WO2022011323A1 - Traitement de maladie oculaire à l'aide d'anticorps anti-facteur tissulaire - Google Patents

Traitement de maladie oculaire à l'aide d'anticorps anti-facteur tissulaire Download PDF

Info

Publication number
WO2022011323A1
WO2022011323A1 PCT/US2021/041191 US2021041191W WO2022011323A1 WO 2022011323 A1 WO2022011323 A1 WO 2022011323A1 US 2021041191 W US2021041191 W US 2021041191W WO 2022011323 A1 WO2022011323 A1 WO 2022011323A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
set forth
sequence
sequence set
Prior art date
Application number
PCT/US2021/041191
Other languages
English (en)
Inventor
Jacques GAUDREAULT
Gabriela BURIAN
Thi-Sau Migone
William Greene
Original Assignee
Iconic Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Iconic Therapeutics, Inc. filed Critical Iconic Therapeutics, Inc.
Priority to TW110125559A priority Critical patent/TW202216197A/zh
Publication of WO2022011323A1 publication Critical patent/WO2022011323A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/75—Agonist effect on 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
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • TF Tissue factor
  • FVIIa serine protease factor Vila
  • the TF/FVIIa complex catalyzes conversion of the inactive protease factor X (FX) into the active protease factor Xa (FXa).
  • FXa and its co-factor FVa form the prothrombinase complex, which generates thrombin from prothrombin.
  • Thrombin converts soluble fibrinogen into insoluble strands of fibrin and catalyzes many other coagulation-related processes.
  • AMD age related macular degeneration
  • AMD is the most prevalent form of macular degeneration. It is a degenerative disease of the retina and is the leading cause of irreversible blindness and vision impairment in people over 65 years of age in the developed world. (Tomany etal ., Ophthalmology , 2004, which is incorporated by reference in its entirety). AMD occurs in up to 8% of individuals over the age of 60, and its prevalence increases with age. The U.S. is anticipated to have nearly 22 million cases of AMD by the year 2050, while global cases of AMD are expected to be nearly 288 million by the year 2040.
  • AMD is characterized by progressive loss of visual acuity in the central portion of the visual field, changes in color vision, and abnormal dark adaptation and sensitivity.
  • the two main forms of AMD are dry AMD and wet AMD (or neovascular AMD).
  • Dry AMD is associated with atrophic cell death of the central retina or macula, which is required for the fine vision used for activities such as reading, driving, and recognizing faces.
  • dry AMD can progress to wet AMD - a less common form of AMD that accounts for approximately 85% of legally blind AMD cases.
  • Wet AMD is characterized by aberrant vascularization under the macula (choroid neovascularization) or vascular leakage, which displaces the retina and/or results in hemorrhage and scarring.
  • CNV Choroidal neovascularization
  • Diabetic retinopathy is also a major cause of visual impairment in the United States and results from microvascular decompensation that begins with basement membrane thickening and ultimately results in vascular occlusion and angiogenesis. DR is observed in about 28% of diabetes patients over 40 years of age, and 4.4% suffer or are at risk of loss of vision due to DR (Zhang et al., 2010, JAMA. 304: 649-656, which is incorporated by reference in its entirety). Diabetic macular edema (DME) is a symptom of DR and the most frequent cause of blindness in young and middle-aged adults (Klein et ah, 1984, Ophthalmology , 91: 1464-1474 and Moss etal. , 1998, Ophthalmology, 105: 998-1003, each of which is incorporated by reference in its entirety).
  • DME Diabetic macular edema
  • Anti-vascular endothelial growth factor (anti-VEGF) therapies are the standard therapeutic treatment for ocular diseases such as AMD and DME with neovascularization.
  • ocular diseases such as AMD and DME with neovascularization.
  • VEGF anti-vascular endothelial growth factor
  • TF Tissue Factor
  • a method of treating an ocular disease in a subject in need thereof comprising administering to the subject an isolated antibody wherein the antibody binds to the extracellular domain of human Tissue Factor (TF), wherein the antibody binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa.
  • the antibody is an isolated human antibody.
  • the antibody is administered as a dose via intraocular injection and wherein if the dose administered to the subject is greater than 6 mg of antibody/eye, the dose is administered in two or more injections.
  • the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 28 days. In some embodiments, the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 15 days. In some embodiments, the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 20 days.
  • the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 30 days. In some embodiments, the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 40 days.
  • the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 or 42 days.
  • the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 15-20 days, 15-25 days, 15-30 days, 15-35 days, 15-40 days, 20-25 days, 20-30 days, 20-35 days, 20-40 days, 25-30 days, 25-35 days, 25-40 days, 30-35 days, 30-40 days, 35-40 days or a range of days therein.
  • the antibody is administered as a dose via intraocular injection, wherein if the dose administered to the subject is greater than 6 mg of antibody/eye, the dose is administered in two or more injections, and wherein the antibody is administered to the subject via a plurality of doses comprising at least a first dose and at least a second dose and wherein the time between the first and second doses is at least 28 days.
  • the antibody is administered via intravitreal injection.
  • the antibody (i) specifically binds to rabbit TF or (ii) is cross reactive with rabbit TF. In some embodiments, the antibody (i) specifically binds pig TF or (ii) is cross-reactive with pig TF. [0015] In some embodiments, the antibody does not inhibit human thrombin generation as determined by thrombin generation assay (TGA). In some embodiments, the isolated human antibody does not inhibit or inhibits human thrombin generation to a lesser extent, as determined by thrombin generation assay (TGA), compared to a comparator therapy. In some embodiments, the comparator therapy is an anti-TF protein, an anti-TF antibody, an anti-TF immunoconjugate or an anti-TF drug.
  • the comparator therapy is ICON-1. In some embodiments, the comparator therapy is an antibody comprising a VH sequence of SEQ ID NO:821 and a VL sequence of SEQ ID NO:822. In some embodiments, binding between the isolated antibody and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the isolated antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the isolated antibody relative to an isotype control in a live cell staining assay.
  • the antibody comprises all three heavy chain Complementary Determining Regions (CDRs) and all three light chain CDRs from an antibody group in Table 35, wherein the all three heavy chain CDRs and the all three light chain CDRs are from the same antibody group.
  • the antibody comprises all three heavy chain Complementary Determining Regions (CDRs) and all three light chain CDRs from an antibody in any one of Tables 15-34, wherein the all three heavy chain CDRs and the all three light chain CDRs are from the same antibody.
  • the antibody comprises all three heavy chain CDRs and all three light chain CDRs from: the antibody designated 25A, the antibody designated 25A5, the antibody designated 25A5-T, the antibody designated 25G, the antibody designated 25G1, the antibody designated 25G9, the antibody designated 43B, the antibody designated 43B1, the antibody designated 43B7, the antibody designated 43D, the antibody designated 43D7, the antibody designated 43D8, the antibody designated 43E, or the antibody designated 43Ea.
  • the antibody comprises all three heavy chain CDRs and all three light chain CDRs from: the antibody designated 25A, the antibody designated 25A5, the antibody designated 25A5-T, the antibody designated 25G, the antibody designated 25G1, or the antibody designated 25G9.
  • the antibody comprises all three heavy chain CDRs and all three light chain CDRs from: the antibody designated 43B, the antibody designated 43B1, the antibody designated 43B7, the antibody designated 43D, the antibody designated 43D7, the antibody designated 43D8, the antibody designated 43E, or the antibody designated 43Ea.
  • the antibody comprises a VH Domain sequence and VL domain sequence from Table 14, wherein the VH and VL domain sequences are from the same group in Table 14.
  • the antibody comprises a VH Domain sequence and VL domain sequence from Table 13, wherein the VH and VL domain sequences are from the same clone in Table 13.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:779; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:780; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:781; a VL-CDRl comprising the sequence set forth in SEQ ID NO:782; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:783; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:784.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:872; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:873; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:874; a VL-CDRl comprising the sequence set forth in SEQ ID NO:875; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:876; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:877.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:884; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:885; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:886; a VL-CDRl comprising the sequence set forth in SEQ ID NO:887; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:888; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:889.
  • the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:868 and a VL sequence comprising the sequence set forth in SEQ ID NO:869.
  • the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO: 189 and a VL sequence comprising the sequence set forth in SEQ ID NO: 190. In some embodiments, the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:836 and a VL sequence comprising the sequence set forth in SEQ ID NO:837. In some embodiments, the antibody comprises: a heavy chain comprising the sequence set forth in SEQ ID NO:920 and a light chain comprising the sequence set forth in SEQ ID NO:921.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:878; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:879; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:880; a VL-CDR1 comprising the sequence set forth in SEQ ID NO:881; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:882; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:883.
  • the antibody comprises: a VH-CDR1 comprising the sequence set forth in SEQ ID NO:267; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:268; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:269; a VL-CDR1 comprising the sequence set forth in SEQ ID NO:270; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:271; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:272.
  • the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:870 and a VL sequence comprising the sequence set forth in SEQ ID NO:871. In some embodiments, the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:303 and a VL sequence comprising the sequence set forth in SEQ ID NO:304. In some embodiments, the antibody comprises: a heavy chain comprising the sequence set forth in SEQ ID NO:922 and a light chain comprising the sequence set forth in SEQ ID NO:923.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:797; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:798; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:799; a VL-CDR1 comprising the sequence set forth in SEQ ID NO:800; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:801; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:802.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:571; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:572; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:573; a VL-CDR1 comprising the sequence set forth in SEQ ID NO:574; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:575; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:576.
  • the antibody comprises: a VH-CDRl comprising the sequence set forth in SEQ ID NO:609; a VH-CDR2 comprising the sequence set forth in SEQ ID NO:610; a VH-CDR3 comprising the sequence set forth in SEQ ID NO:611; a VL-CDR1 comprising the sequence set forth in SEQ ID NO:612; a VL-CDR2 comprising the sequence set forth in SEQ ID NO:613; and a VL-CDR3 comprising the sequence set forth in SEQ ID NO:614.
  • the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:769 and a VL sequence comprising the sequence set forth in SEQ ID NO:770. In some embodiments, the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:569 and a VL sequence comprising the sequence set forth in SEQ ID NO:570. In some embodiments, the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:607 and a VL sequence comprising the sequence set forth in SEQ ID NO:608.
  • the antibody comprises: a VH sequence comprising the sequence set forth in SEQ ID NO:645 and a VL sequence comprising the sequence set forth in SEQ ID NO:646. In some embodiments, the antibody comprises: a heavy chain comprising the sequence set forth in SEQ ID NO:924 and a light chain comprising the sequence set forth in SEQ ID NO: 925. In some embodiments, the antibody comprises: a heavy chain comprising the sequence set forth in SEQ ID NO:926 and a light chain comprising the sequence set forth in SEQ ID NO:927.
  • the antibody competes for binding to human TF with the antibody designated 25A, the antibody designated 25A5, the antibody designated 25A5-T, the antibody designated 25G, the antibody designated 25G1, the antibody designated 25G9, the antibody designated 43B, the antibody designated 43B1, the antibody designated 43B7, the antibody designated 43D, the antibody designated 43D7, the antibody designated 43D8, the antibody designated 43E, or the antibody designated 43Ea.
  • the antibody competes for binding to human TF with the antibody designated 25A, the antibody designated 25A5, the antibody designated 25A5-T, the antibody designated 25G, the antibody designated 25G1, or the antibody designated 25G9. In some embodiments, the antibody competes for binding to human TF with the antibody designated 43B, the antibody designated 43B1, the antibody designated 43B7, the antibody designated 43D, the antibody designated 43D7, the antibody designated 43D8, the antibody designated 43E, or the antibody designated 43Ea.
  • the antibody binds to the same human TF epitope bound by the antibody designated 25A, the antibody designated 25A5, the antibody designated 25A5-T, the antibody designated 25G, the antibody designated 25G1, the antibody designated 25G9, the antibody designated 43B, the antibody designated 43B1, the antibody designated 43B7, the antibody designated 43D, the antibody designated 43D7, the antibody designated 43D8, the antibody designated 43E, or the antibody designated 43Ea.
  • the antibody binds to the same human TF epitope bound by the antibody designated 25A, the antibody designated 25A5, the antibody designated 25A5-T, the antibody designated 25G, the antibody designated 25G1, or the antibody designated 25G9. In some embodiments, the antibody binds to the same human TF epitope bound by the antibody designated 43B, the antibody designated 43B1, the antibody designated 43B7, the antibody designated 43D, the antibody designated 43D7, the antibody designated 43D8, the antibody designated 43E, or the antibody designated 43Ea.
  • the antibody does not inhibit human thrombin generation as determined by thrombin generation assay (TGA), does not reduce the thrombin peak on a thrombin generation curve (Peak Ila) compared to an isotype control, does not increase the time from the assay start to the thrombin peak on a thrombin generation curve (ttPeak) compared to an isotype control, does not decrease the endogenous thrombin potential (ETP) as determined by the area under a thrombin generation curve compared to an isotype control, allows human thrombin generation as determined by thrombin generation assay (TGA), maintains the thrombin peak on a thrombin generation curve (Peak Ila) compared to an isotype control, maintains the time from the assay start to the thrombin peak on a thrombin generation curve (ttPeak) compared to an isotype control, preserves the endogenous thrombin potential
  • the three heavy chain CDRs and the three light chain CDRs are determined using exemplary, Rabat, Chothia, AbM, Contact, or IMGT numbering.
  • the affinity of the antibody to human TF is greater compared to a comparator therapy.
  • the comparator therapy is an anti-TF protein, an anti-TF antibody, an anti-TF immunoconjugate or an anti-TF drug.
  • the comparator therapy is ICON-1.
  • the antibody comprises an Fc region.
  • the Fc region of the antibody and comprises one or more mutations that result in enhanced effector functionan increased immune response.
  • the antibody interacts with an Fey Receptor (FcyR) expressed by an immune cell via the Fc domain, and wherein the antibody increases an immune response in the subject upon specific binding between the antibody and one or more TF-expressing cells, thereby treating the ocular disease.
  • the immune response is enhanced Fc effector function.
  • the immune response increases compared to a comparator therapy.
  • the immune response is any one of: (i) increased T cell activity, (ii) increased activation of ab and/or gd T cells, (iii) increased cytotoxic T cell activity, (iv) increased NK and/or NKT cell activity, (v) alleviation of ab and/or gd T-cell suppression, (vi) increased pro-inflammatory cytokine secretion, (vii) increased IL-2 secretion; (viii) increased interferon-y production, (ix) increased Thl response, (x) decreased Th2 response, (xi) decreased or eliminated cell number and/or activity of at least one of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), immature myeloid cells (iMCs), mesenchymal stromal cells, or TIE2-expressing monocytes, (xii) reduced regulatory cell activity, and/or the reduced activity of one or more of myeloid T cells, and/or the reduced activity
  • the immune response is any one of: (i) antibody-dependent cellular cytotoxicity (ADCC), (ii) antibody-dependent cellular phagocytosis (ADCP), and (iii) Clq binding to activate complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • the comparator therapy is an anti-TF protein, an anti-TF antibody, an anti-TF immunoconjugate or an anti-TF drug. In some embodiments, the comparator therapy is ICON-1.
  • the method comprises administering the antibody of any one of the preceding claims to the subject by intravitreal injection, suprachoroidal injection or subretinal injection. In some embodiments, the method comprises administering the antibody of any one of the preceding claims to the subject by intravitreal injection.
  • the ocular disease is neovascularization, age-related macular degeneration (AMD), retinal vein occlusion, retinopathy of prematurity or diabetic retinopathy.
  • the diabetic retinopathy is proliferative diabetic retinopathy or non-proliferative diabetic retinopathy.
  • the AMD is neovascular AMD.
  • the neovascularization is macular neovascularization.
  • the AMD is wet AMD.
  • the AMD is dry AMD.
  • the antibody upon administration to a subject, decreases the total leakiness area on the retina relative to baseline levels. In some embodiments, upon administration to a subject, decreases the total leakiness area on the retina relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug. In some embodiments, upon administration to a subject, the antibody does not alter the total leakiness area on the retina relative to baseline levels. In some embodiments, the total leakiness area is determined using fluorescein angiography or OCT-angiography.
  • the antibody upon administration to a subject, the antibody reduces choroidal neovascularization (CNV) area relative to baseline levels. In some embodiments, upon administration to a subject, the antibody reduces choroidal neovascularization (CNV) area relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug. In some embodiments, the CNV area is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%. In some embodiments, the CNV area is determined using fluorescein angiography or OCT-angiography [0047] In some embodiments, upon administration to a subject, the antibody does not result in ocular inflammation.
  • the antibody upon administration to a subject, results in reduced ocular inflammation relative to baseline levels. In some embodiments, upon administration to a subject, the antibody results in reduced ocular inflammation relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug . In some embodiments, the ocular inflammation is as determined using direct or indirect ophthalmoscopy. In some embodiments, upon administration to a subject, the antibody results in reduced retinal thickness relative to baseline levels.
  • the antibody upon administration to a subject, results in reduced retinal thickness relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug.
  • the retinal thickness is reduced by at least about 50 pm, at least about 100 pm, at least about 150 pm, at least about 175 pm, at least about 200 pm, at least about 225 pm or at least about 250 pm.
  • the retinal thickness is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%.
  • the reduced retinal thickness is reduced central retinal subfield thickness (CST), reduced center point thickness (CPT), or reduced central foveal thickness (CFT).
  • the retinal thickness is central retina thickness (CRT).
  • the CRT value increases to a value within the range of 200-270 pM.
  • the retinal thickness is measured by spectral domain optical coherence tomography (SD OCT).
  • the antibody upon administration to a subject, results in reduced mean foveal thickness relative to baseline levels. In some embodiments, upon administration to a subject, the antibody results in reduced mean foveal thickness relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug . In some embodiments, the mean foveal thickness is measured by SD OCT.
  • the antibody upon administration to a subject, results in reduced macular edema relative to baseline levels.
  • the macular edema is determined by fluorescein angiography, or optical coherence tomography (OCT).
  • the antibody upon administration to a subject, results in reduced leakage into the macula relative to baseline levels.
  • the leakage into the macula is determined by fluorescein angiography, or optical coherence tomography (OCT).
  • the antibody upon administration to a subject, results in reduced or absent biomarkers of inflammation relative to baseline levels. In some embodiments, upon administration to a subject, the antibody results in reduced or absent biomarkers of inflammation relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug. In some embodiments, upon administration to a subject, the antibody results in reduced or absent biomarkers of inflammation, as compared to a a comparator therapy.
  • the comparator therapy is an anti-TF protein, an anti-TF antibody, an anti-TF immunoconjugate or an anti-TF drug. In some embodiments, the comparator therapy is ICON-1.
  • the biomarkers of inflammation comprise one or more inflammatory cytokines.
  • the inflammatory cytokines comprise at least one of: IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF or GMCSF).
  • the biomarkers of inflammation are measured by ELISA.
  • the antibody upon administration to a subject, results in reduced or equal anti-drug antibody (ADA) levels relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug .
  • ADA levels are systemic ADA levels.
  • the ADA levels are within vitreous humor and/or aqueous humor.
  • the ADA levels are measured by ECL bridging assay.
  • the antibody upon administration to a subject, does not significantly alter intraocular pressure (IOP) relative levels to baseline levels.
  • IOP intraocular pressure
  • the IOP is measured by tonometry.
  • the antibody upon administration to a subject, results in reduced mononuclear cell infiltration relative to baseline levels. In some embodiments, upon administration to a subject, the antibody results in reduced mononuclear cell infiltration relative to a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug.
  • the mononuclear cell infiltration comprises Ml or M2 macrophages. In some embodiments, the mononuclear cell infiltration is infiltration of mononuclear cells into one or more of: retina, choroid, vitreous body, iris, ciliary body, limbus, and conjunctiva. In some embodiments, the mononuclear cell infiltration is measured by immunohistochemical (IHC) analysis.
  • IHC immunohistochemical
  • the exposure to the antibody in target tissues is comparable to that from a different anti-TF antibody, anti- TF drug, anti-VEGF antibody or anti-VEGF drug.
  • the concentration of the antibody in the vitreous humor is significantly higher than the concentration in the serum for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days following the administration.
  • the antibody upon administration to a subject, results in less severe adverse ocular changes relative to baseline levels, wherein the adverse ocular changes comprise the presence of one or more of: anterior chamber cells, flare and fibrin, keratic precipitates/corneal endothelial cells, cellular deposits on the anterior lens capsule, incomplete pupil dilation, cells on corneal endothelium and lens capsule, vitreous haze, vitreous cells, retinal sheathing, and chorio-retinal hemorrhages.
  • the adverse ocular changes comprise the presence of one or more of: anterior chamber cells, flare and fibrin, keratic precipitates/corneal endothelial cells, cellular deposits on the anterior lens capsule, incomplete pupil dilation, cells on corneal endothelium and lens capsule, vitreous haze, vitreous cells, retinal sheathing, and chorio-retinal hemorrhages.
  • the antibody upon administration to a subject, results in less severe adverse ocular changes relative to a different anti-TF antibody, anti-TF drug, anti- VEGF antibody or anti-VEGF drug , wherein the adverse ocular changes comprise the presence of one or more of: anterior chamber cells, flare and fibrin, keratic precipitates/corneal endothelial cells, cellular deposits on the anterior lens capsule, incomplete pupil dilation, cells on corneal endothelium and lens capsule, vitreous haze, vitreous cells, retinal sheathing, and chorio-retinal hemorrhages.
  • the different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug comprises: aflibercept, ranibizumab, brolucizumab, pegaptanib, bevacizumab, abicipar pegol, RG7716, KSI-301 or ICON-1.
  • the antibody binds human TF with a KD less than 10 7 M. [0063] The method of any one of the preceding claims, wherein the antibody binds human TF with a KD between 10 7 M and 10 11 M.
  • KD for mouse TF as measured by ForteBio is 360 nM or less.
  • KD for mouse TF as measured by ForteBio is between 6.12 nM and 360 nM.
  • KD for mouse TF as measured by ForteBio is 21 nM or less.
  • KD for mouse TF as measured by ForteBio is between about 2.4 nM and 21 nM.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the administration is intraocular. In some embodiments, the antibody is administered by intravitreal injection.
  • the antibody is administered once a month or once every two months. In some embodiments, the antibody is administered once every week, two weeks, three weeks or four weeks. In some embodiments, the antibody is administered biweekly. In some embodiments, the antibody is administered weekly.
  • the antibody is administered at 0.6 mg of antibody/eye. In some embodiments, the antibody is administered at less than 0.6 mg of antibody/eye. In some embodiments, the antibody is administered at 0.6, 2, 3.6, 4, or 6 mg of antibody/eye. In some embodiments, the antibody is administered in a single injection of the antibody/eye. In some embodiments, the antibody is administered at 11.9 or 12 mg of antibody/eye. In some embodiments, the antibody is administered at less than 11.9 or 12 mg of antibody/eye. In some embodiments, the antibody is administered in a single injection of the antibody/eye. In some embodiments, the antibody is administered at less than 0.6, 2, 3.6, 4, 6, 11.9 or 12 mg of antibody/eye.
  • FIG. 1 includes a schematic showing the experimental design for swine laser CNV model studies. ( See Examples).
  • FIGs. 2A and 2B includes plots showing the efficacy of the indicated anti-TF antibodies, ICON1, and adalimumab in a swine CNV model.
  • FIG. 2A shows the mean Corrected Total Lesion Fluorescence (CTLF) at day 7 (treatment day), day 14, and day 28.
  • CTLF Corrected Total Lesion Fluorescence
  • FIG. 2B shows the percent change in CTLF relative to baseline levels (day 7).
  • FIG. 3 includes a plot showing the efficacy of the indicated anti-TF antibodies, Eylea, and combination therapy in swine CNV model. WT denotes the wild-type of the antibody.
  • FIG. 4 includes plots showing the percent change in CTLF from the indicated anti-TF antibodies, Eylea, and combination therapy relative to baseline levels in swine CNV model. WT denotes the wild-type of the antibody.
  • FIG. 5 includes a plot showing the efficacy of the indicated anti-TF antibodies, and ICON1 in swine CNV model.
  • DE denotes the antibody having two mutations in the human IgGl Fc region (S239D and I332E, as determined by Kabat numbering).
  • FIG. 6 includes a plot showing the percent change in CTLF from the indicated anti- TF antibodies, and ICON1 relative to baseline in swine CNV model.
  • DE denotes the antibody having two mutations in the human IgGl Fc region (S239D and I332E, as determined by Kabat numbering).
  • FIG. 7 includes a plot showing the efficacy of the indicated anti-TF antibodies, anti- TF antigen-biding fragment, Eylea and combination therapy in swine CNV model.
  • DE denotes the antibody having two mutations in the human IgGl Fc region (S239D and I332E, as determined by Kabat numbering).
  • LALAPG denotes the antibody having the combination of L234A, L235A, and P329A (LALA-PG) substitutions in the human IgGl heavy chain constant region (according to Kabat numbering).
  • FIG. 8 includes a plot showing the percent change in CTLF from the indicated anti- TF antibodies, anti-TF antigen-biding fragment, Eylea and combination therapy relative to baseline in swine CNV model.
  • DE denotes the antibody having two mutations in the human IgGl Fc region (S239D and I332E, as determined by Kabat numbering).
  • LALAPG denotes the antibody having the combination of L234A, L235A, and P329A (LALA-PG) substitutions in the human IgGl heavy chain constant region (according to Kabat numbering).
  • FIG. 9 includes a plot showing the efficacy of different doses of Eylea and 25A5-T anti-TF antibodies in swine CNV model.
  • FIG. 10 includes a plot showing the percent change in CTLF from different doses of Eylea and 25A5-T anti-TF antibodies relative to baseline in swine CNV model.
  • FIG. 11 includes a plot showing the mean concentration profiles for 25A5-T in rabbit vitreous humor in a PK study.
  • FIG. 12A includes a plot showing the mean concentration profiles for 25A5-T at 600 ug dose in rabbit ocular tissues and serum in a PK study.
  • FIG. 12B includes a plot showing the mean concentration profiles for 25A5-T at 3600 ug dose in rabbit ocular tissues and serum in a PK study.
  • FIG. 13 includes a plot showing the mean concentration profiles for ICON-1 at 600 ug dose in rabbit ocular tissues and plasma in a PK study.
  • FIG. 14 includes plots showing the mean concentration profiles for 25A5-T in the vitreous humor of cynomolgus monkey in a PK study.
  • FIG. 15A includes a plot showing the mean concentration profiles for 25A5-T in the ocular tissues and serum of cynomolgus monkey in a PK study.
  • FIG. 15B includes a plot showing the mean concentration profiles for 25A5-T in the ocular tissues and serum of cynomolgus monkey in a PK study.
  • FIG. 16 includes a plot showing the mean (SD) 25A5-T serum profiles after single IVT administration of 0.6 and 12 mg/eye.
  • FIG. 17 includes a schematic showing the experimental design for the pilot toxicology study conducted in cynomolgus monkeys.
  • FIG. 18 includes plots showing the mean concentration profiles for 25A5-T in the serum from the pilot toxicology study conducted in cynomolgus monkeys.
  • FIG. 19 includes plots showing the mean concentration profiles for 25A5-T in the serum from the pilot toxicology study conducted in cynomolgus monkeys.
  • FIG. 20 includes a plot showing the effects of 25A5-T vs. ICON-1 on the coagulation cascade, assessed using a thrombin generation assay. Peak Ila (highest thrombin concentration generated on the thrombin generation curve [nM]) is reported relative to plasma in the absence of antibody.
  • FIGS. 21A and 21B include plots showing the inhibition of cytokine secretion resulting from treatment of TF-expressing cells with 25A5-T and ICON-1.
  • FIG. 21A shows the GM-CSF measurements and
  • FIG. 2 IB shows the IL-8 measurements.
  • FIGS. 22A and 22B include plots showing the effect of 25A5-T vs. ICON-1 on antibody-dependent cell-mediated cytotoxicity (ADCC; FIG. 22A) and antibody-dependent cellular phagocytosis (ADCP; FIG. 22B).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • FIG. 23 includes a plot showing the serum concentration-time profile following IVT administration of 25A5-T.
  • FIGS. 24A-24B include plots showing summary (mean ⁇ SD) serum 25A5-T concentrations following bilateral intravitreal injection of 0.6, 2.0 and 6.0 mg/eye 25A5-T on Day 1 in Male (FIG. 24A) and Female (FIG. 24B) cynomolgus monkeys.
  • FIGS. 25A-25B include plots showing summary (mean ⁇ SD) serum 25A5-T concentrations following bilateral intravitreal injection of 0.6, 2.0 and 6.0 mg/eye 25A5-T on Day 1 in Male (FIG. 25A) and Female (FIG. 25B) cynomolgus monkeys.
  • FIG. 26 includes sensorgrams from TF binding kinetics experiments of 25A5-T binding to the indicated TF proteins.
  • the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ⁇ one standard deviation of that value(s).
  • tissue Factor tissue Factor
  • platelet tissue factor factor III
  • thromboplastin thromboplastin
  • CD 142 tissue Factor
  • the TF protein is a TF protein naturally expressed by a primate (e.g., a monkey or a human), a rodent (e.g, a mouse or a rat), a dog, a camel, a cat, a cow, a goat, a horse, a pig or a sheep.
  • the TF protein is human TF (hTF; SEQ ID NO:809).
  • the TF protein is cynomolgus TF (cTF; SEQ ID NO:813).
  • the TF protein is mouse TF (mTF; SEQ ID NO:817).
  • the TF protein is pig TF (pTF; SEQ ID NO:824).
  • TF is a cell surface receptor for the serine protease factor Vila. It is often times constitutively expressed by certain cells surrounding blood vessels and in some disease settings.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • the cytotoxic agent can be an anti-angiogenic agent, a pro-apoptotic agent, an anti-mitotic agent, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioactive isotope.
  • cytotoxic agents include calicheamycin, camptothecin, carboplatin, irinotecan, SN-38, carboplatin, camptothecan, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, doxorubicin, etoposide, idarubicin, topotecan, vinca alkaloid, maytansinoid, maytansinoid analog, pyrrolobenzodiazepine, taxoid, duocarmycin, dolastatin, auristatin, and derivatives thereof.
