WO2024254441A1 - Anticorps bispécifiques dirigés contre siglec-6 et siglec-8 et leurs procédés d'utilisation - Google Patents

Anticorps bispécifiques dirigés contre siglec-6 et siglec-8 et leurs procédés d'utilisation Download PDF

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WO2024254441A1
WO2024254441A1 PCT/US2024/032993 US2024032993W WO2024254441A1 WO 2024254441 A1 WO2024254441 A1 WO 2024254441A1 US 2024032993 W US2024032993 W US 2024032993W WO 2024254441 A1 WO2024254441 A1 WO 2024254441A1
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amino acid
seq
acid sequence
hvr
antibody
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John Leung
Wouter Korver
Julia SCHANIN
Bradford Andrew Youngblood
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Allakos Inc
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Allakos Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/528—CH4 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • Domain 3 of the extracellular domain of human Siglec-6 comprises the amino acid sequence of SEQ ID NO: 123.
  • the first antigen binding domain is a humanized antigen binding domain.
  • the first VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and wherein the first VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 127, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 128, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 129.
  • the second antibody heavy chain comprises a CHI region comprising substitution of one or more native, non-positively charged amino acid(s) to positively charged amino acid(s); wherein the second antibody light chain comprises a CL region comprising substitution of one or more native, non-negatively charged amino acid(s) to negatively charged amino acid(s); wherein the second VL region replaces the second VH region on the second antibody heavy chain; wherein the second VH region replaces the second VL region on the second antibody light chain; wherein the first antibody heavy chain comprises a CHI region comprising substitution of one or more native, non-negatively charged amino acid(s) to negatively charged amino acid(s); and wherein the first antibody light chain comprises a CL region comprising substitution of one or more native, non-positively charged amino acid(s) to positively charged amino acid(s).
  • the second antibody light chain comprises a Q124E substitution; wherein the first antibody light chain comprises E123K and Q124K substitutions; and wherein the first antibody heavy chain comprises K147E and K213E substitutions; wherein the amino acid residues are numbered according to the EU index as in Kabat.
  • the first antibody heavy chain comprises one or more substitutions encoding a protuberance
  • the second antibody heavy chain comprises one or more substitutions encoding a cavity
  • the protuberance of the first antibody heavy chain is positionable in the cavity of the second antibody heavy chain.
  • the first antibody heavy chain comprises T366W and S354C substitutions and wherein the second antibody heavy chain comprises Y349C, T366S, L368A, and Y407V substitutions, wherein the amino acid residues are numbered according to the EU index as in Kabat.
  • the second antibody heavy chain comprises one or more substitutions encoding a protuberance, wherein the first antibody heavy chain comprises one or more substitutions encoding a cavity, and wherein the protuberance of the second antibody heavy chain is positionable in the cavity of the first antibody heavy chain.
  • the second antibody heavy chain comprises T366W and S354C substitutions and wherein the first antibody heavy chain comprises Y349C, T366S, L368A, and Y407V substitutions, wherein the amino acid residues are numbered according to the EU index as in Kabat.
  • the first antibody heavy chain comprises H435R and Y436F substitutions, wherein the amino acid residues are numbered according to the EU index as in Kabat.
  • the second antibody heavy chain comprises H435R and Y436F substitutions, wherein the amino acid residues are numbered according to the EU index as in Kabat.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:272, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:273 or 274, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:275, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:276 or 277.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:281, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:282 or 283, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:278, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:279 or 280.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285 or 286, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:287, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:288 or 289.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:293, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:294 or 295, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:290, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:291 or 292.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:272, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:273, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:275, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:276.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:281, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:282, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:278, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:279.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:287, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:288.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:293, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:294, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:290, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:291.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285 or 286, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:298, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:299 or 300.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:298, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:299.
  • binding of the bispecific antibody to an extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of Siglec-6 on the cell surface.
  • binding of the bispecific antibody to an extracellular domain of human Siglec-8 when expressed on a surface of a human mast cell leads to a reduced level of Siglec-8 on the cell surface.
  • binding of the bispecific antibody to an extracellular domain of human Siglec-6 inhibits activation of a mast cell that expresses the human Siglec-6.
  • binding of the bispecific antibody to an extracellular domain of human Siglec-8 inhibits activation of a mast cell that expresses the human Siglec-8.
  • binding of the bispecific antibody to an extracellular domain of human Siglec-8 depletes eosinophils expressing human Siglec-8 in vivo.
  • a composition comprising the bispecific antibody according to any one of the embodiments described herein.
  • the bispecific antibody comprises a Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, wherein less than 50% of the N-glycoside-linked carbohydrate chains contain a fucose residue.
  • substantially none of the N-glycoside-linked carbohydrate chains contain a fucose residue.
  • a pharmaceutical composition comprising the bispecific antibody according to any one of the embodiments described herein and a pharmaceutically acceptable carrier.
  • a polynucleotide encoding the bispecific antibody according to any one of the embodiments described herein.
  • a kit of polynucleotides comprising a first polynucleotide encoding the first antibody heavy chain and first antibody light chain according to any one of the embodiments described herein (e.g., a first antibody arm) and a second polynucleotide encoding the second antibody heavy chain and second antibody light chain according to any one of the embodiments described herein (e.g., a second antibody arm).
  • a vector comprising the polynucleotide according to any one of the embodiments described herein.
  • kits of vectors comprising a first vector encoding the first antibody heavy chain and first antibody light chain according to any one of the embodiments described herein (e.g., a first antibody arm) and a second vector encoding the second antibody heavy chain and second antibody light chain according to any one of the embodiments described herein (e.g., a second antibody arm).
  • a host cell e.g., an isolated host cell
  • the host cell comprises the polynucleotide, vector, kit of polynucleotides, or kit of vectors according to any one of the embodiments described herein.
  • the host cell is a mammalian or insect cell.
  • the host cell is Chinese hamster ovary (CHO) cell.
  • the host cell comprises a Fut8 knockout.
  • the host cell overexpresses GnT-III.
  • the host cell additionally overexpresses Manll.
  • provided herein is a method of producing a bispecific antibody, comprising culturing the host cell according to any one of the embodiments described herein under a condition that produces the bispecific antibody. In some embodiments, the method further comprises recovering the bispecific antibody produced by the host cell. In another aspect, provided herein is a bispecific antibody produced by the method according to any one of the embodiments described herein. [0020] In another aspect, provided herein is a method of treating a disease or condition characterized by increased activity and/or number of mast cells expressing Siglec-6 and/or Siglec-8 in a subject, comprising administering to the subject an effective amount of the bispecific antibody or composition according to any one of the embodiments described herein.
  • provided herein is a method of inhibiting activation of mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof, comprising administering to the subject an effective amount of the bispecific antibody or composition according to any one of the embodiments described herein.
  • a method of depleting mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof comprising administering to the subject an effective amount of the bispecific antibody or composition according to any one of the embodiments described herein.
  • provided herein is a method of treating a disease or condition mediated by cells expressing Siglec-8 in a subject (e.g., in need thereof), comprising administering to the subject an effective amount of the bispecific antibody or composition according to any one of the embodiments described herein.
  • an effective amount of the bispecific antibody or composition according to any one of the embodiments described herein for use in a method of treating a disease or condition characterized by increased activity and/or number of mast cells expressing Siglec-6 and/or Siglec-8 in a subject, inhibiting activation of mast cells expressing Siglec-6 and/or Siglec- 8 in a subject in need thereof, depleting mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof, and/or treating a disease or condition mediated by cells expressing Siglec-8 in a subject (e.g., in need thereof).
  • provided herein is the use of the bispecific antibody or composition according to any one of the embodiments described herein in the manufacture of a medicament for treating a disease or condition characterized by increased activity and/or number of mast cells expressing Siglec-6 and/or Siglec-8 in a subject, inhibiting activation of mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof, depleting mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof, and/or treating a disease or condition mediated by cells expressing Siglec-8 in a subject (e.g., in need thereof).
  • administration of the bispecific antibody or composition results in a decrease in one or more symptoms in the subject, as compared to the one or more symptoms in the subject prior to the administration.
  • the one or more symptoms are selected from the group consisting of nausea, cramping, constipation, abdominal pain, bloating, vomiting, diarrhea, fatigue, eye pain, light sensitivity, redness, discharge, runny nose, headache, dizziness, brain fog, itching, flushing, sweating, hives, hypotension, shortness of breath, bone pain, joint pain, weight loss, osteoporosis, angioedema, chest pain, anxiety, depression, rapid heartbeat, bronchoconstriction, and general pain.
  • the disease or condition is mastocytosis, mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs.
  • NASH nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • GSH host disease
  • colitis hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria, atopic dermatitis, contact dermatitis, angioedema, pruigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma, allergy, keloid, chronic rhinosinusitis, aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic
  • the subject has or has been diagnosed with mastocytosis, mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs.
  • NASH nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • GSH host disease
  • colitis hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria, atopic dermatitis, contact dermatitis, angioedema, pruigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma, allergy, keloid, chronic rhinosinusitis, aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic
  • the disease or disorder is selected from the group consisting of allergic rhinitis, nasal polyposis, pauci granulocytic asthma, acute or chronic airway hypersensitivity, eosinophilic esophagitis, eosinophilic leukemia, Churg-Strauss syndrome, inflammation associated with a cytokine, inflammation associated with cells expressing Siglec-8, malignancy associated with cells expressing Siglec-8, physical urticaria, cold urticaria, pressure-urticaria, bullous pemphigoid, food allergy, chronic rhinosinusitis with concomitant asthma, aspirin-exacerbated respiratory disease, adult onset non-atopic asthma with sinus disease, chronic obstructive pulmonary disease, fibrotic disease, pre-fibrotic disease, mastocytosis, advanced systemic mastocytosis, indolent systemic mastocytosis (ISM), inflammatory bowel disease (IBD), eosinophilic esophagitis (EO)
  • FIGS. 1A-1C show exemplary bispecific anti-Siglec-6/anti-Siglec-8 (Siglec-6/8) antibody configurations, in accordance with some embodiments.
  • FIG. 1A shows a design using an alternative interchain disulfide bond in the anti-Siglec-8 arm to promote proper light chain pairing.
  • the anti-Siglec-8 arm includes an F126C mutation in the heavy chain CHI region and an S121C mutation in the light chain constant (CL) region to introduce the alternative interchain disulfide, a C220V mutation in the heavy chain and a C214V mutation in the light chain to remove native interchain disulfide, as well as H435R and Y436F mutations in the heavy chain.
  • FIG. IB shows a design using swapping of CHI and CL regions to promote proper light chain pairing, along with the knobs-into-holes scheme shown in FIG. 1A.
  • Left panel shows anti-Siglec-8 on left and anti- Sigec-6 on right with CH1/CL swap on anti-Siglec-6 arm; right panel shows anti-Siglec-8 on right and anti-Sigec-6 on left with CH1/CL swap on anti-Siglec-8 arm.
  • FIG. 1C shows a design using swapping of VH and VL regions and introduced electrostatic pairing of CHI and CL to promote proper light chain pairing, along with the knobs-into-holes scheme shown in FIG. 1A.
  • Left panel shows anti-Siglec-8 on left and anti-Sigec-6 on right with CH1/CL swap on anti- Siglec-6 arm; right panel shows anti-Siglec-8 on right and anti-Sigec-6 on left with CH1/CL swap on anti-Siglec-8 arm.
  • Electrostatic pairing mutations include E123K and Q124K on the light chain and K147E and K213E on the heavy chain. Designs in FIGS. IB & 1C also include the mutations to introduce the alternative interchain disulfide and remove native interchain disulfide described in FIG. 1A.
