Antibodies that bind to human fibrin or fibrinogen gamma C domain and methods of use - Patents.com

Abstract

Described herein are novel and improved antibodies that bind to human fibrin or fibrinogen γC domain, and methods of using the same. In certain aspects, described herein are methods of inhibiting microglial activation. In certain aspects, described herein are pharmaceutical compositions that include antibodies that bind to fibrin or fibrinogen γC domain. In certain aspects, the antibodies and methods described herein are used for the treatment of degenerative neuropathies involving inflammatory demyelination. TIFF2024525188000039.tif92170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 212,414, filed June 18, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing Not applicable. [Background technology]

[0003] background Degenerative neurological disorders such as multiple sclerosis (MS) can involve inflammatory demyelination and autoimmune responses. Microglia, particularly perivascular microglia, are thought to be necessary for the maintenance as well as the development of inflammatory demyelination in central nervous system (CNS) autoimmune diseases. Microglial activation contributes to the death of both neurons and oligodendrocytes through the release of cytokines and nitric oxide. In MS, the inflammatory process is associated with the destruction of the myelin sheath and may be accompanied by axonal damage that can lead to permanent functional impairments such as paralysis and vision loss. Resident microglia are involved in demyelination through their ability to phagocytose myelin and secrete pro-inflammatory cytokines.

[0004] In MS lesions, perivascular activation of microglia colocalizes with areas of blood-brain barrier (BBB) ​​disruption, and in vivo imaging studies have shown that BBB disruption causes immediate and localized activation of microglia. One of the earliest events associated with BBB disruption in MS is leakage of the blood protein fibrinogen in the nervous system, which leads to perivascular fibrin deposition. Fibrinogen is absent in the healthy CNS and only leaks into the brain after BBB disruption, thus acting as an environmental "danger" signal. Once fibrinogen is converted to fibrin, the CD11b / CD18 integrin receptor (also known as Mac-1, aMfl 2, and complement receptor 3) binds to fibrin and induces microglial activation leading to inflammatory demyelination. CD11b is the alpha chain of the receptor that regulates the phagocytosis of myelin during inflammatory demyelination. Immobilized fibrinogen and insoluble fibrin, but not soluble fibrinogen, have been identified as the physiological, high-affinity ligands of Mac-1.

[0005] The γ377-395 epitope of the fibrin or fibrinogen γC domain is the binding epitope for CDIIb of fibrin. 377~395 The peptide functions as an inhibitor of microglial activation by blocking the binding of fibrin to Mac-1. Fibrin binds to platelet integrin α IIb Because CD11b mediates blood clotting by binding to the β3 receptor via a distinct epitope, therapeutic agents (including antibodies) that block the CD11b binding epitope to fibrin can reduce the damaging effects of fibrin in the nervous system without affecting its beneficial effects in blood clotting. Therefore, safe and effective antibodies that inhibit fibrin-induced microglial activation without affecting its beneficial effects in blood clotting are needed as therapeutic agents for degenerative neuropathies involving inflammatory demyelination. Summary of the Invention

[0006] overview In certain aspects, described herein is an isolated antibody that binds to a human fibrin or fibrinogen γC domain, comprising a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises the sequence set forth in SEQ ID NOs: 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, or 229; and CDR-H2 comprises the sequence set forth in SEQ ID NOs: 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, or 230; CDR-H3 comprises the sequence set forth in SEQ ID NO: 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 159, 171, 183, 195, 207, 219, or 231; CDR-L1 comprises the sequence set forth in SEQ ID NO: CDR-L2 comprises the sequence set forth in SEQ ID NO: 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 161, 173, 185, 197, 209, 221, or 233; and CDR-L3 comprises the sequence set forth in SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, or 234.

[0007] In certain embodiments, the antibody comprises a VH sequence selected from the sequence set forth in one of SEQ ID NOs: 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 163, 175, 187, 199, 211, 223, or 235. In certain embodiments, the antibody comprises a VL sequence selected from the sequence set forth in SEQ ID NOs: 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 166, 178, 190, 202, 214, 226, or 238.

[0008] In certain embodiments, the antibody comprises a VH sequence selected from the sequences set forth in one of SEQ ID NO:7, and a VL sequence set forth in SEQ ID NO:10. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:19 and a VL sequence set forth in SEQ ID NO:22. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:31 and a VL sequence set forth in SEQ ID NO:34. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:43 and a VL sequence set forth in SEQ ID NO:46. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:55 and a VL sequence set forth in SEQ ID NO:58. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:67 and a VL sequence set forth in SEQ ID NO:70. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:79 and a VL sequence set forth in SEQ ID NO:82. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:91 and a VL sequence set forth in SEQ ID NO:94. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:103 and a VL sequence set forth in SEQ ID NO:106. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:115 and a VL sequence set forth in SEQ ID NO:118. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:127 and a VL sequence set forth in SEQ ID NO:130. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:139 and a VL sequence set forth in SEQ ID NO:142. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:151 and a VL sequence set forth in SEQ ID NO:154. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in SEQ ID NO: 166. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 175 and a VL sequence set forth in SEQ ID NO: 178.In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 187 and a VL sequence set forth in SEQ ID NO: 190. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 199 and a VL sequence set forth in SEQ ID NO: 202. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 211 and a VL sequence set forth in SEQ ID NO: 214. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 223 and a VL sequence set forth in SEQ ID NO: 226. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 235 and a VL sequence set forth in SEQ ID NO: 238.

[0009] In certain embodiments, the antibody comprises a humanized antibody, a human antibody, or a chimeric antibody. In certain embodiments, the antibody comprises a humanized antibody. In certain embodiments, the antibody comprises a heavy chain human constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. In certain embodiments, the human Fc region comprises a human heavy chain constant region of a class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the human Fc region comprises a wild type human IgG1 Fc. In certain embodiments, the human Fc domain comprises a sequence set forth in SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, or 236.

[0010] In certain embodiments, the heavy chain comprises a constant heavy chain sequence described by SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, or 236. In certain embodiments, the light chain comprises a constant light chain sequence described by SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153, 165, 177, 189, 201, 213, 225, or 237.

[0011] In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:7, and a VL sequence set forth in SEQ ID NO:10; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:19, and a VL sequence set forth in SEQ ID NO:22; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:31, and a VL sequence set forth in SEQ ID NO:34; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:43, and a VL sequence set forth in SEQ ID NO:46; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:55, and a VL sequence set forth in SEQ ID NO:58; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:67, and a VL sequence set forth in SEQ ID NO:70; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:79, and a VL sequence set forth in SEQ ID NO:82; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO:91, and a VL sequence set forth in SEQ ID NO:94; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 103, and a VL sequence set forth in SEQ ID NO: 106; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 115, and a VL sequence set forth in SEQ ID NO: 118; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 127, and a VL sequence set forth in SEQ ID NO: 130; and the human Fc region comprises wild-type human IgG1 Fc.In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 139, and a VL sequence set forth in SEQ ID NO: 142; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 151, and a VL sequence set forth in SEQ ID NO: 154; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 163, and a VL sequence set forth in SEQ ID NO: 166; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 175, and a VL sequence set forth in SEQ ID NO: 178; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 187, and a VL sequence set forth in SEQ ID NO: 190; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 199, and a VL sequence set forth in SEQ ID NO: 202; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 211, and a VL sequence set forth in SEQ ID NO: 214; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the antibody comprises a VH sequence set forth in SEQ ID NO: 223, and a VL sequence set forth in SEQ ID NO: 226; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the antibody comprises the VH sequence set forth in SEQ ID NO:235, and the VL sequence set forth in SEQ ID NO:238; and the human Fc region comprises wild-type human IgG1 Fc.

[0012] In certain embodiments, the Fc region comprises one or more amino acid substitutions, wherein the one or more substitutions result in increased antibody half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more substitutions. In certain embodiments, the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

[0013] In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody binds to the γ377-395 epitope of the fibrin or fibrinogen γC domain. In certain embodiments, the antibody has a specific binding affinity of about 1, 2, 3, 4, 5, 6, 7, or 8×10 as measured by a surface plasmon resonance (SPR) single cycle kinetics (SCK) assay. -5 K below M D In certain embodiments, the antibody binds to a peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241, and 249-253. In certain embodiments, the antibody binds to a peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241, and 249-253, and has a binding affinity of about 8×10 as measured by a surface plasmon resonance (SPR) single cycle kinetics (SCK) assay. -5 K below M D The antibody binds to a peptide comprising the sequence of the γ377-395 epitope of the human fibrin or fibrinogen γC domain at a specific concentration. In certain embodiments, the antibody inhibits Mac-1 binding to the fibrin or fibrinogen γC domain. In certain embodiments, the antibody exhibits inhibition of microglial adhesion to the fibrin or fibrinogen γC domain.

[0014] In certain aspects, described herein is an isolated antibody according to any one of the preceding claims for use in the treatment of a degenerative disorder of the nervous system.

[0015] In certain aspects, described herein is an isolated polynucleotide or set of polynucleotides, optionally cDNA, encoding an antibody, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof, according to any of the preceding claims.

[0016] In certain aspects, described herein is a vector or set of vectors that comprises a polynucleotide or set of polynucleotides described herein.

[0017] In certain aspects, described herein is a host cell comprising a polynucleotide or set of polynucleotides or a vector or set of vectors described herein.

[0018] In certain aspects, described herein are methods of producing an antibody, the methods comprising expressing the antibody in a host cell described herein and isolating the expressed antibody.

[0019] In certain aspects, described herein are pharmaceutical compositions comprising an antibody described herein and a pharma- ceutically acceptable excipient.

[0020] In certain aspects, described herein are kits comprising any of the compositions described herein or any of the pharmaceutical compositions described herein and instructions for use.

[0021] In certain aspects, described herein are methods for treating a degenerative disorder of the nervous system, comprising administering to a mammalian subject a therapeutically effective amount of an antibody described herein or a pharmaceutical composition described herein. In certain embodiments, the degenerative disorder of the nervous system is selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0022] In certain aspects, described herein are methods for treating pathologies associated with Mac-1 binding to fibrin or fibrinogen, the methods comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein.

[0023] In certain aspects, described herein are methods of inhibiting microglial activation, the methods comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein.

[0024] In certain aspects, described herein are methods of preventing degenerative disorders of the nervous system, the methods comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein.

[0025] In certain aspects, described herein are methods of treating colitis in a subject in need thereof, comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein. In certain aspects, described herein are methods of preventing colitis in a subject in need thereof, comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein. In certain aspects, described herein are methods of treating an ocular inflammatory condition in a subject in need thereof, comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein. In certain aspects, described herein are methods of preventing an ocular inflammatory condition in a subject in need thereof, comprising administering to a mammalian subject a therapeutically effective amount of an antibody or pharmaceutical composition described herein. In certain embodiments, the ocular inflammatory condition is uveitis.

[0026] In certain aspects, described herein are isolated antibodies that bind to human fibrin or fibrinogen γC domain, wherein the antibody binds to human fibrin at any one of amino acid residues Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421. In certain embodiments, wherein the antibody binds to human fibrin at at least 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of amino acid residues Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421.

[0027] In certain embodiments, described herein are antibodies comprising a VH region that comprises a paratope comprising any one of amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Leu 50, Asp 52, Asp 54, Tyr 56, Ala 93, Ser 94, Ser 95, Lys 96 or Asp 96, Pro 97 or Ala 97, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that includes a paratope that includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Leu 50, Asp 52, Asp 54, Tyr 56, Ala 93, Ser 94, Ser 95, Lys 96 or Asp 96, Pro 97 or Ala 97, Gly 101, Gly 102, and Trp 103. In certain embodiments, the antibody comprises a VH region that includes a paratope that includes amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that includes a paratope that includes amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ala 93, Ser 94, Lys 96, Pro 97, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that comprises a paratope comprising amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Asp 52, Asp 54, Tyr 56, Ser 94, Ser 95, Asp 96, Ala 97, Gly 101, Gly102, and Trp 103.

[0028] In certain embodiments, described herein is an isolated antibody comprising a VL region that includes a paratope that includes any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gln 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that includes a paratope that includes at least 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gln 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that comprises a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gln 50, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that comprises a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gln 50, Asn 91, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that comprises a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gln 50, Ala 91, Leu 92, Leu 94, and Leu 96.