  • a “linker” refers to a molecule that connects one composition to another, e.g, an antibody to an agent.
  • Linkers described herein can conjugate an antibody to a cytotoxic agent.
  • Exemplary linkers include a labile linker, an acid labile linker, a photolabile linker, a charged linker, a disulfide-containing linker, a peptidase-sensitive linker, a b-glucuronide-linker, a dimethyl linker, a thio-ether linker, and a hydrophilic linker.
  • a linker can be cleavable or non-cleavable.
  • immunoglobulin refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI, Cm, and Cm. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.
  • antibody is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.
  • An antibody specifically includes intact antibodies (e.g, intact immunoglobulins), antibody fragments, and multi-specific antibodies.
  • alternative scaffold refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope.
  • the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of an antibody.
  • Exemplary alternative scaffolds include those derived from fibronectin (e.g, AdnectinsTM), the b-sandwich (e.g, iMab), lipocalin (e.g, Anticalins ® ), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g, Kunitz domains), thioredoxin peptide aptamers, protein A (e.g, Affibody ® ), ankyrin repeats (e.g, DARPins), gamma-B-crystallin/ubiquitin (e.g, Affilins), CTLD3 (e.g, Tetranectins), Fynomers, and (LDLR-A module) (e.g, Avimers).
  • fibronectin e.g, AdnectinsTM
  • the b-sandwich e.g, iMab
  • lipocalin e.g, Anticalins
  • antigen-binding domain means the portion of an antibody that is capable of specifically binding to an antigen or epitope.
  • an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer of an antibody.
  • Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin.
  • Antigen-binding domains can be found in various contexts including antibodies and chimeric antigen receptors (CARs), for example CARs derived from antibodies or antibody fragments such as scFvs.
  • CARs chimeric antigen receptors
  • full length antibody is an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region.
  • a “full length antibody” is an antibody that comprises two heavy chains and two light chains.
  • Fc region means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system.
  • the structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini, ./. Allergy Clin. Immunol ., 2010, 125:S41-52, incorporated by reference in its entirety.
  • the Fc region may be a naturally occurring Fc region, or an Fc region modified as described in the art or elsewhere in this disclosure.
  • the VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved.
  • the more conserved regions are called framework regions (FRs).
  • Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Rabat et al. , Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.
  • a “Complementary Determining Region (CDR)” refers to one of three hypervariable regions (HI, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH b-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL b-sheet framework.
  • CDRs are variable region sequences interspersed within the framework region sequences. CDRs are well recognized in the art and have been defined by, for example, Rabat as the regions of most hypervariability within the antibody variable (V) domains.
  • CDRs have also been defined structurally by Chothia as those residues that are not part of the conserved b-sheet framework, and thus are able to adapt different conformations. See Chothia and Lesk, JMol Biol , 1987, 196:901-917, incorporated by reference in its entirety. Both the Kabat and Chothia nomenclatures are well known in the art.
  • AbM, Contact and IMGT also defined CDRs. CDR positions within a canonical antibody variable domain have been determined by comparison of numerous structures.
  • a number of hypervariable region delineations are in use and are included herein.
  • the Kabat CDRs are based on sequence variability and are the most commonly used. See Kabat et al. (1992) Sequences of Proteins of Immunological Interest , DIANE Publishing: 2719, incorporated by reference in its entirety. Chothia refers instead to the location of the structural loops (Chothia and Lesk, supra).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • the Contact hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted in Table 1.
  • IMGT ImMunoGeneTics
  • IMGT Information System
  • IG immunoglobulins
  • TR T cell receptors
  • MHC major histocompatibility complex
  • the light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (l), based on the sequence of its constant domain.
  • the heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, d, e, g, and m, respectively.
  • the IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • constant region refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the CHI, Cm and Cm domains of the heavy chain and the CL domain of the light chain.
  • EU numbering scheme is generally used when referring to a residue in an antibody heavy chain constant region ( e.g ., as reported in Kabat etal ., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.
  • an “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody.
  • Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab’)2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments.
  • Fv fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.
  • Fab fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody.
  • F(ab’)2 fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds.
  • F(ab’)2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody.
  • the F(ab’) fragments can be dissociated, for example, by treatment with B-mercaptoethanol.
  • Single-chain Fv or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain.
  • the VH and VL are generally linked by a peptide linker.
  • Any suitable linker may be used.
  • the linker is a (GGGGS)n(SEQ ID NO:823).
  • n 1, 2,
  • scFv-Fc fragments comprise an scFv attached to an Fc domain.
  • an Fc domain may be attached to the C-terminal of the scFv.
  • the Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH -VL or VL - VH). Any suitable Fc domain known in the art or described herein may be used.
  • single domain antibody refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain.
  • Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al. , FEBS Letters , 1998, 414:521-526 and Muyldermans el al. , Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety.
  • Single domain antibodies are also known as sdAbs or nanobodies.
  • a “multispecific antibody” is an antibody that comprises two or more different antigen-binding domains that collectively specifically bind two or more different epitopes.
  • the two or more different epitopes may be epitopes on the same antigen (e.g, a single TF molecule expressed by a cell) or on different antigens (e.g, a TF molecule and a non-TF molecule).
  • a multi-specific antibody binds two different epitopes (i.e., a “bispecific antibody”).
  • a multi-specific antibody binds three different epitopes (i.e., a “trispecific antibody”).
  • a multi-specific antibody binds four different epitopes (i.e., a “quadspecific antibody”). In some aspects, a multi-specific antibody binds five different epitopes (i.e., a “quintspecific antibody”). In some aspects, a multi specific antibody binds 6, 7, 8, or more different epitopes. Each binding specificity may be present in any suitable valency. Examples of multispecific antibodies are provided elsewhere in this disclosure.
  • a “monospecific antibody” is an antibody that comprises one or more binding sites that specifically bind to a single epitope.
  • An example of a monospecific antibody is a naturally occurring IgG molecule which, while divalent (i.e., having two antigen-binding domains), recognizes the same epitope at each of the two antigen-binding domains.
  • the binding specificity may be present in any suitable valency.
  • the term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies.
  • a population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts.
  • a monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones.
  • the selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
  • affinity maturation affinity maturation
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • a humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
  • Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function.
  • a “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences ( e.g ., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
  • an “isolated antibody” or “isolated nucleic acid” is an antibody or nucleic acid that has been separated and/or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials.
  • an isolated antibody is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator.
  • an isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain.
  • an isolated antibody may include an antibody in situ within recombinant cells, since at least one component of the antibody’s natural environment is not present.
  • an isolated antibody or isolated nucleic acid is prepared by at least one purification step.
  • an isolated antibody or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by weight.
  • an isolated antibody or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume.
  • an isolated antibody or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% antibody or nucleic acid by weight.
  • an isolated antibody or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% antibody or nucleic acid by volume.
  • affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g, an antibody) and its binding partner (e.g, an antigen or epitope).
  • affinity refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).
  • the affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below.
  • VEGF vascular endothelial growth factor
  • SPR surface plasmon resonance
  • BIACORE ® BIACORE ®
  • biolayer interferometry e.g, FORTEBIO ®
  • VEGF/VEGF-A human vascular endothelial growth factor
  • VEGF is a homodimeric glycoprotein that is known to promote angiogenesis. It includes 165-amino acid human vascular endothelial growth factor (human VEGF165) and related 121, 189 and 206 vascular endothelial growth factor isoforms (Uniprot ID No: PI 5692).
  • the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g ., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule).
  • Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule.
  • Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule.
  • the affinity of a TF antibody for a non-target molecule is less than about 50% of the affinity for TF. In some aspects, the affinity of a TF antibody for a non target molecule is less than about 40% of the affinity for TF. In some aspects, the affinity of a TF antibody for a non-target molecule is less than about 30% of the affinity for TF. In some aspects, the affinity of a TF antibody for a non-target molecule is less than about 20% of the affinity for TF. In some aspects, the affinity of a TF antibody for a non-target molecule is less than about 10% of the affinity for TF.
  • the affinity of a TF antibody for a non-target molecule is less than about 1% of the affinity for TF. In some aspects, the affinity of a TF antibody for a non-target molecule is less than about 0.1% of the affinity for TF. [00133] In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 1 nM. In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 10 nM. In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 50 nM. In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 100 nM. In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 200 nM.
  • specifically binding refers to an antibody binding with an affinity of less than 300 nM. In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 200 nM, 300 nM, 400 nM or 500 nM. In some embodiments, specifically binding refers to an antibody binding with an affinity of less than 0 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM.
  • k a (M 1 sec 1 ), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the k 0n value.
  • affinity of an antibody is described in terms of the KD for an interaction between such antibody and its antigen. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.
  • an “affinity matured” antibody is an antibody with one or more alterations (e.g ., in one or more CDRs or FRs) relative to a parent antibody (i.e., an antibody from which the altered antibody is derived or designed) that result in an improvement in the affinity of the antibody for its antigen, compared to the parent antibody which does not possess the alteration(s).
  • a parent antibody i.e., an antibody from which the altered antibody is derived or designed
  • an affinity matured antibody has nanomolar or picomolar affinity for the target antigen.
  • Affinity matured antibodies may be produced using a variety of methods known in the art. For example, Marks et al. Bio/Technology , 1992, 10:779-783, incorporated by reference in its entirety) describes affinity maturation by VH and VL domain shuffling.
  • Random mutagenesis of CDR and/or framework residues is described by, for example, Barbas et al, Proc. Nat. Acad. Sci. U.S.A., 1994, 91:3809-3813; Schier et al., Gene, 1995, 169:147-155; Yelton etal., J. Immunol., 1995, 155:1994-2004; Jackson etal., J. Immunol., 1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992, 226:889-896; each of which is incorporated by reference in its entirety.
  • the antibodies of the present disclosure have enhanced Fc effector function.
  • Fc effector functions refer to those biological activities mediated by the Fc region of an antibody, which activities may vary depending on the antibody isotype.
  • Non limiting examples of antibody effector functions include Clq binding to activate complement dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP).
  • the term “competes with” or “cross-competes with” indicates that the two or more antibodies compete for binding to an antigen (e.g., TF).
  • TF is coated on a surface and contacted with a first TF antibody, after which a second TF antibody is added.
  • first a TF antibody is coated on a surface and contacted with TF, and then a second TF antibody is added. If the presence of the first TF antibody reduces binding of the second TF antibody, in either assay, then the antibodies compete with each other.
  • the term “competes with” also includes combinations of antibodies where one antibody reduces binding of another antibody, but where no competition is observed when the antibodies are added in the reverse order.
  • the first and second antibodies inhibit binding of each other, regardless of the order in which they are added.
  • one antibody reduces binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • concentrations of the antibodies used in the competition assays based on the affinities of the antibodies for TF and the valency of the antibodies.
  • the assays described in this definition are illustrative, and a skilled artisan can utilize any suitable assay to determine if antibodies compete with each other. Suitable assays are described, for example, in Cox etal. , “Immunoassay Methods,” in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih.gov/books/NBK92434/; accessed September 29, 2015); Silman etal. , Cytometry , 2001, 44:30-37; and Finco etal. , J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated by reference in its entirety. As provided in Example 8, antibodies of group 25 and antibodies of group 43 compete with each other for binding to human TF, while antibodies from groups 1, 29, 39, and 54 do not compete for binding to human TF with antibodies of groups 25 and 43.
  • an antibody that binds specifically to a human antigen is considered to bind the same antigen of mouse origin when a KD value can be measured on a ForteBio Octet with the mouse antigen.
  • An antibody that binds specifically to a human antigen is considered to be “cross-reactive” with the same antigen of mouse origin when the KD value for the mouse antigen is no greater than 20 times the corresponding KD value for the respective human antigen.
  • the antibody Ml 593 described in U.S Pat. Nos. 8,722,044, 8,951,525, and 8,999,333 each of which is herein incorporated by reference for all purposes, the humanized 5G9 antibody described in Ngo et al, 2007, Int J Cancer,
  • TF antibodies from groups 25 and 43 bind to mouse TF, e.g., the TF antibodies 25G, 25G1, 25G9, and 43D8 are cross-reactive with mouse TF.
  • an antibody that binds specifically to a human antigen is considered to be “cross-reactive” with the same antigen of cynomolgus monkey origin when the KD value for the cynomolgus monkey antigen is no greater than 15 times the corresponding KD value for the respective human antigen.
  • all tested antibodies from groups 1, 25, 29, 39, 43, and 54 are cross-reactive with cynomolgus monkey TF.
  • epitope means a portion of an antigen that is specifically bound by an antibody. Epitopes frequently include surface-accessible amino acid residues and/or sugar side chains and may have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination such as, for example, testing for antibody binding to TF variants with different point-mutations, or to chimeric TF variants.
  • Percent “identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent 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, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE 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.
  • a “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of an amino acid with a chemically or functionally similar amino acid.
  • Conservative substitution tables providing similar amino acids are well known in the art.
  • the groups of amino acids provided in Tables 2-4 are, in some embodiments, considered conservative substitutions for one another.
  • Table 2 Selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.
  • Table 3 Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.
  • Table 4 Further selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.
  • amino acid refers to the twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • host cell refers to cells into which an exogenous nucleic acid has been introduced, and the progeny of such cells.
  • Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), which each include the primary transformed or transfected cell and progeny derived therefrom.
  • Such progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations.
  • treating refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the term “treat” refers to improving the results for a subject in the early treatment of diabetic retinopathy (EDTRS) on the eye chart from the start of therapy.
  • ETRS diabetic retinopathy
  • the term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.
  • An effective amount is sufficient to effect a desired results or benefit in a subject.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the terms “baseline levels” and “baseline” refer to the levels for a parameter (e.g ., retinal thickness) immediately prior to treatment or at the time of treatment.
  • the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs and sheep. In certain embodiments, the subject is a human. In some embodiments the subject has a disease or condition that can be treated with an antibody provided herein. In some aspects, the disease or condition involves neovascularization or vascular inflammation. In certain aspects, the disease or condition involving neovascularization is age-related macular degeneration (AMD), diabetic retinopathy, or other ocular diseases.
  • AMD age-related macular degeneration
  • AMD diabetic retinopathy
  • the phrase “subject in need thereof’ refers to a subject that exhibits and/or is diagnosed with one or more symptoms or signs of ocular disease as described herein.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g ., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
  • a “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer.
  • Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.
  • cytostatic agent refers to a compound or composition which arrests growth of a cell either in vitro or in vivo.
  • a cytostatic agent is an agent that reduces the percentage of cells in S phase.
  • a cytostatic agent reduces the percentage of cells in S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.
  • modulate and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
  • increase and activate refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
  • reduce and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
  • the term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor.
  • An “agonist” is an entity that binds to and agonizes a receptor.
  • the term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor.
  • An “antagonist” is an entity that binds to and antagonizes a receptor.
  • the TF is hTF (SEQ ID NO:809). In some aspects, the TF is cTF (SEQ ID NO:813). In some aspects, the TF is mTF (SEQ ID NO:817). In some aspects, the TF is rabbit TF (SEQ ID NO:832). In some aspects, the TF is pTF (SEQ ID NO:824).
  • the antibodies provided herein specifically bind to hTF (SEQ ID NO: 809), cTF (SEQ ID NO:813), mTF (SEQ ID NO:817), rabbit TF (SEQ ID NO:832), and pTF (SEQ ID NO:824).
  • the antibodies provided herein specifically bind to hTF (SEQ ID NO:809), cTF (SEQ ID NO:813), mTF (SEQ ID NO:817), and pTF (SEQ ID NO:824).
  • the antibodies provided herein specifically bind to hTF (SEQ ID NO:809), cTF (SEQ ID NO:813), and mTF (SEQ ID NO:817).
  • the antibodies provided herein specifically bind to hTF (SEQ ID NO: 809) and cTF (SEQ ID NO:813). In some embodiments, the antibodies provided herein do not bind mTF (SEQ ID NO:817). In some embodiments, the antibodies provided herein do not bind pTF (SEQ ID NO:824). In some embodiments, the antibodies provided herein do not bind rabbit TF (SEQ ID NO:832).
  • the antibodies provided herein specifically bind to the extracellular domain of human TF (SEQ ID NO:810).
  • the binding between an antibody provided herein and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is K149N.
  • the binding between an antibody provided herein and a variant TF extracellular domain comprising a mutation at amino acid residue 68 of the sequence shown in SEQ ID NO:810 is greater than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the mutation at amino acid residue 68 of the sequence shown in SEQ ID NO:810 is K68N.
  • the binding between an antibody provided herein and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 are N171H and T197K.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 1-77 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 1-76 of the sequence shown in SEQ ID NO:838 is greater than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 39-77 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 38-76 of the sequence shown in SEQ ID NO:838 is greater than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 94-107 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 99-112 of the sequence shown in SEQ ID NO:838 is greater than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 146-158 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 151-163 of the sequence shown in SEQ ID NO:838 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 159-219 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 164-224 of the sequence shown in SEQ ID NO:838 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 159-189 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 164-194 of the sequence shown in SEQ ID NO:838 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 159-174 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 164-179 of the sequence shown in SEQ ID NO:838 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a human TF extracellular domain with amino acid residues 167-174 of the sequence shown in SEQ ID NO:810 replaced by rat TF extracellular domain amino acid residues 172-179 of the sequence shown in SEQ ID NO:838 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a rat TF extracellular domain with amino acid residues 141-194 of the sequence shown in SEQ ID NO: 838 replaced by human TF extracellular domain amino acid residues 136-189 of the sequence shown in SEQ ID NO:810 is greater than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • the binding between an antibody provided herein and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810; the binding between an antibody provided herein and a variant TF extracellular domain comprising a mutation at amino acid residue 68 of the sequence shown in SEQ ID NO:810 is greater than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810; the binding between an antibody provided herein and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody provided herein and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810; the binding between an antibody provided herein and a human TF extracellular domain with amino acid residue
  • the mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is K149N; the mutation at amino acid residue 68 of the sequence shown in SEQ ID NO:810 is K68N; and the mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 are N171H and T197K.
  • the antibodies provided herein are inert in inhibiting human thrombin generation as determined by thrombin generation assay (TGA) compared to a reference antibody Ml 593, wherein the reference antibody Ml 593 comprises a VH sequence of SEQ ID NO:821 and a VL sequence of SEQ ID NO:822.
  • TGA thrombin generation assay
  • the antibodies provided herein do not inhibit human thrombin generation as determined by thrombin generation assay (TGA).
  • the antibodies provided herein allow human thrombin generation as determined by thrombin generation assay (TGA).
  • the antibodies provided herein bind human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX. In certain embodiments, the antibodies provided herein do not interfere with the ability of TF:FVIIa to convert FX into FXa.
  • the antibodies provided herein bind human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa. In certain embodiments, the antibodies provided herein do not compete for binding to human TF with human FVIIa.
  • the antibodies provided herein bind to the extracellular domain of human TF, bind human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa, bind human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX, and allow human thrombin generation as determined by thrombin generation assay (TGA).
  • TGA thrombin generation assay
  • the antibodies provided herein bind to the extracellular domain of human TF, do not inhibit human thrombin generation as determined by thrombin generation assay (TGA), do not interfere with the ability of TF:FVIIa to convert FX into FXa, and do not compete for binding to human TF with human FVIIa.
  • TGA thrombin generation assay
  • the antibodies provided herein bind to the extracellular domain of human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa, do not inhibit human thrombin generation as determined by thrombin generation assay (TGA), allow human thrombin generation as determined by thrombin generation assay (TGA), bind to human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX, do not interfere with the ability of TF :FVIIa to convert FX into FXa, and do not compete for binding to human TF with human FVIIa.
  • the antibodies provided herein inhibit FVIIa-dependent TF signaling.
  • the antibodies provided herein reduce lesion size in a swine choroidal neovascularization (CNV) model.
  • TF is aberrantly expressed in choroidal neovascularization (CNV) found in wet age-related macular degeneration.
  • the antibodies provided herein bind to the extracellular domain of human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa, do not inhibit human thrombin generation as determined by thrombin generation assay (TGA), allow human thrombin generation as determined by thrombin generation assay (TGA), bind to human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX, do not interfere with the ability of TF :FVIIa to convert FX into FXa, do not compete for binding to human TF with human FVIIa, and bind to cynomolgus and mouse TF.
  • TGA thrombin generation assay
  • TGA thrombin generation assay
  • the antibodies provided herein bind to the extracellular domain of human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa, do not inhibit human thrombin generation as determined by thrombin generation assay (TGA), allow human thrombin generation as determined by thrombin generation assay (TGA), bind to human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX, do not interfere with the ability of TF :FVIIa to convert FX into FXa, do not compete for binding to human TF with human FVIIa, bind to cynomolgus, mouse, and pig TF, and reduce lesion size in a swine choroidal neovascularization (CNV) model.
  • TGA thrombin generation assay
  • TGA thrombin generation assay
  • TGA thrombin generation assay
  • TGA thrombin generation assay
  • TGA
  • the antibodies provided herein bind to the extracellular domain of human TF, inhibit FVIIa-dependent TF signaling, and bind to cynomolgus TF.
  • an antibody provided herein comprises a VH sequence selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:37.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:75.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:l 13.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO: 151.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO: 189. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:836. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:227. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:265. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:303. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:341. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:379.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:417. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:455. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:493. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:531. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:569. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:607. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:645.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:683. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:721. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:759.
  • an antibody provided herein comprises a VH sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VH sequence provided in SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759.
  • an antibody provided herein comprises a VH sequence provided in SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, with up to 1, 2, 3, 4, 5,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a VL sequence selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO:38.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO:76.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO: 114.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO: 152.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO: 190. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:837. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:228. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:266. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:304. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:342. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:380.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO:418. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:456. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:494. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:532. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:570. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:608. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:646.
  • an antibody provided herein comprises a VL sequence of SEQ ID NO:684. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:722. In some embodiments, an antibody provided herein comprises a VL sequence of SEQ ID NO:760.
  • an antibody provided herein comprises a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VL sequence provided in SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • an antibody provided herein comprises a VL sequence provided in SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760, with up to 1, 2, 3, 4, 5,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a VH sequence selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759 and a VL sequence selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:37 and a VL sequence of SEQ ID NO:38.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:75 and a VL sequence of SEQ ID NO:76. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO: 113 and a VL sequence of SEQ ID NO: 114. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:151 and a VL sequence of SEQ ID NO: 152. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO: 189 and a VL sequence of SEQ ID NO: 190. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:836 and a VL sequence of SEQ ID NO:837.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:227 and a VL sequence of SEQ ID NO:228. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:265 and a VL sequence of SEQ ID NO:266. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:303 and a VL sequence of SEQ ID NO:304. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:341 and a VL sequence of SEQ ID NO:342. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:379 and a VL sequence of SEQ ID NO:380.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO:417 and a VL sequence of SEQ ID NO:418. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:455 and a VL sequence of SEQ ID NO:456. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:493 and a VL sequence of SEQ ID NO:494. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:531 and a VL sequence of SEQ ID NO:532. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO: 569 and a VL sequence of SEQ ID NO:570.
  • an antibody provided herein comprises a VH sequence of SEQ ID NO: 607 and a VL sequence of SEQ ID NO: 608. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:645 and a VL sequence of SEQ ID NO:646. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:683 and a VL sequence of SEQ ID NO:684. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:721 and a VL sequence of SEQ ID NO:722. In some embodiments, an antibody provided herein comprises a VH sequence of SEQ ID NO:759 and a VL sequence of SEQ ID NO:760.
  • an antibody provided herein comprises a VH sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VH sequence provided in SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, and a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VL sequence provided in SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • an antibody provided herein comprises a VH sequence provided in SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions, and a VL sequence provided in SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises one to three CDRs of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759.
  • an antibody provided herein comprises two to three CDRs of a VH domain selected from SEQ ID NOs:
  • an antibody provided herein comprises three CDRs of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759.
  • the CDRs are Exemplary CDRs.
  • the CDRs are Rabat CDRs.
  • the CDRs are Chothia CDRs.
  • the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.
  • the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759.
  • the CDR-H1 is a CDR-H1 of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, with up to 1, 2, 3, 4, or 5 amino acid substitutions.