  • FIG. 2A & 2B show that both fucosylated and afucosylated bispecific Siglec-6/8 huIgGl antibodies induces internalization of both Siglec-6 and Siglec-8 on human tissue mast cells.
  • Human lung mast cells were cultured with varying concentrations of the bispecific Siglec- 6/8 huIgGl (diamond), afucosylated bispecific Siglec-6/8 huIgGl (open diamond), anti-Siglec-6 huIgGl (triangle), anti-Siglec-8 afucosylated huIgGl (square), or an isotype control (circle) for 18h at 37°C. Shown are Siglec-6 expression (dMFI; FIG. 2A) and Siglec-8 expression (dMFI; FIG. 2B) as a function of drug concentration (ug/mL).
  • FIG. 3 shows that bispecific Siglec-6/8 huIgGl antibody inhibits activation of human tissue mast cells.
  • Human lung mast cells were stimulated with an anti-FcsRI (lOug/mL) in the presence of the bispecific Siglec-6/8 huIgGl (diamond), anti-Siglec-6 huIgGl (triangle), anti- Siglec-8 huIgGl (square), or an isotype control (circle) at indicated concentrations for 20 min at 37°C. Shown is % CD63+ mast cells as a function of drug concentration (ug/mL).
  • FIGS. 4A & 4B show that both fucosylated and afucosylated bispecific Siglec-6/8 huIgGl antibodies induce Siglec-8 receptor internalization and deplete mouse blood eosinophils in vivo.
  • Siglec-6/Siglec-8 double transgenic mice (expressing human Siglec-6 and human Siglec-8) were intraperitoneally dosed with 5mg/kg of bispecific Siglec-6/8 huIgGl, afucosylated bispecific Siglec-6/8 huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control for 24h (as indicated). Shown are Siglec-8 expression on eosinophils (dMFI; FIG. 4A) and % eosinophils out of CD45+ cells (FIG. 4B) as a function of drug type. [0026] FIG.
  • FIGS. 5A & 5B show that both fucosylated and afucosylated bispecific Siglec-6/8 huIgGl antibodies internalize Siglec-6 and Siglec-8 on mouse tissue mast cells in vivo.
  • Siglec- 6/Siglec-8 double transgenic mice (expressing human Siglec-6 and human Siglec-8) were intraperitoneally dosed with 5mg/kg of bispecific Siglec-6/8 huIgGl, afucosylated bispecific Siglec-6/8 huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control for 24h (as indicated). Shown are Siglec-6 expression on mast cells (dMFI; FIG. 5A) and Siglec-8 expression on mast cells (dMFI; FIG. 5B) as a function of drug type.
  • FIG. 6A shows that both fucosylated and afucosylated bispecific Siglec-6/8 huIgGl antibodies reduce tissue mast cell activation in vivo.
  • Siglec-6/Siglec-8 double transgenic mice (expressing human Siglec-6 and human Siglec-8) were intraperitoneally dosed with 5mg/kg of bispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control for 24h (as indicated). Shown is CD63 expression on mast cells (MFI) as a function of drug type.
  • MFI mast cells
  • FIG. 6B shows that both fucosylated and afucosylated bispecific Siglec-6/8 huIgGl antibodies inhibit activation of mouse peritoneal mast cells.
  • Siglec-6/Siglec-8 double transgenic mice (expressing human Siglec-6 and human Siglec-8) were intravenously dosed with lOmg/kg ofbispecific Siglec-6/8 huIgGl (diamond), afucosylated bispecific Siglec-6/8 huIgGl (open diamond), anti-Siglec-6 huIgGl (triangle), or an isotype control (circle).
  • peritoneal mast cells were harvested and stimulated with an anti-FcsRI at indicated concentrations for 20 min at 37°C. Shown is % CD63+ mast cells as a function of anti-FcsRI concentration (ng/mL).
  • FIG. 7 shows that both fucosylated and afucosylated bispecific Siglec-6/8 huIgGl antibodies induce reduction of human tissue mast cells.
  • Human lung mast cells were cultured with monocyte-derived macrophages and treated with varying concentrations of the bispecific Siglec-6/8 huIgGl (diamond), afucosylated bispecific Siglec-6/8 huIgGl (open diamond), anti- Siglec-6 huIgGl (triangle), anti-Siglec-8 afucosylated huIgGl (square), or an isotype control (circle) for 18h at 37°C. Shown is % mast cells out of CD45+ cells as a function of drug concentration (ug/mL).
  • FIGS. 8A & 8B show the interactome of membrane and intracellular proteins that interact with Siglec-6 and Siglec-8 in mast cells.
  • FIG. 8A shows a schematic of the process for analysis of Siglec co-immunoprecipitated protein complexes from primary mast cells by liquid chromatography-mass spectrometry (LC-MS).
  • FIG. 8B shows the results of STRING analysis of the Siglec-6 and Siglec-8 interactomes.
  • Ubiquitin ligase machinery 6. Interferon-induced proteins 7. TGFP signaling 8.
  • Pyruvate dehydrogenases 9. PPP6 phosphatase complex 10. Pyrophosphate metabolism.
  • aspects and embodiments of the present disclosure include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.
  • 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).
  • immunoglobulin Ig
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • 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.
  • 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 y chains and four CH domains for p and 8 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 (CHI). 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.
  • 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, 5, 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.
  • 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).
  • 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.
  • 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.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, z.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.
  • 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.
  • 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.
  • 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.
  • 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.
  • naked antibody refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
  • 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.
  • 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.
  • the intact antibody may have one or more effector functions.
  • an “antibody fragment” comprises a portion of an intact antibody, the antigen binding and/or the variable region of the intact antibody.
  • 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.
  • 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 (CHI).
  • VH variable region domain of the H chain
  • CHI first constant domain of one heavy chain
  • 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 CHI 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.
  • 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.
  • FcR Fc receptors
  • “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.
  • 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.
  • 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.
  • “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.
  • antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • 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 etal., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • 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 homolog
  • 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.
  • “humanized antibody” is used as a subset of “chimeric antibodies.”
  • “Humanized” forms of non-human e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • 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.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • 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.
  • 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.
  • 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.
  • Fc immunoglobulin constant region
  • 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.
  • variable region 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.
  • hypervariable region refers to the regions of an antibody-variable domain that are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3).
  • 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.
  • H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • 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).
  • 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.
  • variable-domain residues HVR residues and framework region residues
  • HVR residues and framework region residues are numbered according to Kabat et al., supra.
  • “Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.
  • 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.
  • 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.
  • 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.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat el al.. Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • the “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
  • 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.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • 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.
  • binding of a bispecific antibody described herein e.g., an antibody that binds to human Siglec-8 and human Siglec-6 to an unrelated non-Siglec-8 or -Siglec-6 polypeptide is less than about 10% of the antibody binding to Siglec-8 and/or Siglec-6 as measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)).
  • ELISA enzyme-linked immunosorbent assay
  • a bispecific antibody that binds to a Siglec-8 and Siglec-6 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' 8 M to 10' 13 M, e.g., from 10' 9 M to 10' 13 M) for one or both of Siglec-6 and Siglec- 8.
  • Kd dissociation constant
  • 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.
  • Siglec-8 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.
  • 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.
  • Siglec-6 refers to a human Siglec-6 protein.
  • the term also includes naturally occurring variants of Siglec-6, including splice variants or allelic variants.
  • Siglec-6, the sialic acid binding Ig-like lectin 6, is also known as CD327, CD33L, OBBP1, CD33L1, CD33L2, and CDW327.
  • a human Siglec-6 protein is any protein or polypeptide expressed by a human SIGLEC6 gene.
  • An exemplary human SIGLEC6 gene is described by NCBI Ref. Seq. Gene ID No. 946. Amino acid sequences of exemplary human Siglec-6 proteins and domains thereof are described herein.
  • a human Siglec-6 protein comprises an extracellular domain (ECD) comprising the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEE VQEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRV MALTHRPNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTI TPRPQDHSTNLTCQVTFPGAGVTMERTIQLNVSYAPQKVAISIFQGNSAAFKILQNTSSLP VLEGQALRLLCDADGNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQ HPLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO: 120).
  • ECD extracellular domain
  • anti-Siglec-6 antibody or “an antibody that binds to human Siglec-6” refers to an antibody that binds to a polypeptide or an epitope of human Siglec-6 without substantially binding to any other polypeptide or epitope of an unrelated non-Siglec-6 polypeptide.
  • 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).
  • apoptotic bodies For example, the apoptotic activity of the bispecific antibodies (e.g., an antibody that binds to human Siglec-8 and human Siglec-6) of the present disclosure can be shown by staining cells with annexin V.
  • 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.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., natural killer (NK) cells, neutrophils and macrophages
  • NK cells natural killer cells
  • monocytes express FcyRI, FcyRII and FcyRIII.
  • an antibody e.g., a bispecific antibody that binds to human Siglec-8 and Siglec-6
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • fucosylation refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of an antibody.
  • 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.
  • 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).
  • a composition of a "fully fucosylated antibody” essentially all oligosaccharides comprise fucose residues, i.e. are fucosylated.
  • a composition of a fully fucosylated antibody has a degree of fucosylation of at least about 90%.
  • an individual antibody in such a composition typically comprises fucose residues in each of the two N-linked oligosaccharides in the Fc region.
  • 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.
  • a composition of a fully non- fucosylated antibody has a degree of fucosylation of less than about 10%.
  • 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.
  • 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%).
  • Binding affinity 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).
  • a binding affinity of an antibody for a Siglec-8 or Siglec-6 polypeptide can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.
  • Binding avidity refers to the binding strength of multiple binding sites of a molecule (e.g, an antibody) and its binding partner (e.g, an antigen).
  • 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.
  • composition 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.
  • 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.
  • 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, di saccharides, 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 TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin,
  • treatment 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., mast cell gastritis, mast cell esophagitis, mast cell colitis, mast cell enteritis, and/or mast cell gastroenteritis) are mitigated or eliminated.
  • a disease e.g., mast cell gastritis, mast cell esophagitis, mast cell colitis, mast cell enteritis, and/or mast cell gastroenteritis
  • 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.
  • conjunction with refers to administration of one treatment modality in addition to another treatment modality.
  • in conjunction with refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.
  • prevention 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.
  • bispecific antibodies e.g., an antibody that binds to human Siglec-8 and human Siglec-6 described herein are used to delay development of a disease or disorder of the present disclosure.
  • an individual “at risk” of developing a disease may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein.
  • “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of the disease (e.g., eosinophilic esophagitis), as known in the art. An individual having one or more of these risk factors has a higher probability of developing the disease than an individual without one or more of these risk factors.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the individual or subject is a human.
  • multispecific antibodies that bind (e.g., specifically bind) human Siglec-6 and human Siglec-8.
  • the multispecific (e.g., bispecific) antibodies comprise one or more antigen binding domain(s) that bind to human Siglec-6 and one or more antigen binding domain(s) that bind to human Siglec-8.
  • bispecific antibodies that comprise an antigen binding domain that binds to human Siglec-6 and an antigen binding domain that binds to human Siglec-8. Any of the exemplary antigen binding domains that bind to human Siglec-6 or human Siglec-8 described infra may find use in a bispecific and/or multispecific antibody of the present disclosure.
  • an anti-Siglec-8 antigen binding domain (or bispecific antibody comprising said antigen binding domain) 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
  • 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, a bispecific 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, a bispecific antibody described herein binds to a human Siglec-8 expressed on mast cells and inhibits mast cell-mediated activity.
