCL2, IP-10, IL-8, VEGF, histamine, leukotriene C4, leukotriene E4, prostaglandin D2, eotaxin, periostin, eosinophil peroxidase (EPX), IL-6, TNF, C-reactive protein (CRP), ferritin, major basic protein (MBP), eosinophil- derived neurotoxin (EDN), and IFN-y.

27. The method of any one of claims 1-26, wherein a serum sample obtained from the individual prior to administration of the composition has an elevated level of one or more polypeptide(s) expressed by mast cells, as compared to a reference or reference value.

28. The method of claim 27, wherein the one or more polypeptide(s) expressed by mast cells are selected from the group consisting of chymase, p-tryptase, and CPA3.

29. The method of any one of claims 1-28, wherein a lung-derived sample obtained from the individual prior to administration of the composition has: an elevated level of one or more polypeptide(s) expressed by mast cells, pro- inflammatory cytokine(s), or chemokine(s), as compared to a reference or reference value; or an elevated level of one or more polynucleotides encoding one or more polypeptide(s) expressed by mast cells, pro-inflammatory cytokine(s), or chemokine(s), as compared to a reference or reference value.

30. The method of claim 29, wherein the one or more polypeptide(s) expressed by mast cells and/or pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, CCL4, IL-8, IP-10, TPSB2, TPSAB1, and FCER1G.

31. The method of any one of claims 21, 24, 29, and 30, wherein the lung-derived sample is a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample.

32. The method of any one of claims 1-31, wherein, prior to administration of the composition, the individual has eosinopenia in peripheral blood.

33. The method of any one of claims 1-32, wherein a serum sample obtained from the individual prior to administration of the composition has an elevated level of one or more polypeptide(s) expressed by eosinophils, as compared to a reference or reference value.

34. The method of claim 33, wherein the one or more polypeptide(s) expressed by eosinophils comprises EDN.

35. The method of any one of claims 1-34, wherein a lung-derived sample obtained from the individual prior to administration of the composition has: an elevated level of one or more polypeptide(s) expressed by eosinophils, as compared to a reference or reference value; or an elevated level of one or more polynucleotides encoding one or more polypeptide(s) expressed by eosinophils, as compared to a reference or reference value.

36. The method of claim 35, wherein the one or more polypeptide(s) expressed by eosinophils are selected from the group consisting of EDN and Gal ectin- 10.

37. The method of claim 35 or claim 36, wherein the lung-derived sample is a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample.

38. The method of any one of claims 1-37, wherein one or more symptom(s) of viral infection in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition.

39. The method of any one of claims 1-37, wherein one or more symptom(s) of inflammation in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition.

40. The method of any one of claims 11-37, wherein one or more symptom(s) of ARDS in the individual are reduced after administration of the composition as compared to a baseline level before administration of the composition.

41. The method of any one of claims 1-37, wherein level of one or more pro-inflammatory cytokine(s) or chemokine(s) in a serum sample obtained from the individual are reduced after administration of the composition, as compared to a baseline level in a serum sample obtained before administration of the composition.

42. The method of claim 41, wherein the one or more pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, IP-10, IL-6, ferritin, C-reactive protein (CRP), and TNF.

43. The method of any one of claims 1-42, wherein level of one or more polypeptide(s) expressed by mast cells in a serum sample obtained from the individual are reduced after

-139- administration of the composition, as compared to a baseline level in a serum sample obtained before administration of the composition.

44. The method of claim 43, wherein the one or more polypeptide(s) expressed by mast cells comprises chymase.

45. The method of any one of claims 1-44, wherein level of one or more pro-inflammatory cytokine(s) or chemokine(s) or one or more polynucleotides encoding one or more pro- inflammatory cytokine(s) or chemokine(s)in a lung-derived sample obtained from the individual are reduced after administration of the composition, as compared to a baseline level in a lung- derived sample obtained before administration of the composition.

46. The method of claim 45, wherein the one or more pro-inflammatory cytokine(s) or chemokine(s) are selected from the group consisting of CCL2, IL-6, CXCL2, and IL-ip.

47. The method of any one of claims 1-46, wherein number(s) of eosinophils, monocytes, and/or neutrophils in a peripheral blood sample obtained from the individual are reduced after administration of the composition, as compared to a baseline level in a peripheral blood sample obtained before administration of the composition.

48. The method of any one of claims 1-46, wherein number(s) of eosinophils, monocytes, and/or neutrophils in a lung-derived sample obtained from the individual are reduced after administration of the composition, as compared to a baseline level in a lung-derived sample obtained before administration of the composition.

