Multispecific antigen-binding proteins that bind to human fibrin or fibrinogen gamma C domain and vascular endothelial growth factor and methods of use

Abstract

Described herein are multispecific antigen-binding proteins that bind to human fibrin or fibrinogen γC domains and vascular endothelial growth factor, and methods of using the same. In certain aspects, described herein are pharmaceutical compositions comprising multispecific antigen-binding proteins that bind to fibrin or fibrinogen γC domains and vascular endothelial growth factor. In certain aspects, the antibodies and methods described herein are used to treat ocular disorders or conditions. TIFF2025533065000031.tif61157

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 412,836, filed October 3, 2022, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] background Microglia are resident immune cells of the central nervous system (CNS), including the retina. Microglia have been implicated in many degenerative eye disorders, including retinitis pigmentosa, age-related macular degeneration (AMD), glaucoma, diabetic retinopathy, uveitis, and retinal detachment. While these diseases differ in their underlying etiology, they are all characterized by the loss of photoreceptors or retinal ganglion cells (RGCs), leading to visual impairment and, in some cases, blindness. Microglial activation contributes to both neuronal and oligodendrocyte death through the release of cytokines and nitric oxide. Interventions targeting activated microglia may potentially reduce photoreceptor and RGC death, thereby helping to preserve vision in patients.

[0003] The γ377-395 epitope of the fibrin or fibrinogen γC domain is the binding epitope of fibrin to CDIIb. 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 the beneficial effects of fibrin in blood clotting.

[0004] Neovascular ocular conditions, such as retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration, are the leading causes of irreversible vision loss in developed countries. Evidence suggests that vascular endothelial growth factor (VEGF) promotes angiogenesis in each of these conditions.

[0005] Therefore, safe and effective therapeutic agents that inhibit fibrin-induced microglial activation and also block VEGF without affecting the beneficial effects of fibrin on blood clotting are needed for the treatment of ocular disorders and conditions. Summary of the Invention

[0006] overview In certain aspects, described herein are multispecific antigen-binding proteins comprising at least two different antigen-binding regions, wherein a first antigen-binding region specifically binds to human fibrin or fibrinogen γC domain and a second antigen-binding region specifically binds to vascular endothelial growth factor (VEGF).

[0007] In certain embodiments, the first antigen-binding region comprises 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 NO: 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, 229, or 258; and CDR-H2 comprises the sequence set forth in SEQ ID NO: CDR-H3 comprises the sequence set forth in SEQ ID NOs: 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 159, 171, 183, 195, 207, 219, 231, or 260; CDR-L1 comprises the sequence set forth in SEQ ID NOs: CDR-L2 comprises the sequence set forth in SEQ ID NOs: 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 160, 172, 184, 196, 208, 220, 232, or 262; CDR-L2 comprises the sequence set forth in SEQ ID NOs: 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 161, 173, 185, 197, 209, 221, 233, or 263; and CDR-L3 comprises the sequence set forth in SEQ ID NOs: 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234 or 264.

[0008] In certain embodiments, the multispecific antigen-binding protein 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.

[0009] In certain embodiments, the multispecific antigen-binding protein comprises a VL sequence selected from the sequences 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.

[0010] In certain embodiments, the first antigen-binding region comprises a VH sequence set forth in one of SEQ ID NO:7 and a VL sequence set forth in SEQ ID NO:10. In certain embodiments, the first antigen-binding region 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 first antigen-binding region 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 first antigen-binding region 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 first antigen-binding region 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 first antigen-binding region 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 first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79 and the VL sequence set forth in SEQ ID NO: 82. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91 and the VL sequence set forth in SEQ ID NO: 94. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103 and the VL sequence set forth in SEQ ID NO: 106. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115 and the VL sequence set forth in SEQ ID NO: 118. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127 and the VL sequence set forth in SEQ ID NO: 130. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139 and the VL sequence set forth in SEQ ID NO: 142. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151 and the VL sequence set forth in SEQ ID NO: 154. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163 and the VL sequence set forth in SEQ ID NO: 166.In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175 and the VL sequence set forth in SEQ ID NO: 178. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187 and the VL sequence set forth in SEQ ID NO: 190. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199 and the VL sequence set forth in SEQ ID NO: 202. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211 and the VL sequence set forth in SEQ ID NO: 214. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223 and the VL sequence set forth in SEQ ID NO: 226. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 235 and the VL sequence set forth in SEQ ID NO: 238. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO:256 and the VL sequence set forth in SEQ ID NO:257.