  • the invention provides antigen binding domains that bind to a human Siglec-8.
  • the human Siglec-8 comprises an amino acid sequence of SEQ ID NO:72.
  • the human Siglec-8 comprises an amino acid sequence of SEQ ID NO:73.
  • the anti-Siglec-8 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain described herein binds to a linear epitope in the extracellular domain of human Siglec-8. In some embodiments, the anti-Siglec-8 antigen binding domain described herein binds to a conformational epitope in the extracellular domain of human Siglec-8.
  • a bispecific antibody described herein binds to a human Siglec-8 expressed on eosinophils and induces apoptosis of eosinophils. In some embodiments, a bispecific antibody described herein binds to a human Siglec-8 expressed on mast cells and depletes mast cells. In some embodiments, a bispecific antibody described herein binds to a human Siglec-8 expressed on mast cells and inhibits mast cell-mediated activity. In some embodiments, a bispecific antibody described herein binds to a human Siglec-8 expressed on mast cells and kills mast cells by ADCC activity. In some embodiments, a bispecific antibody described herein depletes mast cells and inhibits mast cell activation.
  • a bispecific antibody herein depletes activated eosinophils and inhibits mast cell activation.
  • a bispecific antibody herein e.g., a non-fucosylated bispecific antibody
  • an antibody herein e.g., a non-fucosylated bispecific antibody
  • an anti-Siglec-8 antigen binding domain that binds to human Siglec- 8 and non-human primate Siglec-8. Identification of antigen binding domains with primate cross-reactivity would be useful for preclinical testing in non-human primates.
  • the invention provides anti-Siglec-8 antigen binding domains that bind to a non-human primate Siglec-8.
  • the invention provides anti-Siglec-8 antigen binding domains that bind to a human Siglec-8 and a non-human primate Siglec-8.
  • the non-human primate Siglec-8 comprises an amino acid sequence of SEQ ID NO: 118 or a portion thereof.
  • the non-human primate Siglec-8 comprises an amino acid sequence of SEQ ID NO: 119 or a portion thereof.
  • the non-human primate is a baboon (e.g., Papio Anubis).
  • the anti-Siglec-8 antigen binding domain 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.
  • Domain 1 of human Siglec-8 comprises the amino acid sequence of SEQ ID NO: 112.
  • the anti-Siglec-8 antigen binding domain 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.
  • Domain 3 of human Siglec-8 comprises the amino acid sequence of SEQ ID NO: 114.
  • the anti-Siglec-8 antigen binding domain that binds to a human Siglec-8 and a non-human primate Siglec-8 is a humanized, chimeric, or a human antigen binding domain.
  • the anti- Siglec-8 antigen binding domain that binds to a human Siglec-8 and a non-human primate Siglec-8 is a murine antibody.
  • anti-Siglec-8 antigen binding domains that compete with murine 2E2 antibody and murine 2C4 antibody binding to Siglec-8 are provided.
  • Anti-Siglec-8 antigen binding domains 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.
  • anti-Siglec-8 antigen binding domains 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 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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 antigen binding domain 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 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • the anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain described herein binds to human Siglec-8 and non-human primate Siglec-8.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • the anti-Siglec-8 antigen binding domain 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 anti-Siglec-8 antigen binding domain described herein binds to human Siglec-8 and non-human primate Siglec-8.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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
  • 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • 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.
  • An anti-Siglec-8 antigen binding domain described herein may comprise any suitable framework variable domain sequence, provided that the antibody retains the ability to bind human Siglec-8.
  • heavy chain framework regions are designated “HC-FR1-FR4”
  • light chain framework regions are designated “LC-FR1-FR4.”
  • the anti-Siglec-8 antigen binding domain 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).
  • the anti-Siglec-8 antigen binding domain 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 antigen binding domain 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).
  • an anti-Siglec-8 antigen binding domain 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.
  • 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;
  • an anti-Siglec-8 antigen binding domain 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.
  • 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),
  • an anti-Siglec-8 antigen binding domain 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;
  • the HVR-L3 comprises an amino acid sequence of SEQ ID NO:66.
  • an anti-Siglec-8 antigen binding domain 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;
  • the HVR-L3 comprises an amino acid sequence of SEQ ID NO:71.
  • 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 anti-Siglec-8 antigen binding domains, 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 anti-Siglec-8 antigen binding domains, 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.
  • 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 anti-Siglec-8 antigen binding domains, 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 anti-Siglec-8 antigen binding domains, 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.
  • 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:63).
  • DGSSPYYYSMDY SEQ ID NO: 68
  • DGSSPYYYSMEV SEQ ID NO:69
  • DGSSPYYYGMDV SEQ ID NO:70
  • 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)).
  • HVR-H1 SYAMS (SEQ ID NO:88); DYYMY (SEQ ID NO:89); or SSWMN (SEQ ID NO: 90)
  • HVR-H2 IISSGGSYTYYSDSVKG (SEQ ID NO:91); RIA
  • the heavy chain FR sequences comprise the following: a) HC-FR1 (EVQLVESGGGLVQPGGSLRLSCAASGFSLT (SEQ ID NO:26);
  • 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);
  • 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)).
  • 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)).
  • 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)).
  • HVR-L1 SASSSVSYMH (SEQ ID NO:97); RASQDITNYLN (SEQ ID NO:98); or SASSSVSYMY (SEQ ID NO:99)
  • HVR-L2 DTSKLAY (SEQ ID NO: 100); FTSRLHS (SEQ ID NO: 101); or
  • the anti-Siglec-8 antigen binding domain 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
  • 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
  • an anti-Siglec-8 antigen binding domain that binds to human Siglec-8, wherein the anti-Siglec-8 antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises:
  • HC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:26-29;
  • HC-FR2 comprising the amino acid sequence selected from SEQ ID NOs:31-36;
  • HC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:38-43;
  • an HC-FR4 comprising the amino acid sequence selected from SEQ ID NOs:45-46, and/or
  • an LC-FR1 comprising the amino acid sequence selected from SEQ ID NOs:48-49;
  • an LC-FR3 comprising the amino acid sequence selected from SEQ ID NOs:55-58;
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti- Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti- Siglec-8 antigen binding domain 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.
  • the anti-Siglec-8 antigen binding domain is a human, humanized, or chimeric antigen binding domain.
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable domain of SEQ ID NO: 106 and/or comprising a light chain variable domain of SEQ ID NO: 109.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable domain of SEQ ID NO: 107 and/or comprising a light chain variable domain of SEQ ID NO: 110.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable domain of SEQ ID NO: 108 and/or comprising a light chain variable domain of SEQ ID NO: 111.
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti- Siglec-8 antigen binding domain 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.
  • 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 anti-Siglec-8 antigen binding domain comprising that amino acid sequence retains the ability to bind to human Siglec-8.
  • the substitutions, insertions, or deletions e.g., 1, 2, 3, 4, or 5 amino acids
  • an anti-Siglec-8 antigen binding domain comprises a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO:6.
  • an anti-Siglec-8 antigen binding domain comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 106-108.
  • an anti-Siglec-8 antigen binding domain 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.
  • an anti- Siglec-8 antigen binding domain 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.
  • 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 anti-Siglec-8 antigen binding domain comprising that amino acid sequence retains the ability to bind to human Siglec-8.
  • an anti-Siglec-8 antigen binding domain 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 antigen binding domain comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 109-111.
  • the present disclosure provides an anti-Siglec-8 antigen binding domain 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.
  • the present disclosure provides an anti-Siglec-8 antigen binding domain 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.
  • the present disclosure provides an anti-Siglec-8 antigen binding domain 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.
  • an anti-Siglec-8 antigen binding domain 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.
  • CDR or HVR sequences of an antibody variable domain are known in the art and may be used to describe an antibody of the present disclosure, e.g., by CDR/HVR sequences.
  • antibody CDR/HVR sequences are defined as in Kabat (see, e.g., Kabat el al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)).
  • antibody CDR/HVR sequences are defined as in Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • antibody CDR/HVR sequences are defined as in IMGT (see, e.g., Lefranc, M.P. (1999) The Immunologist 7: 132-136).
  • CDR/HVR sequences of a single antibody are defined as by mixing two or more definitions, e.g., Kabat, Chothia, and/or IMGT.
  • an anti-Siglec-8 antigen binding domain comprising a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises (i) HVR-H1 comprising the amino acid sequence of GFSLTIY (SEQ ID NO:296), (ii) HVR-H2 comprising the amino acid sequence of WAGGS (SEQ ID NO:297), and (iii) HVR-H3 comprising the amino acid sequence of DGSSPYYYSMEY (SEQ ID NO:63); and/or wherein the light chain variable region comprises (i) HVR-L1 comprising the amino acid sequence of SATSSVSYMH (SEQ ID NO:64), (ii) HVR-L2 comprising the amino acid sequence of STSNLAS (SEQ ID NO:65), and (iii) HVR-L3 comprising the amino acid sequence of QQRSSYPFT (SEQ ID NO:66).
  • the antibody HVR sequences are defined as in Cho
  • an anti-Siglec-8 antigen binding domain or bispecific 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.
  • an anti-Siglec-8 antigen binding domain or bispecific 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.
  • an anti-Siglec-8 antigen binding domain or bispecific 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.
  • the anti-Siglec-8 antigen binding domain or bispecific 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.
  • the binding affinity of the anti-Siglec-8 antigen binding domain or bispecific antibody can be determined by a surface plasmon resonance assay.
  • the Kd or Kd value can be measured by using a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ⁇ 10 response units (RU).
  • carboxymethylated dextran biosensor chips (CM5, BIAcore® Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions.
  • Capture antibodies e.g., anti-human-Fc
  • 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.
  • biolayer interferometry may be used to determine the affinity of anti-Siglec-8 antigen binding domains or bispecific antibodies against Siglec-8.
  • 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).
  • the binding affinity of the anti-Siglec-8 antigen binding domain or bispecific 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).
  • the binding avidity of the anti-Siglec-8 antigen binding domain or bispecific antibody can be determined by a surface plasmon resonance assay.
  • 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
  • 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.
  • 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).
  • a cell e.g., a mast cell
  • an anti-Siglec-8 antigen binding domain or bispecific 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).
  • an anti-Siglec-8 antigen binding domain or bispecific 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).
  • an anti-Siglec-8 antigen binding domain or bispecific 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).
  • a cell e.g., a mast cell
  • an anti-Siglec-8 antigen binding domain or bispecific antibody 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.
  • Tm melting temperature
  • 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.
  • the anti-Siglec-8 antigen binding domain or bispecific antibody has a similar or higher Tm as compared to mouse 2E2 antibody and/or mouse 2C4 antibody.
  • the anti-Siglec-8 antigen binding domain or bispecific 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.
  • the anti-Siglec-8 antigen binding domain or bispecific antibody has the same or higher Tm as compared to a chimeric 2C4 antibody.
  • the anti- Siglec-8 antigen binding domain or bispecific 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.
  • a bispecific 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.
  • a bispecific antibody described herein induces ADCC activity.
  • a bispecific antibody described herein kills eosinophils expressing Siglec-8 by ADCC activity.
  • a composition comprises non-fucosylated (j.e., afucosylated) bispecific antibodies.
  • a composition comprising non- fucosylated bispecific 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.
  • effector cells and target cells are used.
  • 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., mast cell gastritis, mast cell esophagitis, mast cell colitis, mast cell enteritis, mast cell duodenitis, and/or mast cell gastroenteritis).