49. The method of any one of claims 1-48, wherein level of one or more polypeptide(s) expressed by eosinophils in a serum sample obtained from the individual are reduced after administration of the composition, as compared to a baseline level in a serum sample obtained before administration of the composition.

50. The method of any one of claims 1-49, wherein level of one or more polypeptide(s) expressed by eosinophils or one or more polynucleotides encoding one or more polypeptide(s) expressed by eosinophils in a lung-derived sample obtained from the individual are reduced after administration of the composition, as compared to a baseline level in a lung-derived sample obtained before administration of the composition.

-140-

51. The method of claim 49 or claim 50, wherein the one or more polypeptide(s) expressed by eosinophils comprise eosinophil peroxidase (EPX).

52. The method of any one of claims 1-51, wherein eosinophil activation in the individual is reduced after administration of the composition, as compared to a baseline level before administration of the composition.

53. The method of claim 52, wherein eosinophil activation in peripheral blood is reduced after administration of the composition.

54. The method of claim 52 or claim 53, wherein eosinophil activation in a lung-derived sample is reduced after administration of the composition.

55. The method of any one of claims 1-54, wherein mast cell activation in the individual is reduced after administration of the composition, as compared to a baseline level before administration of the composition.

56. The method of claim 55, wherein mast cell activation in peripheral blood is reduced after administration of the composition.

57. The method of claim 55 or claim 56, wherein mast cell activation in a lung-derived sample is reduced after administration of the composition.

58. The method of any one of claims 45, 46, 48, 50, 51, 54, and 57, wherein the lung-derived sample is a lung biopsy sample, sputum sample, or bronchoalveolar lavage (BAL) sample.

59. The method of any one of claims 1-58, wherein the composition is administered by subcutaneous injection.

60. The method of any one of claims 1-58, wherein the composition is administered by intravenous infusion.

61. The method of any one of claims 1-60, wherein the antibody comprises a Fc region and N-gly coside-linked carbohydrate chains linked to the Fc region, wherein less than 50% of the N- gly coside-linked carbohydrate chains of the antibody in the composition contain a fucose residue.

-141-

62. The method of claim 61, wherein substantially none of the N-gly coside-linked carbohydrate chains of the antibody in the composition contain a fucose residue.

63. The method of any one of claims 1-62, wherein the antibody is produced in a cell line having a alphal,6-fucosyltransferase (Fut8) knockout.

64. The method of any one of claims 1-62, wherein the antibody is produced in a cell line overexpressing pi,4-N-acetylglucosminyltransferase III (GnT-III).

65. The method of claim 64, wherein the cell line additionally overexpresses Golgi p- mannosidase II (Manll).

66. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

67. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence selected from SEQ ID NOs: 67-70; and wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71.

68. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 16 or 21.

-142-

69. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence selected from SEQ ID NOs: 11-14; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs:23- 24.

70. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence selected from SEQ ID NOs:2-14; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 16- 24.

71. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence selected from SEQ ID NOs:2-10; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 16- 22.

72. The method of any one of claims 1-65, wherein the antibody comprises:

(a) heavy chain variable region comprising:

(1) an HC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:26-29;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61;

(3) an HC-FR2 comprising the amino acid sequence selected from SEQ ID NOs:31-36;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62;

(5) an HC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:38-43;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and

(7) an HC-FR4 comprising the amino acid sequence selected from SEQ ID NOs:45-46, and

(b) a light chain variable region comprising:

(1) an LC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:48-49;

(2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64;

-143- (3) an LC-FR2 comprising the amino acid sequence selected from SEQ ID

NOs:51-53;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65;

(5) an LC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:55-58;

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66; and

(7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60.

73. The method of any one of claims 1-65, wherein the antibody comprises:

(a) heavy chain variable region comprising:

(1) an HC-FR1 comprising the amino acid sequence of SEQ ID NO:26;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61;

(3) an HC-FR2 comprising the amino acid sequence of SEQ ID NO:34;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62;

(5) an HC-FR3 comprising the amino acid sequence of SEQ ID NO:38;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and

(7) an HC-FR4 comprising the amino acid sequence of SEQ ID NOs:45; and

(b) a light chain variable region comprising:

(1) an LC-FR1 comprising the amino acid sequence of SEQ ID NO:48;

(2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64;

(3) an LC-FR2 comprising the amino acid sequence of SEQ ID NO:51;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65;

(5) an LC-FR3 comprising the amino acid sequence of SEQ ID NO:55;

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66; and

(7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60.