[0029] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief description of the drawings]

[0030] [Figure 1] FIG. 13 is a plot from a FACS analysis of an antibody library that binds to N-terminally biotinylated fibrin P2 gamma peptide showing the results of three rounds of antibody affinity maturation with one library created from one of three parent humanized antibodies (clone 56657). [Figure 2A]Graph showing the results of an enzyme-linked immunosorbent assay (ELISA) performed with the indicated humanized antibody variants and P2 peptide. A = clone 60143; B = clone 61278; C = clone 61278 (duplicate); D = parent antibody. [Figure 2B] Graph showing the results of enzyme-linked immunosorbent assay (ELISA) performed on the indicated humanized antibody variants and FGG (fibrinogen). A = clone 60143; B = clone 61278; C = clone 61278 (duplicate); D = parent antibody. [Figure 2C] Graph showing the results of enzyme-linked immunosorbent assay (ELISA) performed with the indicated humanized antibody variants and fibrin. A = clone 60143; B = clone 61278; C = clone 61278 (duplicate); D = parent antibody. [Figure 3A] 3A and 3B are graphs showing the results of an assay demonstrating the clot lysis times of samples in the presence of variant humanized antibodies. A = clone 56666; B = clone 56657; C = clone 60143; D = clone 60181; E = clone 60175; F = clone 60163; G = clone 60173; H = clone 60184; I = clone 60141; J = clone 60179; K = clone 60140; L = clone 60183. [Figure 3B] See legend to Figure 3A. [Figure 4A-1] 1 is a graph showing the results of a ForteBio KD measurement as described herein on a solution of N-terminally biotinylated fibrin P2 gamma peptide (100 nM) conjugated to IgG or N-terminally biotinylated fibrin P2 gamma peptide (100 nM) conjugated to FAB (monovalent).The antibody clones tested are indicated. [Figure 4A-2] See legend to Figure 4A-1. [Figure 4B-1] See legend to Figure 4A-1. [Figure 4B-2] See legend to Figure 4A-1. [Figure 4C] See legend to Figure 4A-1. [Figure 4D-1] See legend to Figure 4A-1. [Figure 4D-2] See legend to Figure 4A-1. [Diagram 5] 13 is a graph showing the results of octet Fab binding to N-terminally biotinylated fibrin P2 gamma peptide on a SA sensor using a 100 mM Fab solution.The antibody clones tested are shown. [Figure 6] Graph showing staining of brain tissue sections from a fibrinogen-induced encephalomyelitis (FIE) mouse model injected IV with artificial cerebrospinal fluid (acsf), fibrinogen only, or fibrinogen and the indicated antibody clones at either 10 mg / Kg ("10") or 30 mg / Kg ("30"). Sections were stained with either Iba-1 (left) (a microglia marker at 1:750 dilution) or Mac-2 (right) (a macrophage infiltration marker at 1:750 dilution). [Figure 7] Graph showing clinical scores of mice in an experimental autoimmune encephalomyelitis (EAE) model subjected to prophylactic injections of PBS only, IgG1 only, antibody clone 60143, antibody clone 61278, or dexamethasone. Antibodies were injected by intraperitoneal injection at 5 mg / kg every 3 days. [Figure 8] Graphs of disease onset (left) and paralysis rate (right) are shown for mice in an experimental autoimmune encephalomyelitis (EAE) model subjected to prophylactic injections of PBS only, IgG1 only, antibody clone 60143, antibody clone 61278, or dexamethasone. Antibodies were injected by intraperitoneal injection at 5 mg / kg every 3 days. [Figure 9] Graph showing clinical scores in mice of an experimental autoimmune encephalomyelitis (EAE) model subjected to prophylactic injections of PBS alone, dexamethasone, antibody clone 60143 (left) or control antibody human IgG1 (right). [Figure 10]Graph showing percentage of paralyzed mice (complete paralysis - left) or paralyzed mice (partial hind limb paralysis - right) in an experimental autoimmune encephalomyelitis (EAE) model subjected to prophylactic injections of PBS only, dexamethasone (dexa), antibody clone 6043 (indicated concentrations; 5=5 mg / kg, 1=1 mg / kg and 0.2=0.2 mg / kg) or control antibody human IgG1 (5 mg / kg). [Figure 11] Schematic showing the workflow of gene expression assays of BMDM cell lines. [Figure 12] Graph showing interleukin (IL)-12b expression in BMDM cells following incubation with either 50 ug / mL (left) or 10 ug / ml (right) antibody along with fibrinogen and fibrin, IgG1, antibody clone 60143, and antibody clone 61278. [Figure 13] Graph showing interleukin (IL)-12b expression in BMDM cells following incubation with the indicated concentrations of fibrinogen and antibody clone 61278 (left) or antibody clone 60143 (right). [Figure 14] FIG. 1 is a graph showing reduction in physiological symptoms of colitis in animals injected intravenously with 5 mg / kg or 30 mg / kg of antibody clone 60143 or isotype control antibody human IgG1 in a dextran sulfate sodium (DSS)-induced colitis mouse model. [Figure 15] Figures 15A and 15B show the uptake (%ID) of [I]SIB-60143 and [I]SIB-61278 injected at 10 mg / kg and 30 mg / kg in mouse blood (A) and plasma (B), corrected for theoretical blood and plasma volumes. [Figure 16A] Figures 16A, 16B, 16C, and 16D show the ex vivo biodistribution over time in mice of [I]SIB-60143 (A and B) and [I]SIB-61278 (C and D) injected at 10 mg / kg (A and C) and 30 mg / kg (B and D). [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17] Schematic depicting the crystal structures of the Fabs of antibody clone 60143 (ADI60143) and antibody clone 61278 (ADI61278) in complex with the P2 peptide (co-crystal - right). The structure of fibrinogen (FGG) and the location of the P2 peptide are also shown (bottom left). [Figure 18] Schematic diagram depicting the superposition of the Fabs of antibody clone 60143 (ADI60143) and antibody clone 61278 (ADI61278) in complex with P2 peptide. [Figure 19] 2 is a graph depicting the binding affinity of antibody clone 60143 (ADI60143) and antibody clone 61278 (ADI61278). The binding of ADI-60143 and ADI-61278 Fab to the P2 peptide was determined using Octet RED384 (FIGS. 21 and 22), and similar binding profiles were observed for the three P2 peptides from different species. However, the three extended P2 peptides did not bind well to ADI-60143 Fab (FIG. 22). [Figure 20] Figure 20A is a graph depicting the binding of ADI-60143 Fab to rat, mouse or human P2 peptides as determined by ELISA. Figure 20B is a graph depicting the binding of ADI-60143 IgG to rat, mouse or human P2 peptides as determined by ELISA. [Figure 21] 1 is a graph depicting binding of ADI-60143 IgG to rat, mouse or human extended P2 peptide as determined by ELISA. [Figure 22]Figure 22A is a schematic diagram depicting the experimental protocol carried out to determine microglial activation and macrophage recruitment in a fibrin-induced encephalitis (FIE) mouse model. Figure 22B is a graph depicting the area percent of Iba-1 positive staining to determine microglial activation in brain tissue sections from FIE mice administered acsf (artificial cerebrospinal fluid), fibrinogen, IgG isotype control (30 mg / Kg), parent humanized antibody clone THN227 (not affinity matured) (10 or 30 mg / Kg), and affinity matured antibody clone ADI-60143 (10 or 30 mg / Kg). FIG. 22C is a graph depicting the area percent of Mac-2 positive staining to determine macrophage infiltration in brain tissue sections from FIE mice administered acsf (artificial cerebrospinal fluid), fibrinogen, IgG isotype control (30 mg / Kg), parental humanized antibody clone THN227 (not affinity matured) (10 or 30 mg / Kg), and affinity matured antibody clone ADI-60143 (10 or 30 mg / Kg). [Figure 23] 13 is an image showing tissue sections from the spinal cord of healthy and EAE mice stained for ADI-60143. [Figure 24] Figure 24A is a graph depicting demylination as determined by MBP staining of tissue sections from the spinal cord of EAE mice administered PBS, dexamethasone (DEXA), and 0.2, 1, or 5 mg / Kg of antibody clone ADI-60143. Figure 24B is a graph depicting the percentage of EAE mice showing complete hind limb paralysis administered PBS, dexamethasone (DEXA), and 0.2, 1, or 5 mg / Kg of antibody clone ADI-60143. [Diagram 25] Graph depicting clinical scores of EAE mice administered isotype control, antibody clone ADI-60143 IgG, and antibody clone ADI-60143 with an Fc stabilizing LALA mutation, as well as naive mice without EAE induction. [Figure 26]Figure 26A is a graph depicting the average number of total inflammatory foci / spinal cord tissue section for EAE mice administered isotype control, antibody clone ADI-60143 IgG, and antibody clone ADI-60143 with Fc stabilizing LALA mutation, as well as naive mice without EAE induction. Figure 26B is a graph depicting the CD11b+ area percent per tissue section for EAE mice administered isotype control, antibody clone ADI-60143 IgG, and antibody clone ADI-60143 with Fc stabilizing LALA mutation, as well as naive mice without EAE induction. [Figure 27] Graph depicting uveitis clinical scores in rats administered intravitreally with isotype control, murine antibody clone ADI-60143 with Fc stabilizing LALA mutation (low dose = 10 ug / eye; high dose = 50 ug / eye), positive control FTY-720 (administered by oral gavage at a dose of 0.3 mg / kg) and naive mice without EAE induction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description definition Unless otherwise defined, all technical terms, notations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and conditions, unless otherwise noted.

[0032] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0033] It is understood that the aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0034] For all compositions described herein and all methods of using the compositions described herein, the composition can include the recited components or steps or can "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" recited components, the composition can include the recited components and other components that do not substantially affect the condition being treated, but do not include any other components that do not substantially affect the condition being treated, other than the components explicitly recited, or when the composition includes additional components other than the recited components that substantially affect the condition being treated, the composition does not include the additional components in a concentration or amount sufficient to substantially affect the condition being treated. When a method is described as "consisting essentially of" recited steps, the method can include the recited steps and other steps that do not substantially affect the condition being treated, but the method does not include any other steps that substantially affect the condition being treated, other than the steps explicitly recited. As a non-limiting example, when a composition is described as "consisting essentially of" components, the composition can further include any amount of a pharma- ceutically acceptable carrier, vehicle, or diluent, and such other components that do not substantially affect the condition being treated.

[0035] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0036] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which 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 include the primary transformed or transfected cells and their derived progeny, respectively. Such antibodies may not be completely identical in nucleic acid content to the parent cell and may contain mutations. "Recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells.

[0037] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocotyledons, dicotyledons, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0038] As used herein, the term "prokaryote" refers to prokaryotic organisms. For example, non-eukaryotes may be from the Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or Archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, e.g., Haloferax volcanii, etc.) phylogenetic domain. volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

[0039] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic compound, such as an anti-fibrin (FIBRIN) antibody, administered to an individual, either as a single dose or as part of a series of doses, that is effective to produce or contribute to a desired therapeutic effect, either alone or in combination with another therapy. Examples of desired therapeutic effects are enhanced immune response, slowing or delaying tumor development; stabilization of disease; amelioration of one or more symptoms. An effective amount can be given in one or multiple doses.

[0040] The term "treat" (and variations thereof, such as "treat" or "treatment") refers to a clinical intervention that seeks to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed during the course of clinical pathology. Desirable effects of treatment include inhibiting recurrence of disease, alleviating symptoms, reducing direct or indirect pathological consequences of disease, inhibiting metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and remission or improving prognosis.

[0041] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate an immune response in a subject.

[0042] As used herein, the term "subject" or "individual" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, 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 the antibodies provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0043] The term "in vitro" refers to processes carried out within living cells that are grown apart from an organism, for example, in tissue culture.

[0044] The term "in vivo" refers to a process that takes place within a living organism.

[0045] The term "package insert" is used to refer to instructions customarily included in a commercial package (e.g., a kit) of a therapeutic or diagnostic agent that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic or diagnostic agent.

[0046] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective in treating a subject, and that does not contain additional ingredients that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0047] The terms "co-administration," "co-administering," and "in combination with" include administration of two or more therapeutic agents simultaneously, concurrently, or sequentially without specific time limitations. In one embodiment, the agents are present in a cell or in a subject's body at the same time or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to administration of a second therapeutic agent.

[0048] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0049] The terms "increase" and "activate" refer to a 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 increase in the recited variable.

[0050] The terms "reduce" and "inhibit" refer to a 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 decrease in the recited variable.

[0051] The term "about" connotes and encompasses the indicated value and a range above and below that value. In certain embodiments, the term "about" refers to the indicated value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "about" refers to the indicated value ± one standard deviation of that value.

[0052] The term "agonize" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to a receptor and agonizes the receptor.

[0053] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0054] For any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0055] The term "optionally," when used consecutively, means the inclusion of one to all of the listed combinations, and contemplates all subcombinations.

[0056] The term "amino acid" refers to the 20 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 (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; 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).

[0057] The term "affinity" refers to the strength of the sum 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). Unless otherwise indicated, as used herein, "affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope).

[0058] As used herein, "k d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is k off Also called value.

[0059] As used herein, "k a " " -1 ×sec -1 The term k ) refers to the binding rate constant of a particular antibody-antigen interaction. This value is k on Also called value.

[0060] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. D = k d / k a In some embodiments, the affinity of an antibody is determined by the K D For clarity, as known in the art, K D A smaller value indicates a higher affinity interaction, and K D Higher values ​​indicate lower affinity interactions.

[0061] As used herein, "K A " " -1 The term K ) refers to the binding equilibrium constant of a particular antibody-antigen interaction. A = k a / k d .

[0062] The term "antibody" is used herein in its broadest sense and includes certain immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0063] A "fibrin antibody", "anti-fibrin antibody" or "fibrin-specific antibody" as provided herein is an antibody that specifically binds to the antigen fibrin. In some embodiments, the antibody binds to the extracellular domain of fibrin. In certain embodiments, the fibrin antibody provided herein binds to an epitope of fibrin that is conserved between or within fibrin proteins from different species.

[0064] The term "epitope" refers to a portion of an antigen that specifically binds to an antibody.

[0065] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops").

[0066] The term "antigen-binding domain" means a part of an antibody capable of specifically binding to an antigen or epitope.

[0067] The term "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, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0068] The term "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0069] The term "humanized antibody" refers to a protein having a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions such that the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response when administered to a human subject, as compared to the non-human species antibody.

[0070] The term "multispecific antibody" refers to an antibody that comprises two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes.

[0071] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is a natural IgG molecule that is bivalent (i.e., has two antigen-binding domains) but recognizes the same epitope at each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.

[0072] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope, except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present in small amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, 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 modified, for example, to improve its 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.

[0073] The term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in a single chain Fab molecule. As described in more detail herein, an scFv has a variable domain of the light chain (VL) connected from its C-terminus to the N-terminus of the variable domain of the heavy chain (VH) by a polypeptide chain. Alternatively, an scFv comprises a polypeptide chain in which the C-terminus of the VH is connected to the N-terminus of the VL by a polypeptide chain.

[0074] The "Fab fragment" (also called fragment antigen-binding) contains the variable domains VL and VH of the light and heavy chains, respectively, as well as the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The variable domains contain the complementarity determining loops (CDRs, also called hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.

[0075] An "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0076] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0077] "Single-chain Fv" or "sFv" or "scFv" comprises the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0078] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. The Fc domain can 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 can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0079] The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et 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. sdabs are fairly stable and easy to express as fusion partners with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Appl. Microbiol Biotechnol. 77(1): 13-22).

[0080] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain including an Fc region. For example, when used to refer to an IgG molecule, a "full length antibody" is an antibody that contains two heavy chains and two light chains.

[0081] The term "antibody fragment" refers to an antibody that contains a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0082] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions.

[0083] The term "substantially purified" refers to constructs described herein, or variants thereof, which may be substantially or essentially free from components normally associated with or interacting with the protein as found in its naturally occurring environment, i.e., in a native cell, or, in the case of recombinantly produced heteromultimers, in a host cell, and in certain embodiments are substantially free of cellular material, including preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein.