  • a disease of interest e.g., mast cell gastritis, mast cell esophagitis, mast cell colitis, mast cell enteritis, mast cell duodenitis, and/or mast cell gastroenteritis.
  • 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 51 CrO4). See, e.g., Immunology, 14, 181 (1968); J. Immunol. Methods., 172, 227 (1994); and J. Immunol. Methods., 184, 29 (1995).
  • 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.
  • NK natural killer
  • 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.
  • a bispecific antibody described herein inhibits mast cell-mediated activities.
  • Mast cell tryptase has been used as a biomarker for total mast cell number and activation.
  • 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.
  • Certain aspects of the present disclosure relate to antigen binding domains that bind to human Siglec-6, i.e., anti-Siglec-6 antigen binding domains.
  • Antigen binding domains from any of the antibodies that bind to human Siglec-6 described in International Publication No. W02023044390 may find use in a bispecific antibody of the present disclosure.
  • the anti-Siglec-6 antigen binding domain is a humanized antigen binding domain that binds to Domain 1 of an extracellular domain of human Siglec-6, e.g., comprising the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEE VQEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRV MALTHR (SEQ ID NO: 121).
  • the anti-Siglec-6 antigen binding domain binds to Domain 2 of an extracellular domain of human Siglec-6, e.g., comprising the amino acid sequence PNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITPRPQDH STNLTCQVTFPGAGVTMERTIQLNVSYA (SEQ ID NO: 122).
  • the antigen binding domain is a humanized or human antigen binding domain.
  • the anti-Siglec-6 antigen binding domain binds to Domain 3 of an extracellular domain of human Siglec-6, e.g., comprising the amino acid sequence PQKVAISIFQGNSAAFKILQNTSSLPVLEGQALRLLCDADGNPPAHLSWFQGFPALNATP ISNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO: 123).
  • the antigen binding domain is a humanized or human antigen binding domain.
  • the anti-Siglec-6 antigen binding domain comprises 1, 2, 3, 4, 5, or all 6 HVR sequences of a single anti-Siglec-6 antigen binding domain as set forth in Table 6. In some embodiments, the anti-Siglec-6 antigen binding domain comprises a VH region comprising 1, 2, or all 3 HVR sequences of a VH region of a single anti-Siglec-6 antigen binding domain as set forth in Table 6. In some embodiments, the anti-Siglec-6 antigen binding domain comprises a VL region comprising 1, 2, or all 3 HVR sequences of a VL region of a single anti- Siglec-6 antigen binding domain as set forth in Table 6. In some embodiments, the anti-Siglec-6 antigen binding domain is a human, humanized, or chimeric antigen binding domain.
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:208, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:209, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:210; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:211, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:212, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:213.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:208, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:209, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:210
  • the VL region comprises an HVR-L1 comprising
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 127, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 128, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 129.
  • VH heavy chain variable
  • VL light chain variable
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:202, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:203, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:204; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:205, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:206, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:207.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:202, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:203, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:204
  • the VL region comprises an HVR-L1 comprising the amino
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 130, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 132; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 133, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 134, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 135.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 130, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 132
  • the VL region comprises an HVR-
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 196, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 197, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 198; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 199, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:200, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:201.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 196, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 197, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 198; and wherein the VL region comprises an
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 136, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 137, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 138; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 139, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14L
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 190, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 192; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 193, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 194, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 195.
  • VH heavy chain variable
  • VL light chain variable
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 172, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 173, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 174; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 175, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 176, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 177.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 172, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 173, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 174; and wherein the VL region comprises an H
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 148, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 149, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 150; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 151, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 152, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.
  • VH heavy chain variable
  • VL light chain variable
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 142, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 143, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 144; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 145, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 147.
  • VH heavy chain variable
  • VL light chain variable
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 154, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 155, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 156; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 157, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 158, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 154, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 155, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 156; and wherein the VL region
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 160, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 162; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 163, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 164, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 165.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 160, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 162
  • the VL region comprises an HVR-L1
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 166, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 167, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 168; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 169, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 170, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 171.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 166, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 167, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 168; and wherein the VL region comprises
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 178, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 179, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 180; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 181, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 182, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 183.
  • VH heavy chain variable
  • VL light chain variable
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 184, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 185, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 186; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 187, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 188, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 189.
  • VH heavy chain variable
  • VL light chain variable
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:214, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:215, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:216; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:217, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:218, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:219.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:214, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:215, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:216
  • the VL region comprises an HVR-L1 compris
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:220, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:221, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:222; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:223, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:224, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:225.
  • VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:220, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:221, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:222
  • the VL region comprises an HVR-L1 comprising
  • the anti-Siglec-6 antigen binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:226, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:227, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:228; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:229, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:230, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:23L
  • an anti-Siglec-6 antigen binding domain provided herein competes for binding to human Siglec-6 e.g., an ECD or sub-domain thereof of a human Siglec- 6 protein) with a reference antibody, e.g., an anti-Siglec-6 antibody of the present disclosure.
  • an anti-Siglec-6 antigen binding domain provided herein competes for binding to human Siglec-6 (e.g., an ECD or sub-domain thereof of a human Siglec-6 protein) with one or more of the following anti-Siglec-6 antibodies described herein: AK04, AK05, AK02, AK14, AK11, AK15, AK13, AK12, AKIO, AK09, AK08, AK07, AK06, AK03, AK01, AK16, AK17, and AK18.
  • human Siglec-6 e.g., an ECD or sub-domain thereof of a human Siglec-6 protein
  • an anti-Siglec-6 antigen binding domain provided herein competes for binding to Domain 1 of human Siglec-6 with one or more of the following anti-Siglec-6 antibodies described herein: AK04, AK05, AK02, AK07, AK06, AK03, and AK01.
  • an anti-Siglec-6 antigen binding domain provided herein competes for binding to Domain 2 of human Siglec-6 with one or more of the following anti- Siglec-6 antibodies described herein: AKIO and AK11.
  • an anti-Siglec-6 antigen binding domain provided herein competes for binding to Domain 3 of human Siglec-6 with one or more of the following anti-Siglec-6 antibodies described herein: AK09, AK08, AK12, AK13, AK14, and AK15.
  • an anti-Siglec-6 antigen binding domain described herein binds to an extracellular domain (ECD) of a human Siglec-6 protein.
  • the Siglec-6 ECD comprises the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEE VQEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRV MALTHRPNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTI TPRPQDHSTNLTCQVTFPGAGVTMERTIQLNVSYAPQKVAISIFQGNSAAFKILQNTSSLP VLEGQALRLLCDADGNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQ HPLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO: 120).
  • an anti-Siglec-6 antigen binding domain described herein binds to Domain 1, Domain 2, or Domain 3 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein). In some embodiments, an anti-Siglec-6 antigen binding domain described herein binds to Domain 1 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein).
  • Domain 1 comprises the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEE VQEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRV MALTHR (SEQ ID NO: 121).
  • an anti-Siglec-6 antigen binding domain described herein binds to Domain 2 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein).
  • Domain 2 comprises the amino acid sequence PNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITPRPQDH STNLTCQVTFPGAGVTMERTIQLNVSYA (SEQ ID NO: 122).
  • an anti-Siglec-6 antigen binding domain described herein binds to Domain 3 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein).
  • Domain 3 comprises the amino acid sequence PQKVAISIFQGNSAAFKILQNTSSLPVLEGQALRLLCDADGNPPAHLSWFQGFPALNATP ISNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO: 123).
  • an anti-Siglec-6 antigen binding domain provided herein binds the same epitope on human Siglec-6 (e.g., an ECD or sub-domain thereof of a human Siglec-6 protein) as an anti-Siglec-6 antibody of the present disclosure.
  • an anti- Siglec-6 antigen binding domain provided herein binds the same epitope on human Siglec-6 Domain 1, 2, or 3 as an anti-Siglec-6 antibody of the present disclosure.
  • CDR or HVR sequences of an antibody variable domain are known in the art and may be used to describe an antibody of the present disclosure, e.g., by CDR/HVR sequences.
  • antibody CDR/HVR sequences are defined as in Kabat (see, e.g., Kabat el al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)).
  • antibody CDR/HVR sequences are defined as in Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • antibody CDR/HVR sequences are defined as in IMGT (see, e.g., Lefranc, M.P. (1999) The Immunologist 7: 132-136).
  • CDR/HVR sequences of a single antibody are defined as by mixing two or more definitions, e.g., Kabat, Chothia, and/or IMGT.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:232 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:233.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK15 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK15 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:234 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 235.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK14 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK14 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:236 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:237.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK13 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK13 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK12 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK12 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:240 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 241.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK11 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK11 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:242 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:243.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AKIO as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AKIO as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK09 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK09 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:246 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:247.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK07 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK07 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:252 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:253.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK05 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK05 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:254 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 255.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK04 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK04 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:256 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:257.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK03 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK03 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:258 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:259.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK02 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK02 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:260 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 261.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK01 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK01 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:262 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:263.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK16 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK16 as described herein (see, e.g., Table 7).
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:264 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 265.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:266 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:267.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK18 as described herein (see, e.g., Table 7) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK18 as described herein (see, e.g., Table 7).
  • the antibody is a humanized antibody.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:238 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:239.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:240 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:241.
  • an anti-Siglec- 6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:242 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:243.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:244 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:245. In some embodiments, an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:246 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:247. In some embodiments, an anti-Siglec- 6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:248 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:249.
  • an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:256 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:257. In some embodiments, an anti-Siglec-6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:258 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:259. In some embodiments, an anti-Siglec- 6 antigen binding domain of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO:260 and/or a VL domain comprising the amino acid sequence of SEQ ID NO:261.
  • the bispecific antibody comprises an anti-Siglec-6 antigen binding domain comprising a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:208, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:209, and an HVR- H3 comprising the amino acid sequence of SEQ ID NO:210 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:211, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:212, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:213 and an anti-Siglec-8 antigen binding domain comprising a VH region comprising (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 a VL
  • the anti-Siglec-6 antigen binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:254 and a VL region comprising the amino acid sequence of SEQ ID NO:255
  • the anti-Siglec-8 antigen binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:6 and a VL region comprising the amino acid sequence of SEQ ID NO: 16 or 21.
  • an anti-Siglec-6 antigen binding domain of the present disclosure binds to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell.
  • binding of the bispecific antibody to the extracellular domain of human Siglec-6 inhibits activation of the mast cell.
  • Assays for assessing mast cell activation are known in the art and exemplified herein.
  • binding of the antibody to the extracellular domain of human Siglec-6 inhibits CD63 expression, e.g., of a human mast cell.
  • a bispecific antibody described herein inhibits one or more mast cell-mediated activities.
  • binding of the antibody to the extracellular domain of human Siglec-6 inhibits expression and/or release (e.g., by a human mast cell) of one or more of the following inflammatory mediators: proteases (e.g., pan-tryptase, active or beta-tryptase, chymase, CP A3, heparin, etc.), leukotrienes (e.g., leukotriene C4 or B4, platelet activating factor, prostaglandin D2 or E2, etc.), amines (e.g., histamine, serotonin, dopamine, polyamines, etc.), growth factors (e.g., SCF, GM-CSFG-CSF, FGF, EGF, NGF, VEGF, PDGF, etc ), cytokines (e.g., TNF, IL-lb, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13,
  • proteases
  • 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.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-6 on the cell surface (z.e., of the human mast cell). Binding of the antibody can lead to reduced levels of Siglec-6 on the cell membrane, e.g., through Siglec-6 internalization, endocytosis, shedding, cleaving, etc. Assays for assessing levels of Siglec-6 surface expression are known in the art and exemplified herein. In some embodiments, Siglec-6 surface expression is measured by flow cytometry, e.g., using an anti-Siglec-6 antibody with a detectable (e.g., fluorescent) tag.