  • the CDR-H2 is a CDR-H2 of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, with up to 1,
  • the CDR-H3 is a CDR- H3 of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises one to three CDRs of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760. In some embodiments, an antibody provided herein comprises two to three CDRs of a VL domain selected from SEQ ID NOs:
  • an antibody provided herein comprises three CDRs of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • the CDRs are Exemplary CDRs.
  • the CDRs are Rabat CDRs.
  • the CDRs are Chothia CDRs.
  • the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.
  • the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • the CDR-L1 is a CDR-L1 of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760, with up to 1, 2, 3, 4, or 5 amino acid substitutions.
  • the CDR- L2 is a CDR-L2 of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760, with up to 1, 2, 3, 4, 5,
  • the CDR-L3 is a CDR-L3 of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises one to three CDRs of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759 and one to three CDRs of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • an antibody provided herein comprises two to three CDRs of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759 and two to three CDRs of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • an antibody provided herein comprises three CDRs of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759 and three CDRs of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760.
  • the CDRs are Exemplary CDRs.
  • the CDRs are Rabat CDRs.
  • the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs. [00204] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759 and at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418,
  • the CDR-H1 is a CDR-H1 of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455, 493, 531, 569, 607, 645, 683, 721, and 759, with up to 1, 2, 3, 4, or 5 amino acid substitutions;
  • the CDR-H2 is a CDR-H2 of a VH domain selected from SEQ ID NOs: 37, 75,
  • the CDR-H3 is a CDR-H3 of a VH domain selected from SEQ ID NOs: 37, 75, 113, 151, 189, 227, 265, 303, 341, 379, 417, 455,
  • the CDR-L1 is a CDR-L1 of a VL domain selected from SEQ ID NOs: 38, 76,
  • the CDR-L2 is a CDR-L2 of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456,
  • the CDR-L3 is a CDR-L3 of a VL domain selected from SEQ ID NOs: 38, 76, 114, 152, 190, 228, 266, 304, 342, 380, 418, 456, 494, 532, 570, 608, 646, 684, 722, and 760, with up to 1, 2, 3, 4, or 5 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • an antibody provided herein comprises a CDR-H3 selected from SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535,
  • the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H3 of SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535, 573, 611, 649, 687, and 725.
  • the CDR-H3 is a CDR-H3 selected from SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-H2 selected from SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344, 382, 420, 458, 496, 534,
  • the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344, 382, 420, 458, 496, 534, 572, 610, 648, 686, and 724.
  • the CDR-H2 is a CDR-H2 selected from SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344, 382, 420, 458, 496, 534,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-H1 selected from SEQ ID NOs: 1, 39, 77, 115, 153, 191, 229, 267, 305, 343, 381, 419, 457, 495, 533,
  • the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H1 of SEQ ID NOs: 1, 39, 77, 115, 153, 191, 229, 267, 305, 343, 381, 419, 457, 495, 533, 571, 609, 647, 685, and 723.
  • the CDR-H1 is a CDR-H1 selected from SEQ ID NOs: 1, 39, 77, 115, 153, 191, 229, 267, 305, 343, 381, 419, 457, 495, 533,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-H3 selected from SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535,
  • an antibody provided herein comprises a CDR-H3 selected from SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535, 573, 611, 649, 687, and 725, a CDR-H2 selected from SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344,
  • the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H3 of SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535, 573, 611, 649, 687, and 725
  • the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NOs: 2, 40,
  • the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR- H1 of SEQ ID NOs: 1, 39, 77, 115, 153, 191, 229, 267, 305, 343, 381, 419, 457, 495, 533, 571, 609, 647, 685, and 723.
  • the CDR-H3 is a CDR-H3 selected from SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535, 573, 611, 649, 687, and 725, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions;
  • the CDR-H2 is a CDR-H2 selected from SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344, 382,
  • the CDR-H1 is a CDR-H1 selected from SEQ ID NOs: 1, 39, 77, 115, 153, 191, 229, 267, 305, 343, 381, 419, 457, 495, 533, 571, 609, 647, 685, and 723, with up to 1, 2, 3, 4, or 5 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibody described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538,
  • the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L3 of SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538, 576, 614, 652, 690, and 728.
  • the CDR-L3 is a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-L2 selected from SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537,
  • the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537, 575, 613, 651, 689, and 727.
  • the CDR-L2 is a CDR-L2 selected from SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-L1 selected from SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270, 308, 346, 384, 422, 460, 498, 536,
  • the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1 of SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270, 308, 346, 384, 422, 460, 498, 536, 574, 612, 650, 688, and 726.
  • the CDR-L1 is a CDR-L1 selected from SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270, 308, 346, 384, 422, 460, 498, 536,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538,
  • an antibody provided herein comprises a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538, 576, 614, 652, 690, and 728, a CDR-L2 selected from SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347,
  • the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L3 of SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538, 576, 614, 652, 690, and 728, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NOs: 5, 43,
  • the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR- LI of SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270, 308, 346, 384, 422, 460, 498, 536,
  • the CDR-L3 is a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538, 576, 614, 652, 690, and 728, with up to 1, 2, 3, 4, or 5 amino acid substitutions
  • the CDR-L2 is a CDR- L2 selected from SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537, 575, 613, 651, 689, and 727, with up to 1, 2, 3, or 4 amino acid substitutions
  • the CDR-L1 is a CDR-L1 selected from SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270,
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-H3 selected from SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535,
  • a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538, 576, 614, 652, 690, and 728
  • a CDR-L2 selected from SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537,
  • CDR-L1 selected from SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270, 308, 346, 384, 422, 460, 498, 536, 574, 612, 650, 688, and 726.
  • the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H3 of SEQ ID NOs: 3, 41, 79, 117, 155, 193, 231, 269, 307, 345, 383, 421, 459, 497, 535, 573, 611, 649, 687, and 725
  • the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344, 382, 420, 458, 496, 534, 572, 610, 648, 686, and 724
  • the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H1 of SEQ ID NOs: 1, 39,
  • the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L3 of SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538, 576, 614, 652, 690, and 728
  • the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537, 575, 613, 651, 689, and 727
  • the CDR-L1 has at least about 50%,
  • the CDR-H2 is a CDR-H2 selected from SEQ ID NOs: 2, 40, 78, 116, 154, 192, 230, 268, 306, 344, 382, 420, 458, 496, 534, 572, 610, 648, 686, and 724, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions
  • the CDR-H1 is a CDR-H1 selected from SEQ ID NOs: 1, 39, 77, 115, 153, 191, 229, 267, 305, 343, 381, 419, 457, 495, 533, 571, 609, 647, 685, and 723, with up to 1, 2, 3, 4, or 5 amino acid substitutions
  • the CDR-L3 is a CDR-L3 selected from SEQ ID NOs: 6, 44, 82, 120, 158, 196, 234, 272, 310, 348, 386, 424, 462, 500, 538,
  • the CDR-L2 is a CDR-L2 selected from SEQ ID NOs: 5, 43, 81, 119, 157, 195, 233, 271, 309, 347, 385, 423, 461, 499, 537, 575, 613, 651, 689, and 727, with up to 1, 2, 3, or 4 amino acid substitutions
  • the CDR-L1 is a CDR-L1 selected from SEQ ID NOs: 4, 42, 80, 118, 156, 194, 232, 270, 308, 346, 384, 422, 460, 498, 536, 574, 612, 650, 688, and 726, with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibodies described in this paragraph are referred to herein as “variants.”
  • such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.
  • such variants are not derived from a sequence provided herein and may, for example, be isolated de novo according to the methods provided herein for obtaining antibodies.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO:2, a CDR-H3 of SEQ ID NO:3, a CDR-L1 of SEQ ID NO:4, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:6, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:39, a CDR-H2 of SEQ ID NO:40, a CDR-H3 of SEQ ID NO:41, a CDR-L1 of SEQ ID NO:42, a CDR-L2 of SEQ ID NO:43, and a CDR-L1 of SEQ ID NO:44, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO: 77, a CDR-H2 of SEQ ID NO: 78, a CDR-H3 of SEQ ID NO: 79, a CDR-L1 of SEQ ID NO: 80, a CDR-L2 of SEQ ID NO: 81, and a CDR-L1 of SEQ ID NO: 82, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO: 115, a CDR-H2 of SEQ ID NO:l 16, a CDR-H3 of SEQ ID NO:117, a CDR-L1 of SEQ ID NO: 118, a CDR-L2 of SEQ ID NO: 119, and a CDR-L1 of SEQ ID NO: 120, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO: 153, a CDR-H2 of SEQ ID NO:154, a CDR-H3 of SEQ ID NO:155, a CDR-L1 of SEQ ID NO: 156, a CDR-L2 of SEQ ID NO: 157, and a CDR-L1 of SEQ ID NO: 158, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:884, a CDR-H2 of SEQ ID NO:885, a CDR-H3 of SEQ ID NO:886, a CDR-L1 of SEQ ID NO:887, a CDR-L2 of SEQ ID NO:888, and a CDR-L1 of SEQ ID NO:889, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO : 191 , a CDR-H2 of SEQ ID NO : 192, a CDR-H3 of SEQ ID NO : 193 , a CDR-L 1 of SEQ ID NO: 194, a CDR-L2 of SEQ ID NO: 195, and a CDR-L1 of SEQ ID NO: 196, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:229, a CDR-H2 of SEQ ID NO:230, a CDR-H3 of SEQ ID NO:231, a CDR-L1 of SEQ ID NO:232, a CDR-L2 of SEQ ID NO:233, and a CDR-L1 of SEQ ID NO:234, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:267, a CDR-H2 of SEQ ID NO:268, a CDR-H3 of SEQ ID NO:269, a CDR-L1 of SEQ ID NO:270, a CDR-L2 of SEQ ID NO:271, and a CDR-L1 of SEQ ID NO:272, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:305, a CDR-H2 of SEQ ID NO:306, a CDR-H3 of SEQ ID NO:307, a CDR-L1 of SEQ ID NO: 308, a CDR-L2 of SEQ ID NO: 309, and a CDR-L 1 of SEQ ID NO: 310, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:343, a CDR-H2 of SEQ ID NO:344, a CDR-H3 of SEQ ID NO:345, a CDR-L1 of SEQ ID NO:346, a CDR-L2 of SEQ ID NO:347, and a CDR-L1 of SEQ ID NO:348, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:381, a CDR-H2 of SEQ ID NO:382, a CDR-H3 of SEQ ID NO:383, a CDR-L1 of SEQ ID NO:384, a CDR-L2 of SEQ ID NO:385, and a CDR-L1 of SEQ ID NO:386, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:419, a CDR-H2 of SEQ ID NO:420, a CDR-H3 of SEQ ID NO:421, a CDR-L1 of SEQ ID NO:422, a CDR-L2 of SEQ ID NO:423, and a CDR-L1 of SEQ ID NO:424, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:457, a CDR-H2 of SEQ ID NO:458, a CDR-H3 of SEQ ID NO:459, a CDR-L1 of SEQ ID NO:460, a CDR-L2 of SEQ ID NO:461, and a CDR-L1 of SEQ ID NO:462, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:495, a CDR-H2 of SEQ ID NO:496, a CDR-H3 of SEQ ID NO:497, a CDR-L1 of SEQ ID NO:498, a CDR-L2 of SEQ ID NO:499, and a CDR-L1 of SEQ ID NO:500, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:533, a CDR-H2 of SEQ ID NO:534, a CDR-H3 of SEQ ID NO:535, a CDR-L1 of SEQ ID NO:536, a CDR-L2 of SEQ ID NO:537, and a CDR-L1 of SEQ ID NO:538, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:571, a CDR-H2 of SEQ ID NO:572, a CDR-H3 of SEQ ID NO:573, a CDR-L1 of SEQ ID NO:574, a CDR-L2 of SEQ ID NO:575, and a CDR-L1 of SEQ ID NO:576, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:609, a CDR-H2 of SEQ ID NO:610, a CDR-H3 of SEQ ID NO:611, a CDR-L1 of SEQ ID NO:612, a CDR-L2 of SEQ ID NO:613, and a CDR-L1 of SEQ ID NO:614, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:647, a CDR-H2 of SEQ ID NO:648, a CDR-H3 of SEQ ID NO:649, a CDR-L1 of SEQ ID NO:650, a CDR-L2 of SEQ ID NO:651, and a CDR-L1 of SEQ ID NO:652, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:685, a CDR-H2 of SEQ ID NO:686, a CDR-H3 of SEQ ID NO:687, a CDR-L1 of SEQ ID NO:688, a CDR-L2 of SEQ ID NO:689, and a CDR-L1 of SEQ ID NO:690, as determined by the Exemplary numbering system.
  • an antibody provided herein comprises a CDR-H1 of SEQ ID NO:723, a CDR-H2 of SEQ ID NO:724, a CDR-H3 of SEQ ID NO:725, a CDR-L1 of SEQ ID NO: 726, a CDR-L2 of SEQ ID NO: 727, and a CDR-L1 of SEQ ID NO: 728, as determined by the Exemplary numbering system.
  • a first family of antibodies comprising the following six CDR sequences: (a) a CDR-H1 having the sequence G-F-T-F-S-X1-Y-A-M-X2, wherein Xi is D or S and X2 is A or G (SEQ ID NO:773); (b) a CDR-H2 having the sequence X3-I-S-G-S-G-G-L-T-Y-Y-A-D-S-V-K-G, wherein X3 is A or T (SEQ ID NO:774); (c) a CDR-H3 having the sequence APYGYYMDV (SEQ ID NO:775); (d) a CDR-L1 having the sequence RASQSISSWLA (SEQ ID NO:776); (e) a CDR-L2 having the sequence KASSLES (SEQ ID NO:777); and (f) a CDR-
  • an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence G-Y-T-F-X1-X2-Y-G-I-S, wherein Xi is D or R and X2 is S or V (SEQ ID NO: 779); (b) a CDR-H2 having the sequence W-X3-A-P-Y-X4-G-N-T-N-Y-A-Q-K-L-Q- G, wherein X3 is I or V and X4 is S or N (SEQ ID NO:780); (c) a CDR-H3 having the sequence D-A-G-T-Y-S-P-Xs-G-Y-G-M-D-V, wherein X 5 is F or Y (SEQ ID NO:781); (d) a CDR-L1 having the sequence X6-A-S-X
  • an antibody of such family comprises a VH sequence of SEQ ID NO:763 and a VL sequence of SEQ ID NO:764. In some embodiments, provided herein is an antibody within such second family.
  • an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence G-F-T-F-X1-S-X2-G-M-H, wherein Xi is H or R and X2 is R or Y (SEQ ID NO:785); (b) a CDR-H2 having the sequence VITYDGINKYYADSVEG (SEQ ID NO:786); (c) a CDR-H3 having the sequence DGVYYGVYDY (SEQ ID NO: 787); (d) a CDR-L1 having the sequence KSSQSVLFSSNNKNYLA (SEQ ID NO:788); (e) a CDR-L2 having the sequence WASTRES (SEQ ID NO:789); and (f) a CDR-L3 having the sequence QQFHSYPLT (SEQ ID NO:790).
  • an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence G-
  • an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence GGTFSSNAIG (SEQ ID NO:791); (b) a CDR-H2 having the sequence SIIPIIGF ANY AQKF Q G (SEQ ID NO: 792); (c) a CDR-H3 having the sequence DSGYYYGASSFGMDV (SEQ ID NO:793); (d) a CDR-L1 having the sequence RASQSVSSNLA (SEQ ID NO:794); (e) a CDR-L2 having the sequence GASTRAT (SEQ ID NO:795); and (f) a CDR-L3 having the sequence EQYNNLPLT (SEQ ID NO:796).
  • an antibody of such family comprises a VH sequence of SEQ ID NO:767 and a VL sequence of SEQ ID NO:768.
  • an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence G-G-S-X1-S-S-G-X2-Y-W-S, wherein Xi is I or L and X2 is Q or Y (SEQ ID NO:797); (b) a CDR-H2 having the sequence E-I-X3-X4-S-G-S-T-R-Y-N-P-S-L-K-S, wherein X3 is Y or G and X4 is Y or A (SEQ ID NO:798); (c) a CDR-H3 having the sequence D-Xs-P-Y-Y-Y-Xe-G-G-Y-Y-Y-Y-M-D-V, wherein X 5 is T or A and Xe is E, G, or D (SEQ ID NO:799); (d)
  • an antibody of such family comprises a VH sequence of SEQ ID NO:769 and a VL sequence of SEQ ID NO:770. In some embodiments, provided herein is an antibody within such fifth family. [00240] In some embodiments, provided herein is a sixth family of antibodies, wherein an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence GYTFANYYMH (SEQ ID NO:803); (b) a CDR-H2 having the sequence IINPSGGITVYAQKFQG (SEQ ID NO:804); (c) a CDR-H3 having the sequence GGSKVAALAFDI (SEQ ID NO: 805); (d) a CDR-L1 having the sequence QASQDISNSLN (SEQ ID NO: 806); (e) a CDR-L2 having the sequence DASNLET (SEQ ID NO: 807); and (f) a CDR-L3 having the sequence QQYNFHP
  • a seventh family of antibodies wherein an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence G-Y-T-F-D-Xi-Y-G-I-S, wherein Xi is V or A (SEQ ID NO:872); (b) a CDR-H2 having the sequence W-I-A-P-Y-X2-G-N-T-N-Y-A-Q-K-L-Q-G, wherein X2 is N or S (SEQ ID NO: 873); (c) a CDR-H3 having the sequence D-A-G-T-Y-S-P-F-G-Y-G-M-D-V (SEQ ID NO:874); (d) a CDR-L1 having the sequence X3-A-S-X4-S-I-X5-X6-W-L-A, wherein X3 is R or Q, X
  • an antibody of such family comprises the following six CDR sequences: (a) a CDR-H1 having the sequence G-Y-T-F-R-S-Y-G-I-S (SEQ ID NO:878); (b) a CDR-H2 having the sequence W-V-A-P-Y-Xi-G-N-T-N-Y-A-Q-K-L-Q-G, wherein Xi is S or N (SEQ ID NO:879); (c) a CDR-H3 having the sequence D-A-G-T-Y-S-P-Y-G-Y-G-M-D-V (SEQ ID NO:880); (d) a CDR-L1 having the sequence X2-A-S-X3-S-I-X4-S-W-L-A, wherein X2 is R or Q, X3 is Q or H, X4 is S or D
  • antibody variants defined based on percent identity to an illustrative antibody sequence provided herein, or substitution of amino acid residues in comparison to an illustrative antibody sequence provided herein.
  • a variant of an antibody provided herein has specificity for hTF. In some embodiments, a variant of an antibody provided herein has specificity for cTF. In some embodiments, a variant of an antibody provided herein has specificity for mTF. In some embodiments, a variant of an antibody provided herein has specificity for hTF and cTF. In some embodiments, a variant of an antibody provided herein has specificity for hTF and mTF. In some embodiments, a variant of an antibody provided herein has specificity for cTF and mTF. In some embodiments, a variant of an antibody provided herein has specificity for hTF, cTF and mTF.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for hTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for cTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for mTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for both hTF and cTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for both hTF and mTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for both cTF and mTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody that is derived from an illustrative antibody sequence provided herein retains affinity, as measured by KD, for all three of hTF, cTF and mTF that is within 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 the affinity of such illustrative antibody.
  • a variant of an antibody provided herein retains the ability to inhibit TF signaling, as measured by one or more assays or biological effects described herein. In some embodiments, a variant of an antibody provided herein retains the normal function of TF in the blood coagulation processes.
  • a variant of an antibody provided herein competes for binding to TF with an antibody selected from IF, 1G, 25A, 25A3, 25A5, 25A5-T, 25G,
  • a variant of an antibody provided herein competes for binding to TF with an antibody selected from 25 A, 25 A3,
  • a variant of an antibody provided herein competes for binding to TF with an antibody selected from 43B, 43B1,
  • a variant of an antibody provided herein competes for binding to TF with an antibody selected from 25 A, 25 A3,
  • a variant of an antibody provided herein competes for binding to TF with an antibody selected from IF, 1G, 29D, 29E, 39A, or 54E.
  • a variant of an antibody provided herein allows human thrombin generation as determined by thrombin generation assay (TGA). In some embodiments, a variant of an antibody provided herein does not inhibit human thrombin generation as determined by thrombin generation assay (TGA).
  • a variant of an antibody provided herein binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX. In some embodiments, a variant of an antibody provided herein does not interfere with the ability of TF :F Vila to convert FX into FXa.
  • a variant of an antibody provided herein binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa. In some embodiments, a variant of an antibody provided herein does not compete for binding to human TF with human FVIIa.
  • a variant of an antibody provided herein inhibits FVIIa-dependent TF signaling.
  • a variant of an antibody provided herein binds mouse TF (SEQ ID NO:817). In some embodiments, a variant of an antibody provided herein binds mouse TF with an affinity lower (as indicated by higher KD) than the affinity of the antibody for hTF. In some embodiments, a variant of an antibody provided herein does not bind mTF. [00253] In some embodiments, a variant of an antibody provided herein binds pig TF (SEQ ID NO:824). In some embodiments, a variant of an antibody provided herein binds pig TF with an affinity lower (as indicated by higher KD) than the affinity of the antibody for hTF. In some embodiments, a variant of an antibody provided herein does not bind pTF. [00254] In some embodiments, a variant of an antibody provided herein binds the same epitope of TF as such antibody.
  • an antibody provided herein has one or more of the characteristics listed in the following (a)-(dd): (a) binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; (b) does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); (c) does not reduce the thrombin peak on a thrombin generation curve (Peak Ila) compared to an isotype control; (d) does not increase the time from the assay start to the thrombin peak on a thrombin generation curve (ttPeak) compared to an isotype control; (e) does not decrease the endogenous thrombin potential (ETP) as determined by the area under a thrombin generation curve compared to an isotype control; (f) allows human thrombin generation as determined by thrombin generation assay (TGA); (g) maintains the thrombin peak
  • an antibody provided herein has two or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has three or more of the characteristics listed in the foregoing (a)- (dd). In some embodiments, an antibody provided herein has four or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has five or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has six or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has seven or more of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has eight or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has nine or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has ten or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has eleven or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twelve or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has thirteen or more of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has fourteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has fifteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has sixteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has seventeen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has eighteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has nineteen or more of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has twenty or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-one or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-two or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-three of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-four of the characteristics listed in the foregoing (a)- (dd). In some embodiments, an antibody provided herein has twenty-five of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has twenty-six of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-seven of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-eight of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-nine of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has all thirty of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has one or more of the characteristics listed in the following (a)-(dd): (a) binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; (b) does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); (c) does not reduce the thrombin peak on a thrombin generation curve (Peak Ila) compared to an isotype control; (d) does not increase the time from the assay start to the thrombin peak on a thrombin generation curve (ttPeak) compared to an isotype control; (e) does not decrease the endogenous thrombin potential (ETP) as determined by the area under a thrombin generation curve compared to an isotype control; (f) allows human thrombin generation as determined by thrombin generation assay (TGA); (g) maintains the thrombin peak
  • an antibody provided herein has two or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has three or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has four or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has five or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has six or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has seven or more of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has eight or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has nine or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has ten or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has eleven or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twelve or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has thirteen or more of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has fourteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has fifteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has sixteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has seventeen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has eighteen or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has nineteen or more of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has twenty or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-one or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-two or more of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-three of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-four of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-five of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein has twenty-six of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-seven of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-eight of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has twenty-nine of the characteristics listed in the foregoing (a)-(dd). In some embodiments, an antibody provided herein has all thirty of the characteristics listed in the foregoing (a)-(dd).
  • an antibody provided herein exhibits a combination of characteristics comprising two or more of characteristics listed in the following (a)-(dd): (a) binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; (b) does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); (c) does not reduce the thrombin peak on a thrombin generation curve (Peak Ila) compared to an isotype control; (d) does not increase the time from the assay start to the thrombin peak on a thrombin generation curve (ttPeak) compared to an isotype control; (e) does not decrease the endogenous thrombin potential (ETP) as determined by the area under a thrombin generation curve compared to an isotype control; (f) allows human thrombin generation as determined by thrombin generation assay (TGA); (g)
  • an antibody provided herein exhibits a combination of characteristics comprising two or more of characteristics listed in the following (a)-(dd): (a) binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; (b) does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); (c) does not reduce the thrombin peak on a thrombin generation curve (Peak Ila) compared to an isotype control; (d) does not increase the time from the assay start to the thrombin peak on a thrombin generation curve (ttPeak) compared to an isotype control; (e) does not decrease the endogenous thrombin potential (ETP) as determined by the area under a thrombin generation curve compared to an isotype control; (f) allows human thrombin generation as determined by thrombin generation assay (TGA); (g)
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); and the binding between the antibody and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); and the binding between the antibody and a variant TF extracellular domain comprising mutations N171H and T197K of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; allows human thrombin generation as determined by thrombin generation assay (TGA); and the binding between the antibody and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; allows human thrombin generation as determined by thrombin generation assay (TGA); and the binding between the antibody and a variant TF extracellular domain comprising mutations N171H and T197K of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay.
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); the binding between the antibody and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810,
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); the binding between the antibody and a variant TF extracellular domain comprising a mutation K149N of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations N171H and T197K of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the sequence shown
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; allows human thrombin generation as determined by thrombin generation assay (TGA); the binding between the antibody and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; allows human thrombin generation as determined by thrombin generation assay (TGA); the binding between the antibody and a variant TF extracellular domain comprising a mutation K149N of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations N171H and T197K of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluor
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); binds to cynomolgus TF; the binding between the antibody and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; does not inhibit human thrombin generation as determined by thrombin generation assay (TGA); binds to cynomolgus TF; the binding between the antibody and a variant TF extracellular domain comprising a mutation K149N of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations N171H and T197K of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; allows human thrombin generation as determined by thrombin generation assay (TGA); binds to cynomolgus TF; the binding between the antibody and a variant TF extracellular domain comprising a mutation at amino acid residue 149 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations at amino acid residues 171 and 197 of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF
  • an antibody provided herein exhibits a combination of the characteristics listed in the following: binds human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa; allows human thrombin generation as determined by thrombin generation assay (TGA); binds to cynomolgus TF; the binding between the antibody and a variant TF extracellular domain comprising a mutation K149N of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO: 810, as determined by the median fluorescence intensity value of the antibody relative to an isotype control in a live cell staining assay; and the binding between the antibody and a variant TF extracellular domain comprising mutations N171H and T197K of the sequence shown in SEQ ID NO:810 is less than 50% of the binding between the antibody and the extracellular domain of TF of the sequence shown in SEQ ID NO:
  • the affinity of an antibody provided herein for TF as indicated by KD is less than about 10 5 M, less than about 10 6 M, less than about 10 7 M, less than about 10 8 M, less than about 10 9 M, less than about 10 10 M, less than about 10 11 M, or less than about 10 12 M.
  • the affinity of the antibody is between about 10 7 M and 10 12 M.
  • the affinity of the antibody is between about 10 7 M and 10 11 M.
  • the affinity of the antibody is between about 10 7 M and 10 10 M.
  • the affinity of the antibody is between about 10 7 M and 10 9 M.
  • the affinity of the antibody is between about 10 7 M and 10 8 M.
  • the affinity of the antibody is between about 10 8 M and 10 12 M.
  • the affinity of the antibody is between about 10 8 M and 10 11 M. In some embodiments, the affinity of the antibody is between about 10 9 M and 10 11 M. In some embodiments, the affinity of the antibody is between about 10 10 M and 10 11 M. In some embodiments, the affinity of the antibody is less than about 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM or 1.0 nM.
  • the KD value of an antibody provided herein for cTF is no more than 15x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for cTF is no more than 10* of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for cTF is no more than 8x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for cTF is no more than 5x of the KD value of the antibody for hTF.
  • the KD value of an antibody provided herein for cTF is no more than 3x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for cTF is no more than 2x of the KD value of the antibody for hTF. [00273] In some embodiments, the KD value of an antibody provided herein for mTF is no more than 20x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for mTF is no more than 15x of the KD value of the antibody for hTF.
  • the KD value of an antibody provided herein for mTF is no more than 10x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for mTF is no more than 5x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for mTF is no more than 2x of the KD value of the antibody for hTF.
  • the affinity of an antibody provided herein for hTF as indicated by KD measured by Biacore, as set forth in Table 5 is selected from about 0.31 nM, about 6.20 nM, about 0.36 nM, about 0.08 nM, about 23.0 nM, about 0.94 nM, about 13.3 nM, about 0.47 nM, about 0.09 nM, about 1.75 nM, about 0.07 nM, about 0.14 nM, about 2.09 nM, about 0.06 nM, about 0.15 nM, about 1.46 nM, about 1.60 nM, and about 0.42 nM.
  • such affinity as indicated by KD ranges from about 23.0 nM to about 0.06 nM. In some embodiments, such is about 23.0 nM or less.
  • the affinity of an antibody provided herein for hTF as indicated by KD measured by ForteBio, as set forth in Table 5 is selected from about 1.28 nM, about 2.20 nM, about 8.45 nM, about 1.67 nM, about 0.64 nM, about 21.9 nM, about 3.97 nM, about 35.8 nM, about 3.30 nM, about 2.32 nM, about 0.83 nM, about 2.40 nM, about 0.96 nM, about 0.86 nM, about 3.84 nM, about 1.02 nM, about 1.61 nM, about 2.52 nM, about 2.28 nM, and about 1.59 nM.
  • such affinity as indicated by KD ranges from about 35.8 nM to about 0.64 nM. In some embodiments, such KD is about 35.8 nM or less.
  • the affinity of an antibody provided herein for cTF as indicated by KD measured by Biacore, as set forth in Table 5 is selected from about 0.26 nM, about 5.42 nM, about 0.21 nM, about 0.04 nM, about 18.0 nM, about 0.78 nM, about 16.4 nM, about 5.06 nM, about 0.08 nM, about 5.64 nM, about 0.12 nM, about 0.24 nM, about 5.66 nM, about 0.39 nM, about 5.69 nM, about 6.42 nM, and about 1.83 nM.