[0084] The term percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., using publicly available computer software such as BLAST, BLASTP, BLASTN, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software or other algorithms available to those of skill in the art) or by visual inspection. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or can exist over the entire length of the two sequences being compared.

[0085] In sequence comparison, one sequence usually serves as reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinate is designated if necessary, and sequence algorithm program parameter is designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on designated program parameter.

[0086] Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0087] It is understood that the ranges recited herein are shorthand for all values ​​within the range, including the recited end points.For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0088] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0089] Antifibrin antibodies antibody structure The present application provides antibodies and compositions comprising the antibodies that bind to fibrin proteins.

[0090] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, several of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

[0091] An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50-70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The IgG1 heavy chain is composed of the VH, CH1, CH2 and CH3 domains, respectively, in the N-terminal to C-terminal direction. The light chain is composed of the VL and CL domains in the N-terminal to C-terminal direction. The IgG1 heavy chain includes a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin construct includes at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to a therapeutic polypeptide. In some embodiments, the immunoglobulin domain found in the antibody provided herein is from or derived from an immunoglobulin-based construct, such as a diabody or nanobody.In certain embodiments, the immunoglobulin construct described herein comprises at least one immunoglobulin domain from a heavy chain antibody, such as a camelid antibody.In certain embodiments, the immunoglobulin construct provided herein comprises at least one immunoglobulin domain from a mammalian antibody, such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody, or any chimeric antibody.

[0092] In some embodiments, the antibody provided herein comprises a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0093] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0094] Typically, a native four-chain antibody contains six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and / or complementarity determining regions (CDRs), the latter of which are most highly sequence variable and / or involved in antigen recognition. Except for CDR1 in VH, CDRs typically contain amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable regions that form the antigen-binding region. This particular region has been described by Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983), and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is intended that the application of either definition to refer to the CDR of an antibody or its variants is within the scope of the term as defined and used herein.The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues constitute a particular CDR given the variable region amino acid sequence of an antibody.

[0095] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of several known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme); each of which is incorporated by reference in its entirety.

[0096] Table A shows the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0097] CDRs can be assigned using antibody numbering software such as Abnum, available at www.bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, which is incorporated by reference in its entirety.

[0098] (Table A) Residues in the CDRs according to the Kabat and Chothia numbering scheme. TIFF2024525188000002.tif46158 * The C-terminus of CDR-H1, numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the CDR.

[0099] The "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise indicated, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0100] An example of an antigen-binding domain is the antigen-binding domain formed by the VH-VL dimer of an antibody.Another example of an antigen-binding domain is the antigen-binding domain formed by diversifying a specific loop from the tenth fibronectin type III domain of an adnectin.An antigen-binding domain can include CDRs 1, 2 and 3, in that order, from the heavy chain; and CDRs 1, 2 and 3, in that order, from the light chain.

[0101] Epitopes often consist of surface accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former may be lost in the presence of denaturing solvents, but the binding to the latter is not lost. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination, such as, for example, testing the binding of the antibody to fibrin variants with different point mutations or chimeric fibrin variants.

[0102] To screen for antibodies that bind to an epitope on a target antigen (e.g., fibrin) to which an antibody of interest binds, a routine cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, or in addition, epitope mapping can be performed by methods known in the art.

[0103] A chimeric antibody has a portion of the heavy and / or light chain derived from a particular source or species, and the remainder of the heavy and / or light chain derived from a different source or species.

[0104] A human antibody is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0105] A humanized antibody has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions such that the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response when administered to a human subject, compared to the non-human species antibody. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the immunogenic potential of the non-human antibody when administered to a human subject, either the altered amino acid residues are not important for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of methods for making humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0106] The two or more different epitopes may be epitopes on the same antigen (e.g., a single fibrin molecule expressed by a cell) or may be epitopes on different antigens (e.g., different fibrin molecules, or a fibrin molecule and a non-fibrin molecule, expressed by the same cell). In some aspects, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some aspects, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody").

[0107] Anti-fibrin antibodies can include those described herein, such as the clones listed in the figures and / or tables. In some embodiments, the antibody comprises an alternative scaffold. In some embodiments, the antibody consists of an alternative scaffold. In some embodiments, the antibody consists essentially of an alternative scaffold. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the antibody consists of an antibody fragment. In some embodiments, the antibody consists essentially of an antibody fragment.

[0108] In some embodiments, the antibody is a monoclonal antibody.

[0109] In some embodiments, the antibody is a polyclonal antibody.

[0110] In some embodiments, the antibody is produced by a hybridoma, hi other embodiments, the antibody is produced by a recombinant cell that has been engineered to express the desired variable and constant domains.

[0111] In some embodiments, the antibody may be a single chain antibody or other antibody derivative or variant thereof that retains the antigen specificity and lower hinge region.

[0112] In some embodiments, the antibody may be a polyfunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment or variant thereof. In certain embodiments, the antibody fragment or derivative thereof is selected from a Fab fragment, a Fab'2 fragment, a CDR and a ScFv.

[0113] In some embodiments, the antibody can form an immune complex, for example, the immune complex can be a tumor cell coated with the antibody.

[0114] In sequence comparison, one sequence usually serves as reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinate is designated if necessary, and sequence algorithm program parameter is designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on designated program parameter.

[0115] Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0116] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0117] Fibrin antibody sequence V H domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 7. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 8. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 11. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 13. H In some embodiments, the antibodies provided herein comprise the V HIn some embodiments, the antibodies provided herein comprise the V H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 16. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 17. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 18. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 19. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 20. H Contains arrays.

[0118] In some embodiments, the antibodies provided herein are selected from the exemplary VV ... H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H In some embodiments, the antibodies provided herein comprise a V or VL sequence as provided in SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 2 ... HThe antibody may be a nucleotide sequence of the antibody provided herein. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibody described in this paragraph is referred to herein as a "variant". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0119] V L domain In some embodiments, the antibody provided herein comprises a VL sequence selected from SEQ ID NO:21.

[0120] In some embodiments, the antibodies provided herein comprise a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity with the exemplary VL sequence provided in SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise a VL sequence provided in SEQ ID NO: 21 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. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0121] VH-VL combinations In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. H and V selected from SEQ ID NO: 21. L Contains arrays.

[0122] In some embodiments, the antibody provided herein comprises the V H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 8. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 9. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 10. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 11. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 12. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 13. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the V H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the V H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 16. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 17. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 18. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 19. H Sequence and V of SEQ ID NO: 21 L In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 20. H Sequence and V of SEQ ID NO: 21 L Contains arrays.

[0123] In certain aspects, any of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 can be combined with any of SEQ ID NOs: 21.

[0124] In some embodiments, the antibodies provided herein are selected from the exemplary VV ... H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence Hand V sequences having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary V sequences provided in SEQ ID NO: 21. L In some embodiments, the antibodies provided herein comprise a VH sequence as provided in SEQ ID NO: 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 as provided in SEQ ID NO: 21, 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. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0125] CDR In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. H In some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. HIn some embodiments, the antibodies provided herein comprise 2-3 CDRs of a VH domain selected from SEQ ID NOs: 37, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some aspects, the CDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, 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.

[0126] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, the CDR-H1 is the CDR-H1 of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is a CDR-H2 of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 is a CDR-H3 of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., by the methods provided herein for obtaining antibodies.

[0127] In some embodiments, the antibodies provided herein comprise one to three CDRs of the VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise two to three CDRs of the VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise three CDRs of the VL domain of SEQ ID NO: 21. In some aspects, the CDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, 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.

[0128] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NO: 21. In some embodiments, CDR-L1 is CDR-L1 of the VL domain of SEQ ID NO: 21 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, CDR-L2 is CDR-L2 of the VL domain of SEQ ID NO: 21 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR-L3 is CDR-L3 of the VL domain of SEQ ID NO: 21 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0129] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and one to three CDRs of a VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and two to three CDRs of a VL domain of SEQ ID NO: 21. In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and three CDRs of a VL domain of SEQ ID NO: 21. In some aspects, the CDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. In some aspects, 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.

[0130] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 24, 25, 26, 27, 28, 29, and 30. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 24, 25, 26, 27, 28, 29, and 30 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, e.g., by the methods provided herein for obtaining antibodies.

[0131] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 24. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 24. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0132] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 25. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 25. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0133] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 26. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 26. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0134] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 27. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 27. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 27 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0135] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 28. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 28. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0136] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 29. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 29. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 29 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0137] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 30. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 30. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 30 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0138] In some embodiments, the antibodies provided herein comprise a selected CDR-H3 of SEQ ID NO: 3. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H3 of SEQ ID NO: 3. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NO: 3 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0139] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1. In some aspects, the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H1 is a CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0140] In some embodiments, the antibodies provided herein comprise a selected CDR-H2 of SEQ ID NO: 2. In some aspects, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-H2 of SEQ ID NO: 2. In some embodiments, the CDR-H2 is a selected CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0141] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 24 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 24, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0142] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 25, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0143] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 26 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 26, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 26, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 26 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0144] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 27 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 27, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 27, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 27 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0145] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 28 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 28, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 28, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 28 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0146] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 29, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H3 of SEQ ID NO: 29, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 29 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0147] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30 and a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30, a CDR-H2 of SEQ ID NO: 2, and a CDR-H1 of SEQ ID NO: 1. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 30, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 30 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0148] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 6. In some aspects, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-L3 of SEQ ID NO: 6. In some embodiments, the CDR-L3 is a CDR-L3 of SEQ ID NO: 6 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0149] In some embodiments, the antibodies provided herein comprise a CDR-L2 of SEQ ID NO: 5. In some aspects, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-L2 of SEQ ID NO: 5. In some embodiments, the CDR-L2 is a CDR-L2 of SEQ ID NO: 5 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0150] In some embodiments, the antibodies provided herein comprise a CDR-L1 of SEQ ID NO: 4. In some aspects, the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-L1 is a CDR-L1 of SEQ ID NO: 4 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0151] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 6 and a CDR-L2 of SEQ ID NO: 5. In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 6, a CDR-L2 of SEQ ID NO: 5, and a CDR-L1 of SEQ ID NO: 4. In some embodiments, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO:6, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L2 of SEQ ID NO:5, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO:4. In some embodiments, the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with up to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with up to 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0152] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:24, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 24, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 24 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0153] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:25, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 25, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6, and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 25 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0154] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:26, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 26, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 26 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0155] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:27, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 27, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 27 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0156] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:28, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 28, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 28 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0157] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO:29, a CDR-H2 of SEQ ID NO:2, a CDR-H1 of SEQ ID NO:1, a CDR-L3 of SEQ ID NO:6, a CDR-L2 of SEQ ID NO:5, and a CDR-L1 of SEQ ID NO:4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 29, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 29 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0158] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 30, a CDR-H2 of SEQ ID NO: 2, a CDR-H1 of SEQ ID NO: 1, a CDR-L3 of SEQ ID NO: 6, a CDR-L2 of SEQ ID NO: 5, and a CDR-L1 of SEQ ID NO: 4. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO: 30, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO: 2, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO: 1, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: 6 and CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO: and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 30 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 2 with a maximum of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and the CDR-L1 is CDR-L1 of SEQ ID NO: 4 with a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, and may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0159] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 24, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0160] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 25, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0161] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 26, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0162] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 27, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0163] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 28, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0164] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 29, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0165] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 30, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0166] Epitope In certain embodiments, described herein are isolated antibodies that bind to human fibrin or fibrinogen γC domain, binding to human fibrin at any one of amino acid residues Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421. In certain embodiments, the antibodies bind to human fibrin at at least 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of amino acid residues Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421. In certain embodiments, the isolated antibody binds to human fibrin at amino acid residues Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421. In certain embodiments, amino acid residues of a human fibrin or fibrinogen γC domain epitope bind to the paratope of the antibody at a distance of less than 5 angstroms or less, 4 angstroms or less, 3 angstroms or less, or 2 angstroms or less.

[0167] Paratope In certain embodiments, the antibodies described herein comprise a VH region that comprises a paratope that binds to a human fibrin or fibrinogen γC domain, wherein the paratope comprises any one of amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Leu 50, Asp 52, Asp 54, Tyr 56, Ala 93, Ser 94, Ser 95, Lys 96 or Asp 96, Pro 97 or Ala 97, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that includes a paratope that includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Leu 50, Asp 52, Asp 54, Tyr 56, Ala 93, Ser 94, Ser 95, Lys 96 or Asp 96, Pro 97 or Ala 97, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that includes a paratope that includes amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that includes a paratope that includes amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ala 93, Ser 94, Lys 96, Pro 97, Gly 101, Gly102, and Trp 103. In certain embodiments, the antibody comprises a VH region that comprises a paratope comprising amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Asp 52, Asp 54, Tyr 56, Ser 94, Ser 95, Asp 96, Ala 97, Gly 101, Gly102, and Trp 103.

[0168] In certain embodiments, the antibody comprises a VL region that includes a paratope that includes any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gln 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that includes a paratope that includes at least 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gln 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that comprises a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gln 50, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that comprises a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gln 50, Asn 91, Leu 92, Leu 94, and Leu 96. In certain embodiments, the antibody comprises a VL region that comprises a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gln 50, Ala 91, Leu 92, Leu 94, and Leu 96.

[0169] In certain embodiments, the paratope of the antibody binds to amino acid residues of a human fibrin or fibrinogen γC domain epitope at a distance of less than 5 angstroms or less, 4 angstroms or less, 3 angstroms or less, or 2 angstroms or less.

[0170] Fc area The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or a modified Fc region as described in the art or elsewhere in this disclosure.

[0171] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, the Fc polypeptide of a dimeric IgG Fc includes IgG CH2 and IgG CH3 constant domain sequences. Fc may be of IgA, IgD, IgE, IgG, and IgM classes, some of which may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0172] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, an FcR can be a native sequence human FcR. Generally, FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibitory receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes can also be bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0173] Modification of CH2 domain can affect FcR binding to Fc. Some amino acid modifications in Fc region are known in the art to selectively modify the affinity of Fc to different Fc gamma receptors. In some aspects, Fc comprises one or more modifications to promote selective binding to Fc-gamma receptors.