  • a detectable e.g., fluorescent
  • mast cells can be contacted with an anti-Siglec-6 antibody, and surface expression can be measured by flow cytometry with a fluorescent-tagged anti-Siglec-6 antibody that binds to a different epitope on Siglec-6 than the test antibody.
  • Reduced fluorescence in the presence of the test antibody can indicate a reduced level of Siglec-6 surface expression.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-6 surface expression regardless of the presence/absence of an antibody Fc region, or regardless of the ability of the antibody Fc region to bind an Fc receptor (e.g., expressed on an effector cell).
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to an expression level Siglec-6 on the cell membrane that is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, e.g., as compared to surface expression of Siglec-6 in the absence of an antibody, or in the absence of an antibody that does not bind to Siglec-6.
  • binding of the bispecific antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-6 on the cell membrane (i.e., of the human mast cell) in the presence of a cell (e.g., an effector cell) expressing an Fc receptor.
  • the antibody comprises an Fc region (e.g., an active Fc region).
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 dependent upon interaction between the antibody Fc region (e.g., an active Fc region) and an Fc receptor or an effector cell, e.g., that expresses an Fc receptor.
  • the antibody Fc region e.g., an active Fc region
  • an Fc receptor or an effector cell e.g., that expresses an Fc receptor.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 only when the antibody is a full-length antibody comprising an Fc region (e.g., an active Fc region), e.g., in the presence of an Fc receptor or an effector cell (e.g., expressing an Fc receptor).
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 when the antibody is an antibody fragment, e.g., lacking an Fc region.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 when the antibody is a full-length antibody comprising an Fc region that does not bind an Fc receptor (e.g, an inactive or dead Fc region lacking effector function).
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 when the antibody is a full-length antibody comprising an Fc region (e.g, an active Fc region), e.g., in the presence of an Fc receptor or an effector cell (e.g., expressing an Fc receptor), but not when the antibody is an antibody fragment lacking an Fc region or comprising an Fc region that does not bind an Fc receptor (e.g., an inactive or dead Fc region).
  • an Fc region e.g, an active Fc region
  • an effector cell e.g., expressing an Fc receptor
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to an expression level Siglec-6 on the cell membrane that is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% when the antibody comprises an active Fc region, e.g., as compared to surface expression of Siglec-6 using an antibody that does not comprise an Fc region, or does not comprise an active Fc region.
  • binding of the bispecific antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces dimerization and/or internalization of Siglec-6.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces dimerization and internalization of Siglec-6.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces endocytosis of Siglec-6.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces shedding of Siglec-6 (e.g, the Siglec-6 ECD). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces cleaving of Siglec-6 (e.g., the Siglec-6 ECD).
  • a bispecific antibody described herein depletes mast cells expressing human Siglec-6 in vitro and/or in vivo, e.g., in the presence of effector cells.
  • binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) activity, e.g., in the presence of effector cells in vitro and/or in vivo.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • an anti-Siglec-6 antibody described herein kills mast cells expressing Siglec-6 by ADCC activity in vitro and/or in vivo.
  • a bispecific antibody described herein induces phagocytosis of mast cells expressing Siglec-6 and/or Siglec-8 by ADCP activity in vitro and/or in vivo.
  • a composition comprises non-fucosylated (i.e., afucosylated) bispecific antibodies.
  • a composition comprising non-fucosylated bispecific antibodies described herein enhances ADCC activity as compared to a composition comprising partially fucosylated bispecific antibodies.
  • Assays for assessing ADCC activity are well known in the art and described herein.
  • effector cells and target cells are used, e.g., at a specific ratio (e.g, 1 :50 target cells: effector cells) in the presence of an antibody to be evaluated.
  • 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.
  • the target cell is any cell which expresses on the cell surface antigens that antibodies to be evaluated can recognize.
  • target cell is a mast cell which expresses Siglec-6 on the cell surface.
  • Target cells are labeled with a reagent that enables detection of cytolysis.
  • reagents for labeling include a radio-active substance such as sodium chromate (Na2 51 CrO4) and carboxyfluorescein succinimidyl ester. See, e.g., Immunology, 14, 181 (1968); J. Immunol. Methods, 172, 227 (1994); and J. Immunol. Methods, 184, 29 (1995).
  • the number of remaining target cells can then be assessed (e.g., by flow cytometry) after co-incubation and normalized (e.g., to number of remaining target cells co-incubated with effector cells treated with an antibody that does not bind to Siglec-6 or treated with no antibody).
  • target cells can be co-cultured with macrophages (e.g., human monocyte-derived macrophages), monocytes, or PBMCs at a specific ratio (e.g., 1 :50 target cells: macrophages) in the presence of an antibody to be evaluated.
  • macrophages e.g., human monocyte-derived macrophages
  • monocytes e.g., monocytes
  • PBMCs e.g., PBMCs at a specific ratio (e.g., 1 :50 target cells: macrophages) in the presence of an antibody to be evaluated.
  • the number of remaining target cells can then be assessed (e.g., by flow cytometry) after co-incubation and normalized (e.g., to number of remaining target cells co-incubated with macrophages treated with an antibody that does not bind to Siglec-6, or treated with no antibody).
  • binding of a bispecific antibody of the present disclosure to an extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of Siglec-6 on the cell surface.
  • binding of a bispecific antibody of the present disclosure to an extracellular domain of human Siglec-6 inhibits activation of a mast cell that expresses the human Siglec-6.
  • binding of a bispecific antibody of the present disclosure to an extracellular domain of human Siglec-8 when expressed on a surface of a human mast cell leads to a reduced level of Siglec-8 on the cell surface.
  • binding of a bispecific antibody of the present disclosure to an extracellular domain of human Siglec-8 when expressed on a surface of a human eosinophil leads to a reduced level of Siglec-8 on the cell surface.
  • binding of a bispecific antibody of the present disclosure to an extracellular domain of human Siglec-8 inhibits activation of a mast cell that expresses the human Siglec-8.
  • an antigen binding domain of the present disclosure is a singledomain antigen-binding domain.
  • a single-domain antigen-binding domain 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.
  • a singledomain antigen-binding domain is a human single-domain antigen-binding domain (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl).
  • a single- domain antigen-binding domain consists of all or a portion of the heavy chain variable domain of an antigen-binding domain.
  • amino acid sequence modification(s) of the antigen-binding domains described herein are contemplated.
  • 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.
  • a useful method for identification of certain residues or regions of the antigen-binding domain that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • variants are 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.
  • 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)):
  • 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.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody
  • 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).
  • scanning mutagenesis e.g., alanine scanning
  • hypervariable region residues contributing significantly to antigen binding.
  • 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.
  • 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.
  • a bispecific antibody described herein is an antibody fragment (including antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab ' ⁇ fragment.
  • a bispecific antibody described herein comprises an antibody fragment (including antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab ' ⁇ fragment.
  • an antibody fragment including antigen-binding fragment
  • Fab fragment
  • Fab'-SH fragment
  • Fv fragment
  • scFv fragment
  • Fab ' ⁇ fragment fragment
  • any of the bispecific antibodies described herein are purified.
  • IgA immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • IgG immunoglobulins
  • IgG2 immunoglobulins
  • IgG3 immunoglobulins
  • IgA2 immunoglobulins
  • IgA2 immunoglobulins
  • IgG3 immunoglobulins
  • IgA2 immunoglobulins
  • 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.
  • the bispecific antibody may comprise a heavy chain Fc region, e.g., an Fc region comprising a human IgG Fc region.
  • the human IgG Fc region comprises a human IgGl or IgG4.
  • the bispecific antibody is an IgGl antibody.
  • the bispecific antibody is an IgG2 antibody.
  • the bispecific antibody is an IgG4 antibody.
  • the human IgG4 comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat.
  • 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. [0188] 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.
  • the Fc region variant comprises a human IgG4 Fc region.
  • 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.
  • 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.
  • the antibody may comprise one or more Fc mutations that extend half-life, e.g., in vivo.
  • 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 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 human IgGl Fc region comprises one or more mutation(s) that reduce effector function.
  • the human IgGl Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) L234 and/or L235; (b) A327, A330, and/or P331; (c) E233, L234, L235, and/or G236; (d) E233, L234, and/or L235; (e) E233, L234, L235, G236, A327, A330, and/or P331; (f) E233, L234, L235, A327, A330, and/or P331; (g) N297; (h) L242, N297, and/or K334; (i) A287, N297, and/
  • the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index: (a) L234A and/or L235A; (b) A327G, A330S, and/or P33 IS; (c) E233P, L234V, L235A, and/or G236del; (d) E233P, L234V, and/or L235A; (e) E233P, L234V, L235A, G236del, A327G, A330S, and/or P331S; (f) E233P, L234V, L235A, A327G, A330S, and/or P33 IS; (g) N297A; (h) N297G; (i) N297Q; (j) L242C, N297C, and/or K334C; (k) A287C, N297G, and/or L306C; (1) R292
  • the antibody heavy chain comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:268 or 269.
  • the human IgGl Fc region comprises one or more mutation(s) that increase or enhance effector function.
  • the human IgGl Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) F243, R292, Y300, V305, and/or P396; (b) S239 and/or 1332; (c) S239, 1332, and/or A330; (d) S298, E333, and/or K334; (e) G236, S239, and/or 1332; (f) K326 and/or E333; (g) S267, H268, and/or S324; or (h) E345, E430, and/or S440.
  • the human IgGl Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) F243L, R292P, Y300L, V305I, and/or P396L; (b) S239D and/or I332E; (c) S239D, I332E, and/or A330L; (d) S298A, E333A, and/or K334A; (e) G236A, S239D, and/or I332E; (f) K326W and/or E333S; (g) S267E, H268F, and/or S324T; or (h) E345R, E430G, and/or S440Y.
  • the human IgG2 Fc region comprises one or more mutation(s) that reduce effector function.
  • the human IgG2 Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) A330 and/or P331; (b) V234, G237, P238, H268, V309, A330, and/or P331; or (c) V234, G237, H268, V309, A330, P331, C232, C233, S267, L328, M252, S254, and/or T256.
  • the human IgG2 Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) A330S and/or P331S; (b) V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S; or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and/or T256E. See, e.g, Armour, K.L. et al. (2003) Afo/. Immunol.
  • the human IgG4 Fc region comprises one or more mutation(s) that reduce effector function.
  • the human IgG4 Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) E233, F234, L235, and/or G236; (b) E233, F234, and/or L235; or (c) S228 and/or L235.
  • the human IgG4 Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) E233P, F234V, L235A, and/or G236del; (b) E233P, F234V, and/or L235A; (c) S228P and/or L235E; or (d) S228P and/or L235A.
  • EU index e.g., Schlothauer, T. et al. (2016) Protein Eng. Des. Sei. 29:457-466; and Armour, K.L. et al. (2003) Mol. Immunol. 40:585-593.
  • the antibody heavy chain comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:270.
  • a bispecific antibody of the present disclosure comprises an antibody light chain comprising a light chain constant (CL) domain, e.g., a human kappa or lambda CL domain.
  • CL domain is a human kappa CL domain.
  • the CL domain comprises the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:271).