  • such affinity as indicated by KD ranges from about 18.0 nM to about 0.04 nM.
  • such KD is about 18.0 nM or less.
  • the affinity of an antibody provided herein for cTF as indicated by KD measured by ForteBio, as set forth in Table 5 is selected from about 1.43 nM, about 2.70 nM, about 7.65 nM, about 1.36 nM, about 0.76 nM, about 17.5 nM, about 4.99 nM, about 42.9 nM, about 12.0 nM, about 15.0 nM, about 0.57 nM, about 3.40 nM, about 1.05 nM, about 0.94 nM, about 4.12 nM, about 1.11 nM, about 1.96 nM, about 4.07 nM, about 2.71 nM, and about 4.16 nM.
  • such affinity as indicated by KD ranges from about 42.9 nM to about 0.57 nM. In some embodiments, such KD is about 42.9 nM or less.
  • the affinity of an antibody provided herein for mTF as indicated by KD measured by Biacore, as set forth in Table 5 is selected from about 5.4 nM, about 2.9 nM, about 21 nM, and about 2.4 nM. In some embodiments, such affinity as indicated by KD ranges from about 21 nM to about 2.4 nM. In some embodiments, such KD is about 21 nM or less.
  • the affinity of an antibody provided herein for mTF as indicated by KD measured by ForteBio, as set forth in Table 5 is selected from about 263 nM, about 131 nM, about 188 nM, about 114 nM, about 34.2 nM, about 9.16 nM, about 161 nM, about 72.1 nM, about 360 nM, about 281 nM, about 41.4 nM, about 6.12 nM, about 121 nM, and about 140 nM.
  • such affinity as indicated by KD ranges from about 360 nM to about 6.12 nM. In some embodiments, such KD is about 360 nM or less.
  • the affinity of an antibody provided herein for hTF as indicated by ECso measured with human TF-positive HCT-116 cells is selected from about 50 pM, about 58 pM, about 169 pM, about 77 pM, about 88 pM, about 134 pM, about 85 pM, about 237 pM, about 152 pM, about 39 pM, about 559 pM, about 280 pM, about 255 pM, about 147 pM, about 94 pM, about 117 pM, about 687 pM, about 532 pM, and about 239 pM.
  • such affinity ranges from about 687 pM to about 39 pM.
  • such ECso is about 687 pM
  • the affinity of an antibody provided herein for mTF as indicated by ECso measured with mouse TF-positive CHO cells is selected from about 455 nM, about 87 nM, about 11 nM, about 3.9 nM, about 3.0 nM, about 3.4 nM, about 255 nM, about 2.9 nM, about 3.6 nM, and about 4.0 nM.
  • such affinity ranges from about 455 nM to about 2.9 nM.
  • such ECso is about 455 pM or less.
  • the KD value of an antibody provided herein for pTF is no more than 20x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for pTF is no more than 15* of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for pTF is no more than 10* of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for pTF is no more than 5x of the KD value of the antibody for hTF. In some embodiments, the KD value of an antibody provided herein for pTF is no more than 2x of the KD value of the antibody for hTF.
  • the affinity of an antibody provided herein for pTF as indicated by KD measured by Biacore, as set forth in Table 40 is 3.31 nM or 12.9 nM.
  • the TF antibodies provided herein do not inhibit human thrombin generation as determined by thrombin generation assay (TGA). In certain embodiments, the TF antibodies provided herein allow human thrombin generation as determined by thrombin generation assay (TGA).
  • the percent peak thrombin generation is at least 40% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 50% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 60% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 70% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 80% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 90% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 95% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 99% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 40% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 50% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 60% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 70% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 80% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 90% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 95% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 99% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 60% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 70% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 80% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila is at least 90% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 95% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % Peak Ila is at least 99% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % Peak Ila in the presence of 100 nM TF antibody, as set forth in Table 6 and Table 37 is selected from about 99%, about 100%, about 103%, about 64%, about 52%, about 87%, about 96%, about 98%, and about 53% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) without antibody pre-incubation.
  • TGA thrombin generation assay
  • such % Peak Ila ranges from about 52% to about 103%.
  • such % Peak Ila is about 52% or more.
  • the % Peak Ila in the presence of 50 nM TF antibody is selected from about 99%, about 100%, about 103%, about 67%, about 58%, about 89%, about 96%, about 98%, about 68%, about 62%, and about 88% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) without antibody pre-incubation.
  • TGA thrombin generation assay
  • Peak Ila ranges from about 58% to about 103%. In some embodiments, such % Peak Ila is about 58% or more.
  • the % Peak Ila in the presence of 10 nM TF antibody, as set forth in Table 6 and Table 37 is selected from about 100%, about 99%, about 103%, about 87%, about 83%, about 95%, about 98%, about 86%, and about 96% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) without antibody pre-incubation.
  • TGA thrombin generation assay
  • such % Peak Ila ranges from about 83% to about 103%.
  • such % Peak Ila is about 83% or more.
  • the % Peak Ila in the presence of 100 nM TF antibody, as set forth in Table 7 and Table 38 is selected from about 108%, about 105%, about 111%, about 58%, about 47%, about 91%, about 103%, about 109%, about 107%, and about 45% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) with 10 min antibody pre-incubation.
  • TGA thrombin generation assay
  • such % Peak Ila ranges from about 45% to about 111%.
  • such % Peak Ila is about 45% or more.
  • the % Peak Ila in the presence of 50 nM TF antibody is selected from about 107%, about 104%, about 114%, about 62%, about 49%, about 87%, about 105%, about 109%, about 55%, and about 92% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) with 10 min antibody pre-incubation.
  • TGA thrombin generation assay
  • such % Peak Ila ranges from about 49% to about 114%.
  • such % Peak Ila is about 49% or more.
  • the % Peak Ila in the presence of 10 nM TF antibody is selected from about 105%, about 114%, about 76%, about 68%, about 94%, about 108%, about 104%, about 74%, and about 93% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) with 10 min antibody pre-incubation.
  • TGA thrombin generation assay
  • such % Peak Ila ranges from about 68% to about 114%.
  • such % Peak Ila is about 68% or more.
  • the percent endogenous thrombin potential is at least 80% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 90% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 95% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 99% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % ETP is at least 80% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 90% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 95% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 99% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % ETP is at least 80% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 90% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 95% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA). In some embodiments, the % ETP is at least 99% in the presence of no less than 10 nM TF antibody compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA).
  • the % ETP in the presence of 100 nM TF antibody, as set forth in Table 6 and Table 37 is selected from about 108%, about 103%, about 109%, about 100%, about 96%, about 102%, about 105%, and about 92% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) without antibody pre-incubation.
  • TGA thrombin generation assay
  • such % ETP ranges from about 92% to about 109%.
  • such % ETP is about 92% or more.
  • the % ETP in the presence of 50 nM TF antibody is selected from about 108%, about 103%, about 111%, about 101%, about 97%, about 104%, about 106%, about 93%, about 96%, and about 105% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) without antibody pre-incubation.
  • TGA thrombin generation assay
  • such % ETP ranges from about 93% to about 111%. In some embodiments, such % ETP is about 93% or more.
  • the % ETP in the presence of 10 nM TF antibody, as set forth in Table 6 and Table 37 is selected from about 106%, about 109%, about 105%, about 104%, about 107%, about 99%, about 101%, and about 102% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) without antibody pre-incubation.
  • TGA thrombin generation assay
  • such % ETP ranges from about 99% to about 109%. In some embodiments, such % ETP is about 99% or more.
  • the % ETP in the presence of 100 nM TF antibody, as set forth in Table 7 and Table 38 is selected from about 110%, about 104%, about 106%, about 98%, about 95%, about 108%, about 107%, about 96%, about 92%, and about 103% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) with 10 min antibody pre-incubation.
  • TGA thrombin generation assay
  • such % ETP ranges from about 92% to about 110%. In some embodiments, such % ETP is about 92% or more.
  • the % ETP in the presence of 50 nM TF antibody is selected from about 110%, about 106%, about 108%, about 103%, about 96%, about 109%, about 102%, about 104%, about 94%, and about 98% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) with 10 min antibody pre-incubation.
  • TGA thrombin generation assay
  • such % ETP ranges from about 94% to about 110%.
  • such % ETP is about 94% or more.
  • the % ETP in the presence of 10 nM TF antibody, as set forth in Table 7 and Table 38 is selected from about 107%, about 106%, about 110%, about 103%, about 100%, about 105%, about 102%, and about 101% compared to the control conditions without the antibody, as determined by thrombin generation assay (TGA) with 10 min antibody pre-incubation.
  • TGA thrombin generation assay
  • such % ETP ranges from about 100% to about 110%. In some embodiments, such % ETP is about 100% or more.
  • the antibodies provided herein bind human TF at a human TF binding site that is distinct from a human TF binding site bound by human FX. In certain embodiments, the antibodies provided herein do not interfere with the ability of TF:FVIIa to convert FX into FXa.
  • the percentage of FXa conversion is at least 75% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 80% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 85% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 90% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 95% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody.
  • the % FXa is at least 75% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 80% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 85% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 90% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 95% in the presence of no less than 50 nM TF antibody compared to the control conditions without the antibody.
  • the % FXa is at least 75% in the presence of no less than 25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 80% in the presence of no less than 25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 85% in the presence of no less than 25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 90% in the presence of no less than 25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 95% in the presence of no less than 25 nM TF antibody compared to the control conditions without the antibody.
  • the % FXa is at least 75% in the presence of no less than 12.5 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 80% in the presence of no less than 12.5 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 85% in the presence of no less than 12.5 nM TF antibody compared to the control conditions without the antibody. In some embodiments, % FXa is at least 90% in the presence of no less than 12.5 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FXa is at least 95% in the presence of no less than 12.5 nM TF antibody compared to the control conditions without the antibody.
  • the % FXa in the presence of 100 nM TF antibody, as set forth in Table 8 is selected from about 89%, about 96%, about 116%, about 108%, about 117%, about 105%, about 112%, about 106%, about 103%, about 111%, about 98%, and about 101% compared to the control conditions without the antibody. In some embodiments, such % FXa ranges from about 89% to about 117%. In some embodiments, such % FXa is about 89% or more.
  • the % FXa in the presence of 50 nM TF antibody, as set forth in Table 8 is selected from about 94%, about 93%, about 78%, about 102%, about 99%, about 104%, about 105%, about 108%, about 107%, about 97%, and about 106% compared to the control conditions without the antibody. In some embodiments, such % FXa ranges from about 78% to about 108%. In some embodiments, such % FXa is about 78% or more.
  • the % FXa in the presence of 25 nM TF antibody, as set forth in Table 8 is selected from about 81%, about 89%, about 85%, about 109%, about 96%, about 97%, about 108%, about 104%, about 103%, about 112%, and about 89% compared to the control conditions without the antibody. In some embodiments, such % FXa ranges from about 81% to about 112%. In some embodiments, such % FXa is about 81% or more.
  • the % FXa in the presence of 12.5 nM TF antibody, as set forth in Table 8 is selected from about 87%, about 89%, about 82%, about 99%, about 101%, about 98%, about 113%, about 106%, about 115%, about 110%, about 120%, about 85%, and about 108% compared to the control conditions without the antibody.
  • such % FXa ranges from about 82% to about 120%. In some embodiments, such % FXa is about 82% or more.
  • the antibodies provided herein bind human TF at a human TF binding site that is distinct from a human TF binding site bound by human FVIIa. In certain embodiments, the antibodies provided herein do not compete for binding to human TF with human FVIIa.
  • the percentage of FVIIa binding is at least 75% in the presence of no less than 250 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 80% in the presence of no less than 250 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 85% in the presence of no less than 250 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 90% in the presence of no less than 250 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 95% in the presence of no less than 250 nM TF antibody compared to the control conditions without the antibody.
  • the % FVIIa is at least 75% in the presence of no less than 83 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 80% in the presence of no less than 83 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 85% in the presence of no less than 83 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 90% in the presence of no less than 83 nM TF antibody compared to the control conditions without the antibody.
  • the % FVIIa is at least 95% in the presence of no less than 83 nM TF antibody compared to the control conditions without the antibody. [00315] In some embodiments, the % FVIIa is at least 75% in the presence of no less than 28 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 80% in the presence of no less than 28 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 85% in the presence of no less than 28 nM TF antibody compared to the control conditions without the antibody.
  • the % FVIIa is at least 90% in the presence of no less than 28 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 95% in the presence of no less than 28 nM TF antibody compared to the control conditions without the antibody.
  • the % FVIIa is at least 75% in the presence of no less than 9.25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 80% in the presence of no less than 9.25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 85% in the presence of no less than 9.25 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the % FVIIa is at least 90% in the presence of no less than 9.25 nM TF antibody compared to the control conditions without the antibody.
  • the % FVIIa is at least 95% in the presence of no less than 9.25 nM TF antibody compared to the control conditions without the antibody.
  • the % FVIIa in the presence of 250 nM TF antibody, as set forth in Table 9 is selected from about 98%, about 87%, about 80%, about 92%, about 95%, about 89%, about 91%, about 97%, about 94%, about 101%, and about 96% compared to the control conditions without the antibody.
  • such % FVIIa ranges from about 80% to about 101%. In some embodiments, such % FVIIa is about 80% or more.
  • the % FVIIa in the presence of 83 nM TF antibody, as set forth in Table 9 is selected from about 97%, about 88%, about 77%, about 93%, about 94%, about 91%, about 98%, about 100%, and about 92% compared to the control conditions without the antibody. In some embodiments, such % FVIIa ranges from about 77% to about 100%. In some embodiments, such % FVIIa is about 77% or more.
  • the % FVIIa in the presence of 28 nM TF antibody, as set forth in Table 9 is selected from about 101%, about 87%, about 79%, about 96%, about 93%, about 95%, about 98%, about 100%, about 102%, about 99%, about 92%, and about 91% compared to the control conditions without the antibody. In some embodiments, such % FVIIa ranges from about 79% to about 102%. In some embodiments, such % FVIIa is about 79% or more.
  • the % FVIIa in the presence of 9.25 nM TF antibody, as set forth in Table 9 is selected from about 100%, about 90%, about 76%, about 97%, about 93%, about 99%, about 98%, about 102%, about 101%, and about 95% compared to the control conditions without the antibody. In some embodiments, such % FVIIa ranges from about 76% to about 102%. In some embodiments, such % FVIIa is about 76% or more.
  • the antibodies provided herein inhibit FVIIa-dependent TF signaling.
  • the inhibition of FVIIa-dependent TF signaling is measured by the reduction of IL8.
  • the inhibition of FVIIa-dependent TF signaling is measured by the reduction of GM-CSF.
  • the Interleukin 8 concentration is reduced by at least 70% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 80% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 90% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody.
  • the IL8 cone is reduced by at least 70% in the presence of no less than 40 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 80% in the presence of no less than 40 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 90% in the presence of no less than 40 nM TF antibody compared to the control conditions without the antibody.
  • the IL8 cone is reduced by at least 60% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 70% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 80% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 90% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody.
  • the IL8 cone is reduced by at least 50% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 60% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 70% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 80% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the IL8 cone is reduced by at least 90% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody.
  • the Granulocyte-Macrophage Colony-Stimulating Factor concentration is reduced by at least 70% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 80% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 90% in the presence of no less than 100 nM TF antibody compared to the control conditions without the antibody.
  • the GM-CSF cone is reduced by at least 70% in the presence of no less than 40 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 80% in the presence of no less than 40 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 90% in the presence of no less than 40 nM TF antibody compared to the control conditions without the antibody.
  • the GM-CSF cone is reduced by at least 60% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 70% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 80% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 90% in the presence of no less than 16 nM TF antibody compared to the control conditions without the antibody.
  • the GM-CSF cone is reduced by at least 50% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 60% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 70% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 80% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody. In some embodiments, the GM-CSF cone is reduced by at least 90% in the presence of no less than 6.4 nM TF antibody compared to the control conditions without the antibody.
  • the percentage of Interleukin 8 (% IL8) in the presence of 100 nM TF antibody, as set forth in Table 10 is selected from about 2%, about 9%, about 8%, about 6%, about 13%, about 1%, about 3%, about 4%, and about 5% compared to the control conditions without the antibody. In some embodiments, such % IL8 ranges from about 1% to about 13%. In some embodiments, such % IL8 is about 13% or less.
  • the % IL8 in the presence of 40 nM TF antibody, as set forth in Table 10 is selected from about 2%, about 8%, about 7%, about 10%, about 14%, about 4%, about 5%, and about 6% compared to the control conditions without the antibody. In some embodiments, such % IL8 ranges from about 2% to about 14%. In some embodiments, such % IL8 is about 14% or less.
  • the % IL8 in the presence of 16 nM TF antibody, as set forth in Table 10 is selected from about 2%, about 3%, about 10%, about 8%, about 7%, about 16%, about 9%, about 15%, about 5%, and about 6% compared to the control conditions without the antibody. In some embodiments, such % IL8 ranges from about 2% to about 16%. In some embodiments, such % IL8 is about 16% or less.
  • the % IL8 in the presence of 6.4 nM TF antibody, as set forth in Table 10 is selected from about 3%, about 4%, about 11%, about 9%, about 14%, about 22%, about 12%, about 6%, about 5%, about 15%, about 21%, and about 8% compared to the control conditions without the antibody.
  • such % IL8 ranges from about 3% to about 22%. In some embodiments, such % IL8 is about 22% or less.
  • the percentage of Granulocyte-Macrophage Colony- Stimulating Factor (% GM-CSF) in the presence of 100 nM TF antibody, as set forth in Table 11 is selected from about 6%, about 7%, about 22%, about 20%, about 12%, about 19%, about 17%, about 25%, about 5%, about 14%, about 11%, and about 10% compared to the control conditions without the antibody.
  • % GM-CSF ranges from about 5% to about 25%. In some embodiments, such % GM-CSF is about 25% or less.
  • the % GM-CSF in the presence of 40 nM TF antibody, as set forth in Table 11 is selected from about 6%, about 7%, about 19%, about 15%, about 18%, about 16%, about 26%, about 5%, about 13%, about 11%, and about 10% compared to the control conditions without the antibody. In some embodiments, such % GM-CSF ranges from about 5% to about 26%. In some embodiments, such % GM-CSF is about 26% or less.
  • the % GM-CSF in the presence of 16 nM TF antibody, as set forth in Table 11 is selected from about 6%, about 7%, about 22%, about 19%, about 14%, about 32%, about 17%, about 26%, about 5%, about 12%, about 13%, about 9%, about 11%, and about 15% compared to the control conditions without the antibody.
  • such % GM-CSF ranges from about 5% to about 32%. In some embodiments, such % GM-CSF is about 32% or less.
  • the % GM-CSF in the presence of 6.4 nM TF antibody, as set forth in Table 11 is selected from about 8%, about 9%, about 24%, about 20%, about 18%, about 39%, about 34%, about 15%, about 21%, about 16%, about 17%, and about 10% compared to the control conditions without the antibody.
  • such % GM- CSF ranges from about 8% to about 39%. In some embodiments, such % GM-CSF is about 39% or less.
  • the antibodies provided herein reduce lesion size in a swine choroidal neovascularization (CNV) model.
  • the reduction in lesion size is measured by Fluorescein Angiography (FA).
  • the lesion size in a swine CNV model is reduced by at least 5% 7 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 10% 7 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 20% 7 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 40% 7 days after administration of the anti- TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 60% 7 days after administration of the anti-TF antibody.
  • the lesion size in a swine CNV model is reduced by at least 10% 21 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 20% 21 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 40% 21 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 60% 21 days after administration of the anti-TF antibody. In some embodiments, the lesion size in a swine CNV model is reduced by at least 80% 21 days after administration of the anti-TF antibody.
  • the antibodies provided herein may comprise any suitable VH and VL germline sequences.
  • the VH region of an antibody provided herein is from the VH3 germline. In some embodiments, the VH region of an antibody provided herein is from the Vni germline. In some embodiments, the VH region of an antibody provided herein is from the VH4 germline.
  • the VH region of an antibody provided herein is from the VH3-23 germline. In some embodiments, the VH region of an antibody provided herein is from the VH1-18 germline. In some embodiments, the VH region of an antibody provided herein is from the VH3-30 germline. In some embodiments, the VH region of an antibody provided herein is from the VH1-69 germline. In some embodiments, the VH region of an antibody provided herein is from the VH4-31 germline. In some embodiments, the VH region of an antibody provided herein is from the VH4-34 germline. In some embodiments, the VH region of an antibody provided herein is from the VH1-46 germline.
  • the VL region of an antibody provided herein is from the VK1 germline. In some embodiments, the VL region of an antibody provided herein is from the VK4 germline. In some embodiments, the VL region of an antibody provided herein is from the VK3 germline
  • the VL region of an antibody provided herein is from the VK1-05 germline. In some embodiments, the VL region of an antibody provided herein is from the VK4-01 germline. In some embodiments, the VL region of an antibody provided herein is from the VK3-15 germline. In some embodiments, the VL region of an antibody provided herein is from the VK3-20 germline. In some embodiments, the VL region of an antibody provided herein is from the VK1-33 germline.
  • the antibodies provided herein are monospecific antibodies.
  • the antibodies provided herein are multispecific antibodies.
  • a multispecific antibody provided herein binds more than one antigen.
  • a multispecific antibody binds two antigens.
  • a multispecific antibody binds three antigens.
  • a multispecific antibody binds four antigens.
  • a multispecific antibody binds five antigens.
  • a multispecific antibody provided herein binds more than one epitope on a TF antigen. In some embodiments, a multispecific antibody binds two epitopes on a TF antigen. In some embodiments, a multispecific antibody binds three epitopes on a TF antigen.
  • the multispecific antibody comprises an immunoglobulin comprising at least two different heavy chain variable regions each paired with a common light chain variable region (i.e., a “common light chain antibody”).
  • the common light chain variable region forms a distinct antigen-binding domain with each of the two different heavy chain variable regions.
  • the multispecific antibody comprises an immunoglobulin comprising an antibody or fragment thereof attached to one or more of the N- or C-termini of the heavy or light chains of such immunoglobulin. See Coloma and Morrison, Nature Biotechnol. , 1997, 15:159-163, incorporated by reference in its entirety. In some aspects, such antibody comprises a tetravalent bispecific antibody.
  • the multispecific antibody comprises a hybrid immunoglobulin comprising at least two different heavy chain variable regions and at least two different light chain variable regions. See Milstein and Cuello, Nature , 1983, 305:537- 540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA , 1986, 83:1453-1457; each of which is incorporated by reference in its entirety.
  • the multispecific antibody comprises immunoglobulin chains with alterations to reduce the formation of side products that do not have multispecificity.
  • the antibodies comprise one or more “knobs-into-holes” modifications as described in U.S. Pat. No. 5,731,168, incorporated by reference in its entirety.
  • the multispecific antibody comprises immunoglobulin chains with one or more electrostatic modifications to promote the assembly of Fc hetero- multimers. See WO 2009/089004, incorporated by reference in its entirety.
  • the multispecific antibody comprises a bispecific single chain molecule. See Traunecker et aI., EMBO J., 1991, 10:3655-3659; and Gruber et al, J. Immunol ., 1994, 152:5368-5374; each of which is incorporated by reference in its entirety. [00357] In some embodiments, the multispecific antibody comprises a heavy chain variable domain and a light chain variable domain connected by a polypeptide linker, where the length of the linker is selected to promote assembly of multispecific antibodies with the desired multispecificity.
  • monospecific scFvs generally form when a heavy chain variable domain and light chain variable domain are connected by a polypeptide linker of more than 12 amino acid residues. See U.S. Pat. Nos. 4,946,778 and 5,132,405, each of which is incorporated by reference in its entirety.
  • reduction of the polypeptide linker length to less than 12 amino acid residues prevents pairing of heavy and light chain variable domains on the same polypeptide chain, thereby allowing pairing of heavy and light chain variable domains from one chain with the complementary domains on another chain.
  • the resulting antibodies therefore have multispecificity, with the specificity of each binding site contributed by more than one polypeptide chain.
  • Polypeptide chains comprising heavy and light chain variable domains that are joined by linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabodies) and tetramers (termed tetrabodies) are favored. However, the exact type of oligomerization appears to depend on the amino acid residue composition and the order of the variable domain in each polypeptide chain (e.g ., VH- linker-VL vs. VL-linker-VH), in addition to the linker length. A skilled person can select the appropriate linker length based on the desired multispecificity.
  • the multispecific antibody comprises a diabody. See Hollinger et al. , Proc. Natl. Acad. Sci. USA , 1993, 90:6444-6448, incorporated by reference in its entirety. In some embodiments, the multispecific antibody comprises a triabody. See Todorovska etal. , J. Immunol. Methods , 2001, 248:47-66, incorporated by reference in its entirety. In some embodiments, the multispecific antibody comprises a tetrabody. See id., incorporated by reference in its entirety.
  • the multispecific antibody comprises a trispecific F(ab’)3 derivative. See Tutt et al. J. Immunol., 1991, 147:60-69, incorporated by reference in its entirety. [00360] In some embodiments, the multispecific antibody comprises a cross-linked antibody. See U.S. Patent No. 4,676,980; Brennan etal. , Science , 1985, 229:81-83; Staerz, et al. Nature , 1985, 314:628-631; and EP 0453082; each of which is incorporated by reference in its entirety.
  • the multispecific antibody comprises antigen-binding domains assembled by leucine zippers. See Kostelny etal. , J. Immunol ., 1992, 148:1547- 1553, incorporated by reference in its entirety.
  • the multispecific antibody comprises complementary protein domains.
  • the complementary protein domains comprise an anchoring domain (AD) and a dimerization and docking domain (DDD).
  • AD and DDD bind to each other and thereby enable assembly of multispecific antibody structures via the “dock and lock” (DNL) approach.
  • DNL dimerization and docking domain
  • Antibodies of many specificities may be assembled, including bispecific antibodies, trispecific antibodies, tetraspecific antibodies, quintspecific antibodies, and hexaspecific antibodies.
  • Multispecific antibodies comprising complementary protein domains are described, for example, in U.S. Pat. Nos. 7,521,056; 7,550,143; 7,534,866; and 7,527,787; each of which is incorporated by reference in its entirety.
  • the multispecific antibody comprises a dual action Fab (DAF) antibody as described in U.S. Pat. Pub. No. 2008/0069820, incorporated by reference in its entirety.
  • DAF dual action Fab
  • the multispecific antibody comprises an antibody formed by reduction of two parental molecules followed by mixing of the two parental molecules and reoxidation to assembly a hybrid structure. See Carlring et al ., PLoS One , 2011, 6:e22533, incorporated by reference in its entirety.
  • the multispecific antibody comprises a DVD-IgTM.
  • a DVD-IgTM is a dual variable domain immunoglobulin that can bind to two or more antigens. DVD-IgsTM are described in U.S. Pat. No. 7,612,181, incorporated by reference in its entirety.
  • the multispecific antibody comprises a DARTTM.
  • DART sTM are described in Moore et al. , Blood , 2011, 117:454-451 , incorporated by reference in its entirety.
  • the multispecific antibody comprises a DuoBody ® .
  • DuoBodies ® are described in Labrijn etal, Proc. Natl. Acad. Sci. USA, 2013, 110:5145- 5150; Gramer etal., mAbs, 2013, 5:962-972; and Labrijn et al, Nature Protocols, 2014, 9:2450-2463; each of which is incorporated by reference in its entirety.
  • the multispecific antibody comprises an antibody fragment attached to another antibody or fragment.
  • the attachment can be covalent or non-covalent. When the attachment is covalent, it may be in the form of a fusion protein or via a chemical linker.
  • multispecific antibodies comprising antibody fragments attached to other antibodies
  • tetravalent bispecific antibodies where an scFv is fused to the C-terminus of the Cro from an IgG. See Coloma and Morrison, Nature Biotechnol. , 1997, 15:159-163.
  • Other examples include antibodies in which a Fab molecule is attached to the constant region of an immunoglobulin. See Miler etal. , J. Immunol ., 2003, 170:4854- 4861, incorporated by reference in its entirety. Any suitable fragment may be used, including any of the fragments described herein or known in the art.
  • the multispecific antibody comprises a CovX-Body.
  • CovX-Bodies are described, for example, in Doppalapudi etal. , Proc. Natl. Acad. Sci. USA , 2010, 107:22611-22616, incorporated by reference in its entirety.
  • the multispecific antibody comprises an Fcab antibody, where one or more antigen-binding domains are introduced into an Fc region.
  • Fcab antibodies are described in Wozniak-Knopp etal., Protein Eng. Des. Sel, 2010, 23:289-297, incorporated by reference in its entirety.
  • the multispecific antibody comprises a TandAb ® antibody.