[0174] Exemplary mutations that alter binding of FcR to Fc are listed below: S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu Y, Vernes JM, Chiang N, et al. J Immunol Methods. 2011 Feb 28;365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen JB, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep 15;67(18):8882-90; Nordstrom JL, Gorlatov S, Zhang W, et al. Breast Cancer Res. 2011 Nov 30;13(6):R123); F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Sel. 2011 Sep;24(9):671-8.), S298A / E333A / K334A (Shields RL, Namenuk AK, Hong K, et al. J Biol Chem. 2001 Mar 2;276(9):6591-604); S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S, et al. Proc Natl Acad Sci US A. 2006 Mar 14;103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S, et al. Mol Immunol. 2008 Sep;45(15):3926-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other mutations listed in WO2011 / 120134 and WO2011 / 120135, which are incorporated herein by reference. Therapeutic Antibody Engineering (by William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012) lists the mutations on page 283.

[0175] In some embodiments, the antibody described herein comprises modifications to improve its ability to mediate effector function.Such modifications are known in the art and include defucosylation or engineering the affinity of Fc to activating receptors, mainly FCGR3a for ADCC and C1q for CDC.The following Table B summarizes the various designs reported in the literature for engineering effector function.

[0176] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn 297 EU numbering) without modifying the amino acid sequence are well known in the art. GlymaX® technology (ProBioGen AG) is based on the introduction of a gene for an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the addition of the sugar "fucose" to the N-linked antibody carbohydrate moiety by the antibody producing cells. (von Horsten et al. (2010) Glycobiology. 2010 Dec; 20 (12):1607-18. Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Pat. No. 8,409,572, which teaches that cell lines for producing antibodies are selected for their ability to produce lower levels of antibody fucosylation; the antibodies can be fully defucosylated (meaning that they contain no detectable fucose) or they can be partially defucosylated, meaning that the isolated antibody contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose normally found for a similar antibody produced by a mammalian expression system.

[0177] Thus, in one embodiment, the antibodies described herein can comprise a dimeric Fc that contains one or more amino acid modifications as described in Table B that confer improved effector function. In another embodiment, the antibodies can be defucosylated to improve effector function.

[0178] (Table B) CH2 domain and effector function engineering TIFF2024525188000003.tif86148

[0179] Fc modifications that reduce FcgR and / or complement binding and / or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curr Opin Biotech 20:685-691, and Strohl, WR and Strohl LM, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp 225-249). These strategies include reducing effector function by modifying glycosylation, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 regions of Fc. For example, U.S. Patent Application Publication No. 2011 / 0212087 (Strohl), International Patent Application Publication No. WO 2006 / 105338 (Xencor), U.S. Patent Application Publication No. 2012 / 0225058 (Xencor), U.S. Patent Application Publication No. 2012 / 0251531 (Genentech), and Strop et al ((2012) J. Mol. Biol. 420: 204-219) describe specific modifications that reduce binding of FcgR or complement to Fc.

[0180] Specific non-limiting examples of amino acid modifications known to reduce binding of FcgR or complement to Fc include those identified in Table C below.

[0181] (Table C) Modifications that reduce FcgR or complement binding to Fc TIFF2024525188000004.tif114128

[0182] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn 297 EU numbering) without modifying the amino acid sequence are well known in the art. GlymaxX® technology (ProBioGen AG) is based on the introduction of a gene for an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the addition of the sugar "fucose" to the N-linked antibody carbohydrate moiety by the antibody producing cells. (von Horsten et al. (2010) Glycobiology. 2010 Dec; 20 (12):1607-18.) Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD) (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells, which are deficient in protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Application Publication No. 2003 / 0157108; WO 2004 / 056312; These include knockout cell lines, such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (see Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107; each of which is incorporated by reference in its entirety). Another approach to obtaining antibodies with reduced levels of fucosylation can be found in U.S. Patent No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to produce lower levels of antibody fucosylation.

[0183] Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD) (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHO cells that are deficient in protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Patent Application Publication No. 2003 / 0157108; WO 2004 / 056312; each of which is incorporated by reference in its entirety), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003 / 085107; each of which is incorporated by reference in its entirety.

[0184] The antibodies can be completely defucosylated (meaning that they contain no detectable fucose) or they can be partially defucosylated, meaning that the isolated antibody contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15% or less than 5% of the amount of fucose typically detected for a similar antibody produced in a mammalian expression system.

[0185] In some aspects, the antibodies provided herein comprise an IgG1 domain with reduced fucose content at Asn 297 compared to naturally occurring IgG1 domains. Such Fc domains are known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, which is incorporated by reference in its entirety. In some aspects, such antibodies do not comprise fucose at Asn 297. The amount of fucose can be determined using any suitable method, for example, as described in WO 2008 / 077546, which is incorporated by reference in its entirety.

[0186] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of Fc region positions 298, 333, and 334. In some embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions at positions 239, 332, and 330, as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006,103:4005-4010, which is incorporated by reference in its entirety.

[0187] Other exemplary glycosylation variants that can be incorporated into the antibodies provided herein are described, e.g., in U.S. Patent Application Publication Nos. 2003 / 0157108, 2004 / 0093621, 2003 / 0157108, 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0132140, 2004 / 0110704, 2004 / 0110282, 2004 / 0109865; Nos. 2000 / 61739, 2001 / 29246, 2003 / 085119, 2003 / 084570, 2005 / 035586, 2005 / 035778; 2005 / 053742, 2002 / 031140; Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249; and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of which is incorporated by reference in its entirety.

[0188] In some embodiments, the antibody provided herein comprises an Fc region with at least one galactose residue in the oligosaccharide attached to the Fc region.Such antibody variants can have improved CDC function.Examples of such antibody variants are described in, for example, WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764; each of which is incorporated by reference in its entirety.

[0189] In some embodiments, the antibodies provided herein contain one or more modifications that improve or attenuate C1q binding and / or CDC. See U.S. Patent No. 6,194,551; WO 99 / 51642; and Idusogie et al., J. Immunol., 2000, 164:4178-4184; each of which is incorporated by reference in its entirety.

[0190] join The affinity of a molecule X for its partner Y is expressed as the dissociation equilibrium constant (K D The affinity can be expressed by the following equation: . The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0191] With respect to antibody binding to a target molecule, the terms "bind", "specifically binding", "specifically binds", "specific to", "selectively binds" and "selective to" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from non-specific or non-selective interactions (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 an epitope recognized on the target molecule. In that case, specific binding is indicated if binding of the antibody to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 50% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 40% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 30% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 20% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 10% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 1% of its affinity to fibrin. In some embodiments, the affinity of the fibrin antibody to the non-target molecule is less than about 0.1% of its affinity to fibrin.

[0192] When used herein in the context of two or more antibodies, the term "competes with" or "cross-competes with" indicates that two or more antibodies compete for binding to an antigen (e.g., fibrin). In one exemplary assay, fibrin is coated on a surface and contacted with a first fibrin antibody, followed by the addition of a second fibrin antibody. In another exemplary assay, a first fibrin antibody is coated on a surface and contacted with fibrin, followed by the addition of a second fibrin antibody. In either assay, antibodies compete with each other if the presence of the first fibrin antibody reduces the binding of the second fibrin antibody. The term "competes with" also includes antibody combinations in which one antibody reduces the binding of another antibody, but no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit each other's binding regardless of the order in which they are added. In some embodiments, an antibody reduces the 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%, at least 95%, or at least 99% when measured in a competitive binding assay. A person skilled in the art can select the concentration of antibody used in a competitive assay based on the affinity of the antibody for fibrin and the valency of the antibody. The assays described in this definition are exemplary, and a person skilled in the art can use any suitable assay to determine whether antibodies compete with each other.Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated by reference in its entirety.

[0193] A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits or blocks the binding of the reference antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when measured in a competitive binding assay. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody for steric hindrance to occur. For example, a second competing antibody can be identified that competes with the first antibody described herein for binding to fibrin. In some cases, the second antibody can block or inhibit the binding of the first antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when measured in a competitive binding assay. In some cases, the second antibody can replace the first antibody by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0194] In some embodiments, the anti-fibrin antibody does not substantially bind to myeloid cells present outside the cancer tissue. In some embodiments, the anti-fibrin antibody does not substantially bind to stimulatory myeloid cells present within the cancer tissue.

[0195] In some embodiments, the anti-fibrin antibody binds to residues γ377-395 of the fibrin or fibrinogen γC domain of human fibrin (SEQ ID NO: 31). The binding epitope includes residues within a numerical range (e.g., residues 377-395 of fibrin), the beginning residue of each range (e.g., residues 377-394 of human fibrin) and the ending residue of each range (e.g., residues 378-395 of human fibrin), or any combination thereof.

[0196] In some embodiments, the antibodies provided herein have a molecular mass of about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 x 10 as measured by a Biacore assay. -6 K below M D In some embodiments, the K of the antibodies provided herein binds to human fibrin. D is approximately 0.001-0.01, 0.01-0.1, 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-1, 0.25-0.75, 0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-2, 1-5, 2-7, 3-8, 3-5, 4-6, 5-7, 6-8, 7-9, 7-10, or 5-10 x 10 as measured by Biacore assay. -6 In some embodiments, the antibodies provided herein have a titer of about 1×10 -5 M, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, or 1×10 -9 K below M D It binds to human fibrin.

[0197] In some embodiments, the antibodies provided herein have a molecular mass of about 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001 x 10 as measured by a Biacore assay. -5 K less than or equal to M D In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 5-3, 4-2, 3-1, 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, 1.9-1.5, 1.5-1, 1-0.8, 1-0.5, 0.9-0.6, 0.7-0.4, 0.6-0.2, 0.5-0.3, 0.3-0.2, 0.2-0.1, 0.1-0.01, 0.01-0.001, or 0.001-0.0001 x 10 as measured by a Biacore assay. -5 K of M D In some embodiments, the antibodies provided herein bind to human fibrin at about 10, 9.56, 9.5, 9.0, 8.88, 8.84, 8.5, 8, 7.5, 7.32, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 x 10 as measured by a Biacore assay. -4 (1 / s) or less, or K smaller d In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 7-10, 7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8.-8.5, 8.5-9, 9-9.5, or 9.5-10 x 10 as measured by a Biacore assay. -4 (1 / s)K dIn some embodiments, the antibodies provided herein bind to human fibrin at about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 45, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 7, 8, 9, or 10×10 as measured by a Biacore assay. 5 K greater than or equal to (1 / Ms) a In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 4-7, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10 x 10 as measured by a Biacore assay. 5 (1 / Ms)K a It binds to human fibrin (FIBRIN).

[0198] function "Effector function" refers to the biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include receptor ligand blocking, receptor agonism, or antagonism, C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the effector function of the fibrin antibodies described herein is antagonism, blocking Mac-1 receptor binding to fibrin.

[0199] Pharmaceutical Compositions The present application provides compositions comprising antibodies, including pharmaceutical compositions comprising any one or more of the antibodies described herein together with one or more pharma- ceutically acceptable excipients.In some embodiments, the compositions are sterile.The pharmaceutical compositions typically comprise an effective amount of antibodies.

[0200] These compositions may contain, in addition to one or more of the antibodies disclosed herein, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other materials may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal.

[0201] The pharmaceutical composition for oral administration can be in the form of tablet, capsule, powder or liquid. Tablets can contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions usually contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0202] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity and stability.Those skilled in the art can fully prepare appropriate solutions using isotonic vehicles such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary.

[0203] The anti-fibrin antibody given for administration to an individual is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (although in some cases prevention can also be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. The determination of treatment prescription, such as dosage, is within the responsibility of general practitioners and other physicians, and will usually take into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0204] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated.

[0205] method Preparation method The antibodies described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an antibody described herein. Such a nucleic acid can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of an antibody (e.g., the light and / or heavy chain of an antibody), or an amino acid sequence comprising the VHH of a single domain antibody. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In one embodiment, the nucleic acid is provided in a multicistronic vector. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antigen-binding polypeptide construct. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody is provided, the method comprising culturing a host cell comprising an antibody-encoding nucleic acid as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0206] For recombinant production of an antibody, for example, nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0207] When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein is present in certain embodiments at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1% of the dry weight of the cells or less. When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein is present in the medium in certain embodiments at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L of the dry weight of the cells or less. In certain embodiments, the "substantially purified" heteromultimers produced by the methods described herein have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically at least about 75%, 80%, 85%, and more specifically at least about 90%, at least about 95%, at least about 99% or more, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0208] Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0209] A recombinant host cell, or host cell, is a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for creating a recombinant host cell. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or may be integrated into the host genome. The host cell may include CHO, a derivative of CHO, NS0, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0210] For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, antibodies may be isolated in a soluble fraction from bacterial cell paste and can be further purified.

[0211] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0212] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. A number of baculovirus strains have been identified that can be used for transfection of insect cells, particularly Spodoptera frugiperda cells.

[0213] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0214] Vertebrate cells may be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); see, e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0215] In one embodiment, the antibodies described herein are produced in stable mammalian cells by a method comprising transfecting at least one stable mammalian cell with nucleic acids encoding the antibodies in a predetermined ratio; and expressing the nucleic acids in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that results in the highest percentage of antibody in the expression product.

[0216] In some embodiments, there are methods of producing antibodies in stable mammalian cells as described herein, wherein the expression product of at least one stable mammalian cell comprises a greater proportion of the desired glycosylated antibody as compared to monomeric heavy or light chain polypeptides, or other antibodies.

[0217] In some embodiments, there is a method of producing a glycosylated antibody in a stable mammalian cell as described herein, the method comprising identifying and purifying a desired glycosylated antibody, in some embodiments, the identification is by one or both of liquid chromatography and mass spectrometry.

[0218] If necessary, the antibody can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins can find use in the present invention for purification of antibodies. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification is often made possible by the specific fusion partner. For example, glutathione resin is used when GST fusions are utilized, Ni when His tags are utilized, etc. +2 The antibody can be purified using affinity chromatography or, if a flag tag is used, using immobilized anti-flag antibodies. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994, which is incorporated by reference in its entirety. The degree of purification required varies depending on the use of the antibody. In some cases, no purification is necessary.

[0219] In certain embodiments, antibodies are purified using anion exchange chromatography, including but not limited to, chromatography on Q-Sepharose, DEAE Sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0220] In certain embodiments, the proteins described herein are purified using cation exchange chromatography, including, but not limited to, SP Sepharose, CM Sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and equivalents.