  • the bispecific antibody comprises a first antibody heavy chain comprising a first VH region and a first Fc region, a first antibody light chain comprising a first VL region, a second antibody heavy chain comprising a second VH region and a second Fc region, and a second antibody light chain comprising a second VL region; wherein the first VH and VL regions form the first antigen binding domain; and wherein the second VH and VL regions form the second antigen binding domain.
  • one or both heavy chains comprises a modification to favor heterodimerization of the heavy chains (e.g., association of one arm comprising one antigen binding domain and another arm comprising a different antigen binding domain) and/or disfavor homodimerization of the heavy chains (e.g., association of two arms comprising the same antigen binding domain).
  • an “arm” of a bispecific antibody may refer to a half-antibody comprising the antibody heavy chain and antibody light chain that makes up one of the antigen binding domains of the present disclosure, e.g., in which the VH region of the heavy chain and VL region of the light chain form an anti-Siglec-6 or anti-Siglec-8 antigen binding domain of the present disclosure.
  • a bispecific antibody of the present disclosure comprises two arms: one forming an anti-Siglec-6 antigen binding domain of the present disclosure, and the other forming an anti-Siglec-8 antigen binding domain of the present disclosure.
  • Another such format has been described as the DuetMab format (Medimmune LLC). See, e.g., U.S. Pat. No. 9,527,927.
  • this format one or more interchain cysteines have been relocated which, in some aspects, results in the relocation of an interchain disulphide linkage at the HC-LC interface.
  • this involves modification of one heavy chain and the corresponding light chain within an antibody whereby a native cysteine is substituted by a noncysteine amino acid, and a native non-cysteine amino acid is substituted by a cysteine amino acid.
  • the antibodies provided herein are bispecific which means they contain variable domains with different antigen and/or epitope specificities.
  • the modified heavy and light chain duplex for a given antibody contains a variable domain with a certain antigen specificity and the unmodified heavy and light chain duplex within the same antibody contains a variable domain with a different antigen specificity.
  • Another format with engineered disulfide bonds between heavy and light chains is the BIClonal format; see, e.g., Litvak-Greenfeld, D. et al. (2019) Methods Mol Biol 1904:431-454.
  • one of the antibody heavy chains comprises a first constant heavy chain (CHI) region that comprises a substitution of a native cysteine to a non-cysteine amino acid and a substitution of a native non-cysteine amino acid to a cysteine amino acid
  • the corresponding antibody light chain comprises a constant light chain (CL) region that comprises a substitution of a native cysteine to a non-cysteine amino acid and a substitution of a native non-cysteine amino acid to a cysteine amino acid, such that the substituted cysteines on the CHI region and the CL region on the corresponding heavy and light chains form a disulfide bond.
  • CHI constant heavy chain
  • CL constant light chain
  • the other antibody heavy chain and antibody light chain do not contain said substitutions.
  • the arm comprising the anti-Siglec-6 antigen binding domain comprises the substituted CHI and CL regions, and the arm comprising the anti- Siglec-8 antigen binding domain does not.
  • the arm comprising the anti- Siglec-8 antigen binding domain comprises the substituted CHI and CL regions, and the arm comprising the anti-Siglec-6 antigen binding domain does not.
  • An example of this format is provided in FIG. 1A.
  • the substituted CHI region comprises an F126C substitution
  • the substituted CL region comprises an S121C substitution
  • the substituted CHI region further comprises a C220V substitution
  • the substituted CL region further comprises a C214V substitution.
  • CrossMab format Another such format has been described as the CrossMab format (Roche). See, e.g., International Publication No. W02009080253.
  • this format the CHI and CL regions in one arm are replaced by each other, leaving the CHI region in the light chain and the CL region in the heavy chain, while the other arm retains its native configuration.
  • the ratio of a desired bivalent, bispecific antibody compared to undesired side products can be improved by the replacement of certain domains in only one pair of heavy chain and light chain (HC/LC).
  • the second of the two HC/LC pairs originates from an antibody specifically binding to a second antigen , and is altered by the following replacement: light chain: replacement of the constant light chain domain CL by the constant heavy chain domain CHI of said antibody specifically binding to a second antigen , and heavy chain: replacement of the constant heavy chain domain CHI by the constant light chain domain CL of said antibody specifically binding to a second antigen.
  • bivalent, bispecific antibodies are artificial antibodies which comprise a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen; wherein said light chain (of an antibody specifically binding to a second antigen) contains a constant domain CHI instead of CL wherein said heavy chain(of an antibody specifically binding to a second antigen) a constant domain CL instead of CHI.
  • the arm comprising the anti-Siglec-6 antigen binding domain comprises an antibody heavy chain that comprises a constant light chain (CL) region that replaces a native first constant heavy chain (CHI) region and an antibody light chain that comprises a CHI region that replaces a native CL region, and the arm comprising the anti- Siglec-8 does not.
  • the arm comprising the anti-Siglec-8 antigen binding domain comprises an antibody heavy chain that comprises a constant light chain (CL) region that replaces a native first constant heavy chain (CHI) region and an antibody light chain that comprises a CHI region that replaces a native CL region, and the arm comprising the anti- Siglec-6 does not. Examples of these formats are illustrated in FIG. IB.
  • the light chain on one arm comprises a Q124E
  • the heavy chain on the other arm comprises K147E and K213E substitutions and its corresponding light chain comprises E123K and Q124K substitutions.
  • bispecific antibodies One approach known in the art for making bispecific antibodies is the “knobs-into- holes” or “protuberance-into-cavity” approach (see, e.g., US Pat. No. 5,731,168 and Ridgway, J.B. et al. (1996) Protein Eng 9(7):617-621).
  • two immunoglobulin polypeptides e.g., heavy chain polypeptides each comprise an interface.
  • An interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide (e.g., the heavy chain of the other arm of bispecific antibody of the present disclosure), thereby allowing the two immunoglobulin polypeptides to associate in an assembled, heterodimeric bispecific antibody.
  • These interfaces may be engineered such that a “knob” or “protuberance” (these terms may be used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds with a “hole” or “cavity” (these terms may be used interchangeably herein) located in the interface of the other immunoglobulin polypeptide.
  • the hole is of identical or similar size to the knob and suitably positioned such that when the two interfaces interact, the knob of one interface is positionable in the corresponding hole of the other interface.
  • this is thought to stabilize the heteromultimer and favor formation of the heteromultimer over other species, for example homomultimers.
  • this approach may be used to promote the heteromultimerization of two different immunoglobulin polypeptides, creating a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes. Examples of this approach are shown in FIGS. 1A-1C.
  • a knob may be constructed by replacing a small amino acid side chain with a larger side chain.
  • a hole may be constructed by replacing a large amino acid side chain with a smaller side chain.
  • Knobs or holes may exist in the original interface, or they may be introduced synthetically. For example, knobs or holes may be introduced synthetically by altering the nucleic acid sequence encoding the interface to replace at least one “original” amino acid residue with at least one “import” amino acid residue.
  • Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art.
  • the side chain volumes of various amino acid residues are shown in the following table.
  • original residues have a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and import residues for forming a knob are naturally occurring amino acids and may include arginine, phenylalanine, tyrosine, and tryptophan.
  • original residues have a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and import residues for forming a hole are naturally occurring amino acids and may include alanine, serine, threonine, and valine.
  • a large side chain volume e.g., arginine, phenylalanine, tyrosine, and tryptophan
  • import residues for forming a hole are naturally occurring amino acids and may include alanine, serine, threonine, and valine.
  • 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.
  • 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.
  • 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).
  • the carbohydrate attached thereto may be altered.
  • 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 (Kyowa Hakko 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.
  • a glycosylation variant comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose.
  • Such variants have improved ADCC function.
  • 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 1,4-N- acetylglycosminyltransferase III (GnT-III) and Golgi p-mannosidase II (Manll).
  • knockout cell lines such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane- Ohnuki et al
  • 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.
  • 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).
  • native CHO cells e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene.
  • 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.
  • 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.
  • the antibody is altered to improve its serum half-life.
  • a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example.
  • 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).
  • bispecific antibody described herein is prepared using techniques available in the art for generating bispecific antibodies, exemplary methods of which are described in more detail infra.
  • Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens.
  • bispecific antibodies are human or humanized antibodies.
  • one of the binding specificities is for Siglec-8 and the other is for Siglec-6.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Siglec-8 and/or Siglec-6.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2bispecific antibodies).
  • Bispecific antibodies include cross-linked or “heteroconjugate” antibodies.
  • 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.
  • all four polypeptides comprising a bispecific antibody are produced in the same cell.
  • the heavy chain and light chain forming one arm are produced in one cell, the heavy chain and light chain forming the other arm are produced in a different cell, and the resulting arms are purified separately, then joined, e.g., via reduction.
  • knob- and hole-bearing immunoglobulin polypeptides may be expressed in host cells in co-culture and purified together as a heteromultimer, or they may be expressed in single cultures, separately purified, and assembled in vitro.
  • two strains of bacterial host cells one expressing an immunoglobulin polypeptide with a knob, and the other expressing an immunoglobulin polypeptide with a hole
  • the two strains may be mixed in a specific ratio, e.g., so as to achieve equal expression levels in culture.
  • bispecific antibodies may be purified using standard techniques including cationexchange chromatography and measured using standard techniques including size exclusion chromatography. For a more detailed description of these methods, see Speiss et al., Nat Biotechnol 31 :753-8, 2013.
  • modified immunoglobulin polypeptides may be expressed separately in CHO cells and assembled in vitro using the methods described above.
  • one of the two arms of the bispecific antibody comprises a heavy chain that comprises H435R and Y436F substitutions (numbering according to EU index).
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:281, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:282, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:278, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:279.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:287, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:288.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:293, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:294, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:290, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:291.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:281, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:282 or 283, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:278, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:279 or 280.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285 or 286, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:287, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:288 or 289.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:293, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:294 or 295, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:290, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:291 or 292.
  • a bispecific antibody comprising a first antibody light chain comprising the amino acid sequence of SEQ ID NO:284, a first antibody heavy chain comprising the amino acid sequence of SEQ ID NO:285 or 286, a second antibody light chain comprising the amino acid sequence of SEQ ID NO:298, and a second antibody heavy chain comprising the amino acid sequence of SEQ ID NO:299 or 300.
  • 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.
  • 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.
  • a single polynucleotide encodes all 4 polypeptide chains for a bispecific antibody of the present disclosure.
  • a single polynucleotide encodes the heavy chain and light chain of one arm, and another polynucleotide encodes the heavy chain and light chain of the other arm.
  • each of the 4 polypeptide chains for the bispecific antibody are encoded by a separate polynucleotide.
  • a kit comprising two polynucleotides, each encoding an arm (e.g., encoding a heavy chain polypeptide and corresponding light chain polypeptide) of a bispecific antibody of the present disclosure.
  • a kit comprising four polynucleotides, each encoding one of the four polypeptides of a bispecific antibody of the present disclosure.
  • 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.
  • 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.
  • 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.
  • 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.
  • bacteriophage such as ZGEM.TM.-l 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • 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.
  • Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the P- galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter.
  • trp tryptophan
  • other promoters that are functional in bacteria such as other known bacterial or phage promoters
  • 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.
  • each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane.
  • 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.
  • 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.
  • STII heat-stable enterotoxin II
  • LamB, PhoE, PelB, OmpA and MBP are STII signal sequences or variants thereof.
  • 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.
  • TIR translational initiation region
  • 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-Dalgarno 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.
  • 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.
  • Prokaryotic host cells suitable for expressing antibodies of the present disclosure include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms.
  • 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.
  • gram-negative cells are used.