  • TandAb ® antibodies are described in Kipriyanov etal, J. Mol. Biol., 1999, 293:41-56 and Zhukovsky etal, Blood, 2013, 122:5116, each of which is incorporated by reference in its entirety.
  • the multispecific antibody comprises a tandem Fab. Tandem Fabs are described in WO 2015/103072, incorporated by reference in its entirety. [00373] In some embodiments, the multispecific antibody comprises a ZybodyTM. ZybodiesTM are described in LaFleur etal, mAbs, 2013, 5:208-218, incorporated by reference in its entirety.
  • administration of the multispecific antibody results in cell- mediated cytotoxicity.
  • an antibody provided herein may be altered to increase, decrease or eliminate the extent to which it is glycosylated.
  • Glycosylation of polypeptides is typically either “N-linked” or “O-linked.”
  • N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
  • O-linked glycosylation refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5 -hydroxy lysine may also be used.
  • Addition or deletion of N-linked glycosylation sites to or from an antibody provided herein may be accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences is created or removed.
  • Addition or deletion of O-linked glycosylation sites may be accomplished by addition, deletion, or substitution of one or more serine or threonine residues in or to (as the case may be) the sequence of an antibody.
  • an antibody provided herein comprises a glycosylation motif that is different from a naturally occurring antibody. Any suitable naturally occurring glycosylation motif can be modified in the antibodies provided herein.
  • the structural and glycosylation properties of immunoglobulins, for example, are known in the art and summarized, for example, in Schroeder and Cavacini, J. Allergy Clin. Immunol ., 2010, 125:S41-52, incorporated by reference in its entirety.
  • an antibody provided herein comprises an IgGl Fc region with modification to the oligosaccharide attached to asparagine 297 (Asn 297).
  • Naturally occurring IgGl antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn 297 of the Cm domain of the Fc region. See Wright etal. , TIBTECH , 1997, 15:26-32, incorporated by reference in its entirety.
  • the oligosaccharide attached to Asn 297 may include various carbohydrates such as mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • various carbohydrates such as mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • the oligosaccharide attached to Asn 297 is modified to create antibodies having altered ADCC. In some embodiments, the oligosaccharide is altered to improve ADCC. In some embodiments, the oligosaccharide is altered to reduce ADCC.
  • an antibody provided herein comprises an IgGl domain with reduced fucose content at position Asn 297 compared to a naturally occurring IgGl domain. Such Fc domains are known to have improved ADCC. See Shields et al. , ./. Biol. Chem ., 2002, 277:26733-26740, incorporated by reference in its entirety. In some aspects, such antibodies do not comprise any fucose at position Asn 297. The amount of fucose may be determined using any suitable method, for example as described in WO 2008/077546, incorporated by reference in its entirety.
  • an antibody provided herein comprises a bisected oligosaccharide, such as a biantennary oligosaccharide attached to the Fc region of the antibody that is bisected by GlcNAc.
  • a bisected oligosaccharide such as a biantennary oligosaccharide attached to the Fc region of the antibody that is bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; U.S. Pat. No. 6,602,684; and U.S. Pat. Pub. No. 2005/0123546; each of which is incorporated by reference in its entirety.
  • an antibody provided herein comprises an Fc region with at least one galactose residue in the oligosaccharide attached to the Fc region.
  • Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764; each of which is incorporated by reference in its entirety.
  • Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells, which are deficient in protein fucosylation ( see Ripka et al. , Arch.
  • an antibody provided herein is an aglycosylated antibody.
  • An aglycosylated antibody can be produced using any method known in the art or described herein.
  • an aglycosylated antibody is produced by modifying the antibody to remove all glycosylation sites.
  • the glycosylation sites are removed only from the Fc region of the antibody.
  • an aglycosylated antibody is produced by expressing the antibody in an organism that is not capable of glycosylation, such as E. coli , or by expressing the antibody in a cell-free reaction mixture.
  • an antibody provided herein has a constant region with reduced effector function compared to a native IgGl antibody.
  • the affinity of a constant region of an Fc region of an antibody provided herein for Fc receptor is less than the affinity of a native IgGl constant region for such Fc receptor.
  • an antibody provided herein comprises an Fc region with one or more amino acid substitutions, insertions, or deletions in comparison to a naturally occurring Fc region.
  • substitutions, insertions, or deletions yield antibodies with altered stability, glycosylation, or other characteristics.
  • substitutions, insertions, or deletions yield aglycosylated antibodies.
  • the Fc region of an antibody provided herein is modified to yield an antibody with altered affinity for an Fc receptor, or an antibody that is more immunologically inert.
  • the antibody variants provided herein possess some, but not all, effector functions. Such antibodies may be useful, for example, when the half-life of the antibody is important in vivo , but when certain effector functions (e.g, complement activation and ADCC) are unnecessary or deleterious.
  • the Fc region of an antibody provided herein is a human IgG4 Fc region comprising one or more of the hinge stabilizing mutations S228P and L235E. See Aalberse et al. , Immunology , 2002, 105:9-19, incorporated by reference in its entirety.
  • the IgG4 Fc region comprises one or more of the following mutations: E233P, F234V, and L235A. See Armour etal.,Mol. Immunol ., 2003, 40:585-593, incorporated by reference in its entirety.
  • the IgG4 Fc region comprises a deletion at position G236.
  • the Fc region of an antibody provided herein is a human IgGl Fc region comprising one or more mutations to reduce Fc receptor binding.
  • the one or more mutations are in residues selected from S228 (e.g, S228A), L234 (e.g., L234A), L235 (e.g, L235A), D265 (e.g, D265A), andN297 ( e.g , N297A).
  • the antibody comprises a PVA236 mutation.
  • PVA236 means that the amino acid sequence ELLG (SEQ ID NO: 928), from amino acid position 233 to 236 of IgGl or EFLG (SEQ ID NO: 929) of IgG4, is replaced by PVA. See U.S. Pat. No. 9,150,641, incorporated by reference in its entirety.
  • the Fc region of an antibody provided herein is modified as described in Armour et al., Eur. J. Immunol ., 1999, 29:2613-2624; WO 1999/058572; and/or U.K. Pat. App. No. 98099518; each of which is incorporated by reference in its entirety.
  • the Fc region of an antibody provided herein is a human IgG2 Fc region comprising one or more of mutations A330S and P331S.
  • the Fc region of an antibody provided herein has an amino acid substitution at one or more positions selected from 238, 265, 269, 270, 297, 327 and 329. See U.S. Pat. No. 6,737,056, incorporated by reference in its entirety.
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 with alanine. See U.S. Pat. No. 7,332,581, incorporated by reference in its entirety.
  • the antibody comprises an alanine at amino acid position 265. In some embodiments, the antibody comprises an alanine at amino acid position 297.
  • an antibody provided herein comprises an Fc region with one or more amino acid substitutions which improve ADCC, such as a substitution at one or more of positions 298, 333, and 334 of the Fc region.
  • an antibody provided herein comprises an Fc region with one or more amino acid substitutions at positions 239, 332, and 330, as described in Lazar etal. , Proc. Natl. Acad. Sci. USA , 2006,103:4005-4010, incorporated by reference in its entirety.
  • an antibody provided herein has one or more of S239D, A330L, and I332E substitutions in the Fc region, as determined by Kabat numbering.
  • the FC region is a human IgGl Fc region.
  • an antibody provided herein comprises the combination of L234A, L235A, and P329A (LALA-PG) substitutions in the human IgGl heavy chain constant region (as determined by Kabat numbering) that have been shown to effectively silence the effector function of human IgGl antibodies.
  • L234A, L235A, and P329A LALA-PG substitutions in the human IgGl heavy chain constant region (as determined by Kabat numbering) that have been shown to effectively silence the effector function of human IgGl antibodies.
  • LALA-PG L234A, L235A, and P329A substitutions in the human IgGl heavy chain constant region (as determined by Kabat numbering) that have been shown to effectively silence the effector function of human IgGl antibodies.
  • an antibody provided herein comprises one or more alterations to increase half-life.
  • Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) are described, for example, in Hinton et al. , J. Immunol ., 2006, 176:346-356; and U.S. Pat. Pub. No. 2005/0014934; each of which is incorporated by reference in its entirety.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340,
  • an antibody provided herein comprises one or more Fc region variants as described in U.S. Pat. Nos. 7,371,826, 5,648,260, and 5,624,821; Duncan and Winter, Nature , 1988, 322:738-740; and WO 94/29351; each of which is incorporated by reference in its entirety.
  • antibodies comprising a polypeptide sequence having a pE residue at the N-terminal position. In some embodiments, provided herein are antibodies comprising a polypeptide sequence in which the N-terminal residue has been converted from Q to pE. In some embodiments, provided herein are antibodies comprising a polypeptide sequence in which the N-terminal residue has been converted from E to pE.
  • cysteine engineered antibodies also known as “thioMAbs,” in which one or more residues of the antibody are substituted with cysteine residues.
  • the substituted residues occur at solvent accessible sites of the antibody.
  • reactive thiol groups are introduced at solvent accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, for example, to create an immunoconjugate.
  • any one or more of the following residues may be substituted with cysteine: V205 of the light chain; A118 of the heavy chain Fc region; and S400 of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, for example, in U.S. Pat. No. 7,521,541, which is incorporated by reference in its entirety.
  • the antibodies of the present disclosure are useful for the treatment of ocular diseases.
  • the antibodies described herein have superior properties (e.g ., retinal thickness reduction, macular edema reduction, inflammatory cytokine reduction, etc.) relative to comparator therapies.
  • a comparator therapy is a reference antibody as described herein.
  • These comparator therapies can be, for example, vascular endothelial growth factor (VEGF) inhibitors, VEGF receptor inhibitors, TF inhibitors, platelet derived growth factor (PDGF) inhibitors or PDGF receptor inhibitors, etc.
  • the comparator therapies are anti-TF antibodies, anti-TF drugs, anti- VEGF antibodies, or anti-VEGF drugs.
  • the comparator therapies are anti-TF proteins or anti-TF immunoconjugates. In some embodiments, the comparator therapries are anti-VEGF proteins or anti-VF immunoconjugates.
  • anti-TF antibodies, anti-TF drugs, anti-VEGF antibodies, or anti-VEGF drugs other comparator therapies may be contemplated as well.
  • Non-limiting examples of different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug that may be compared to the anti-TF antibodies of the present disclosure include: aflibercept (Eylea), ranibizumab, brolucizumab, pegaptanib, bevacizumab, abicipar pegol, Faricimab (RG7716), KSI-301, hl-conl (also referred to as “ICONl” or “ICON-1”) and Ml 593.
  • Aflibercept (trade name: Eylea) is a recombinant protein composed of the binding domains of two human vascular endothelial growth factor (VEGF) receptors fused with the Fc region of human immunoglobulin gamma 1 (IgGl). It is a dimeric glycoprotein and is in a class of medications called vascular endothelial growth factor-A (VEGF-A) and placental growth factor (P1GF) antagonists. It functions by stopping abnormal blood vessel growth and leakage in the eye(s).
  • VEGF vascular endothelial growth factor
  • P1GF placental growth factor
  • Aflibercept as an ophthalmic agent, is used in the treatment of macular edema following Central Retinal Vein Occlusion (CRVO) and neovascular Age-Related Macular Degeneration (AMD).
  • CRVO Central Retinal Vein Occlusion
  • ALD Age-Related Macular Degeneration
  • An exemplary sequence for aflibercept is provided below: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSR KGFIISNAT YKEIGLLT CE ATVN GHLYKTNYLTHRQTNTIID VVL SP SHGIELS VGEKL VLNCT ARTELN V GIDFNWEYP S SKHQHKKL VNRDLKTQ SGSEMKKFL STLTIDGVTR SDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTC VVVD V S
  • Ranibizumab (Ranbizumab, trade name: Lucentis®) is a monoclonal antibody fragment derived from the same parental murine antibody as bevacizumab (Avastin) that is approved for the treatment of ocular diseases such as wet AMD. However, it has been affinity matured to provide stronger binding to VEGF-A. Because VEGF-A blockade is associated with some systemic toxicity, ranibizumab lacks the Fc portion that reduces serum half-life and thus systemic toxicity. It is indicated for the treatment of macular edema after retinal vein occlusion, age-related macular degeneration (wet), and diabetic macular edema.
  • Lucentis® is a monoclonal antibody fragment derived from the same parental murine antibody as bevacizumab (Avastin) that is approved for the treatment of ocular diseases such as wet AMD. However, it has been affinity matured to provide stronger binding to VEGF-A. Because VEGF-A block
  • Ranibizumab Light Chain DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG N S QES VTEQD SKD STYSLSSTLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 931).
  • Brolucizumab (trade name: Beovu ® ) is a humanized single chain monoclonal antibody indicated to treat neovascular age related macular degeneration. It is in a class of medications called vascular endothelial growth factor A (VEGF-A) antagonists and has a high affinity to all VEGF-A isoforms. It inhibits the activation of VEGF receptors through the prevention of ligand-receptor interaction resulting in the suppression of endothelial cell proliferation and vascular permeability.
  • VEGF-A vascular endothelial growth factor A
  • An exemplary sequence for brolucizumab is provided below:
  • Pegaptanib (also referred to as macugen) is a polynucleotide aptamer. Pegatinib aids neovascular age-related macular degeneration by binding to VEGF which in order reduces angiogenesis and vessel permeability. It is approved as an aqueous sodium solution for intravitreous injection. Pegaptanib sodium is a covalent conjugate of an oligonucleotide of twenty-eight nucleotides in length that terminates in a pentylamino linker, to which two 20- kilodalton monomethoxy polyethylene glycol (PEG) units are covalently attached via the two amino groups on a lysine residue.
  • PEG polyethylene glycol
  • Bevacizumab (trade name: Avastin) was the first anti angiogenic agent introduced to the US market. It is a humanized monoclonal IgG antibody, and inhibits angiogenesis by binding and neutralizing VEGF-A. Bevacizumab is generally indicated for use in combination with different chemotherapy regimens which are specific to the type, severity, and stage of cancer. Its use in ophthalmology application is off-label, and in such cases is used for the treatment of CNV (in AMD and other diseases), diabetic macular edema (DME), and macular edema due to retinal vein occlusions.
  • CNV in AMD and other diseases
  • DME diabetic macular edema
  • macular edema due to retinal vein occlusions.
  • An exemplary sequence for the light chain and heavy chain of bevacizumab is provided below:
  • Bevacizumab light chain DIQMTQ SP S SL S AS V GDRVTITC S ASQDISNYLNW YQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG N S QES VTEQD SKD STYSLSSTLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 934).
  • Bevacizumab heavy chain E V QL VES GGGL V QPGGSLRL S C A AS G YTF TN Y GMNW VRQ APGKGLEW V GWINT YT GEPT Y A ADFKRRF TF SLD T SK S T A YLQMN SLRAEDT A V Y Y C AK YPHY Y GS SHW YFD VWGQGTL VT V S S ASTKGP S VFPL AP S SKST SGGT AALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICN VN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVV S
  • Abicipar pegol is a designed ankyrin repeat proteins (DARPin) directed to bind all VEGF-A isoforms, similar to ranibizumab. It has a higher affinity and a longer intraocular half-life than ranibizumab (>13 days vs. 7.2 days), making it a potential anti-VEGF therapy with longer duration and need for less frequent injections. ( See Moisseiev, E. & Loewenstein, A. Eye 2020, 34:605-606, incorporated by reference in its entirety). It is an investigational drug indicated for treatment of macular edema and macular degeneration. An exemplary sequence for abicipar pegol is provided below:
  • GSDLDKKLLE A ARAGQDDE VRILM AN GAD VN ARD S T GWTPLHL AAP W GHPEI VE V LLKNGAD VNAADF QGWTPLHL A AAV GHLEIVEVLLK Y GAD VNAQDKF GKT AFDISI DN GNEDL AEILQK A AGGGS GGGS C (SEQ ID NO: 936).
  • Faricimab (also identified as RG7716) is a bispecific antibody designed specifically for the treatment of retinal eye diseases that simultaneously binds to and inactivates Angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A). It is an investigational drug indicated for treatment of wet age-related macular degeneration (AMD).
  • An exemplary sequence for faricimab is provided below:
  • KSI-301 (Kodiac Sciences, Palo Alto, CA) is a full-length anti-VEGF antibody stably linked to a biopolymer to create an antibody-biopolymer conjugate (ABC).
  • biopolymer uses a large molecular structure to bind to and inhibit vascular endothelial growth factor.
  • the biopolymer s high molecular weight is intended to help retain the drug in the vitreous and enhance durability. It is an investigational drug indicated for treatment of wet AMD, diabetic eye disease, DME, and uveitis.
  • ICON1 (also referred to as “hl-conl” or “ICON-l”) is an anti-TF fusion protein. It combines a targeting domain with a modified Factor VII, which binds to TF, with an effector domain that can engage Fc bearing cells.
  • the drug is in development for the treatment of patients with wet age-related macular degeneration (AMD), which is characterized by TF overexpression.
  • AMD wet age-related macular degeneration
  • the sequence for ICON-1 is provided below (underlining indicates the mutant FVII sequence and the bold indicates the Fc hinge region).
  • ICON-1 is described in US Application No. 15/746,545, which is incorporated by reference in its entirety for all purposes.
  • Ml 593 is an anti-TF monoclonal antibody. ( See US Application No.: 14/230,128, which is incorporated by reference in its entirety). The sequences for its light and heavy chains are provided below:
  • the antibodies of present disclosure can be administered at higher doses than comparator therapies (e.g ., those disclosed herein) and result in a better safety profile than the comparator therapies administered at the same dose or a lower dose.
  • the antibody upon administration to a subject, results in less severe adverse ocular changes relative to a different anti-TF antibody, anti-TF drug, anti- VEGF antibody or anti-VEGF drug , wherein the adverse ocular changes comprise the presence of one or more of: inflammation in the anterior and/or posterior chambers of the eye, anterior chamber cells, flare and fibrin, keratic precipitates/comeal endothelial cells, cellular deposits on the anterior lens capsule, incomplete pupil dilation, cells on corneal endothelium and lens capsule, vitreous haze, vitreous cells, retinal sheathing, and chorio retinal hemorrhages.
  • the antibody upon administration to a subject, results in reduced mononuclear cell infiltration than comparator drugs administered at the same dose or a lower dose than the antibody.
  • This mononuclear inflammation may be in the vitreous humor, aqueous humor, retinal tissues, or other tissues within the eye(s). Macrophages play a role in inflammation and in angiogenesis (see JagerMJ, J Ophthalmic Vis Res. 2014;9(1): 1- 2, which is incorporated by reference in its entirety).
  • the predominant type of macrophage is M2 macrophages.
  • the antibody upon administration to a subject, results in reduced infiltration of M2 macrophages than comparator drugs.
  • Ophthalmologic examinations can be conducted on subjects before and after administration of an antibody provided herein.
  • Various tests/examinations that can be included in an ophthalmologic examination are exemplified in the Example section.
  • an ophthalmologic examination may include best-corrected visual acuity testing with Early Treatment Diabetic Retinopathy Study (ETDRS) charts, tonometry (to measure intraocular pressure), slit-lamp biomicroscopy, and fundus examination.
  • EDRS Early Treatment Diabetic Retinopathy Study
  • ocular tissue thickness values e.g., macular thickness, central retinal thickness, foveal thickness, choroidal thickness, central retinal subfield thickness (CST), center point thickness (CPT), or central foveal thickness (CFT)
  • CST central retinal subfield thickness
  • CPT center point thickness
  • CFT central foveal thickness
  • the central area (the macula) of the retina is of interest in ocular diseases (e.g., AMD and DME).
  • An average of this central 1000 pm retina is assessed, the central retina thickness (CRT), and fovea is at the centerpoint of this area.
  • a subject undergoes spectral domain optical coherence tomography (SD OCT) imaging by the Spectralis (Heidelberg Engineering Germany, Version 1.6.4.0), which uses an internal fixation source and centers on the patient's fovea.
  • SD OCT spectral domain optical coherence tomography
  • Retinal thickness in the 9 ETDRS subfields can be analyzed by the RT map analysis protocol.
  • Choroidal thickness can be measured with EDI scans in the center of each ETDRS subfield.
  • an ocular disease is known to increase ocular tissue thickness.
  • the CRT values can be above 300-500 pM.
  • the ocular tissue thickness e.g. CRT
  • the ocular tissue thickness is reduced relative to a comparator therapy and/or baseline levels.
  • the ocular tissue thickness e.g. CRT
  • the ocular tissue thickness is restored to normal (non-diseased) thickness values.
  • an ocular disease is known to reduce ocular tissue thickness.
  • the thickness can decrease below 200 mM.
  • the ocular tissue thickness e.g. CRT
  • the ocular tissue thickness is increased relative to a comparator therapy and/or baseline levels.
  • the ocular tissue thickness e.g. CRT
  • the ocular tissue thickness is restored to normal (non-diseased) thickness values.
  • an antibody provided herein is administered by IVT injection and the IOP is measured immediately prior to administration (to establish baseline levels) and following administration (e.g., approximately 30 minutes after treatment).
  • the IOP measurement prior to treatment can be bilateral (measurement in both eyes), regardless of whether both eyes will receive the IVT injection.
  • the IOP measurement s) following treatment can be in the test eye only ( i.e . the eye receiving IVT injection).
  • the IOP in the eye of a subject is measured about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 1 hour prior to administration.
  • the antibody provided herein does not significantly alter IOP.
  • “significantly alter” refers to a change in IOP levels other than that expected within the first 30 minutes following IVT injection. In cases where IOP is not “significantly altered” the IOP levels are restored to pre-injection levels in less than, for example, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes or 40 minutes.
  • the antibody provided herein upon administration to a subject, result in reduced or absent biomarkers of inflammation relative to baseline levels or a comparator therapy (e.g., a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug).
  • a comparator therapy e.g., a different anti-TF antibody, anti-TF drug, anti-VEGF antibody or anti-VEGF drug.
  • these biomarkers of inflammation include inflammatory cytokines and chemokines (also referred to as proinflammatory cytokines and chemokines), which are proteins secreted from immune cells to promote inflammation either systemically or locally.
  • Non-limiting examples of inflammatory cytokines and chemokines that are reduced upon administration of the antibodies provided herein, relative to baseline levels or a comparator therapy include: IL-1 beta, IL-6, IL-8, IL-9, IL-10, IL-12, MCP-1 (CCL2), MCP- 3 (CCL7), VEGF, TNF, and MMP9. These are examples of cytokines that are elevated in cases of subjects suffering from ocular diseases such as AMD.
  • the antibody provided herein result in reduced or absent inflammatory cytokine relative to baseline levels or a comparator therapy, wherein the inflammatory cytokine is associated with the diseased state for an ocular disease, or wherein the inflammatory cytokine is known to be elevated in a subject having an ocular disease.
  • the biomarkers of inflammation e.g ., inflammatory cytokines, chemokines, and other inflammatory mediators
  • Biomarkers of inflammation can also be measure in samples using ELISA or various Olink panels (e.g. custom Olink panels).
  • the expression levels for biomarkers of inflammation (e.g, inflammatory cytokines) in ocular tissues or samples can be measured using RT-PCR.
  • the TF antigen used for isolation of the antibodies provided herein may be intact TF or a fragment of TF.
  • the TF antigen may be, for example, in the form of an isolated protein or a protein expressed on the surface of a cell.
  • the TF antigen is a non-naturally occurring variant of TF, such as a TF protein having an amino acid sequence or post-translational modification that does not occur in nature.
  • the TF antigen is truncated by removal of, for example, intracellular or membrane-spanning sequences, or signal sequences.
  • the TF antigen is fused at its C-terminus to a human IgGl Fc domain or a polyhistidine tag.
  • Monoclonal antibodies may be obtained, for example, using the hybridoma method first described by Kohler etal, Nature, 1975, 256:495-497 (incorporated by reference in its entirety), and/or by recombinant DNA methods ( see e.g., U.S. Patent No. 4,816,567, incorporated by reference in its entirety). Monoclonal antibodies may also be obtained, for example, using phage-display libraries ( see e.g. , U.S. Patent No. 8,258,082, which is incorporated by reference in its entirety) or, alternatively, using yeast-based libraries (see e.g, U.S. Patent No. 8,691,730, which is incorporated by reference in its entirety).
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Useful myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive media conditions, such as the presence or absence of HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP -21 and MC- 11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, CA), and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, MD).
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. See e.g, Kozbor, J. Immunol., 1984, 133:3001, incorporated by reference in its entirety.
  • hybridoma cells that produce antibodies of the desired specificity, affinity, and/or biological activity
  • selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • DNA encoding the monoclonal antibodies may be 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 monoclonal antibodies).
  • the hybridoma cells can serve as a useful source of DNA encoding antibodies with the desired properties.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as bacteria (e.g., E. coli ), yeast (e.g, Saccharomyces or Pichia sp.), COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to produce the monoclonal antibodies.
  • a chimeric antibody is made by using recombinant techniques to combine a non-human variable region (e.g, a variable region derived from a mouse, rat, hamster, rabbit, or non human primate, such as a monkey) with a human constant region.
  • a non-human variable region e.g, a variable region derived from a mouse, rat, hamster, rabbit, or non human primate, such as a monkey
  • Humanized antibodies may be generated by replacing most, or all, of the structural portions of a non-human monoclonal antibody with corresponding human antibody sequences. Consequently, a hybrid molecule is generated in which only the antigen-specific variable, or CDR, is composed of non-human sequence.
  • Methods to obtain humanized antibodies include those described in, for example, Winter and Milstein, Nature, 1991, 349:293-299; Rader et al. , Proc. Nat. Acad. Sci. U.S. A., 1998, 95:8910-8915; Steinberger et al, J. Biol. Chem., 2000, 275:36073-36078; Queen et al, Proc. Natl. Acad. Sci. U.S. A., 1989, 86:10029-10033; and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370; each of which is incorporated by reference in its entirety.
  • Human antibodies can be generated by a variety of techniques known in the art, for example by using transgenic animals (e.g., humanized mice). See, e.g, Jakobovits etal, Proc. Natl. Acad. Sci. U.S.A., 1993, 90:2551; Jakobovits et al, Nature, 1993, 362:255-258; Bruggermann etal, Year in Immuno., 1993, 7:33; and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807; each of which is incorporated by reference in its entirety.
  • Human antibodies can also be derived from phage-display libraries (see e.g, Hoogenboom et al, J. Mol. Biol., 1991, 227:381-388; Marks etal., J. Mol. Biol., 1991, 222:581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905; each of which is incorporated by reference in its entirety). Human antibodies may also be generated by in vitro activated B cells (see e.g, U.S. Patent. Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety). Human antibodies may also be derived from yeast-based libraries (see e.g., U.S. Patent No.
  • the antibody fragments provided herein may be made by any suitable method, including the illustrative methods described herein or those known in the art. Suitable methods include recombinant techniques and proteolytic digestion of whole antibodies. Illustrative methods of making antibody fragments are described, for example, in Hudson et al, Nat. Med., 2003, 9:129-134, incorporated by reference in its entirety. Methods of making scFv antibodies are described, for example, in Pluckthun, in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458; each of which is incorporated by reference in its entirety.
  • the alternative scaffolds provided herein may be made by any suitable method, including the illustrative methods described herein or those known in the art.
  • methods of preparing AdnectinsTM are described in Emanuel etal. , mAbs , 2011, 3:38-48, incorporated by reference in its entirety.
  • Methods of preparing iMabs are described in U.S. Pat. Pub. No. 2003/0215914, incorporated by reference in its entirety.
  • Methods of preparing Anticalins ® are described in Vogt and Skerra, Chem. Biochem ., 2004, 5:191-199, incorporated by reference in its entirety.
  • Methods of preparing Kunitz domains are described in Wagner et al, Biochem.
  • Methods of preparing Affilins are provided in Ebersbach et al, J. Mol. Biol., 2007, 372:172-185, incorporated by reference in its entirety.
  • Methods of preparing Tetranectins are provided in Graversen etal, J. Biol. Chem., 2000, 275:37390-37396, incorporated by reference in its entirety.
  • Methods of preparing Avimers are provided in Silverman et al, Nature Biotech., 2005, 23:1556-1561, incorporated by reference in its entirety.
  • Methods of preparing Fynomers are provided in Silacci et al, ./. Biol. Chem., 2014, 289:14392-14398, incorporated by reference in its entirety.
  • the multispecific antibodies provided herein may be made by any suitable method, including the illustrative methods described herein or those known in the art. Methods of making common light chain antibodies are described in Merchant et al. , Nature Biotechnol. , 1998, 16:677-681, incorporated by reference in its entirety. Methods of making tetravalent bispecific antibodies are described in Coloma and Morrison, Nature Biotechnol. , 1997, 15:159-163, incorporated by reference in its entirety. Methods of making hybrid immunoglobulins are described in Milstein and Cuello, Nature , 1983, 305:537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci.
  • DART sTM Methods of making DART sTM are described in Moore etal, Blood, 2011, 117 :454-451 , incorporated by reference in its entirety.
  • Methods of making DuoBodies ® are described in Labrijn et al., Proc. Natl. Acad. Sci. USA, 2013, 110:5145-5150; Gramer et al., mAbs, 2013, 5:962-972; and Labrijn et al, Nature Protocols, 2014, 9:2450-2463; each of which is incorporated by reference in its entirety.
  • Fcab antibodies are described in Wozniak- Knopp etal, Protein Eng. Des. Sel, 2010, 23:289-297, incorporated by reference in its entirety.
  • Methods of making TandAb ® antibodies are described in Kipriyanov etal, J. Mol. Biol., 1999, 293:41-56 and Zhukovsky et al, Blood, 2013, 122:5116, each of which is incorporated by reference in its entirety.
  • Methods of making tandem Fabs are described in WO 2015/103072, incorporated by reference in its entirety.
  • Methods of making ZybodiesTM are described in LaFleur etal, mAbs, 2013, 5:208-218, incorporated by reference in its entirety.
  • an antibody provided herein is an affinity matured variant of a parent antibody, which may be generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues may be mutated and the variant antibodies, or portions thereof, displayed on phage and screened for affinity. Such alterations may be made in CDR “hotspots,” or residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see Chowdhury, Methods Mol. Biol ., 2008, 207:179-196, incorporated by reference in its entirety), and/or residues that contact the antigen.
  • Any suitable method can be used to introduce variability into a polynucleotide sequence(s) encoding an antibody, including error-prone PCR, chain shuffling, and oligonucleotide-directed mutagenesis such as trinucleotide-directed mutagenesis (TRIM).
  • TAM trinucleotide-directed mutagenesis
  • CDR residues e.g ., 4-6 residues at a time
  • CDR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • CDR-H3 and CDR-L3 in particular are often targeted for mutation.
  • variable regions and/or CDRs can be used to produce a secondary library.
  • the secondary library is then screened to identify antibody variants with improved affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in Hoogenboom et al, Methods in Molecular Biology, 2001, 178:1-37, incorporated by reference in its entirety.
  • nucleic acids encoding TF antibodies
  • vectors comprising the nucleic acids
  • host cells comprising the vectors and nucleic acids, as well as recombinant techniques for the production of the antibodies.
  • the nucleic acid(s) encoding it may be isolated and inserted into a replicable vector for further cloning (; i.e ., amplification of the DNA) or expression.
  • the nucleic acid may be produced by homologous recombination, for example as described in U.S. Patent No. 5,204,244, incorporated by reference in its entirety.