[0221] Moreover, the antibodies described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids generally include, but are not limited to, D-isomers of the common amino acids, 2,4 diaminobutyric acid, alpha-aminoisobutyric acid, 4 aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6 aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoro-amino acids, designer amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs. Additionally, amino acids can be D (dextrorotatory) or L (levorotatory).

[0222] How to use In one aspect, the application provides a method of contacting fibrin with an anti-fibrin antibody, such as a human or humanized antibody, that results in inhibition of microglial adhesion to fibrin or fibrinogen γC domain.

[0223] In one aspect, the present application provides a method of using the isolated anti-fibrin antibody described herein for the treatment of degenerative disorders of the nervous system.In a particular aspect, the present application provides a method for treating degenerative disorders of the nervous system, comprising administering to a mammalian subject a therapeutically effective amount of the anti-fibrin antibody or a pharmaceutical composition comprising the anti-fibrin antibody described herein.In a particular embodiment, the present application provides a method for treating degenerative disorders of the nervous system selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0224] In certain aspects, described herein are methods for treating a pathology associated with Mac-1 binding to fibrin or Mac-1 binding to fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising an isolated anti-fibrin antibody described herein.

[0225] In certain aspects, described herein are methods of inhibiting microglial activation, the methods comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising the isolated antibody described herein.

[0226] In certain aspects, described herein are methods for preventing degenerative disorders of the nervous system, comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising an isolated anti-fibrin antibody described herein. In certain embodiments, the present application provides a method for preventing a degenerative disorder of the nervous system selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0227] In certain aspects, described herein are methods of treating or preventing colitis comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising an isolated anti-fibrin antibody described herein.

[0228] In certain aspects, described herein are methods for treating or preventing an ocular inflammatory condition comprising administering to a mammalian subject a therapeutically effective amount of an isolated anti-fibrin antibody or a pharmaceutical composition comprising an isolated anti-fibrin antibody described herein. In certain embodiments, the ocular inflammatory condition is uveitis.

[0229] Method of administration In some embodiments, the methods provided herein are useful for treating a degenerative nervous system disorder in an individual. In one embodiment, the individual is a human and the antibody is a fibrin antibody described herein.

[0230] In some embodiments, the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intravitreally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally.An effective amount of anti-fibrin antibody can be administered for the treatment of cancer.The appropriate dosage of anti-fibrin antibody can be determined based on the type of cancer to be treated, the type of anti-fibrin antibody, the severity and course of cancer, the clinical condition of the individual, the clinical history of the individual and response to treatment, and the discretion of the attending physician.

[0231] In some embodiments, the antibody provided herein is administered with at least one additional therapeutic agent.Any suitable additional therapeutic or immunotherapeutic agent can be administered with the antibody provided herein.The additional therapeutic agent includes agents used to treat or prevent degenerative disorders of the nervous system selected from the group consisting of multiple sclerosis, spinal cord injury, stroke, and Alzheimer's disease.

[0232] The additional therapeutic agent can be administered by any suitable means. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in the same pharmaceutical composition. In some embodiments, the antibody provided herein and the additional therapeutic agent are comprised in different pharmaceutical compositions.

[0233] In embodiments in which the antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can be performed prior to administration of the additional therapeutic agent, simultaneously with administration of the additional therapeutic agent, and / or after administration of the additional therapeutic agent. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one month of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one week of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one day of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about 12 hours of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent is performed within about one hour of each other.

[0234] Kits and Articles of Manufacture The present application provides a kit comprising any one or more of the antibody compositions described herein.In some embodiments, the kit further comprises a component selected from any of the following: secondary antibody, immunohistochemistry analysis reagent, pharmaceutically acceptable excipient and instruction manual, and any combination thereof.In one particular embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein together with one or more pharmaceutically acceptable excipients.

[0235] The present application also provides an article of manufacture comprising any one of the antibody compositions or kits described herein. An example of an article of manufacture is a vial (including a sealed vial). EXAMPLES

[0236] Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should of course be allowed for.

[0237] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature, see, for example, TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (East, Pennsylvania: Mack Publishing Company, 1992); and, 1997, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2020, 2030, 2030, 2040, 2040, 2050, 2060, 2070, 2070, 2080, 2090, 2091, 2092, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, rd Ed. (Plenum Press) Vols A and B (1992).

[0238] material and method Antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation kit from Pierce. Goat F(ab')2 anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE), and streptavidin-AF633 (SA-633) were from Southern Biotech, Sigma, and Molecular Probes, respectively. Goat anti-human IgG-PE (Human-PE) was from Southern Biotech. Anti-mouse APC was from Jackson ImmunoResearch.

[0239] Example 1: Humanization of anti-fibrin antibodies The humanization process modifies the binding domain of a non-human antibody to make it more similar to a human binding domain. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve the specificity, affinity, stability, or developability profile of the antibody.

[0240] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0241] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0242] Humanization of 5B8 Humanization of the fibrin antibody (5B8) was performed by characterizing a panel of humanized designs produced in yeast. Briefly, designs were created by grafting CDR mouse sequences onto human framework sequences predicted in silico to be most compatible with the original mouse framework. The following combinations were produced in yeast and characterized for binding to the human fibrinogen P2 peptide antigen: 27 antibodies based on 5B8, representing 9 humanized VH and 3 humanized Vκ combinations.

[0243] Antibody Optimization Optimization of the humanized antibodies was performed by introducing diversity into the heavy and light chain variable regions as described below.

[0244] Library construction: Oligonucleotides containing either CDRH1, CDRH2, or CDRH3, and the flanking regions on either side of the CDRs, were ordered from IDT. Amino acid positions within the CDRs were diversified with NNK diversity introduced into the CDR oligos. The HC (heavy chain) variable region DNA was then DNase treated to generate fragments ranging in size from 50 to 200 bp. The CDRH1, CDRH2, and CDRH3 oligos were then recombined with the DNase treated HC variable regions by overlap extension PCR to incorporate the CDR diversity oligos into the HC variable region sequences. Libraries were then generated by transforming this diversified HC variable sequence and the heavy chain expression vector into yeast already containing the parental light chain plasmid. A similar process was performed to introduce diversity into CDRL1, CDRL2, and CDRL3. Oligonucleotides with CDRL1, CDRH2, and CDRH3 diversity were ordered from IDT and incorporated into the diversified light chain (LC) variable regions as described for the CDRH1, CDRH2, CDRH3 libraries. These diversified LC variable region and light chain expression vectors were transformed into yeast already containing the parental heavy chain plasmid. An additional set of libraries was constructed focusing only on diversity within CDRH3. Walking singlet diversity was introduced into CDRH3 by overlap extension PCR with oligonucleotides carrying VH FR1-FR3 and CDRH3 diversity.

[0245] Selection was performed using three rounds of FACS sorting. Approximately 2×10 7Yeast were pelleted, washed three times with wash buffer, and incubated at 30°C with either affinity pressure using human fibrinogen P2 peptide antigen or with multispecific depletion reagent (PSR) to remove nonspecific antibodies from the selection. For this selection, affinity pressure was applied by preincubating the antigen with parental IgG and then applying the precomplexed mixture to the yeast library for a time sufficient for the selection to reach equilibrium. For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see Y. Xu et al, PEDS 26.10, 663-70 (2013)). Yeast were then washed twice with wash buffer and stained with LC-FITC (1:100 dilution) and either SA-633 (1:500 dilution), EAPE (1:50 dilution), or anti-mouse APC (1:500 dilution) secondary reagents for 15 minutes at 4°C. After washing twice with wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a strainer-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine sorting gates to select for antibodies with the desired properties. Four selection rounds were completed. After the final sorting round, yeast were plated and individual colonies were picked for characterization. Figure 1 shows the results of the first three rounds of selection of a single antibody library from one parent antibody and the increase in affinity of the antibody for fibrin P2 gamma peptide after each round of maturation.

[0246] Antibody production and purification Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid, pH 3.5. Fab fragments were generated by papain digestion and purified with CaptureSelect (Life Technologies).

[0247] Example 2: Characterization and affinity maturation of humanized antibody clones Enzyme-linked immunosorbent assays (ELISAs) were performed with selected humanized antibody clones and fibrin P2 peptide (Figure 2A), fibrinogen (Figure 2B), and fibrin (Figure 2C). A = clone 60143; B = clone 61278; C = clone 61278 (duplicate); D = parent antibody.

[0248] These results confirm that the affinity matured humanized antibody clones bind to the fibrin P2 peptide and to fibrin with improved affinity compared to the parent humanized antibody.

[0249] To assess whether affinity matured antibody clones affected fibrin polymerization or lysis, a clot lysis assay was performed showing the clot lysis time of samples in the presence of variant humanized antibodies (Figure 3). A = clone 56666; B = clone 56657; C = clone 60143; D = clone 60181; E = clone 60175; F = clone 60163; G = clone 60173; H = clone 60184; I = clone 60141; J = clone 60179; K = clone 60140; L = clone 60183. Clot lysis assays were performed by preparing two mixtures: Mixture 1 containing 133 nM antibody, 2 uM fibrinogen was prepared in a 96-well plate, centrifuged at 55 rpm, incubated at 37°C for 0.5 h, and Mixture 2 containing 20 nM plasminogen, 0.1 U thrombin, 4 mM CaCl2, and 1 nM tPA was prepared and transferred to the plate. The clot lysis reaction was started immediately after Mixture 2 was transferred to the wells. The progress of the reaction was measured at 350 nm. Each plate included four controls: a buffer blank without thrombin-tPA-CaCl2 mix, a buffer blank, 100 uM GPRP (polymerization inhibitor), and 10 uM EACA (lysis inhibitor).

[0250] The clot lysis times of all antibody clones tested were not significantly altered compared to the parent humanized antibody or the isotype control antibody (Figure 3).

[0251] These results confirm that the affinity matured humanized antibody clones do not affect fibrin polymerization or fibrinolysis.

[0252] ForteBio K D measurement ForteBio affinity measurements were performed using an Octet RED384 as previously described (see Estep et al, High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG online onto the AHC sensor. The sensor was equilibrated offline for 30 min in assay buffer and then monitored online for 60 s to establish a baseline. The IgG-loaded sensor was exposed to 100 nM antigen for 3 min and then transferred to assay buffer for 3 min for off-rate measurements. For monovalent affinity evaluation, Fab was used instead of IgG. For this evaluation, non-biotinylated Fc-fused antigen was loaded online onto the AHC or AMC sensor. The sensor was equilibrated offline for 30 min in assay buffer and then monitored online for 60 s to establish a baseline. Antigen-loaded sensors were exposed to 100 nM Fab for 3 min, after which they were transferred to assay buffer for 3 min for off-rate measurements. All kinetics were analyzed using a 1:1 binding model.

[0253] Figure 4 shows the ForteBio K antibody using N-terminally biotinylated fibrin P2 peptide (100 nM) conjugated to IgG in solution or N-terminally biotinylated fibrin P2 peptide (100 nM) conjugated to FAB (monovalent) in solution. DThe measurement results are shown in Figure 5. Figure 5 shows the results of an octet Fab solution (100 nM) binding to the N-terminally biotinylated fibrin P2 peptide.

[0254] These results indicate that the affinity matured humanized antibody clones have improved binding affinity to fibrin P2 gamma peptide compared to the parent humanized antibody.

[0255] PSR binding assay PSR assays were performed as previously described (see Xu Y, et al. (2013) Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: A FACS-based, high-throughput selection and analytical tool. Protein Eng Des Sel 26(10):663-670). Briefly, soluble membrane proteins were prepared from CHO cells. The enriched membrane fraction was biotinylated using NHS-LCBiotin (Pierce, Thermo Fisher). This polyspecific reagent was incubated with IgG-presenting yeast, followed by washing. Secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide) was then added to the mixture. Samples were analyzed on a FACSCanto II analyzer (BD Biosciences) using an HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity (MFI) in the R-PE channel to assess nonspecific binding. MFI values ​​were normalized from 0 to 1 based on three reference antibodies exhibiting low, medium, and high PSR MFI values.

[0256] Dynamic Scanning Fluorimetry Add 10 uL of 20x Sypro Orange to 20 uL of 0.2-1 mg / mL mAb or Fab solution. Using an RT-PCR instrument (BioRad CFX96 RT PCR), increase the temperature of the sample plate from 40°C to 95°C in 0.5°C increments and equilibrate for 2 minutes at each temperature. Extract the Tm using the negative of the first derivative of the raw data.

[0257] AC-SINS AC-SINS assays were performed as previously described (see Liu Y, et al. (2014) High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy. MAbs 6(2):483-492). Briefly, gold nanoparticles (Ted Pella Inc.) were coated with 80% capture anti-human goat IgG Fc (Jackson ImmunoResearch) and 20% polyclonal goat non-specific antibody (Jackson ImmunoResearch). The antibody of interest was then incubated with the particles for 2 hours and the wavelength shift was measured using a Molecular Devices SpectraMax M2 with SoftMax Pro6 software. Self-interacting clones show a higher wavelength shift away from the PBS sample.

[0258] HIC (Hydrophobic Interaction Chromatography) The methodology of this assay has been described previously (see Estep P, et al. (2015) An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies. MAbs 7(3):553-561). Briefly, 5 μg of IgG sample (1 mg / mL) was spiked into mobile phase A solution (1.8 M ammonium sulfate and 0.1 M sodium phosphate, pH 6.5) to achieve a final ammonium sulfate concentration of approximately 1 M before analysis. A Sepax Proteomix HIC Butyl-NP5 column was used with a linear gradient of mobile phase A and mobile phase B solution (0.1 M sodium phosphate, pH 6.5) over 20 min at a flow rate of 1 mL / min while monitoring UV absorbance at 280 nm.