  • E. coli cells are used as hosts for the present disclosure. Examples ofE.
  • 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. colik 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.
  • 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.
  • the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
  • 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.
  • 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.
  • suitable media include luria broth (LB) plus necessary nutrient supplements.
  • 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.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • 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.
  • 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.
  • PhoA promoters are used for controlling transcription of the polypeptides.
  • the transformed host cells are cultured in a phosphate-limiting medium for induction.
  • 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.
  • 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.
  • PAGE polyacrylamide gel electrophoresis
  • 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 desired density e.g., an OD550 of about 180-220
  • 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.
  • various fermentation conditions can be modified.
  • 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.
  • 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.
  • the antibody of interest is recovered from the solid phase by elution.
  • 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.
  • 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.
  • an origin of replication component is not needed for mammalian expression vectors.
  • the SV40 origin may typically be used only because it contains the early promoter.
  • Selection genes 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.
  • 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.
  • 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.
  • 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.
  • Mtx methotrexate
  • 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).
  • host cells 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.
  • a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418.
  • 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.
  • 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.
  • any or all of these sequences may be suitably inserted into eukaryotic expression vectors.
  • 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.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,
  • 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.
  • 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.
  • 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.
  • 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.
  • COS-7 monkey kidney CV1 line transformed by SV40
  • 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
  • mice 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 3 A, 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).
  • 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
  • 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.
  • 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 GENTAMYCINTM 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.
  • 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 Amicon 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.
  • 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.
  • 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 ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • 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).
  • one of the two arms of the bispecific antibody comprises a heavy chain that comprises H435R and Y436F substitutions (numbering according to EU index).
  • these substitutions can allow for easier purification of the correctly assembled, heterodimeric bispecific antibody over homodimeric forms using protein A affinity chromatography; see, e.g., Tustian, A. et al. (2016)A 4fe 8(4): 828-838.
  • methods for preparing bispecific antibodies with a reduced degree of fucosylation 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-acetylglycosminyltransf erase 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.
  • Glycosidase inhibitors include a-glucosidase I, a-glucosidase II, and a-mannosidase I.
  • the glycosidase inhibitor is an inhibitor of a-mannosidase I (e.g., kifunensine).
  • 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-glycoside-linked sugar chains bound to the Fc region (or domain) is reduced. As used herein, a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the number of Kabat), although a complex N-glycoside linked sugar chain can also be linked to other asparagine residues.
  • a “complex N-glycoside-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-glycoside-linked sugar chain and complex N-glycoside-linked sugar chain.
  • 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
  • the “complex N-glycoside-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.
  • the present methods typically only a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s).
  • a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s).
  • 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.
  • substantially none (i.e., less than about 0.5%) of the antibody has core fucosylation by fucose in a composition.
  • 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.
  • a bispecific antibody wherein substantially none (i.e., less than about 0.5%) of the N-glycoside-linked carbohydrate chains contain a fucose residue. In some embodiments, provided herein is a bispecific antibody wherein at least one or two of the heavy chains of the antibody is/are 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.
  • an “effective amount” refers to an amount of the analog that is sufficient to decrease fucose incorporation into a complex N-glycoside-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%.
  • 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 fucosylated protein (e.g., lacking core fucosylation), as compared with antibodies produced from the host cells cultured in the absence of a fucose analog.
  • 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.
  • 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.).
  • the methods comprise administering to a subject or individual (e.g., in need thereof) an effective amount of a bispecific antibody of the present disclosure or a composition (e.g., a pharmaceutical composition) of the present disclosure.
  • the individual is a human.
  • a method for treating a disease or condition characterized by increased activity and/or number of mast cells expressing Siglec-6 and/or Siglec-8 comprising administering to a subject or individual (e.g., in need thereof) an effective amount of a bispecific antibody of the present disclosure or a composition (e.g., a pharmaceutical composition) of the present disclosure.
  • a method for inhibiting activation of mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof comprising administering to a subject or individual an effective amount of a bispecific antibody of the present disclosure or a composition (e.g., a pharmaceutical composition) of the present disclosure.
  • a method for depleting mast cells expressing Siglec-6 and/or Siglec-8 in a subject in need thereof comprising administering to a subject or individual an effective amount of a bispecific antibody of the present disclosure or a composition (e.g., a pharmaceutical composition) of the present disclosure.
  • provided herein is a method for treating a disease or condition mediated by cells expressing Siglec-6 and/or Siglec-8 in a subject, comprising administering to a subject or individual (e.g., in need thereof) an effective amount of a bispecific antibody of the present disclosure or a composition (e.g., a pharmaceutical composition) of the present disclosure.
  • a variety of diseases/disorders are contemplated for treatment using the methods of the present disclosure. Exemplary diseases/disorders may be found, e.g., in U.S. Pat. No. 9,546,215 and/or International Publication Nos. W02015089117, W02022087610, and W02023044390.
  • the disease or disorder is mast cell-mediated. In some embodiments, the disease or disorder is eosinophil-mediated.
  • the subject or individual has or has been diagnosed with mastocytosis, mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs.
  • NASH nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • GSH host disease
  • colitis hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria, atopic dermatitis, contact dermatitis, angioedema, pruigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma, allergy, keloid, chronic rhinosinusitis, aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic
  • the subject or individual has or has been diagnosed with allergic rhinitis, nasal polyposis, pauci granulocytic asthma, acute or chronic airway hypersensitivity, eosinophilic esophagitis, eosinophilic leukemia, Churg-Strauss syndrome, inflammation associated with a cytokine, inflammation associated with cells expressing Siglec- 8, malignancy associated with cells expressing Siglec-8, physical urticaria, cold urticaria, pressure-urticaria, bullous pemphigoid, food allergy, chronic rhinosinusitis with concomitant asthma, aspirin-exacerbated respiratory disease, adult onset non-atopic asthma with sinus disease, chronic obstructive pulmonary disease, fibrotic disease, pre-fibrotic disease, mastocytosis, advanced systemic mastocytosis, indolent systemic mastocytosis (ISM), inflammatory bowel disease (IBD), eosinophilic esophagitis (EOE),
  • the disease or disorder is mastocytosis, mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs.
  • NASH nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • GSH host disease
  • colitis hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria, atopic dermatitis, contact dermatitis, angioedema, pruigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma, allergy, keloid, chronic rhinosinusitis, aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hi dradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic
  • the disease or disorder is allergic rhinitis, nasal polyposis, pauci granulocytic asthma, acute or chronic airway hypersensitivity, eosinophilic esophagitis, eosinophilic leukemia, Churg-Strauss syndrome, inflammation associated with a cytokine, inflammation associated with cells expressing Siglec-8, malignancy associated with cells expressing Siglec-8, physical urticaria, cold urticaria, pressure-urticaria, bullous pemphigoid, food allergy, chronic rhinosinusitis with concomitant asthma, aspirin-exacerbated respiratory disease, adult onset non-atopic asthma with sinus disease, chronic obstructive pulmonary disease, fibrotic disease, pre-fibrotic disease, mastocytosis, advanced systemic mastocytosis, indolent systemic mastocytosis (ISM), inflammatory bowel disease (IBD), eosinophilic esophagitis (EOE), eosin
  • administration of a composition or bispecific antibody of the present disclosure results in a decrease in one or more symptoms in the subject, e.g., as compared to a reference, reference value, or the one or more symptoms in the subject prior to the administration.
  • exemplary symptoms include, without limitation, nausea, cramping, constipation, abdominal pain, bloating, vomiting, diarrhea, fatigue, eye pain, light sensitivity, redness, discharge, runny nose, headache, dizziness, brain fog, itching, flushing, sweating, hives, hypotension, shortness of breath, bone pain, joint pain, weight loss, osteoporosis, angioedema, chest pain, anxiety, depression, rapid heartbeat, bronchoconstriction, and general pain.
  • administration of a composition or bispecific antibody of the present disclosure results in a sustained response to treatment. In some embodiments, administration of a composition or antibody of the present disclosure results in a complete response to treatment (e.g., after cessation of treatment, or after a single dose of the antibody or composition).
  • reference or “reference value” used interchangeably herein can refer to a measurement or characterization of a value or symptom in an individual.
  • 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.
  • 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).
  • a reference value refers to a standard or benchmark value in the field.
  • a reference value refers to a value calculated de novo from one or more individuals.
  • 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. 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 can be assessed using any of the methodologies or assays described herein at intervals ranging between every week and every three months.
  • efficacy of treatment 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.
  • efficacy of treatment 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.
  • Bispecific antibodies described herein can be used either alone or in combination with other agents in the methods described herein.
  • a bispecific antibody may be coadministered with one or more (e.g., one or more, two or more, three or more, four or more, etc.) additional therapeutic agents.
  • additional therapeutic agents e.g., one or more, two or more, three or more, four or more, etc.
  • 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.
  • compositions comprising any of the bispecific antibodies described herein.
  • a composition comprising a bispecific antibody described herein, wherein the antibody comprises a Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, wherein less than about 50% of the N-glycoside-linked carbohydrate chains contain a fucose residue.
  • the antibody comprises a Fc region and N-glycoside- 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-glycoside-linked carbohydrate chains contain a fucose residue.
  • a composition comprising a bispecific antibody described herein, wherein the antibody comprises a Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, wherein substantially none of the N-glycoside-linked carbohydrate chains contain a fucose residue.
  • 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 & Wikiins, 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.
  • 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.
  • Tonicity agents can be present to adjust or maintain the tonicity of liquid in a composition.
  • stabilizers 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.
  • tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • 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.
  • 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., inosi
  • Non-ionic surfactants or detergents 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.
  • 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.
  • the formulations 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.
  • 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., inj ection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.
  • 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.
  • active compounds are suitably present in combination in amounts that are effective for the purpose intended.
  • an article of manufacture or kit which comprises a bispecific antibody described herein.
  • the article of manufacture or kit may further comprise instructions for use of the antibody in the methods of the present disclosure.
  • the article of manufacture or kit comprises instructions for the use of a bispecific antibody that binds to human Siglec-8 and human Siglec-6 in methods of the present disclosure.
  • the article of manufacture comprises a medicament comprising a bispecific antibody of the present disclosure and a package insert comprising instructions for administration of the medicament in an individual in need thereof according to any of the methods disclosed herein.
  • the package insert further indicates that the treatment is effective in reducing one or more symptoms in the individual as compared to a baseline level before administration of the medicament.
  • the individual is a human.
  • 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.
  • 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.
  • 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.
  • kits for a single doseadministration unit 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.
  • 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.
  • Fresh human lung tissue was procured and provided by the National Cancer Institute (NCI) Cooperative Human Tissue Network (CHTN) from subjects with no previous history of chronic disease.
  • Human tissues were enzymatically and mechanically dissociated using the gentleMACsTM Dissociator (Miltenyi Biotec), according to manufacturer’s protocol. After the last digestion, the tissue was filtered through a 70um filter and red blood cells were lysed using RBC lysis buffer (Biolegend). Single cells were then washed and resuspended into RPMI media with 10% super low IgG FBS and P/S. Immediately after digestion, cell viability was examined using flow cytometry. From dissociated tissues, only single-cell suspensions that had at least 70% viability were used in subsequent experiments.
  • Mouse peripheral blood eosinophils were identified as CD45+ 7AAD- CD1 lb+ Ly6C- Ly6G- SiglecF+. Data acquisition was performed using a NovoCyte flow cytometer and FlowJo was used for data analysis.