  • the vector components generally include one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Patent No. 5,534,615, incorporated by reference in its entirety.
  • a signal sequence generally include one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Patent No. 5,534,615, incorporated by reference in its entirety.
  • suitable host cells are provided below. These host cells are not meant to be limiting, and any suitable host cell may be used to produce the antibodies provided herein.
  • Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g, yeast), or higher eukaryotic (e.g, mammalian) cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia ( E . coif), Enterohacter , Erwinia, Klebsiella , Proteus , Salmonella (S. typhimurium ), Serratia (S. marcescans ), Shigella , Bacilli (B. subtilis and B. licheniformis ), Pseudomonas (P.
  • eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia ( E . coif), Enterohacter , Erwinia, Klebsiella , Proteus
  • E. coli 294 One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli XI 776, and E. coli W3110 are also suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for TF antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker’s yeast, is a commonly used lower eukaryotic host microorganism.
  • Schizosaccharomyces pombe Kluyveromyces (K. lactis , K. fragilis, K. bulgaricus K. wickeramii , K. waltii , K. drosophilarum , K. thermotolerans , and K.
  • Candida C . albicans
  • Trichoderma reesia Neurospora crassa
  • Schwanniomyces S. occidentalis
  • filamentous fungi such as, for example Penicillium, Tolypocladium , and Aspergillus (A. nidulans and A. nigef).
  • Useful mammalian host cells include COS-7 cells, HEK293 cells, baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO), mouse sertoli cells, African green monkey kidney cells (VERO-76), and the like.
  • the host cells used to produce the TF antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as, for example, Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco’s Modified Eagle’s Medium (DMEM) are suitable for culturing the host cells.
  • MEM Minimal Essential Medium
  • RPMI-1640 RPMI-1640
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • 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, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary 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, in the periplasmic space, 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, is removed, for example, by centrifugation or ultrafiltration.
  • the particulate debris either host cells or lysed fragments.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • the antibody is produced in a cell-free system.
  • the cell-free system is an in vitro transcription and translation system as described in Yin etal. , mAbs , 2012, 4:217-225, incorporated by reference in its entirety.
  • the cell-free system utilizes a cell-free extract from a eukaryotic cell or from a prokaryotic cell.
  • the prokaryotic cell is E. coli.
  • Cell-free expression of the antibody may be useful, for example, where the antibody accumulates in a cell as an insoluble aggregate, or where yields from periplasmic expression are low.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon ® or Millipore ® Pellcon ® 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.
  • 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 particularly useful purification technique.
  • affinity chromatography is a particularly useful purification 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 comprise human g ⁇ , g2, or g4 heavy chains (Lindmark et al ., ./. Immunol. Meth ., 1983, 62:1-13, incorporated by reference in its entirety).
  • Protein Gis useful for all mouse isotypes and for human g3 (Guss et al., EMBO J, 1986, 5:1567-1575, incorporated by reference in its entirety).
  • the matrix to which the affinity ligand is attached is most often 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.
  • the antibody comprises a Cm domain
  • the BakerBond ABX ® resin is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 to about 4.5, generally performed at low salt concentrations (e.g ., from about 0 to about 0.25 M salt).
  • Antigen-binding activity of the antibodies provided herein may be evaluated by any suitable method, including using SPR, BLI, RIA and MSD-SET, as described elsewhere in this disclosure. Additionally, antigen-binding activity may be evaluated by ELISA assays and Western blot assays.
  • the epitope is determined by peptide competition. In some embodiments, the epitope is determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography.
  • Thrombin generation in the presence of the antibodies provided herein can be determined by the Thrombin Generation Assay (TGA), as described elsewhere in this disclosure.
  • Inhibition of TF signaling can be determined by measuring the production of a cytokine regulated by the TF signaling, such as IL8 and GM-CSF. Assays for determining the IL8 and/or GM-CSF level are provided elsewhere in this disclosure and, for example, in Hjortoe et al, Blood , 2004, 103:3029-3037.
  • Effector function following treatment with the antibodies provided herein may be evaluated using a variety of in vitro and in vivo assays known in the art, including those described in Ravetch and Kinet, Annu. Rev. Immunol ., 1991, 9:457-492; U.S. Pat. Nos. 5,500,362, 5,821,337; Hellstrom et al. , Proc. Nat’lAcad. Sci. USA , 1986, 83:7059-7063; Hellstrom et al. , Proc. Nat’l Acad. Sci. USA , 1985, 82:1499-1502; Bruggemann et al, J. Exp. Med., 1987, 166:1351-1361; Clynes et al. , Proc. Nat’lAcad. Sci. USA, 1998, 95:652-656;
  • Immunohistochemistry (IHC) assays can be used to evaluate expression and localization of TF and of proinflammatory markers in patient or animal samples.
  • IHC assays for examining animal tissue e.g. ocular tissue
  • animal tissue e.g. ocular tissue
  • IHC can be used to evaluate the TF expression in patient or animal samples as described in PCT application PCT/US2019/012427 or US utility application number 16/959,652, each of which is incorporated by reference in its entirety.
  • Methods for conducting IHC on ocular tissue are also provided in Zalis MC et al, J Histochem Cytochem. 2017;65(4):223- 239 and Fitch, J.M.
  • Epitope binding differences between the anti-human TF antibodies provided herein can be determined by the chimeric TF construct mapping experiments and the epitope binning assays, as described elsewhere in this disclosure.
  • the antibodies provided herein can be formulated in any appropriate pharmaceutical composition and administered by any suitable route of administration.
  • Suitable routes of administration include, but are not limited to, the intravitreal, subretinal, suprachoroidal, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, topical, pulmonary, and subcutaneous routes.
  • the pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients , Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.
  • parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including infusions and bolus injections), intramuscular, and intraarterial. Because their administration typically bypasses subjects’ natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry ( e.g ., lyophilized) products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • compositions provided herein is a pharmaceutical composition or a single unit dosage form.
  • Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies.
  • the amount of the antibody or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof can vary with the nature and severity of the disease or condition, and the route by which the antibody is administered.
  • the frequency and dosage can also vary according to factors specific for each subject depending on the specific therapy (e.g ., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • an antibody provided herein may optionally be administered with one or more additional agents useful to prevent or treat a disease or disorder.
  • the effective amount of such additional agents may depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors known in the art or described herein.
  • the antibodies provided herein are administered by intravitreal injection and are administered once every month, two months, three months, four months or more. In preferred embodiments, when the antibodies provided herein are administered by intravitreal injection, they are administered no more than once a month.
  • the antibodies of the invention are administered to a subject, generally a mammal, generally a human, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed above.
  • the antibodies of the invention may be administered to a subject intravenously as a bolus or by continuous infusion over a period of time, by intravitreal, subretinal, suprachoroidal, intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, intratumoral, or topical routes.
  • the antibodies are administered to a subject by intravitreal routes (e.g ., intravitreal injection).
  • administration is via intravenous, intramuscular, intratumoral, subcutaneous, intrasynovial, intraocular, intraplaque, or intradermal injection of the antibody or of an expression vector having cDNA encoding the antibody.
  • the vector can be a replication-deficient adenoviral vector, retroviral vector or other viral vectors carrying a cDNA encoding the antibody.
  • the patient is treated by intravenous or intratumoral injection of an effective amount of one or more replication-deficient adenoviral vectors, or one or more adeno-associated vectors carrying cDNA encoding the antibody.
  • An intravitreal injection is a type of intraocular injection. It is a procedure to place a medication directly into the space in the back of the eye called the vitreous cavity, which is filled with a jelly-like fluid called the vitreous humor gel. The procedure is typically performed by a medical profession (e.g., physician or trained retina specialist) in an office setting.
  • a medical profession e.g., physician or trained retina specialist
  • the intravitreal administration route presents challenges for preparing suitable compositions (e.g, compositions that include acceptable levels of active agent and contaminant(s)) due to the sensitive nature of the eye. There are also a limited collection of excipients acceptable for intravitreal administration compared with other delivery routes. In most cases, it is recommended that injection volume is limited to less than 0.10 mL per eye.
  • suitable compositions e.g, compositions that include acceptable levels of active agent and contaminant(s)
  • injection volume is limited to less than 0.10 mL per eye.
  • the antibodies provided herein may be useful for the treatment of ocular diseases involving TF.
  • ocular disease refers broadly to any disease, disorder, injury or condition that impairs normal function of the eye(s).
  • “Ocular disease” also encompasses the state of an eye when it exhibits symptoms including, but not limited to, inflammation, impairment of visual acuity, decreased photoreceptor function, decreased night vision, leakiness, macular edema, abnormal leukocyte infiltration, vitreous detachment, hyperplasia in the ocular tissue (e.g, retina or cornea), blurred vision, eye pain, eye redness, light sensitivity, discoloration of the cornea, irritation in or around the eye, double vision, abnormal tearing, tunnel vision, seeing shadowy areas in your central vision or experiencing unusually fuzzy or distorted vision, loss of central vision, straight lines appearing wavy, blindness, retinal neovascularization, angiogenesis in ocular tissue, vascular leakage, retinal thickening within 500 pm of the foveal center, hard yellow exudates within 500 pm of the foveal center (optionally with adjacent retinal thickening), fog vision, floaters, or loss of contrast.
  • the an ocular disease is a disease
  • any suitable ocular disease can be treated with the antibodies provided herein.
  • diseases that are contemplated for treatment in the present disclosure include intraocular neovascular syndromes such as neovascular AMD, wet AMD, dry AMD, diabetic retinopathy, proliferative diabetic retinopathy (PDR), non-proliferative diabetic retinopathy (NPDR), diabetic macular edema, retinopathy of prematurity, neovascularization glaucoma, retinal vein occlusion, central retinal vein occlusion, macular degeneration, retinitis pigmentosa, retinal angiomatous hyperplasia, macular telangiectasia, ischemic retinopathy, macular neovascularization (choroidal neovascularization), iris neovascularization, eye neovascularization, corneal neovascularization, retinal neovascularization, retinal degeneration, inherited retina
  • the ocular disease is selected from the group consisting of: wet AMD, dry AMD, diabetic macular edema (DME), macula cystic edema (CME), NPDR, PDR, cystoid macular edema, vasculitis (e.g., central retinal vein occlusion), papilledema, retinitis, conjunctivitis, uveitis, choroiditis, multifocal choroiditis, ocular mytoplasmosis, orbital inflammation, dry eye (Hugh Glenn) Disease, Sjogren's disease, and other eye diseases associated with ocular neovascularization, vascular leakage, and/or retinal edema.
  • DME diabetic macular edema
  • CME macula cystic edema
  • NPDR macula cystic edema
  • PDR cystoid macular edema
  • vasculitis e.g., central retina
  • the disease or condition that can benefit from treatment with an anti-TF antibody is a disease or condition involving neovascularization.
  • the disease or condition is AMD, wet AMD or dry AMD.
  • the disease or condition involving neovascularization is diabetic retinopathy, PDR or NPDR.
  • provided herein is a method of treating an ocular disease, for example, a disease or condition involving neovascularization in a subject in need thereof by administering an effective amount of an anti-TF antibody provided herein to the subject.
  • the disease or condition involving neovascularization is AMD, wet AMD or dry AMD.
  • the disease or condition involving neovascularization is diabetic retinopathy, PDR or NPDR.
  • provided herein is a method of preventing the onset of an ocular disease, for example, a disease or condition involving neovascularization in a subject in need thereof by administering an effective amount of an antibody provided herein to the subject.
  • provided herein is a method of delaying the onset of age-related macular degeneration (AMD) in a subject in need thereof by administering an effective amount of an antibody provided herein to the subject.
  • AMD age-related macular degeneration
  • provided herein is a method of preventing the onset of age-related macular degeneration (AMD) in a subject in need thereof by administering an effective amount of an antibody provided herein to the subject.
  • AMD age-related macular degeneration
  • provided herein is a method of delaying the onset of diabetic retinopathy in a subject in need thereof by administering an effective amount of an antibody provided herein to the subject.
  • provided herein is a method of preventing the onset of diabetic retinopathy in a subject in need thereof by administering an effective amount of an antibody provided herein to the subject.
  • an antibody provided herein is administered with at least one additional therapeutic agent.
  • Any suitable additional therapeutic agent may be administered with an antibody provided herein.
  • the additional therapeutic agent is selected from radiation, an ophthalmologic agent, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent, an immunostimulatory agent, an anti-angiogenic agent, and combinations thereof.
  • the antibodies provided herein are combined with the comparator drugs described herein.
  • an antibody provided herein e.g . 25G9, 25A5-T
  • an antibody provided herein may be combined with any one of: aflibercept (Eylea), ranibizumab, brolucizumab, pegaptanib, bevacizumab, abicipar pegol, Faricimab (RG7716), KSI-301, hl-conl (also referred to as “ICONl” or “ICON-1”) and M1593
  • the additional therapeutic agent may be administered by any suitable means.
  • an antibody provided herein and the additional therapeutic agent are included in the same pharmaceutical composition.
  • an antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions.
  • administration of the antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • the method can be used to detect TF in a subject having or suspected of having an ocular disease as described herein.
  • the methods comprise (a) receiving a sample from the subject; and (b) detecting the presence or the level of TF in the sample by contacting the sample with the antibody provided herein.
  • the methods comprise (a) administering to the subject the antibody provided herein; and (b) detecting the presence or the level of TF in the subject.
  • the ocular disease involves neovascularization, and optionally is age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • the disease or condition involving neovascularization is diabetic retinopathy, PDR or NPDR.
  • the methods comprise (a) administering to the subject the antibody provided herein; and (b) detecting the presence or the level of TF in the subject.
  • the disease or condition is an ocular disease as described herein.
  • the antibody provided herein is conjugated with a fluorescent label. In some embodiments, the antibody provided herein is conjugated with a radioactive label. In some embodiments, the antibody provided herein is conjugated with an enzyme label.
  • the relative amount of TF expressed by such cells is determined.
  • the fraction of cells expressing TF and the relative amount of TF expressed by such cells can be determined by any suitable method.
  • flow cytometry is used to make such measurements.
  • fluorescence assisted cell sorting FACS is used to make such measurement.
  • kits comprising the antibodies provided herein.
  • the kits may be used for the treatment, prevention, and/or diagnosis of a disease or disorder, as described herein.
  • the kit comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, and IV solution bags.
  • the containers may be formed from a variety of materials, such as glass or plastic.
  • the container holds a composition that is by itself, or when combined with another composition, effective for treating, preventing and/or diagnosing a disease or disorder.
  • the container may have a sterile access port. For example, if the container is an intravenous solution bag or a vial, it may have a port that can be pierced by a needle.
  • At least one active agent in the composition is an antibody provided herein.
  • the label or package insert indicates that the composition is used for treating the selected condition.
  • the kit comprises (a) a first container with a first composition contained therein, wherein the first composition comprises an antibody provided herein; and (b) a second container with a second composition contained therein, wherein the second composition comprises a further therapeutic agent.
  • the kit in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable excipient.
  • the excipient is a buffer.
  • the kit may further include other materials desirable from a commercial and user standpoint, including filters, needles, and syringes.
  • Example 1 Generation of TF Antibodies
  • Human, cynomolgus monkey, and mouse TF extracellular domain (ECD) fragments were expressed as C-terminal His or Fey fragment fusions.
  • Expi293 cells (ThermoFisher Scientific, Waltham, MA, USA) were transiently transfected as recommended by the manufacturer with pcDNA3.1V5-HisA (ThermoFisher Scientific) encoding human, cynomolgus, or mouse TF ECD-His6 (TF-His; SEQ ID NOs:811, 815, and 819, respectively) or pFUSE-hlgGl-Fc (Invivogen, San Diego, CA, USA) encoding human, cynomolgus or mouse TF ECD-Fc (TF-Fc; SEQ ID NOs:812, 816, and 820, respectively).
  • TF-His6 and TF-Fc proteins were purified by affinity chromatography with a HisTrap HP and MabSelect SuRe column (GE Healthcare Bio-Sciences, Marlborough, MA, USA), respectively.
  • FVII-Fc expressed in Expi293 was purified by affinity chromatography with a MabSelect SuRe column, followed by size exclusion chromatography.
  • the TF-His6 and TF-Fc proteins were biotinylated with a 15x molar excess of Sulfo-NHS-SS-biotin as recommended (ThermoFisher Scientific).
  • the non-labeled and biotinylated proteins were further purified by size exclusion chromatography using a Superdex 200 Increase 10/300 column (GE Healthcare Bio-Sciences).
  • Human antibodies against human TF were generated by AdimabTM yeast-based antibody presentation using the biotinylated recombinant TF proteins as screening antigens, as described below. All antibodies against human TF were evaluated for cross-reactivity with cynomolgus monkey and mouse TF. The binding activity of the antibodies to human, cynomolgus monkey, and mouse TF is shown in Table 5.
  • yeast cells ( ⁇ 10 10 cells/library) were incubated with 10 nM of biotinylated human TF Fc-fusion antigen for 15 min at room temperature in FACS wash buffer PBS with 0.1% BSA.
  • the cell pellet was resuspended in 40 mL wash buffer, and 500 pL Streptavidin MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat # 130-048-101) were added to the yeast and incubated for 15 min at 4°C.
  • the yeast were pelleted, resuspended in 5 mL wash buffer, and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat.# 130-042-401). After the 5 mL was loaded, the column was washed 3 times with 3 mL FACS wash buffer. The column was then removed from the magnetic field, and the yeast were eluted with 5 mL of growth media and then grown overnight.
  • FACS flow cytometry
  • Yeast were then washed twice and stained with LC-FITC diluted 1 : 100 (Southern Biotech, Birmingham, Alabama; Cat# 2062-02) and either SA-633 (Life Technologies, Grand Island, NY; Cat # S21375) diluted 1:500, or EA-PE (Sigma-Aldrich, St Louis; Cat # E4011) diluted 1 :50, secondary reagents for 15 min at 4°C. After washing twice with ice-cold wash buffer, the cell pellets were resuspended in 0.4 mL wash buffer and transferred to strainer-capped sort tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sort gates were determined to select for TF binding.
  • LC-FITC diluted 1 : 100 (Southern Biotech, Birmingham, Alabama; Cat# 2062-02) and either SA-633 (Life Technologies, Grand Island, NY; Cat # S21375) diluted 1:500, or EA-PE (Sigma-Aldrich, St Louis; Cat
  • the mouse- and cyno-selected populations from the first round of FACS were grown out and expanded through sub-culturing in selective media.
  • the second, third, and fourth rounds of FACS involved positive sorts to enrich for TF binders and/or negative sorts to decrease the number of non-specific binders using soluble membrane proteins from CHO cells (see, e.g. , WO2014179363 and Xu et al., PEDS, 2013, 26(10):663-70).
  • yeast were plated and sequenced.
  • the first of selection round utilized Miltenyi MACS beads and 10 nM biotinylated human TF Fc-fusion as antigen. Subsequent to the MACS bead selections, three rounds of FACS sorting were performed as described above using cynomolgus and mouse Fc-fusion TF at 10 nM oreither biotinylated Fc-fusion TF antigens or biotinylated monomeric HIS-forms of human, mouse or cynomolgus TF. Individual colonies from each FACS selection round were sequenced.
  • CDR-H1 and CDR-H2 selection The CDR-H3s from clones selected from either naive or light chain diversification procedure were recombined into a premade library with CDR-H1 and CDR-H2 variants of a diversity of 1 x 10 8 and selections were performed using biotinylated Fc-fusion cynomolgus TF antigen, biotinylated cynomolgus HIS-TF antigen, and/or biotinylated human HIS-TF. Affinity pressures were applied by using decreasing concentrations of biotinylated HIS-TF antigens (down to 1 nM) under equilibrium conditions at room temperature.
  • CDR-H3/CDR-H1/CDR-H2 selections Oligos were ordered from IDT which comprised the CDR-H3 as well as a homologous flanking region on either side of the CDR- H3. Amino acid positions in the CDR-H3 were variegated via NNK diversity at two positions per oligo across the entire CDR-H3. The CDR-H3 oligos were double-stranded using primers which annealed to the flanking region of the CDR-H3. The remaining FR1 to FR3 of the heavy chain variable region was amplified from pools of antibodies with improved affinity that were isolated from the CDR-H1 and CDR-H2 diversities selected above.
  • the library was then created by transforming the double stranded CDR-H3 oligo, the FR1 to FR3 pooled fragments, and the heavy chain expression vector into yeast already containing the light chain of the parent. Selections were performed as during previous cycles using FACS sorting.
  • FACS rounds assessed non-specific binding, species cross-reactivity, and affinity pressure, and sorting was performed to obtain populations with the desired characteristics. Affinity pressures for these selections were performed as described above in the CDR-H1 and CDR- H2 selection.
  • CDR-L3/CDR-L1/CDR-L2 selections Oligos were ordered from IDT which comprised the CDR-L3 as well as a homologous flanking region on either side of the CDR- L3. Amino acid positions in the CDR-L3 were variegated via NNK diversity at one position per oligo across the entire CDR-L3. The CDR-L3 oligos were double-stranded using primers which annealed to the flanking region of the CDR-L3. The remaining FR1 to FR3 of the light chain variable region was amplified from pools of antibodies with improved affinity that were isolated from the CDR-L1 and CDR-L2 diversities selected above.
  • the library was then created by transforming the double stranded CDR-L3 oligo, the FR1 to FR3 pooled fragments, and the light chain expression vector into yeast already containing the heavy chain of the parent. Selections were performed as during previous cycles using FACS sorting.
  • FACS rounds assessed non-specific binding, species cross-reactivity, and affinity pressure, and sorting was performed to obtain populations with the desired characteristics. Affinity pressures included titrations as well as incorporation of the parental Fab in antigen pre- complexation.
  • yeast clones were grown to saturation and then induced for 48 h at 30°C with shaking. After induction, yeast cells were pelleted and the supernatants were harvested for purification. IgGs were purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified over CaptureSelect IgG- CH1 affinity matrix (LifeTechnologies, Cat # 1943200250).
  • Example 2 Efficacy Study in Swine CNV Model
  • An efficacy study in a swine choroidal neovascularization (CNV) model was performed to determine the effect of 4 different anti-TF antibodies in reducing lesion size (or leakiness area).
  • 10-12 week old animals underwent bilateral laser using an 810 nm Diode laser delivered through an indirect ophthalmoscope to create approximately 6 single laser spots between retinal veins in each eye of each animal.
  • a vehicle control group was also included in the study.
  • CTLF Corrected Total Lesion Fluorescence
  • FIG. 2A and FIG. 2B The mean CTLF over time for all groups and percent of CTLF relative to day 7 (baseline) for all groups are shown in FIG. 2A and FIG. 2B, respectively.
  • ICON-1 reduced lesion size by greater than 10% relative to baseline but did not exhibit as much lesion size reduction as the 25 and 43 group antibodies within the same period.
  • anti-hTF antibody 25G9 showed the greatest lesion size reduction of all groups relative to baseline with greater than 40% lesion size reduction relative to baseline.
  • Adalimumab and ICON-1 also showed significant lesion size reduction relative to the vehicle control, however, neither therapies resulted in as much lesion size reduction as the 25 group antibody. There was no significant difference in the % CTLF at 14 days and 28 days relative to baseline, when the 43 family antibody was administered. Throughout the study, the anti-hTF antibodies 1G and 29D did not reduce lesion size significantly as compared to the vehicle control group (FIG. 2B).
  • Example 3 Efficacy of 25-Group Antibodies in Swine CNV Model
  • An efficacy study in a swine choroidal neovascularization (CNV) model was performed to determine the effect of various 25-group anti-TF antibodies in reducing lesion size (or leakiness area).
  • 10-12 week old animals (Swine/Hampshire Cross) underwent bilateral laser using an 810 nm Diode laser delivered through an indirect ophthalmoscope to create approximately 6 single laser spots between retinal veins in each eye of each animal.
  • FA Fluorescein Angiography
  • CTL Corrected Total Lesion Fluorescence
  • the perimeter of the lesion was traced, and an integrated density value was obtained.
  • CTLF was then calculated by subtracting the mean fluorescence background adjacent to the lesion from the integrated density measurement.
  • the CTLF was representative of the total leakiness area or total lesion size.
  • the mean CTLF over time for all groups and percent of CTLF for all groups relative to day 7 (baseline) are shown in FIG. 3 and FIG. 4, respectively.
  • Example 4 Dose-Dependent Response of 25G9 in Swine CNV Model
  • CNV swine choroidal neovascularization
  • FA Fluorescein Angiography
  • CTL Corrected Total Lesion Fluorescence
  • the perimeter of the lesion was traced, and an integrated density value was obtained.
  • CTLF was then calculated by subtracting the mean fluorescence background adjacent to the lesion from the integrated density measurement.
  • the CTLF was representative of the total leakiness area or total lesion size. Percent changes in lesion size from day 7 to day 28 are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • anti-hTF antibody 25G9 reduced lesion size in a dose- dependent manner. 25G9 reduced lesion size by greater than 50% at 4 mg. This data indicates that antibody 25G9 was effective in reducing lesion size in the swine CNV model in a dose- dependent manner.
  • Example 5 Efficacy of Wild-type vs. Mutated 25G9 in Swine CNV Model
  • An efficacy study in a swine choroidal neovascularization (CNV) model was performed to compare different doses of anti-TF antibody wild type 25G9, 25G9DE, ICON1, and anti-TF antibody Ml 593 for their ability to reduce lesion size (or leakiness area).
  • the wild-type 25G9 (25G9WT) is the unmutated form of 25G9 as disclosed herein.
  • Anti-TF antibody 25G9DE is the 25G9 antibody having two mutations in the human IgGl Fc region (S239D and I332E, as determined by Kabat numbering) that result in enhanced Fc effector function (particularly, ADCC function).
  • S239D and I332E as determined by Kabat numbering
  • Fc effector function particularly, ADCC function.
  • 10-12 week old animals underwent bilateral laser using an 810 nm Diode laser delivered through an indirect ophthalmoscope to create approximately 6 single laser spots between retinal veins in each eye.
  • animals were treated intravitreally on Day 7 post-laser treatment with one of the following:
  • FA Fluorescein Angiography
  • CTL Corrected Total Lesion Fluorescence
  • the perimeter of the lesion was traced, and an integrated density value was obtained.
  • CTLF was then calculated by subtracting the mean fluorescence background adjacent to the lesion from the integrated density measurement.
  • the CTLF was representative of the total leakiness area or total lesion size. The mean CTLF for all groups and the percent CTLF for all groups relative to baseline are shown in FIG. 5 and FIG. 6.
  • CNV swine choroidal neovascularization
  • Anti-TF antibody 25G9 was the unmutated form of the 25G9 antibody as disclosed herein.
  • Anti-TF antibody 25G9DE was the 25G9 antibody having two mutations in the Fc region (S239D and I332E according to Kabat numbering) that result in enhanced effector function (particularly ADCC function).
  • Anti-TF antibody 25G9-LALAPG was the 25G9 antibody having the combination of L234A, L235A, and P329A (LALA-PG) substitutions in the human IgGl heavy chain constant region (according to Kabat numbering) that have been shown to effectively silence the effector function of human IgGl antibodies.
  • the antigen-binding fragment (Fab) for the 25G9 antibody was identified as 25G9 FAB (; i.e ., this is an Fc null version of the antibody having a single FAB domain).
  • Smaller antibody fragments such as Fab or Fab (2) are often used for intravitreal injection treatment of ocular diseases because of their low serum half-life and low risk of systemic toxicity. However, this smaller fragment also typically has a lower intravitreal half- life ( e.g ., due to faster diffusion into the serum) and usually must be administered more frequently.
  • FA Fluorescein Angiography
  • CTL Corrected Total Lesion Fluorescence
  • the perimeter of the lesion was traced, and an integrated density value was obtained.
  • CTLF was then calculated by subtracting the mean fluorescence background adjacent to the lesion from the integrated density measurement.
  • the CTLF was representative of the total leakiness area or total lesion size.
  • the mean CTLF for all groups and the percent CTLF for all groups relative to day 7 (baseline) are shown in FIG. 7 and FIG. 8, respectively.
  • Example 7 Effectiveness Comparison of 25A5-T to Eylea in Swine CNV Model
  • CTLF Corrected Total Lesion Fluorescence
  • both Eylea groups showed significant reduction in lesion size relative to baseline and relative to the vehicle control.
  • 200 pg of Eylea resulted in about 50% lesion size reduction relative to baseline
  • 2 mg of Eylea resulted in greater than 40% lesion size reduction relative to baseline.
  • the 200 pg and 2 mg Eylea treatments resulted in over 60% and over 70% lesion size reduction, respectively.
  • the 25A5-T groups by day 14, there was not a significant reduction in lesion size relative to baseline and relative to the vehicle control.
  • the lesion size reduction in the 400 pg 25A5-T group was greater than 50% relative to baseline and greater than the vehicle control (FIG. 10)
  • Example 8 Ocular Pharmacokinetics Study in Rabbits
  • PK pharmacokinetics
  • IVT intravitreal
  • rabbits were treated by IVT injection with either a single dose of 25A5-T or a two doses of 25A5-T, as indicated in Table 46.
  • a naive group of rabbits served as the negative control — they did not receive any 25A5-T dose, and were sacrificed on day 0 for eye tissue collection, sampling, and analysis.
  • Table 46 Experimental design for ocular PK study in rabbits.
  • IVT injections 45 mg/mL 25A5-T in PBS was diluted with PBS to get to the desired concentrations.
  • IVT injections were administered to sedated rabbits using a 30-gauge needle.
  • Table 47 PK results for 25A5-T and ICON-1 in rabbit vitreous humor — single IVT administration. *The AUC was determined using non-compartmental methods.
  • FIG. 12A and FIG. 12B The mean concentration profiles and results for 25A5-T in all serum and tissue samples from rabbits in groups 1 and 2 are shown in FIG. 12A and FIG. 12B.
  • the results showed that the concentration of 25A5-T in the retina was approximately half of that in the vitreous humor. Following single IVT administration, the 25A5-T disposition was dose linear with a vitreous half-life of about 6 days. Concentrations in other ocular compartments declined in parallel with those in the vitreous. The results also showed significantly less 25A5-T in the serum samples than in the ocular tissues. These results indicate higher antibody exposure in the target ocular tissues than in the serum.
  • the mean concentration profiles and results for ICON-1 in all plasma and tissue samples from rabbits in group 3 are shown in FIG. 13.
  • Vitreous PK data were analyzed using a non-compartmental method and are shown in Table 65.