[0259] Example 3: Therapeutic Treatment of Fibrinogen-Induced Encephalomyelitis (FIE) We next evaluated the ability of humanized anti-fibrin antibodies to therapeutically inhibit microglial activation and macrophage infiltration (Figure 6) in a fibrinogen-induced encephalomyelitis (FIE) mouse model. To induce FIE, mice were anesthetized with avertin and placed in a stereotaxic apparatus. Plasminogen-free fibrinogen was dissolved in endotoxin-free distilled water and diluted to 5 mg / ml in ACSF (artificial cerebrospinal fluid). Fibrinogen (1 μl of 5 mg / ml) was injected at a rate of 0.3 μl / min according to Paxinos and Watson using a 10 μl Hamilton syringe attached to a 33-gauge needle at the following intracerebral coordinates: anteroposterior, -1.0 mm; mediolateral, -0.7 mm; dorsoventral, -1.325 mm from bregma.

[0260] For prophylactic intracerebroventricular (icv) injections, 10 ug of antibody was delivered intracerebroventricularly (anteroposterior, -2.0 mm; mediolateral, 0 mm, dorsoventral, -2.0 mm) at a rate of 0.3 ul / min using a 10 ul syringe attached to a 33 gauge needle 30 min prior to fibrinogen injection. For prophylactic intravenous (iv) injections, antibody was injected retroorbitally using a 0.3 ml 29 g insulin syringe 1 h prior to fibrinogen injection.

[0261] Stereotaxic fibrinogen injections were performed into the corpus callosum to induce encephalomyelitis. A total of 78 mice were divided into 13 groups, and then n = 6 mice from each group were iv injected with anti-fibrin humanized antibody at either 10 mg / kg or 30 mg / kg. Brain tissue collection and preparation were performed 3 days after injection. Sample exclusions: 5 mice; found dead on postoperative day 1 (C 10 mg / kg, n =1) and postoperative day 2 (B 10 mg / kg, n =1; D 10 mg / kg, n =1); injected into wrong site (B 10 mg / kg, n =1; D 10 mg / kg, n =1). Blinding and quantification: All FIE experiments, image collection and quantification were performed in a blinded manner. Immunohistochemistry (IHC) and quantification were performed as follows: Samples from 73 mice were included for IHC and quantification. Coronal sections (30 um) were prepared on a cryostat. Tissues were stained for Iba-1 (microglia marker, 1:750 dilution) and Mac-2 (macrophage infiltration marker, 1:750 dilution). Iba-1 (Iba-1+ area) and Mac-2 (Mac-2+ area) immunoreactivity was then calculated. A reduction in both microglia and macrophages was detected in tissues from mice treated with either 10 mg / kg or 30 mg / kg of affinity matured humanized anti-fibrin antibody clones.

[0262] These results demonstrate that the humanized antibody variants described herein are capable of therapeutically reducing microglial and macrophage infiltration in mice with FIE.

[0263] Example 4: Prophylactic treatment of relapsing-remitting experimental autoimmune encephalomyelitis (EAE) The ability of a humanized anti-fibrin antibody to prophylactically treat relapsing-remitting EAE ("PLP139-151 EAE") induced by an epitope at amino acids 139-151 of the proteolipid protein (PLP) was evaluated. EAE was induced in 8-9 week old female SJL / J mice by subcutaneous immunization with 15 ug of PLP139-151 in complete Freund's adjuvant supplemented with 400 ug of heat-inactivated Mycobacterium tuberculosis H37Ra (day 0). Two days after immunization, mice are injected with 5 ng of pertussis toxin via IP administration. Antibodies were administered prophylactically IP at 0.2, 1, or 5 mg / kg twice weekly starting on day 0. Dexamethasone (0.5 mg / kg) was administered IP daily as a positive control. Experimental design: 6 groups: n = 10 mice per group, total of 60 mice. Dose regimen: dexamethasone (5 mg / kg daily), humanized anti-fibrin antibodies (A, B, C, D_5 mg / kg every 3 days). EAE disability scores were monitored daily until study termination. The study was terminated 3 days after peak EAE around study days 14-16, and spinal cords were harvested for histopathological analysis.

[0264] Sample Exclusions: 3 mice; found dead on days 12 (Antibody B, n = 1), 15 (Antibody C, n = 1), or 16 (Antibody A, n = 1). Blinding and Quantification: All EAE experiments (antibody treatments and clinical scores) were performed in a blinded manner. 57 spinal cord samples were prepared for tissue processing.

[0265] Clinical scores of PLP EAE were evaluated in mice prophylactically injected with the antibody (5 mg / kg ip every 3 days) (Figure 7). Clinical scores of mice injected with anti-fibrin humanized antibody were reduced compared to control mice injected with PBS or IgG1 only. Time to disease onset was also evaluated (Figure 8). No mice injected with anti-fibrin humanized antibody had paralysis compared to control mice injected with PBS, IgG1, or dexamethasone only, where 25%-50% or more of the mice had paralysis (Figure 8). Figure 9 shows the clinical scores of mice subjected to prophylactic injection of PBS only, dexamethasone, antibody clone 6043 (left) or control antibody human IgG1 (right). FIG. 10 shows the percentage of paralyzed mice (complete paralysis - left) or (partial hind limb paralysis - right) that were subjected to prophylactic injections of PBS alone, dexamethasone (dexa), antibody clone 6043 (at the indicated concentrations; 5 = 5 mg / kg, 1 = 1 mg / kg and 0.2 = 0.2 mg / kg) or control antibody human IgG1 (5 mg / kg).

[0266] These results indicate that anti-fibrin humanized antibodies are effective for the prophylactic treatment of encephalomyelitis.

[0267] Example 5: Humanized anti-fibrin antibodies reduce fibrin-induced IL-12 expression in BMDM cells The ability of affinity matured humanized anti-fibrin antibodies to alter gene expression of interleukin (IL)-12b in bone marrow derived macrophage (BMDM) cell lines was evaluated (Figures 11-13). Cell culture plates with fibrin-coated wells were pre-incubated with humanized anti-fibrin antibodies for 2 hours prior to plating BMDM cells. Cells were incubated with humanized anti-fibrin antibodies or isotype control, fibrinogen, thrombin and CaCl2 for 6 hours, after which cells were harvested and RNA was isolated for gene expression analysis (Figure 11). Cells incubated with isotype control at 50 ug / mL showed a greater than 30-fold increase in IL-12b expression, whereas cells incubated with antibody clones 60143 and 61278 at a concentration of 50 ug / mL showed a 15-20-fold increase in IL-12b expression (Figure 10). In a separate experiment, cells incubated with 10 ug / mL of isotype control showed approximately a 55-fold increase in IL-12b expression, while cells incubated with antibody clones 60143 and 61278 at a concentration of 10 ug / mL showed approximately a 20-45-fold increase in IL-12b expression (Figure 12). As the concentration of antibody clones 60143 and 61278 increased, the fold change in IL-2b expression also decreased (Figure 13).

[0268] These results confirm that fibrin-induced IL-12b expression is reduced by anti-fibrin antibody blockade in bone marrow-derived macrophages.

[0269] Example 6: Treatment of Neurodegenerative Diseases The purified humanized antibody variants described herein are formulated into pharmaceutical compositions that are administered to patients for the treatment of neurodegenerative diseases (e.g., multiple sclerosis or Alzheimer's disease). The pharmaceutical compositions comprising the humanized antibody variants described herein are administered in a dose sufficient to effectively reduce symptoms of the neurodegenerative disease. The pharmaceutical compositions are well tolerated and do not induce significant adverse side effects in patients.

[0270] Example 7: Humanized antibody variants for the treatment of colitis The ability of the humanized antibody variants to treat colitis in a mouse model of colitis was examined (Figure 14). To initiate the dextran sulfate sodium (DSS)-induced colitis treatment, 8-10 week old female C57BL / 6 mice were acclimated to the animal facility for at least 4 days, weighed, and randomly assigned to treatment groups based on weight. Two types of studies were performed: acute (7 days) and chronic (28 days).

[0271] Acute DSS testing is performed by adding 2.5% DSS to drinking water for 7 days. Antibodies were administered IP at 10 and 30 mg / kg every 2 days (Q2D). Mice were euthanized on day 7 by isoflurane anesthesia, exsanguination, followed by cervical dislocation. Colons were removed and analyzed for histopathology.

[0272] Chronic DSS studies were performed by adding 2.0% DSS to drinking water for 1 week, then replacing it with regular drinking water for 1 week, adding 2% DSS for another week, and then finishing with regular drinking water for another week. Humanized antibody variants described herein were administered IV prophylactically at 30 and 5 mg / kg twice weekly starting on day 0. Mice were euthanized after 28 days by isoflurane anesthesia, exsanguination, followed by cervical dislocation. Colons were removed and analyzed for histopathology.

[0273] These results confirm that the humanized antibody variants described herein are effective in treating colitis.

[0274] Example 8: Pharmacokinetics and ex vivo biodistribution of selected anti-fibrin antibodies material and method [125I]SIB-60143 and [125I]SIB-61278 Labeling Protocol Four rounds of 125I-SIB generation were performed, utilizing 10 μL (35 MBq I-125) in each case. Smaller batches were labeled to increase overall efficiency and reproducibility. Two pairs of two reactions were combined and purified by HPLC. The yield of dry 125I-SIB in each case was 25 MBq.

[0275] The pH of 60143 and 61278 stock solutions (0.5 mL each) was lowered from 8.5 to 8.0 using 2 M HEPES. The pH-adjusted solutions were then added to the dried 125I-SIB, respectively, and incubated at room temperature for 1 h. The labeling efficiency was measured by iTLC and found to be 59% and 77% for 60143 and 61278, respectively. The reaction mixture was then purified on a NAP-5 column and eluted with PBS.

[0276] Fractions 4–8 were combined to give 12.6 MBq of [125I]SIB-60143 and 18.0 MBq of [125I]SIB-61278 in 1.25 mL, respectively. Radiochemical purity was determined by iTLC and SEC-HPLC.

[0277] I-125 labeled proteins were diluted in phosphate buffered saline (PBS) to reach 1.33 mg / mL for low dose stocks and 3.75 mg / mL for high dose stocks.

[0278] Animal models Seventy-two female C57BL / 6 mice were received from Charles River UK.

[0279] In vivo high dose pilot safety study Two female C57BL / 6 mice were injected intravenously with 30 mg / kg 60143 and two were injected intravenously with 30 mg / kg 61278. Animals were monitored continuously for the first hour window (0-1 hour post-injection) and the fourth hour window (4-5 hours post-injection). Mice were examined daily for adverse events at 1 and 2 days post-injection, at which point they were euthanized.

[0280] In vivo testing For pharmacokinetic analysis, 68 female C57BL / 6 mice (19.0 ± 1.3 g) were injected with 10 or 30 mg / kg of [125I]SIB-60143 or [125I]SIB-61278 and sacrificed at various time points (5 min, 30 min, 1 h, 4 h, 1 day, 3 days, 7 days, and 14 days; n=2 per time point). Radioactivity in blood, plasma, protein-free plasma, and protein was counted by gamma counter. Further details regarding the study design are shown in Table 1 below.

[0281] Thirty-two of these animals (18.6 ± 1.3 g) were also included in the biodistribution study. All mice were injected with 10 or 30 mg / kg of [125I]SIB-60143 or [125I]SIB-61278 and sacrificed at various time points (days 1, 3, 7, and 14; n=2 per time point). Further details regarding the study design are shown in Table 2 below.

[0282] Table 1. Study design overview for PK analysis TIFF2024525188000005.tif57128

[0283] Table 2. Overview of biodistribution study design TIFF2024525188000006.tif34166

[0284] All animals in groups 1 and 2 were injected awake on day 1. Biodistribution animals in groups 3 and 4 (days 1, 3, 7, and 14) were also injected awake on day 1. PK only animals in groups 3 and 4 (5 min, 30 min, 1 hr, 4 hr) were injected awake on day 2. The same make of test article formulation was used on the different injection days.

[0285] Dose Administration Each animal was weighed on the day of dose administration. Animal weights ranged from 16.4 to 22.5 g. A single intravenous (IV) dose was administered by using a 0.5 mL syringe to give the appropriate dose of 10 mg / kg for groups 1 and 2 and 30 mg / kg for groups 3 and 4. Dosing syringes were weighed before and after injection to determine the amount administered to each subject.

[0286] Ex vivo sample preparation Animals were sacrificed by cardiac puncture followed by exsanguination prior to organ removal. The following organs were harvested for gamma counting: brain, heart, liver, kidneys, muscle, tail, stomach, large intestine, small intestine, cecum, and spleen.

[0287] analysis Results are presented in units of percent implanted dose (%ID) and percent implanted dose per gram (%ID / g). The definitions of these units can be found in the following formula: The %ID for each analyzed region from the ex vivo gamma counting data can be defined as described in Equation 1: TIFF2024525188000007.tif10128Where, Uptake = Radioactivity (MBq) in a particular gamma counting sample, corrected for decay upon injection. Injected Dose = Radioactivity (MBq) injected into the subject.

[0288] The %ID / g for each analyzed region from the ex vivo gamma counting data can be defined as described in Equation 2: TIFF2024525188000008.tif14128Where, Uptake = radioactivity (MBq) in a particular gamma-counted sample, corrected for decay upon injection. Injected Dose = radioactivity (MBq) injected into the subject. Weight = weight of the gamma-counted tissue sample in g.

[0289] Concentration values ​​in μg / mL can be defined for the ex vivo gamma counting data according to Equation 3: TIFF2024525188000009.tif8128Where, Injected Dose = Mass of antibody injected into subject (μg). Assumption: tissue density 1 g / mL.

[0290] Gamma-ray counting analysis for biodistribution The radioactivity of each harvested tissue was measured in counts per minute (CPM). Triplicate aliquots of the radiotracer were also assayed in a gamma counter to calculate a factor for converting counts to mass of injected material (g / CPM). Values ​​were corrected for background radiation and converted to percent injected dose (%ID) and percent injected dose per gram (%ID / g).

[0291] Gamma ray counting analysis for PK studies The radioactivity of each harvested tissue was measured in counts per minute (CPM). Triplicate aliquots of the radiotracer were also assayed in a gamma counter to calculate a factor for converting counts to mass of injected material (g / CPM). Values ​​were then corrected for background radiation and converted to percent injected dose (%ID) and percent injected dose per gram (%ID / g).