  • Human lung mast cells were cultured with varying concentrations of the bispecific Siglec-6/8 huIgGl, anti-Siglec-6 huIgGl (comprising the VH and VL regions of SEQ ID NO:254 and 255, respectively), anti-Siglec-8 afucosylated huIgGl (comprising the VH and VL regions of SEQ ID NO:6 and 16, respectively), or an isotype control for 18h at 37°C. Internalization was measured by FACS using a fluorescent-tagged Siglec-6 or Siglec-8 mAb that binds to a different epitope on Siglec-6 or Siglec-8.
  • MFI Median fluorescence intensity
  • Human lung mast cells were stimulated with an anti-FcsRI (lOpg/mL) in the presence of the bispecific Siglec-6/8 huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 huIgGl, or an isotype control at indicated concentrations.
  • Bound antibodies were crosslinked with 20 pg/mL secondary antibody AffiniPure F(ab')2 Fragment Goat anti-human IgG heavy chain and light chain (H+L). Cells were incubated for 20 min at 37°C. Activation was assessed by flow cytometry using the activation marker CD63.
  • Siglec-6/ Siglec-8 double transgenic mice were dosed intraperitoneally with 5mg/kg of bispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 afucosylated huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control for 24h.
  • Blood was processed through two rounds of red blood cell lysis, according to manufacturer instructions (Biolegend). The cell pellet was resuspended in RPMI complete media. Internalization was measured by FACS using a fluorescent-tagged Siglec-8 mAb that binds to a different epitope on Siglec-8.
  • bispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 afucosylated huIgGl, and anti-Siglec-8 afucosylated huIgGl antibodies induced Siglec-8 internalization on eosinophils (FIG. 4A) as well as eosinophil depletion (FIG. 4B).
  • human peripheral blood NK cells were purchased from StemCell. The day prior to the ADCC assay, the cells were thawed and cultured overnight in media containing interleukin-2 (5 ng/mL). The next day, human blood eosinophils were purified using a human eosinophil isolation kit from StemCell.
  • Eosinophils were plated in the presence of NK cells at a ratio of 9: 1 NK: eosinophils and cultured for 18h at 37°C with specified concentrations of the bispecific Siglec-6/8 huIgGl, bispecific Siglec 6/8 afucosylated huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control. After incubation, cells were resuspended in AnnexinV binding buffer with AnnexinV and 7AAD. Cells were stained in the dark at room temperature for 30 min and then data were acquired by flow cytometry.
  • Siglec-6/Siglec-8 double transgenic mice were intraperitoneally dosed with 5mg/kg of bispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 afucosylated huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control for 24h.
  • the stomachs were collected, and the intraepithelial layer was released by incubation in Hank’s Balanced Salt Solution (HBSS) without magnesium or calcium (Gibco), with 5 mM EDTA and 5% FBS and passed through a 70um filter.
  • HBSS Hank’s Balanced Salt Solution
  • Gabco magnesium or calcium
  • Example 5 Bispecific Siglec-6/8 antibody inhibits tissue mast cell activation in vivo and ex vivo
  • Siglec-6/Siglec-8 double transgenic mice were intraperitoneally dosed with 5mg/kg of bispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 afucosylated huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control for 24h.
  • the stomachs were collected, and the intraepithelial layer was released by incubation in Hank’s Balanced Salt Solution (HBSS) without magnesium or calcium (Gibco), with 5 mM EDTA and 5% FBS and passed through a 70um filter.
  • HBSS Hank’s Balanced Salt Solution
  • Gabco magnesium or calcium
  • the anti-Siglec-6 huIgGl, bispecific Siglec-6/8 huIgGl, and the bispecific Siglec-6/8 afucosylated huIgGl antibodies induced mast cell inhibition as assessed by reduced expression of the activation marker CD63 on tissue mast cells (FIG. 6A).
  • Siglec-6/Siglec-8 double transgenic mice were intraperitoneally dosed with lOmg/kg ofbispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 afucosylated huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 huIgGl, or an isotype control for 7 days.
  • Mice were sacrificed, and the peritoneal mast cells were harvested. The cells were treated with biotinylated anti-FcsRI and crosslinked with 5 pg/mL Neutravidin. Cells were incubated for 20 min at 37°C.
  • Activation was assessed by flow cytometry using the activation marker CD63.
  • the anti-Siglec-6 huIgGl, bispecific Siglec-6/8 huIgGl, and bispecific Siglec-6/8 afucosylated huIgGl antibody treatments induced mast cell inhibition as assessed by reduced expression of the activation marker CD63 on tissue mast cells (FIG. 6B).
  • CD14 + Monocytes were isolated using the EasySep Human Monocyte Isolation kit from healthy blood donors. Monocytes were cultured in the presence of 50 ng/mL macrophage colony stimulating-factor (M-CSF) and differentiated for 6-8 days. Macrophages were harvested by treating with TrypLE solution for 5 minutes to release cells.
  • M-CSF macrophage colony stimulating-factor
  • Human lung tissue cells were plated in the presence of IFNy (50ng/ml)-activated monocyte-derived macrophages at a ratio of 50: 1 macrophages: mast cells and cultured for 18h at 37C with specified concentrations of bispecific Siglec-6/8 huIgGl, bispecific Siglec-6/8 afucosylated huIgGl, anti-Siglec-6 huIgGl, anti-Siglec-8 afucosylated huIgGl, or an isotype control.
  • ADCP was measured by the loss of the mast cell population.
  • Example 7 Functional bispecific Siglec-6/8 antibodies can be generated in various formats [0324] Different formats for bispecific Siglec-6/8 antibodies were tested for the ability to bind both Siglec-6 and Siglec-8, as well as different configurations for which arm bound to Siglec-6 and which bound to Siglec-8. Activity was assessed by evaluating binding to Siglec-6 and Siglec-8 using ELISA. As shown in Table 9, all bispecific antibodies generated bound to Siglec- 6 and Siglec-8.
  • Example 8 Global protein interactions of Siglec-6 and Siglec-8 reveal distinct differences in regulating mast cell function
  • Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are receptors with broad mast cell (MC) inhibitory activity and therefore potential therapeutic targets in diseases driven by MCs. The functional differences between these closely related receptors are incompletely understood.
  • unbiased proteomic profiling was used to identify membrane and intracellular proteins that interact with Siglec-6 or Siglec-8 (i.e., ‘interactome’) in MCs.
  • Components of Siglec-6 and Siglec-8 protein complexes immuno-precipitated from primary MCs were identified by mass-spectrometry (FIG. 8A). Detailed interaction with KIT/CD117 and inhibition of stem cell factor (SCF)-mediated MC activation was evaluated with anti-Siglec-6 and Siglec-8 antibodies.
  • SCF stem cell factor
  • Siglec-6 and Siglec-8 interactomes were comprised of multiple critical MC activating receptors and signaling molecules, including KIT, the high affinity receptor for IgE, (subunits of) receptors for IL-3, IL-4, and IL-33, the intracellular kinases LYN and JAK1, and the phosphatases CD45, SHP-1 and SHP-2.
  • KIT KIT
  • IgE high affinity receptor for IgE
  • IL-3 intercellular kinases
  • IL-4 intercellular kinases
  • IL-33 the intracellular kinases LYN and JAK1
  • phosphatases CD45 SHP-1 and SHP-2
  • Siglec-6 appeared to have a more substantive regulatory role than Siglec-8.
  • Siglec-6 specifically interacted with multiple subunits of the FcsRI receptor, proteins involved in microtubule dynamics and degranulation, and multiple proteins involved in MC metabolism.
  • the pathways enriched in the Siglec-6, but not Siglec-8 interactome included SCF/KIT, thymic stromal lymphopoietin signaling, pyruvate dehydrogenase activity, and regulation of metabolic processes (FIG. 8B).
  • Siglec-6, but not Siglec-8 interacted with the mature, glycosylated form of KIT which resulted in inhibition of SCF-mediated MC activation with an agonistic anti-Siglec-6 antibody.
  • NYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGT TVTVS S (SEQ ID NO: 6)
  • GSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIK Amino acid sequence of 2E2 RKD light chain variable domain
  • Amino acid sequence of mouse 1C3 heavy chain variable domain (underlined residues comprise CDRs Hl and H2 according to Chothia numbering)
  • Amino acid sequence of mouse 1H10 heavy chain variable domain(underlined residues comprise CDRs Hl and H2 according to Chothia numbering)
  • Amino acid sequence of mouse 4F11 heavy chain variable domain (underlined residues comprise CDRs Hl and H2 according to Chothia numbering)
  • DILILGTLESGHSRNLTC S VPW ACKQGTPPMISWIGAS VS SPGPTTARS S VLTLTPKPQDH GTSLTCQVTLPGTGVTTTSTVRLDVS (SEQ ID NO: 113)
  • NPGLLELLRVHVKDEGEFTCQAENPRGSQHISLSLSLQNEGTGTARPVSEVTLAAVGG SEQ ID NO: 119
  • VSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO:299)
  • VSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO:300

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Abstract

La présente invention concerne des anticorps multispécifiques (par exemple bispécifiques) ciblant siglec-6 et siglec-8, ainsi que des acides nucléiques, des vecteurs, des cellules hôtes, des compositions, des procédés, des utilisations et des kits associés.
PCT/US2024/032993 2023-06-08 2024-06-07 Anticorps bispécifiques dirigés contre siglec-6 et siglec-8 et leurs procédés d'utilisation Ceased WO2024254441A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015089117A1 (fr) * 2013-12-09 2015-06-18 Allakos Inc. Anticorps anti-siglec 8 et leurs méthodes d'utilisation
WO2023044390A1 (fr) * 2021-09-16 2023-03-23 Allakos Inc. Anticorps anti-siglec-6 et leurs méthodes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015089117A1 (fr) * 2013-12-09 2015-06-18 Allakos Inc. Anticorps anti-siglec 8 et leurs méthodes d'utilisation
WO2023044390A1 (fr) * 2021-09-16 2023-03-23 Allakos Inc. Anticorps anti-siglec-6 et leurs méthodes d'utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
O’SULLIVAN JEREMY A, CHANG ALAN T, YOUNGBLOOD BRADFORD A, BOCHNER BRUCE S: "Eosinophil and mast cell Siglecs: From biology to drug target", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 108, no. 1, 1 July 2020 (2020-07-01), US , pages 73 - 81, XP093250766, ISSN: 0741-5400, DOI: 10.1002/JLB.2MR0120-352RR *
ROBIDA PIPER A., RISCHE CLAYTON H., MORGENSTERN NETALI BEN-BARUCH, JANARTHANAM RETHAVATHI, CAO YUN, KRIER-BURRIS REBECCA A., KORVE: "Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells", CELLS, vol. 11, no. 7, pages 1138 - 1138-18, XP093250760, ISSN: 2073-4409, DOI: 10.3390/cells11071138 *
SCHANIN, J. ET AL.: "Discovery of an agonistic Siglec-6 antibody that inhibits and reduces human mast cells", COMMUN BIOL, vol. 5, 2022, pages 1226, XP093051250, DOI: http://doi.org/10.1038/s42003-022- 04207-w *
YOUNGBLOOD BRADFORD A., LEUNG JOHN, FALAHATI RUSTOM, WILLIAMS JASON, SCHANIN JULIA, BROCK EMILY C., SINGH BHUPINDER, CHANG ALAN T.: "Discovery, Function, and Therapeutic Targeting of Siglec-8", CELLS, vol. 10, no. 1, pages 19 - 19-14, XP093250757, ISSN: 2073-4409, DOI: 10.3390/cells10010019 *

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