74. The method of any one of claims 1-65, wherein the antibody comprises:

(a) heavy chain variable region comprising:

(1) an HC-FR1 comprising the amino acid sequence of SEQ ID NO:26;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61;

(3) an HC-FR2 comprising the amino acid sequence of SEQ ID NO: 34;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62;

-144- (5) an HC-FR3 comprising the amino acid sequence of SEQ ID NO:38;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and

(7) an HC-FR4 comprising the amino acid sequence of SEQ ID NOs:45; and

(b) a light chain variable region comprising:

(1) an LC-FR1 comprising the amino acid sequence of SEQ ID NO:48;

(2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64;

(3) an LC-FR2 comprising the amino acid sequence of SEQ ID NO:51;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65;

(5) an LC-FR3 comprising the amino acid sequence of SEQ ID NO:58;

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66; and

(7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60.

75. The method of any one of claims 1-65, wherein the antibody comprises: a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 88, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:94; and a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 103; a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 104; or a heavy chain variable region comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 90, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:96; and a light chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 105.

76. The method of any one of claims 1-65, wherein the antibody comprises:

-145- a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 106; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 109; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 107; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 110; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 108; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111.

77. The method of any one of claims 1-65, wherein the antibody binds to a human Siglec-8 and a non-human primate Siglec-8.

78. The method of claim 77, wherein the non-human primate is a baboon.

79. The method of claim 77, wherein the antibody binds to an epitope in Domain 1 of human Siglec-8, wherein Domain 1 comprises the amino acid sequence of SEQ ID NO: 112.

80. The method of claim 77, wherein the antibody binds to an epitope in Domain 3 of human Siglec-8, wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114.

81. The method of claim 77, wherein the antibody binds to the same epitope as antibody 4F11.

82. The method of any one of claims 1-65, wherein the antibody binds to an epitope in Domain 2 or Domain 3 of human Siglec-8.

83. The method of claim 82, wherein Domain 2 comprises the amino acid sequence of SEQ ID NO: 113.

84. The method of claim 82, wherein the antibody binds to the same epitope as antibody 1C3.

85. The method of claim 82, wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114.

86. The method of claim 82, wherein the antibody binds to the same epitope as antibody 1H10.

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87. The method of any one of claims 1-65, wherein the antibody binds to an epitope in Domain 1 of human Siglec-8 and competes with antibody 4F11 for binding to Siglec-8.

88. The method of claim 87, wherein the antibody does not compete with antibody 2E2 for binding to Siglec-8.

89. The method of claim 88, wherein the antibody is not antibody 2E2.

90. The method of claim 87, wherein Domain 1 comprises the amino acid sequence of SEQ ID NO: 112.

91. The method of any one of claims 66-90, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

92. The method of any one of claims 66-91, wherein the antibody depletes blood eosinophils and inhibits mast cell activation.

93. The method of any one of claims 66-92, wherein the antibody comprises a heavy chain Fc region comprising a human IgG Fc region.

94. The method of claim 93, wherein the human IgG Fc region comprises a human IgGl Fc region.

95. The method of claim 94, wherein the human IgGl Fc region is non-fucosylated.

96. The method of claim 93, wherein the human IgG Fc region comprises a human IgG4 Fc region.

97. The method of claim 96, wherein the human IgG4 Fc region comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat.

98. The method of any one of claims 66-90, wherein the antibody has been engineered to improve antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

99. The method of claim 98, wherein the antibody comprises at least one amino acid substitution in the Fc region that improves ADCC activity.

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100. The method of any one of claims 66-99, wherein at least one or two of the heavy chains of the antibody is non-fucosylated.

101. The method of any one of claims 1-65, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:75; and a light chain comprising the amino acid sequence selected from SEQ ID NO:76 or 77.

102. The method of any one of claims 1-101, wherein the antibody is a monoclonal antibody.

103. The method of any one of claims 1-102, wherein the composition is administered in combination with one or more additional therapeutic agent(s) for treating viral infection and/or inhibiting inflammation.

104. The method of claim 103, wherein the one or more additional therapeutic agent(s) are selected from the group consisting of corticosteroids, hydroxychloroquine, azithromycin, colchicine, remdesivir, IL-6 antagonists, antigen-binding moieties that specifically bind viral spike protein, Ramatroban, convalescent plasma, and favipiravir.

105. The method of any one of claims 1-104, wherein the individual is a human.

106. The method of any one of claims 1-105, wherein the composition is a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.

107. An article of manufacture comprising a medicament comprising a composition comprising an antibody that binds to human Siglec-8 and a package insert comprising instructions for administration of the medicament in an individual in need thereof according to any one of claims 1-106.