  • the non-compartmental vitreous parameter measurements were similar for groups 1 and 2 and were consistent with those estimated using the 1-compartmental model.
  • Serum PK data were analyzed using a non-compartmental method are shown in Table 68 and the serum concentration-time profile is shown in FIG. 23.
  • the 25A5-T serum concentrations increased approximately linearly with increasing dose and were much lower than those in the vitreous humor. Due to the presence of ADA on D16, the 25A5-T serum concentrations might be underestimated at later times
  • Example 9 Ocular Pharmacokinetics Study in Cynomolgus Monkeys [00575]
  • PK pharmacokinetics
  • IVTT intravitreal
  • a topical antibiotic (tobramycin) was applied to both eyes, twice on the day before and twice on the day after each injection. Prior to dosing, the animals were sedated by an intramuscular injection of ketamine and dexmedetomidine. The conjunctivae were flushed with diluted benzalkonium chloride. 1% tropicamide was applied to each eye, as needed.
  • Table 70 Retinal 25A5-T PK data following IVT administration of 0.6 and 12 mg/eye 25A5-T (Non-Compartmental Method)
  • Table 71 Aqueous 25A5-T PK data following IVT administration of 0.6 and 12 mg/eye 25A5-T (Non- Compartmental Method)
  • FIG. 15A and FIG. 15B The mean concentration profiles for 25A5-T in all serum and tissue samples from cyno monkeys are shown in FIG. 15A and FIG. 15B.
  • Table 61 Experimental design for the pilot toxicology study in cyno monkeys a Animals were dosed on Days 1 and 15, kept for a 28-day observation, and euthanized on Day 44.
  • Formulation buffer which was administered to group 1, served as the negative control (vehicle control) and comprised of 20 mM Sodium Acetate, 8% Trehalose, and 0.02% (v/v) Polysorbate 20 (pH 5.5).
  • the anti-TF antibody, 25A5-T was stored in a freezer set to maintain -80°C and thawed on ice prior to use.
  • the 25A5-T antibody was withdrawn from the original vial using a filter needle with 5 micron filter and then transferred into a new sterile vial, mixed well and allowed to warm to ambient temperature prior to dosing.
  • the anti-TF antibody was formulated at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared on each dosing day.
  • dose formulation samples (duplicate 100 pL samples for analysis) were collected from the dosing container of all groups and were stored in a freezer set to maintain -80°C until shipment to the test site for concentration verification.
  • a topical antibiotic tobramycin was applied to both eyes twice on the day before and twice on the day after each injection.
  • animals Prior to dosing, and following an appropriate fasting period, animals received an intramuscular injection of a sedative cocktail of ketamine (5 mg/kg) and dexmedetomidine (0.01 mg/kg).
  • animals received an intramuscular injection of 0.1 mg/kg atipamezole, a reversal agent for dexmedetomidine.
  • the conjunctivae were flushed with diluted benzalkonium chloride (ZephiranTM).
  • Mydriatic drops 1% tropicamide
  • test materials were administered bilaterally by intravitreal injection on Days 1 and 15, using a 1 ⁇ 2 cc syringe with attached 29-gauge, 1 ⁇ 2 inch needle.
  • the dose volume in all group was 100 pL/eye, administered as two 50 pL injections, 10 to 15 minutes apart.
  • Intravitreal injections were performed by a board-certified veterinary ophthalmologist. Both eyes were examined by slit lamp biomicroscopy and/or indirect ophthalmoscopy following completion of each treatment to document any abnormalities (especially to the lens, vitreous and retina) caused by the administration procedure.
  • Table 62 details the parameters that were measured in this pilot tox study.
  • Table 62 Parameters and measurement frequency for the cyno pilot toxicology study.
  • the drug was labeled with biotin as the capture reagent and with Sulfo-TAGTM as the detection reagent.
  • the serum or vitreous humor sample was incubated with the capture and detection reagents.
  • Anti-25A5-T antibodies present in the sample formed a bridge between the biotin-labeled drug and the Sulfo-TAG-labeled drug.
  • the complex was then captured onto a Streptavidin coated MSD plate.
  • an electrical potential causes light emission as ECL from the SULFO-TAGTM labels that are bound.
  • the assay provided a relative anti-25A5-T (titer) antibody concentration.
  • the positive control (PC) antibody used for the screening and titer assays consisted of an affinity purified rabbit polyclonal raised against the Fab portion of 25 A3.
  • Serum 25A5-T concentrations were highly variable, and the serum concentrations increased more than proportionally between the 4 and the 11.9 mg/eye dose groups.
  • 25A5-T vitreous concentrations were quantifiable in all eyes with concentrations ranging from 38.3 ng/g (4 mg/eye animal) to 74572 ng/g (11.9 mg/eye animal).
  • All three 4 mg/eye animals were serum ADA-positive after 25A5-T administration and 2/3 11.9 mg/eye dose group animals developed ADA following 25A5-T administration.
  • the presence of serum ADA was generally associated with lower 25A5-T serum concentrations.
  • All vitreous samples were ADA-positive and there was no relationship between ADA titers and 25A5-T vitreous concentrations.
  • Animal No. 3001 (Administered 11.9 mg/eye): Due to moderate ocular inflammation observed on Day 17 ophthalmic examinations, a mydriatic agent (atropine) was applied topically to both eyes from Days 17 to 19.
  • a mydriatic agent atropine
  • Animal No. 3003 (Administered 11.9 mg/eye): Due to moderate ocular inflammation observed on Day 17 ophthalmic examinations, a mydriatic agent (atropine) was applied topically to both eyes from Days 17 to 19. A brief ophthalmology examination was performed on Day 23 and revealed ocular inflammation that was considered secondary to an immune response. As such, the animal received one subconjunctival injection (100 pL) of Kenalog®-40 (Triamcinolone acetonide, 40 mg/mL) in both eyes on Day 24, an analgesic (Buprenorphine slow-release) on Days 23, 26, and 29, and a topical anti-inflammatory (1% Prednisolone) three times daily from Day 23 to 29.
  • Kenalog®-40 Triamcinolone acetonide, 40 mg/mL
  • Animal No. 2002 (Administered 4 mg/eye): On Day 24, due to signs of pain and discomfort (blepharospasm) noted, a brief ophthalmology examination was performed on the animal and revealed similar inflammation to that seen in high dose Animal No. 3003 on Day 23. Therefore Animal No.
  • Intravitreal administration of 25A5-T was performed on Day 1 and Day 15 and most noteworthy changes were seen subsequent to the second injection. Following the first injection, animals were examined three times and the two most common findings were anterior chamber cells and vitreal cells. Anterior chamber cells are regularly noted following intravitreal administration and usually resolve rapidly if procedure-related. In this study, a dose-dependent response was observed when looking at the presence of those cells as an increased incidence of eyes graded moderate on Day 3 was observed (2 eyes in the 4 mg/eye group and 4 eyes in the 11.9 mg/eye group). By Day 14, anterior chamber cells should have resolved if procedure-related, however, they were observed in most eyes in the high-dose group (5/6 eyes and most of them graded very slight) and none were seen in the low-dose group. Vitreal cells were the most common observation and were noted in nearly all eyes on this study (by Day 14, all were graded very slight except for one reference item animal that was graded slight).
  • Tonometry Intraocular Pressure, IOP
  • Intraocular pressures measured on Days 3, 7, 14, 17, 29, and 43 were generally within normal ranges in all animals.
  • Serum concentrations of 25A5-T were highly variable, were quantifiable at all time points in samples collected from 25A5-T-treated animals on Day 1 and were generally quantifiable up to 168 hours postdose on Day 15.
  • Mean serum concentrations increased with dose and in a greater than dose- proportional manner. At the timepoints evaluated, peak mean serum concentrations occurred at 24 hours postdose in both Days 1 and 15 (FIG. 18 and FIG. 19).
  • Vitreous humor, retina and aqueous humor concentrations of 25A5-T were quantifiable on Day 44 in all 25A5-T-treated animals with the exception of Animal No. 2002 (all tissue samples below LLOQ) and Animal No. 3002 (below LLOQ for aqueous humor samples only).
  • Animal No. 3003 that was euthanized on Day 30 due to severe ocular inflammation, had markedly higher 25A5-T concentrations measured in the vitreous humor, retina and aqueous humor samples on Day 30 compared to the concentrations measured in animals of the same dose group on Day 44, with the greatest concentrations noted in the vitreous humor and retina.
  • Table 63 Summary of Microscopic Findings - Scheduled Euthanasia (Day 44) a Numbers in parentheses represent the number of animals with the finding.
  • anterior and posterior segment changes were observed at both 4 and 11.9 mg/eye with the two most common findings being anterior chamber cells and vitreal cells.
  • the eyes of animals given > 4 mg/eye developed inflammation with a stronger response at 11.9 mg/eye.
  • the ocular inflammation was severe enough to require the early euthanasia of 1/3 animals receiving 11.9 mg/eye despite local steroid treatment.
  • Perivascular retinal opacities are associated with the movement of inflammatory cells from the circulation into the retinal tissue. Both dose groups had 2/3 animals (4/6 eyes) that exhibited this change on Day 29 (2 weeks following the second dose). By Day 43, perivascular retinal opacities were still observed in 2/3 animals at 4 mg/eve, but with improvement of the findings in 1 of the 2 animals. At 11.9 mg/eye, perivascular retinal opacities were only observed in 1/3 animals and there was improvement in the severity of the findings.
  • Cynomolgus monkeys were selected as the species for in vivo toxicology, partly because 25A5-T affinity for monkey and human TF is similar. 25A5-T pharmacokinetics in cynomolgus monkeys is more relevant to 25A5-T disposition in humans. To evaluate the effect of 25A5-T ocular and systemic disposition after multiple doses 40 cynomolgus divided into 4 groups and received IVT administration of 100 uL of 25A5-T, once monthly for a total of 3 doses. The experimental design is provided in Table 73.
  • Table 73 Experimental design for 25A5-T Multi-Dose TK study a Animals were dosed on Days 1, 28 and 56, Main Study animals were euthanized on Day 85 and the
  • Blood samples were collected during each dosing interval for quantification of 25A5-T concentrations and ADA in serum samples.
  • the left eye was collected from all animals on Day 85 (main study) or 113 (recovery animals).
  • Vitreous, aqueous, and retinal samples were analyzed for 25A5-T concentrations, while vitreous was also analyzed for ADA.
  • Serum concentration-time profiles were analyzed by non-compartmental methods. Ocular concentrations at Day 85 and Day 113 were reported individually.
  • FIGS. 24A-24B The serum concentration-time profiles at Day 1 are shown in FIGS. 24A-24B and those for Day 56 are shown in FIGS. 25A-25B.
  • 25A5-T serum concentrations increased with the dose in both males and females and were dose proportional for the two lower doses and more than proportional for the 6 mg/eye dose. There were no significant differences observed between male and female monkeys.
  • 25A5-T serum concentrations were generally lower than following the first administration and could not be quantitated in most animals, therefore no TK parameters were calculated after the 1 st dose.
  • the decrease in 25A5-T concentration over time can most likely be attributed to the development of ADA.
  • vitreous 25A5-T concentrations were ⁇ LLOQ i.e. ⁇ 20 ng/mL for the 0.6 mg/eye dose group and were quantifiable in 3/8 eyes only with concentrations ranging from 37.2-134 ng/g for the animals receiving 2 mg/eye.
  • vitreous concentrations were quantifiable in a total of 4/8 animals with concentrations of 33.2 ng/g and 34.5 ng/g in the two ADA positive animals, and concentrations of 1650 ng/g and 261 ng/g for the two ADA negative animals.
  • Human vitreous volume is ⁇ 4.5 mL, and the cynomolgus vitreous volume is ⁇ 2 mL, thus for any given IVT dose, the maximum vitreous concentrations in humans are expected to be 2.25 lower than in monkeys.
  • Example 12 Effect of 25A5-T on Inflammatory Cytokine Inhibition
  • relevant inflammatory cytokine e.g ., IL-8 and GM-CSF
  • the inflammatory cytokine protein levels are measured as described previously in Hjortoe etal ., Blood , 2004, 103:3029-3037.
  • TF-positive ocular cells undergo a 2 hr serum starvation with Leibovitz’s L-15 medium and are incubated with an 8-point 1:2.5 titration starting at 100 nM of anti-TF antibody 25A5-T.
  • FVIIa (NovoSeven RT, Novo Nordisk, Bagsvaerd, Denmark) is added to the cells at a final concentration of 20 nM. 5 hr later cell culture supernatants is harvested and analyzed by ELISA or an Olink custom panel for the inflammatory cytokines as recommended in manufacturer instructions.
  • IL-1 beta IL-6, IL-8, IL-9, IL-10, IL-12, MCP-1 (CCL2), MCP-3 (CCL7), VEGF, TNF, MMP9.
  • a standard curve using each of the recombinant inflammatory cytokines is used in Prism to calculate cytokine concentrations in the cell culture supernatants. Percent of each of the inflammatory cytokines at reported antibody concentration is calculated relative to a no-antibody control. The results show a significant decrease in measured inflammatory cytokine(s) in the cell culture supernatant relative to the no-antibody control group.
  • the antibody was covalently coupled to a CM5 or Cl chip using an amine-coupling kit (GE Healthcare Bio-Sciences). Association between the anti-TF antibodies and a five-point three-fold titration of TF-His starting at 25 to 500 nM was measured for 300 sec. Subsequently, dissociation between the anti-TF antibody and TF-His was measured for up to 1800 sec. Kinetic data was analyzed and fitted globally using a 1 : 1 binding model. The KD values of the TF antibodies measured by the Biacore- based experiments are shown in Table 5.
  • the affinity of the antibodies for hTF is between 10 7 M and 10 11 M. All anti -hTF antibodies are cross-reactive with cTF. In addition, all anti -hTF antibodies from groups 25 and 43 exhibit binding activity to mTF. The anti-hTF antibodies 25G, 25G1, 25G9, and 43D8 are cross-reactive with mTF. There are no other reported human or humanized anti-hTF monoclonal antibodies that exhibit binding activity and cross-reactivity to mouse TF, indicating that the antibodies from groups 25 and 43 bind to a novel TF epitope.
  • HCT116 cells with endogenous expression of human TF were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA) and were maintained as recommended.
  • Flp-In-CHO cells expressing mouse TF were generated by transfection of Flp- In-CHO cells as recommended with a pcDNA5/FRT vector (ThermoFisher Scientific) encoding full-length mouse TF with a C-terminal FLAG tag.
  • a mouse TF -positive CHO clone was isolated by limiting dilution in tissue culture-treated 96-well plates.
  • All anti-hTF antibodies are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652 exhibit high affinity to human TF-positive HCT-116 cells with an ECso ranging from about 687 pM to about 39 pM.
  • Antibodies from groups 25 and 43 exhibit binding to CHO cells expressing mouse TF with an EC50 ranging from about 455 nM to about 2.9 nM, are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the binding activity to mouse TF is a unique property of the anti-hTF antibodies from groups 25 and 43. This is advantageous for pre-clinical studies of these antibodies with mouse models.
  • the TGA assay was performed using the calibrated-automated-thrombogram (CAT) instrument manufactured and distributed by STAGO.
  • the test method design was equivalent to a standard CAT assay measurement, except that the plasma source was normal pooled plasma (NPP) in citrate/CTI.
  • NPP normal pooled plasma
  • the anti-TF antibodies were titrated at 0, 10, 50 and 100 nM and mixed with NPP collected in 11 mM citrate supplemented with 100 microgram/mL of com trypsin inhibitor (citrate/CTI). Relipidated TF was added to a 96-well assay plate, followed by addition of the antibody /NPP mixture.
  • thrombin generation was initiated by the addition of calcium and the thrombin substrate.
  • the STAGO software was used to report the following parameters: Peak Ila (highest thrombin concentration generated [nM]); Lag Time (time to Ila generation [min]); ETP (endogenous thrombin potential, area under the curve [nM x min]); and ttPeak (time to Peak Ila [min]). Percent peak thrombin generation (% Peak Ila) and percent endogenous thrombin potential (% ETP) in the presence of each antibody relative to a no antibody plasma control on the same plate were also reported.
  • the Peak Ila, Lag Time, ETP, ttPeak, % Peak Ila, and % ETP in the presence of each antibody selected from IF, 25A, 25A3, 25G1, 29E, 39A, 43B1, 43D7, 43Ea, and 54E without antibody incubation prior to addition of calcium and thrombin substrate are shown in Table 6.
  • the Peak Ila, Lag Time, ETP, ttPeak, % Peak Ila, and % ETP in the presence of each antibody selected from IF, 25A, 25A3, 25G1, 29E, 39A, 43B1, 43D7, 43Ea, and 54E with 10 min antibody incubation prior to addition of calcium and thrombin substrate are shown in Table 7.
  • the % Peak Ila in the presence of titrations of anti-TF antibodies without antibody incubation prior to addition of calcium and thrombin substrate are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the % Peak Ila in the presence of titrations of anti-TF antibodies with 10 min antibody incubation prior to addition of calcium and thrombin substrate are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the % Peak Ila is greater than 90% in the presence of antibodies from group 25, including 25A, 25A3, and 25G1.
  • the % ETP is greater than 100% in the presence of antibodies from group 25, including 25 A, 25 A3, and 25G1.
  • the % Peak Ila is greater than 40% in the presence of antibodies from group 43, including 43B1, 43D7, and 43Ea.
  • the % ETP is greater than 90% in the presence of antibodies from group 43, including 43B1, 43D7, and 43Ea.
  • TF :FVIIa To evaluate the ability of TF :FVIIa to convert FX into FXa in the presence of human antibodies against TF, 5xl0 4 MDA-MB-231 cells (ATCC, Manassas, VA, EISA) were plated into tissue culture-treated black 96-well plates (Greiner Bio-One, Monroe, NC, EISA). After removal of the cell culture media and addition of a final concentration of 200 nM of FX in a HEPES buffer with 1.5 mM CaCb, cells were incubated with a titration of the antibodies for 15 min at 37°C.
  • FXa conversion percentages (% FXa) in the presence of an anti-TF antibody titration relative to a no-antibody control are summarized in Table 8 and plotted in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the FXa conversion percentage ranges from about 78% to about 120% in presence of different concentrations of antibodies from groups 25 and 43, including 25 A, 25A3, 25G, 25G1, 25G5, 25G9, 43B, 43B1, 43B7, 43D, 43D7, 43D8, 43E, and 43Ea.
  • FVII-Fc conjugates were generated using Alexa Fluor 488 5-sulfo-dichlorophenol esters (ThermoFisher Scientific). Excess Alexa Fluor dye was removed from the conjugate preparations by gel filtration (ThermoFisher Scientific).
  • TF- positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • TF- positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • a final concentration of 20 nM of FVII-Fc conjugated to Alexa488 was added to the antibody cell mixture.
  • cells were washed, stained with a viability dye, and analyzed by flow cytometry.
  • the Alexa488 fluorescence data from viable cells was summarized using median fluorescence intensity.
  • Percentage of FVIIa binding (% FVIIa) in the presence of an anti-TF antibody titration relative to a no-antibody control is summarized in Table 9 and are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the FVIIa binding percentage ranges from about 76% to about 102% in the presence of antibodies of different concentrations from groups 25 and 43, including 25 A, 25A3, 25G, 25G1, 25G5, 25G9, 43B, 43B1, 43B7, 43D, 43D7, 43D8, 43E, and 43Ea.
  • This data indicates that anti-TF antibodies from groups 25 and 43 do not compete for binding to human TF with FVIIa.
  • This data also indicates that anti-TF antibodies from groups 25 and 43 have a human TF binding site that is distinct from the human TF binding site bound by FVIIa.
  • IL-8 and GM-CSF protein levels were measured as described previously in Hjortoe etal. , Blood , 2004, 103:3029-3037.
  • TF-positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • Leibovitz’s L-15 medium were incubated with an 8-point 1:2.5 titration starting at 100 nM of anti-TF antibody.
  • FVIIa NovoSeven RT, Novo Nordisk, Bagsvaerd, Denmark was added to the cells at a final concentration of 20 nM.
  • the concentration of IL8 with the anti-TF antibody titration are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety and the % IL8 at different antibodies concentrations are shown in Table 10.
  • the concentration of GM-CSF with the anti-TF antibody titration is shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety and the % IL8 at different antibodies concentrations are shown in Table 11.
  • IL8 concentrations were reduced by more than 75% in the presence of the anti-TF antibodies at concentrations greater than or equal to 6.4 nM.
  • GM-CSF concentrations were reduced by more than 60% in the presence of the anti-TF antibodies at concentrations greater than or equal to 6.4 nM.
  • Example 19 Antibody Competition Assay [00668] Alexa Fluor antibodies were generated using Alexa Fluor 488 5-sulfo- dichlorophenol esters (ThermoFisher Scientific). Excess Alexa Fluor dye was removed from the antibody dye conjugate preparations by gel filtration (ThermoFisher Scientific).
  • TF- positive A431 cells ATCC, Manassas, VA, USA
  • TF- positive A431 cells ATCC, Manassas, VA, USA
  • a final concentration of 20 nM of 25A conjugated to Alexa488 was added to the antibody cell mixture.
  • cells were washed, stained with a viability dye, and analyzed by flow cytometry.
  • the Alexa488 fluorescence data from viable cells was summarized using median fluorescence intensity.
  • TF- positive A431 cells ATCC, Manassas, VA, USA
  • a final concentration of 20 nM of 43Ea conjugated to Alexa488 was added to the antibody cell mixture.
  • % 25A binding and % 43Ea binding are shown in Table 12. Antibodies from group 25 and group 43 reduced the % 25 A binding and % 43Ea binding to less than 10%. [00672] This data indicates that antibodies of group 25 and antibodies of group 43 compete with each other for binding to human TF, and may bind the same or an overlapping epitope of human TF.
  • a cytotoxicity assay was conducted. Briefly, cells were plated in 384-well plates (Greiner Bio-One, Monroe, NC, USA) at 4xl0 3 cells per well in 40 m ⁇ of media. Antibodies and secondary anti-human Fc antibodies conjugated to the tubulin inhibitor mono-methyl auristatin F (MMAF) (Moradec, San Diego, CA, USA) were serially diluted starting at 5 and 30 nM, respectively. Plates were incubated for 3 days, followed by lysis in CellTiter-Glo (CTG) assay reagent (Promega, Madison, WI, USA).
  • CTG CellTiter-Glo
  • the TGA assay was performed using the calibrated-automated-thrombogram (CAT) instrument manufactured and distributed by STAGO.
  • the test method design was equivalent to a standard CAT assay measurement, except that the plasma source was normal pooled plasma (NPP) in citrate supplemented with com trypsin inhibitor (citrate/C TI).
  • NPP normal pooled plasma
  • citrate/C TI com trypsin inhibitor
  • the anti-TF antibodies were titrated at 0, 10, 50 and 100 nM and mixed with normal pooled plasma (NPP) collected in 11 mM citrate supplemented with 100 microgram/mL of com trypsin inhibitor (citrate/C TI).
  • Relipidated TF was added to a 96-well assay plate, followed by addition of the antibody /NPP mixture.
  • thrombin generation was initiated by the addition of calcium and the thrombin substrate.
  • the STAGO software was used to report the following parameters: Peak Ila (highest thrombin concentration generated [nM]); Lag Time (time to Ila generation [min]); ETP (endogenous thrombin potential, area under the curve [nM x min]); and ttPeak (time to Peak Ila [min]). Percent peak thrombin generation (% Peak Ila) and percent endogenous thrombin potential (% ETP) in the presence of each antibody relative to a no antibody plasma control on the same plate were also reported.
  • the % Peak Ila in the presence of titrations of anti-TF antibodies without antibody incubation prior to addition of calcium and thrombin substrate are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the % Peak Ila in the presence of titrations of anti-TF antibodies with 10 min antibody incubation prior to addition of calcium and thrombin substrate is shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • the M1593 antibody has a VH sequence of SEQ ID NO:821 and VL sequence of SEQ ID NO:822.
  • the % Peak Ila is 95% or greater in the presence of antibodies from group 25, including 25A, 25A3, and 25A5 without antibody pre-incubation.
  • the % Peak Ila is 100% or greater in the presence of antibodies from group 25, including 25A, 25A3, and 25A5 with 10 min antibody pre-incubation.
  • the % ETP is 99% or greater in the presence of the tested antibodies from group 25.
  • the % Peak Ila is greater than 50% but equal to or less than 96% in the presence of antibodies from group 43, including 43B1, 43D7, and 43Ea and anti-TF antibody M1593 without antibody pre-incubation.
  • the % Peak Ila is greater than 40% but equal to or less than 93% in the presence of antibodies from group 43, including 43B1, 43D7, and 43Ea and anti- TF antibody M1593 with 10 min antibody pre-incubation.
  • the % ETP is 92% or greater in the presence of the tested antibodies from group 43 and Ml 593 antibody.
  • Example 22 Binding Affinity Assay For Pig TF [00681] The ability of certain antibodies was tested for binding to pig TF.
  • a given anti-TF antibody was captured by an anti-human IgG antibody covalently coupled to a CM5 chip (GE Healthcare Bio-Sciences). Association between the anti-TF antibodies and a five-point three-fold titration of pig TF-His starting at 100 nM was measured for 180 to 240 sec. Subsequently, dissociation between the anti-TF antibody and TF-His was measured for 1800 sec.
  • Kinetic data was analyzed and fitted globally using a 1 : 1 binding model. The KD values of the indicated TF antibodies measured by the Biacore-based experiments are shown in Table 40.
  • anti-hTF antibodies from groups 25 and 43, 25G9 and 43D8 exhibit binding activity and cross-reactivity to pig TF.
  • Human TF-positive cancer cell lines A431 and MDA-MB-231 and Macaca mulatta TF-positive cell line RF/6A were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA) and were maintained as recommended.
  • E. coli- derived TF was expressed as a fusion between the OmpA signal sequence and TF ECD-His6, and purified by affinity and anion exchange chromatography.
  • the binding of anti-TF antibodies IF, 25A, 25A3, 25G1, 29E, 39A, 43B1, 43D7, 43Ea, and 54E to Expi293- or E. coli- derived TF was determined by protein ELISA studies. Plates coated with Expi293- or E. coli- derived TF-His were incubated with increasing concentrations of antibodies.
  • TGA assay was performed using the calibrated-automated-thrombogram (CAT) instrument manufactured and distributed by STAGO (Diagnostica Stago SAS, Asnieres sur Seine, France). See Samama etal. , Thromb Res , 2012, 129:e77-82, which is incorporated by reference in its entirety.
  • the test method design was equivalent to a standard CAT assay measurement, except that the plasma source was normal pooled plasma (NPP) collected in 11 mM citrate supplemented with 100 pg/mL of corn trypsin inhibitor (citrate/C TI).
  • NPP normal pooled plasma
  • citrate/C TI corn trypsin inhibitor
  • the anti-TF antibodies were titrated at 0, 10, 50 and 100 nM and mixed with NPP in citrate/CTI.
  • Relipidated TF was added to a 96-well assay plate, followed by addition of the antibody /NPP mixture. After a 10-min incubation or directly after combining the relipidated TF with antibody/NPP, thrombin generation was initiated by the addition of calcium and the thrombin substrate.
  • the STAGO software was used to report the following parameters: Peak Ila (highest thrombin concentration generated on the thrombin generation curve [nM]); Lag Time (time from assay start to the moment 10 nM of thrombin is formed [min]); ETP (endogenous thrombin potential, area under the curve [nM x min]); and ttPeak (time from assay start to Peak Ila [min]).
  • TGA thrombin generation assay
  • the Peak Ila, Lag Time, ETP, ttPeak, % Peak Ila, % ETP, and % ttPeak in the presence of each antibody selected from IF, 25 A, 25A3, 25G1, 29E, 39 A, 43B1, 43D7, 43Ea, 54E, TF-011, 5G9, and 10H10 with 10 min antibody incubation prior to addition of calcium and thrombin substrate are shown in Table 45.
  • the thrombin generation curve in the presence of 100 nM anti-TF antibody without antibody pre incubation are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • Group 43 antibodies and 10H10 exhibited mild interference with the peak Ila concentration: 100 nM of 43B1, 43D7, 43Ea and 10H10 reduced the peak Ila concentration by 33, 44, 13 and 34 %, respectively. In addition, 100 nM of 43B1, 43D7 and 10H10 showed at least a 29 % increase in ttPeak. However, the observed decline in peak Ila concentration and delayed ttPeak for group 43 antibodies and 10H10 did not result in more than a 10 % decline in the ETP. [00692] Similar results are shown in Table 45 under the conditions with 10 min antibody pre-incubation.
  • IF, 29E, 39A, 54E diminished the peak Ila concentration by 93, 72, 93 and 87 %, respectively.
  • 100 nM of 5G9 and TF-011 inhibited peak Ila concentration by 92 % and 91 %, respectively.
  • ETP endogenous thrombin generation
  • antibodies from group 25 did not decrease the peak Ila concentration or increase ttPeak.
  • Group 43 antibodies and 10H10 exhibited mild interference with the peak Ila concentration: 100 nM of 43B1, 43D7, 43Ea and 10H10 reduced the peak Ila concentration by 41, 56, 13 and 48 %, respectively.
  • 100 nM of 43B1, 43D7 and 10H10 showed at least a 33 % increase in ttPeak.
  • the observed decline in peak Ila concentration and delayed ttPeak for group 43 antibodies and 10H10 did not result in more than an 11 % decline in the ETP.
  • TF :FVIIa To evaluate the ability of TF :FVIIa to convert FX into FXa in the presence of human antibodies against TF, a cell-based FX conversion assay was conducted as described in Larsen etal, J Biol Chem, 2010, 285:19959-19966, which is incorporated by reference in its entirety. Briefly, 5xl0 4 MDA-MB-231 cells (ATCC, Manassas, VA, USA) were plated into tissue culture-treated black 96-well plates (Greiner Bio-One, Monroe, NC, USA) and cultured overnight.
  • FXa conversion percentages (% FXa) in the presence of an anti-TF antibody titration relative to a no antibody control are shown in international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety.
  • TF- positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • TF-positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • FVII-Fc conjugated to Alexa488 was added to the antibody-cell mixture at a final concentration of 20 nM.
  • cells were washed, stained with a viability dye, and analyzed by flow cytometry.
  • the Alexa488 fluorescence data from viable cells was summarized using median fluorescence intensity (MFI).
  • TF-011 and 5G9 inhibited FX conversion by 57-59 % and 67-70 % at concentrations of 25, 50, and 100 nM. 10H10 did not significantly inhibit FX conversion at these three concentrations.
  • TF-011 effectively competed with FVII, whereas 5G9 and 10H10 showed less than 25 % and 10 % competition at the highest concentration of antibody, respectively.
  • Example 27 Antibody Competition Assay [00700] Alexa Fluor antibodies were generated using Alexa Fluor 488 5-sulfo- dichlorophenol esters (ThermoFisher Scientific) following manufacturer’s protocol. Excess Alexa Fluor dye was removed from the antibody dye conjugate preparations by gel filtration (ThermoFisher Scientific).
  • TF-positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • TF-positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • a final concentration of 20 nM of 25A3 conjugated to Alexa488 was added to the antibody cell mixture.
  • cells were washed, stained with a viability dye, and analyzed by flow cytometry.
  • the Alexa488 fluorescence data from viable cells was summarized using median fluorescence intensity. 25A3 binding was summarized with %
  • TF-positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • TF-positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • a final concentration of 20 nM of 43D7 conjugated to Alexa488 was added to the antibody cell mixture.
  • cells were washed, stained with a viability dye, and analyzed by flow cytometry.
  • the Alexa488 fluorescence data from viable cells was summarized using median fluorescence intensity.
  • TF- positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • TF- positive MDA-MB-231 cells ATCC, Manassas, VA, USA
  • a final concentration of 20 nM of 39A conjugated to Alexa488 was added to the antibody cell mixture.
  • cells were washed, stained with a viability dye, and analyzed by flow cytometry.
  • the antibody competition assay results indicate that antibodies of groups 1, 29, 39, and 54, and TF-011 may bind to the same or an overlapping epitope of human TF or may affect the TF binding of each other through an allosteric mechanism
  • the chimeric TF construct mapping experiments as described elsewhere in this disclosure demonstrate that the antibodies of groups 29, 39 and 54 bind epitopes distinct from TF-01 l’s epitope.
  • Example 28 Anti-TF Antibody Internalization
  • a cytotoxicity assay was conducted as described in Liao-Chan et al. , PLoS One, 2015, 10:e0124708, which is incorporated by reference in its entirety. Briefly, cells were plated in 384-well plates (Greiner Bio-One, Monroe, NC, USA) at 4xl0 3 cells per well in 40 m ⁇ of media. Antibodies and an anti-human Fc Fab conjugated to the tubulin inhibitor mono-methyl auristatin F (MMAF) (Moradec, San Diego, CA, USA) were serially diluted starting at 5 and 30 nM, respectively.
  • MMAF mono-methyl auristatin F
  • the anti-human Fc Fab conjugated to MMAF consisted of a polyclonal antibody specific to the Fc region of human IgGs with a DAR of 1.2 to 1.5. Plates were incubated for 3 days, followed by lysis in CellTiter-Glo (CTG) assay reagent (Promega, Madison, WI, USA). CTG luminescence was measured on an Envision plate reader and the mean and standard deviation of 4 replicates graphed in Prism (GraphPad, La Jolla, CA, USA). For each anti-TF antibody, the IC50 and its associated 95% confidence interval were calculated in Prism using a 4- parameter binding model.
  • Fab:MMAF binds the Fc region of the TF-specific antibodies, cellular uptake of these complexes can trigger cell death. While the TF-specific antibodies alone had no impact on cell viability in three-day cultures of TF-positive A431 cells, the TF-specific antibodies in complex with Fab:MMAF showed dose-dependent cell killing with IC50 values ranging between 0.07 and 0.14 nM. ( See international PCT application PCT/US2019/012427 and US utility application number 16/959,652, incorporated herein by reference in their entirety).