[0292] For PK analysis, concentrations of radiotracer in blood (%ID / g) by gamma counting of blood subsamples from two mice were pooled and calculated as follows: TIFF2024525188000010.tif48145

[0293] The %ID values ​​of subsampled blood and plasma were also extrapolated to values ​​for total (systemic) blood and plasma, respectively. For the extrapolation, a blood volume / body weight ratio of 0.072 mL / kg was used (Diehl et al., 2001). A hematocrit value of 0.387 was used for the calculation of total plasma.

[0294] Noncompartmental analysis The %ID / mL values ​​were converted to units of μg / mL prior to noncompartmental analysis using the following formula: TIFF2024525188000011.tif10128

[0295] Terminal clearance rate λ z To determine PK parameters, regression was performed on μg / mL values ​​using Phoenix WinNonlin (Certara) rules in Python. PK parameters were calculated as follows: TIFF2024525188000012.tif80128

[0296] result PK analysis A range of PK parameters was calculated for the radiolabeled antibodies [125I]SIB-60143 and [125I]SIB-61278 at 10 mg / kg and 30 mg / kg (Figure 15A, Figure 15B, and Table 3). More than 95% of the radioactivity was protein bound, with no signs of in vivo instability (Figures 15A and 15B). Approximately 50-70% of the test article was cleared from blood and plasma 24 hours after injection at both dose levels for both antibodies. The elimination half-life of [125I]SIB-60143 in blood and plasma at both doses ranged from 275 to 375 hours. The elimination half-life of [125I]SIB-61278 was longer than the other antibodies at both dose levels; 550-600 hours in plasma and 775-825 hours in blood. Clearance values ​​were similar when comparing the two dose levels of each antibody independently in blood and plasma.

[0297] (Table 3) [ 125 I]SIB-60143 and [ 125 I]NCA analysis results of SIB-61278 TIFF2024525188000013.tif80163

[0298] Biodistribution The ex vivo biodistribution of both antibodies was determined at 24, 72, 168, and 336 hours after injection. 125FIG. 16B shows the effect of [I]SIB-60143 at 30 mg / kg in mice over time. 125 FIG. 16C shows the effect of [I]SIB-60143 at 10 mg / kg on the inhibition of IL-1 in mice over time. 125 FIG. 16D shows the effect of [I]SIB-61278 at 30 mg / kg in mice over time. 125 I]SIB-61278 over time. Distribution patterns were similar for both antibodies at both doses, with low brain concentrations (0.39 ± <0.01-0.91 ± 0.51 %ID / g) and uptake in the heart (3.50 ± 0.03-10.69 ± 2.02 %ID / g), kidney (3.28 ± 0.01-8.40 ± 1.37 %ID / g), liver (1.85 ± 0.01-5.12 ± 0.85 %ID / g) and spleen (1.77 ± 0.05-5.27 ± 0.55 %ID / g). No differences in concentrations were observed with increasing doses. Uptake of 61278 at 14 days was higher than 60143 in all organs, consistent with a longer elimination half-life.

[0299] Both antibodies cleared 50-70% of the test article from blood and plasma 24 hours after injection at both dose levels. 125 The elimination half-life of SIB-60143 ranged from 275 to 375 hours. 125 The elimination half-life of I]SIB-61278 was longer than the other antibodies at both dose levels; 550-600 hours in plasma and 775-825 hours in blood. Biodistribution data were comparable between both antibodies at both dose levels. 125 I]SIB-61278 uptake was observed in all organs [ 125 I]SIB-60143, consistent with its longer elimination half-life.

[0300] Example 9: Crystal structure of the Fab of antibody clone 60143 The X-ray crystal structure of the Fab of antibody clone ABI-60143, containing the heavy and light chain variable domains, in complex with fibrinogen gamma peptide P2, was solved at 1.5 Å resolution. The crystals described were grown using the hanging drop method of vapor diffusion in 96-well plates with a precipitant solution containing 0.1 M sodium cacodylate pH 5.5 and 25% PEG 4000. Crystals were freeze-cooled without additional cryoprotectant by capturing them directly from the growth drop onto a loop and injecting them into liquid nitrogen. The data set was collected at beamline X06DA (PXIII) at the Swiss Light Source (SLS).

[0301] Processing of the data with MOSFLM (Battye et al., 2011) (CCP4) and AIMLESS (Evans & Murshudov, 2013) (CCP4) indicated that the most likely space group was P212121 with unit cell dimensions a = 67.9 Å, b = 73.3 Å, c = 93.6 Å and α = β = γ = 90.0°, resulting in a total cell volume of 465741.4 Å3. Calculation of the Matthews coefficient (2.3 Å3 / Da and solvent content of 46.6%) indicated that there was almost certainly one complete Fab-ABI-60143-P2 complex per asymmetric unit. Models for use in molecular replacement (MR) were selected by BLAST searching the sequences of the Fab heavy and light chains against the PDB. The models with the highest sequence identity were 6ani (Fab heavy chain) and 3pp3 (Fab light chain). A large number of Fab crystal structures in the PDB revealed a large variation in the elbow angle present between the variable and constant domains. This variation in elbow angle can lead to significant differences in the overall tertiary structure of two otherwise highly homologous Fab fragments, causing MR to fail. For this reason, the hinge regions between the heavy and light chain variable and constant domains were removed. Four separate MR search ensembles (VH, CH, VL and CL domains) were created. Amino acid residues were trimmed from the CDRs of the heavy and light chain variable domain models after visual inspection in COOT to prevent potential clashes at the interface with P2 that could cause MR to fail. All four of the input search ensembles required to build a complete Fab were correctly positioned by MR using PHASER (McCoy et al., 2007) (CCP4). The MR output model was subjected to 30 cycles of jelly body refinement using REFMAC5 (Vagin et al., 2004) (CCP4). The protein sequence was mutated to correspond to that of the Fab using CHAINSAW (Stein, 2008) (CCP4).The Fab ABI-60143 model was iteratively improved by successive cycles of model building and refinement until all ordered regions of the protein visible in the electron density map were completed. The P2 peptide chain was manually added to the model in COOT and the complete complex model was refined in REFMAC5. The final protein model included residues 410-421 from chain A (peptide P2), residues 1-127 and 134-214 from chain H (Fab heavy chain) and residues 1-213 from chain L (Fab light chain). Final Rwork = 16.8%, Rfree = 20.5%.

[0302] Fab ADI-60143 and peptide P2 were each one copy per asymmetric unit. The canonical structures of Fab ABI-60143 CDRs were analyzed according to the PyIgClassify database (Adolf-Bryfogle et al., 2014). The heavy chain CDRs were classified as follows: H1-13-1 (CDR-length-cluster) and H2-10-1. CDR H3 was not classified. The light chain CDRs were classified as follows: L1-16-1, L2-8-1 and L3-9-cys7-1.

[0303] Each P2 peptide bound to a single Fab, whose fold was similar, but not identical, to that seen in published fibrinogen gamma chain crystal structures, such as PDB ID: 1fzc.

[0304] Table 4: Fab ADI-60143 and peptide P2 interface analysis TIFF2024525188000014.tif82128

[0305] Table 5: Interface residues involved in Fab ADI-60143 TIFF2024525188000015.tif97128

[0306] Table 6. Data collection, processing, and refinement statistics TIFF2024525188000016.tif183136

[0307] Example 10: Crystal structure of the Fab of antibody clone ADI-61278 The X-ray crystal structure of Fab antibody clone ADI61278, containing heavy and light chain variable domains in complex with fibrinogen gamma peptide P2, was solved at 1.8 Å resolution. The crystals described were grown using the hanging drop method of vapor diffusion in 96-well plates with a precipitant solution containing 1% (w / v) tryptone, 0.001 M sodium azide, 0.05 M sodium HEPES pH 7.0, and 20% PEG 3350. Crystals were freeze-cooled by briefly transferring into a solution containing 4 parts precipitant solution and 1 part 100% (v / v) glycerol, then captured in a loop and injected into liquid nitrogen. The data set was collected at beamline i03 at Diamond Light Source (DLS).

[0308] Processing of the data with MOSFLM (Battye et al., 2011) (CCP4) and AIMLESS (Evans & Murshudov, 2013) (CCP4) indicated that the most likely space group was P21221 with unit cell dimensions a = 47.0 Å, b = 79.0 Å, c = 144.3 Å and α = β = γ = 90.0°, resulting in a total cell volume of 534840.2 Å3. Calculation of Matthews coefficients (2.8 Å3 / Da and solvent content of 55.8%) indicated that there is almost certainly one complete Fab-ADI61278-P2 complex per asymmetric unit. The Fab-ADI61278 model was selected for use in molecular replacement (MR). A large number of Fab crystal structures in the PDB revealed a large variability in the elbow angle present between the variable and constant domains. This variation in elbow angle may lead to a significant difference in the overall tertiary structure of two otherwise highly homologous Fab fragments, causing MR to fail. For this reason, the hinge region between the variable and constant domains of the heavy and light chains was removed. Two separate MR search ensembles (VH-VL heterodimer and CH-CL heterodimer) were created. Both input search ensembles, which are necessary to build a complete Fab, were correctly positioned by MR using PHASER (McCoy et al., 2007) (CCP4). The MR output model was subjected to 30 cycles of jelly body refinement using REFMAC5 (Vagin et al., 2004) (CCP4). The protein sequence was mutated to correspond to that of Fab-ADI61278 using CHAINSAW (Stein, 2008) (CCP4). The Fab ADI61278 model was iteratively improved by successive cycles of model building and refinement until all ordered regions of the protein visible in the electron density map were completed. The P2 peptide chain was manually added to the model in COOT and the complete complex model was refined in REFMAC5.The final protein model included residues 410–421 from chain A (peptide P2), residues 1–126 and 133–214 from chain H (Fab heavy chain) and residues 1–213 from chain L (Fab light chain). Final Rwork = 19.1%, Rfree = 24.0%.

[0309] Fab ADI61278 and peptide P2 were one copy each per asymmetric unit. The canonical structures of Fab ADI61278 CDRs were analyzed according to the PyIgClassify database (Adolf-Bryfogle et al., 2014). The heavy chain CDRs were classified as follows: H1-13-1 (CDR-length-cluster), H2-10-1 and H3-8-1. The light chain CDRs were classified as follows: L1-16-1, L2-8-1 and L3-9-cys7-1. Each P2 peptide was bound to a single Fab. This fold is similar, but not identical, to that found in published fibrinogen gamma chain crystal structures, such as PDB ID: 1fzc.

[0310] Table 7. Fab ADI-61278 and peptide P2 interface analysis TIFF2024525188000017.tif119128

[0311] Table 8. List of involved interface residues TIFF2024525188000018.tif98128

[0312] Table 9. Data collection, processing statistics, and refinement statistics TIFF2024525188000019.tif138136

[0313] Example 11: ADI-60143 and ADI-61278-Fab both bind at the C-terminus of the P2-peptide and show the greatest differences in the CDR-3 region The crystal structures of the Fabs of the above-mentioned antibody clones ADI-60143 and ADI-61278 were superimposed and the P2 peptide binding sites were compared as shown in Figures 17 and 18. These results indicate that both ADI-60143 and ADI-61278-Fabs bind to the C-terminal position of the P2 peptide, and also show that most of the differences are located in the CDR-3 region of the two Fabs.

[0314] Binding of ADI-60143 and ADI-61278 Fab to the P2 peptide was determined using Octet RED384 as described in Example 2 (Figure 19). Amino acid mutations in CDR1 and CDR3 explain the difference in binding on- and off-rates between ADI-60143 and ADI-61278-Fab interacting with the P2-peptide.

[0315] Binding of ADI-60143 Fab and full-length ADI-60143 IgG to rat, mouse or human P2 peptides and extended P2 peptides was also measured by ELISA (Figures 20 and 21). Similar binding profiles were observed for the three P2 peptides from different species. However, the three extended P2 peptides did not bind well to ADI-60143 IgG (Figure 21).

[0316] Example 12: Therapeutic treatment with ADI-60143 inhibits microglial activation and macrophage recruitment in the fibrin-induced encephalitis (FIE) PD model A mouse model of fibrin-induced encephalitis (FIE) was used to test the efficacy of affinity matured antibody clones for inhibition of microglial activation and macrophage recruitment in the treatment of neurodegenerative diseases and inflammation. A total of 78 mice, 6 per group, received fibrinogen via stereotactic injection into the corpus callosum. Antibodies were administered via intravenous (IV) injection. Four hours after fibrinogen injection, mice were IV injected with affinity matured anti-fibrin humanized antibody (10 mg / kg or 30 mg / kg). Brain tissue was prepared at 3 days post-injection. Samples from 73 mice were included for immunohistochemistry and quantification. Coronal sections (30 um) were prepared on a cryostat. Sections were incubated with anti-Iba-1 antibody (microglial marker, 1:750) to detect microglial activation and anti-Mac-2 antibody (macrophage infiltration marker, 1:750). Iba-1 (Iba-1+ areas) and Mac-2 (Mac-2+ areas) immunoreactivity was then calculated, and image collection and quantification were performed in a blinded manner.

[0317] As shown in Figure 22, mice injected with 30 mg / kg ADI-60143 showed a significant reduction in both microglial activation and macrophage recruitment. This reduction was greater than that measured in mice injected with the parent humanized 5B8 antibody (THN227) that had not undergone affinity maturation. These results confirm that affinity matured antibody clone 60143 is therapeutically effective in inhibiting microglial activation and macrophage recruitment for the treatment of neurodegenerative diseases or conditions and / or neuroinflammation.

[0318] Example 13: Antibody clone ADI-60143 is effective in preclinical models of multiple sclerosis To test the therapeutic efficacy of affinity matured antibody clones for the treatment of multiple sclerosis (MS), we used experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. EAE is characterized by the induction of PLP on day 0. 139~151 / CFA (Hooke Kit(trademark) PLP 139~151EAE was induced by immunization with PBS alone, isotype control-human IgG1 (huIgG) alone, antibody clone 60143 (ABI-60143), or dexamethasone, injected therapeutically twice weekly at 5 mg / kg intraperitoneally starting on day 2 until day 44 for dexamethasone and continuing until day 33 for all other groups. Spinal cord tissues of mice were harvested from mice with EAE at maximal disease or from healthy mice, and immunohistochemistry (IHC) staining of spinal cord tissue was performed to determine antibody drug distribution to spinal cord tissue and demyelination.