Landscapes

  • Health & Medical Sciences (AREA)
  • Ophthalmology & Optometry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des anticorps qui se lient spécifiquement au facteur tissulaire (TF) humain et qui sont utiles pour le traitement de maladies oculaires. L'invention concerne également des méthodes de traitement de sujets atteints de maladies oculaires par administration des anticorps anti-TF.
PCT/US2021/041191 2020-07-10 2021-07-10 Traitement de maladie oculaire à l'aide d'anticorps anti-facteur tissulaire WO2022011323A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
TW110125559A TW202216197A (zh) 2020-07-10 2021-07-12 使用抗組織因子抗體之眼部疾病治療

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063050643P 2020-07-10 2020-07-10
US63/050,643 2020-07-10
US202163182696P 2021-04-30 2021-04-30
US63/182,696 2021-04-30

Publications (1)

Publication Number Publication Date
WO2022011323A1 true WO2022011323A1 (fr) 2022-01-13

Family

ID=79552116

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/041191 WO2022011323A1 (fr) 2020-07-10 2021-07-10 Traitement de maladie oculaire à l'aide d'anticorps anti-facteur tissulaire

Country Status (2)

Country Link
TW (1) TW202216197A (fr)
WO (1) WO2022011323A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120237528A1 (en) * 2011-03-15 2012-09-20 Juan Carlos Almagro Human Tissue Factor Antibody and Uses Thereof
US20130052672A1 (en) * 2004-03-31 2013-02-28 Baxter Healthcare S.A. Kit for measuring the thrombin generation in a sample of a sample of a patient's blood or plasma

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130052672A1 (en) * 2004-03-31 2013-02-28 Baxter Healthcare S.A. Kit for measuring the thrombin generation in a sample of a sample of a patient's blood or plasma
US20120237528A1 (en) * 2011-03-15 2012-09-20 Juan Carlos Almagro Human Tissue Factor Antibody and Uses Thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAUDREAULT JACQUES, TEA GUNDE; HEATHER S. FLOYD; JOEL ELLIS; JULIA TIETZ; DANIELA BINGGELI; BARBARA KELLER; ANNE SCHMIDT; DOMINIK : "Preclinical Pharmacology and Safety of ESBA1008, a Single- chain Antibody Fragment, Investigated as Potential Treatment for Age Related Macular Degeneration", INVEST. OPHTHALMOL. VIS. SCI. ARVO ANNUAL MEETING ABSTRAC, vol. 53, 1 March 2012 (2012-03-01), pages 3025, XP055898427 *

Also Published As

Publication number Publication date
TW202216197A (zh) 2022-05-01

Similar Documents

Publication Publication Date Title
US11753480B2 (en) Anti-SIRP-alpha antibodies and related methods
US11014983B2 (en) Anti-Tim-3 antibodies, compositions comprising anti-Tim-3 antibodies and methods of making and using anti-Tim-3 antibodies
US11447566B2 (en) Anti-tissue factor antibodies, antibody-drug conjugates, and related methods
US11186644B2 (en) Anti-neuropilin antigen-binding proteins and methods of use thereof
JP2016025863A (ja) Ｔｏｌｌ受容体２へのヒト化抗体およびその使用
US20210269542A1 (en) Anti-gitr antigen-binding proteins and methods of use thereof
AU2013352127A1 (en) Anti-VEGF antibodies and their uses
US11993658B2 (en) Anti-BCMA antibodies and treatment methods
JP2023517345A (ja) 抗インターロイキン－３３抗体及びその使用
US20220106401A1 (en) ANTI-EpCAM ANTIBODIES, COMPOSITIONS COMPRISING ANTI-EpCAM ANTIBODIES AND METHODS OF MAKING AND USING ANTI-EpCAM ANTIBODIES
KR20230048439A (ko) 항-par-2 항체 및 이의 사용 방법
US20240166765A1 (en) Inflammatory disease treatment using anti-tissue factor antibodies
WO2022011323A1 (fr) Traitement de maladie oculaire à l'aide d'anticorps anti-facteur tissulaire
RU2795625C2 (ru) Антигенсвязывающие белки против gitr и способы их применения
WO2023137399A2 (fr) Traitement de maladies inflammatoires faisant appel à des anticorps anti-facteur tissulaire
TW202402810A (zh) 與ｖｅｇｆ—ａ及ｉｌ６結合之抗體及其使用方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21837826

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21837826

Country of ref document: EP

Kind code of ref document: A1