[0319] For IHC staining, freshly harvested unfixed tissues were placed in OCT compound in a cryomold. The cryomold was placed in isopentane mixed with dry ice for freezing, and the frozen tissues were kept at -80°C. Tissue sections were cut at a thickness of 10 μm in a cryostat and mounted on slides. Slides were stained by immediately fixing them in ice-cold 4% paraformaldehyde in PBS for 10 min at 4°C. Nonspecific binding of primary antibodies to tissues was blocked by incubating slides in blocking buffer (3% bovine serum albumin in PBS; Millipore Sigma, A9576) for 30 min at 25°C. To detect biotinylated antibodies injected into tissues before sectioning, Cy3-conjugated streptavidin (1:100; SA1010, Thermo Fischer Scientific) was diluted in PBS, applied to tissue sections, and incubated at 25°C for 30 min. For fibrin staining, antibody (1:2000, rabbit polyclonal anti-fibrinogen) diluted in PBS was added to the tissue sections and incubated for 60 min at 25° C. FITC donkey anti-rabbit IgG (1:500 in PBS, Jackson ImmunoResearch) was then added to each section and incubated for 30 min at 25° C.

[0320] As shown in Figure 24, the affinity matured antibody (ABI-60143) localized to the spinal cord of mice with EAE at maximal disease, confirming that the affinity matured antibody is well distributed to diseased spinal cord tissue.

[0321] Percent demyelination in tissue sections was quantified by determining the percent of area lacking myelin basic protein (MBP) staining. As shown in Figure 24A, the percent demyelination was significantly reduced in a dose-dependent manner in mice treated with antibody clone 60143.

[0322] Mice were also assessed for hind limb paralysis and clinical scores. As shown in Figure 24B, there was a dose-dependent reduction in demyelination and complete hind limb paralysis in EAE mice treated with affinity matured antibody clone 60143.

[0323] The EAE clinical scores were evaluated according to the criteria shown in the table below. TIFF2024525188000020.tif165129

[0324] Both negative control groups (vehicle and isotype control) developed EAE as expected in this model. The mean maximum severity (MMS) of the first wave of EAE was 2.4 and 2.5 in the vehicle and isotype control groups, respectively. The relapse rate was 73% and 60% in the vehicle and isotype control groups, respectively. The MMS for the relapse period was 2.3 and 1.9 in the vehicle and isotype control groups, respectively. The disease in these groups had a course and severity typical of this model. As shown in Figure 25, mice administered ABI-60143-LALA IgG (antibody clone 60143 containing the Fc stabilizing LALA mutation) had reduced disease development as demonstrated by significantly reduced mean EAE clinical scores and no detectable disease at 17 days post-immunization.

[0325] To confirm that affinity matured antibodies reduced inflammation, segments from the cervical, thoracic, and lumbar regions of the spinal cord (3 segments) were prepared and stained with H&E and anti-CD4 / anti-CD11b (double-labeled) antibodies. Inflammatory foci of approximately 20 cells were counted in each H&E stained section. If the inflammatory infiltrate consisted of more than 20 cells, an estimate was made of how many foci of 20 cells were present. As shown in Figure 26A, there was a significant reduction in the number of inflammatory foci in tissue sections from EAE mice treated with antibody clone ABI-60143 IgG or ABI-60143-LALA IgG compared to the isotype control. There was also a significant reduction in the pro-inflammatory marker CD11b in tissue sections from EAE mice treated with antibody clone ABI-60143 IgG or ABI-60143-LALA IgG compared to the isotype control (Figure 26B). These results confirm that the affinity matured antibodies ABI-60143 IgG and ABI-60143-LALA IgG can reduce inflammation in preclinical models of multiple sclerosis.

[0326] Taken together, these results confirm that affinity matured humanized anti-fibrin antibodies are therapeutically effective in preclinical models of multiple sclerosis.

[0327] Example 14: Antifibrin P2 treatment reduces inflammation in a uveitis model Experimental autoimmune uveitis (EAU) is an organ-specific autoimmune disease that targets the neural retina. This autoimmune response is elicited when animals are immunized with a retinal antigen, in this case interphotoreceptor retinoid-binding protein [IRBP]. To confirm the therapeutic role of antifibrin treatment in inflammatory ocular conditions or diseases, we tested the efficacy of antifibrin affinity maturation antibodies in a rat experimental autoimmune uveitis (EAU) model after intravitreal administration of antifibrin antibodies.

[0328] In this study, 52 Lewis rats were divided into six groups: PBS (group 1), isotype control (group 2), ABI-60143 low dose (group 3), ABI-60143 high dose (group 4), FTY-720 positive control (group 5), and naive (group 6). All animals in all groups, except group 6, were immunized with IRBP in complete Freund's adjuvant (CFA) emulsion on day 0. Similarly, animals in groups 1-4 received a single intravitreal injection of the sponsor's test article on day 0. Animals in group 5 received a single daily oral dose of the positive control FTY-720. After 8-10 days, immunized animals developed uveitis in each eye. Clinical evaluations were performed on all animals at baseline, days 4, 7, 11, and 14 to follow the extent of disease developed. Clinical observations were performed as follows: Frequency: Once each test date Procedure: Groups were randomized prior to evaluation to keep examiners blinded. Animals were observed under a dissecting microscope and scored on a scale of 0 to 4 based on their anterior clinical disease. Photographs of the anterior chamber were taken at the time of clinical evaluation. Clinical Observation Scoring: 0-0.5: No disease; the eye is translucent. Some blood vessels in the iris may be dilated. 1: Congestion of the blood vessels in the iris; abnormal constriction (or dilation) of the pupil. 2: Slight haze in the anterior chamber. 3: Moderately opaque anterior chamber, but pupil still visible. 4: Opaque anterior chamber and unclear pupil.

[0329] All animals were euthanized on day 14 and whole eyes (OU) were harvested immediately after euthanasia. Once death was confirmed, both eyes of each animal were carefully enucleated. One eye was harvested for histological analysis and the other eye for cytokine analysis. Eyes for cytokine analysis were hemisected and the retina harvested. Each eye was carefully oriented for optimal microscopic examination before wax embedding. Sections (5 μm) were cut and stained with hematoxylin and eosin for histological examination and scored as described by Caspi, et al. (2012) according to the following scale summarized below. Histological analysis was blinded to the examiner. Clinical scoring / grade of uveitis was determined as follows: 0: No disease, normal retinal structure. 0.5: Trace. Mild inflammatory cell infiltration in less than one-quarter of the retina, with or without photoreceptor damage. 1: mild inflammation and / or photoreceptor outer segment damage in ≥1 / 4. 2: Mild to moderate inflammation in ≥1 / 4 and / or involvement of the outer nuclear layer. 3: Moderate to marked inflammation in ≥1 / 4 and / or involvement of the inner nuclear layer. 4: Severe inflammation and / or full-thickness retinal damage in ≥1 / 4.

[0330] As shown in Figure 27, rats administered low or high doses of murine ADI-60143 - LALA Fc stabilized antibody clone showed significantly reduced clinical uveitis scores on study day 14. These results confirm that affinity matured anti-fibrin antibodies reduce inflammation in subjects with uveitis and are therapeutically effective in preclinical models of ocular conditions associated with ocular vascular disorders, such as uveitis.

[0331] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

[0332] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0333] Unofficial sequence listing TIFF2024525188000021.tif219164TIFF2024525188000022.tif239164TIFF202 4525188000023.tif239164TIFF2024525188000024.tif239164TIFF20245251880 00025.tif239164TIFF2024525188000026.tif241164TIFF2024525188000027.t if239164TIFF2024525188000028.tif239164TIFF2024525188000029.tif239164 TIFF2024525188000030.tif239164TIFF2024525188000031.tif239164TIFF202 4525188000032.tif239164TIFF2024525188000033.tif239164TIFF20245251880 00034.tif239164TIFF2024525188000035.tif241164TIFF2024525188000036.t if241164TIFF2024525188000037.tif241164TIFF2024525188000038.tif116164

Claims

**Claim 1** A heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences CDR-H1, CDR-H2, and CDR-H3, and A light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences CDR-L1, CDR-L2, and CDR-L3 An isolated antibody that binds to the human fibrin γC or fibrinogen γC domain, each a. CDR-H1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 157, 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, and 229, b. CDR-H2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 158, 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, and 230, c. CDR-H3 comprises the amino acid sequence set forth in any one of SEQ ID NO: 159, 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, and 231, d. CDR-L1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 160, 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, and 232 e. CDR-L2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 161, 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 173, 185, 197, 209, 221, and 233, and f. CDR-L3 comprises the amino acid sequence set forth in any one of SEQ ID NO: 162, 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 174, 186, 198, 210, 222, and 234, An isolated antibody. **Claim 2** The antibody according to claim 1, comprising: (a) A VH sequence selected from the amino acid sequences set forth in any one of SEQ ID NO: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, and 235; (b) A VL sequence selected from the amino acid sequences set forth in any one of SEQ ID NO: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, and 238; (c) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 163 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 166; (d) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 7 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 10; (e) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 19 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 22; (f) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 31 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 34; (g) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 43 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 46; (h) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 55 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 58; (i) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 67 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 70; (j) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 79 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 82; (k) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 91 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 94; (l) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 103 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 106; (m) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 115 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 118; (n) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 127 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 130; (o) A VH sequence set forth in the amino acid sequence of SEQ ID NO: 139 and a VL sequence set forth in the amino acid sequence of SEQ ID NO: 142; (p) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 151 and the VL sequence set forth in the amino acid sequence of SEQ ID NO: 154; (q) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 175 and the VL sequence set forth in the amino acid sequence of SEQ ID NO: 178; (r) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 187 and the VL sequence set forth in the amino acid sequence of SEQ ID NO: 190; (s) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 199 and the VL sequence set forth in the amino acid sequence of SEQ ID NO: 202; (t) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 211 and the VL sequence set forth in the amino acid sequence of SEQ ID NO: 214; (u) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 223 and the VL sequence set forth in the amino acid sequence of SEQ ID NO: 226; or (v) the VH sequence set forth in the amino acid sequence of SEQ ID NO: 235 and the VL sequence set forth in the amino acid sequence of SEQ ID NO:

238. Claim 3 The antibody is (a) a humanized antibody, a human antibody, or a chimeric antibody; (b) comprising a heavy chain human constant region of a class selected from IgG, IgA, IgD, IgE and IgM; (c) comprising a human Fc region, and the human Fc region comprises a human heavy chain constant region of a subclass selected from class IgG and IgG1, IgG2, IgG3 and IgG4; (d) comprising a heavy chain comprising a constant heavy chain sequence set forth in any one of the amino acid sequences of SEQ ID NO: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236; (e) comprising a light chain comprising a constant light chain sequence set forth in any one of the amino acid sequences of SEQ ID NO: 165, 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153, 177, 189, 201, 213, 225, and 237; and / or (f) a monoclonal antibody. The antibody according to claim 1. Claim 4 The Fc region is (a) comprising one or more amino acid substitutions, wherein the one or more substitutions result in an increase in antibody half-life, an increase in ADCC activity, an increase in ADCP activity, or an increase in CDC activity as compared to an Fc having no one or more substitutions; and / or (b) binding to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb, The antibody according to claim 3.

5. wherein the antibody (a) binds to the γ377-395 epitope of the fibrin γC or fibrinogen γC domain; (b) When measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay, about 1, 2, 3, 4, 5, 6, 7, or 8 × 10 -7 ~10 -9 nM or less of K D and binds to a peptide comprising the amino acid sequence set forth in at least one of SEQ ID NO: 241 and 249-253; (c) When measured by surface plasmon resonance (SPR) single - cycle kinetics (SCK) assay, about 8×10 -7 ~10 -9 nM or less of K D binds to a peptide containing the sequence of the γ377 - 395 epitope of the human fibrin γC or fibrinogen γC domain; (d) inhibits Mac-1 binding to the fibrin γC or fibrinogen γC domain; and / or (e) exhibits inhibition of microglia adhesion to the fibrin γC or fibrinogen γC domain, The antibody according to claim 1.

6. An isolated polynucleotide or set of polynucleotides encoding the antibody according to claim 1, its VH, its VL, its light chain, its heavy chain, or its antigen-binding portion, optionally wherein the polynucleotide is cDNA.

7. A vector or set of vectors comprising the polynucleotide or set of polynucleotides according to claim 6.

8. A host cell comprising the polynucleotide or set of polynucleotides according to claim 6 or the vector or set of vectors according to claim 7.

9. Expressing the antibody according to any one of claims 1 to 5 in the host cell according to claim 8, and Isolating the expressed antibody A method for producing an antibody, comprising.

10. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

11. A kit comprising the antibody according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 10 and instructions for use.

12. The pharmaceutical composition according to claim 10 for treating or preventing a neurodegenerative disorder in a subject in need thereof.

13. The pharmaceutical composition according to claim 12, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, multiple sclerosis, spinal cord injury, and stroke.

14. The pharmaceutical composition according to claim 10, for treating a condition associated with Mac-1 binding to fibrin or Mac-1 binding to fibrinogen.

15. The pharmaceutical composition according to claim 10, for inhibiting microglial activation.

16. The pharmaceutical composition according to claim 10, for treating or preventing colitis in a subject in need thereof.

17. An isolated antibody that binds to human fibrin at any one position of amino acid residues Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421 with respect to the amino acid sequence set forth in RefSeq accession number NM_000509.6, or binds to the human fibrin γC or fibrinogen γC domain.

18. The antibody according to claim 17, or a pharmaceutical composition comprising the antibody according to claim 17 and a pharmaceutically acceptable excipient, for treating or preventing a neurodegenerative disorder, an ophthalmic inflammatory condition, or colitis in a subject in need thereof.

19. The antibody or pharmaceutical composition according to claim 18, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, multiple sclerosis, spinal cord injury, and stroke, or the ophthalmic inflammatory condition includes uveitis.

20. The antibody according to claim 17, or a pharmaceutical composition comprising the antibody according to claim 17 and a pharmaceutically acceptable excipient, for treating a condition associated with Mac-1 binding to fibrin or Mac-1 binding to fibrinogen, or for inhibiting microglial activation.

21. A composition comprising the antibody according to any one of claims 1 to 5 and 17, for treating a neurodegenerative disorder, an ophthalmic inflammatory condition, or colitis.

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