[0011] In certain embodiments, the first antigen-binding region comprises a humanized antibody, a human antibody, or a chimeric antibody. In certain embodiments, the first antigen-binding region comprises a humanized antibody. In certain embodiments, the first antigen-binding region comprises a human heavy chain 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 the 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.

[0012] 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.

[0013] In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 7 and the VL sequence set forth in SEQ ID NO: 10; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19 and the VL sequence set forth in SEQ ID NO: 22; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 31 and the VL sequence set forth in SEQ ID NO: 34; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43 and the VL sequence set forth in SEQ ID NO: 46; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 55 and the VL sequence set forth in SEQ ID NO: 58; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67 and the VL sequence set forth in SEQ ID NO: 70; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79 and the VL sequence set forth in SEQ ID NO: 82; and the human Fc region comprises wild-type human IgG1 Fc. In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91 and the VL sequence set forth in SEQ ID NO: 94; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103 and the VL sequence set forth in SEQ ID NO: 106; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115 and the VL sequence set forth in SEQ ID NO: 118; and the human Fc region comprises wild-type human IgG1 Fc.In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127 and the VL sequence set forth in SEQ ID NO: 130; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139 and the VL sequence set forth in SEQ ID NO: 142; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151 and the VL sequence set forth in SEQ ID NO: 154; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163 and the VL sequence set forth in SEQ ID NO: 166; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175 and the VL sequence set forth in SEQ ID NO: 178; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187 and the VL sequence set forth in SEQ ID NO: 190; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199 and the VL sequence set forth in SEQ ID NO: 202; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211 and the VL sequence set forth in SEQ ID NO: 214; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223 and the VL sequence set forth in SEQ ID NO: 226; and the human Fc region comprises wild-type human IgG1 Fc. In certain embodiments, the first antigen-binding region 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.In a particular embodiment, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 256 and the VL sequence set forth in SEQ ID NO: 257; and the human Fc region comprises wild-type human IgG1 Fc.

[0014] In certain embodiments, the Fc region comprises one or more amino acid substitutions, wherein the one or more substitutions result in increased 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.

[0015] In certain embodiments, the first antigen-binding region comprises a monoclonal antibody. In certain embodiments, the first antigen-binding region consists of a monoclonal antibody. In certain embodiments, the first antigen-binding region binds to the γ377-395 epitope of the fibrin or fibrinogen γC domain. In certain embodiments, the first antigen-binding region has a γ 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 wherein the first antigen-binding region 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 first antigen-binding region binds to a peptide comprising an amino acid sequence of about 8×10 as measured by a surface plasmon resonance (SPR) single cycle kinetics (SCK) assay. -5 K below M D wherein the first antigen-binding region binds to a peptide comprising the sequence of the γ377-395 epitope of the human fibrin or fibrinogen γC domain. In certain embodiments, the first antigen-binding region inhibits Mac-1 binding to the fibrin or fibrinogen γC domain. In certain embodiments, the first antigen-binding region inhibits microglial adhesion to the fibrin or fibrinogen γC domain.

[0016] In certain embodiments, the first antigen-binding region 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, the first antigen-binding region binds to human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten 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 first antigen-binding region comprises a VH region comprising 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 first antigen-binding region comprises a VH region comprising a paratope comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the following 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 first antigen-binding region comprises a VH region comprising a paratope comprising amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103.In certain embodiments, the first antigen-binding region comprises a VH region comprising a paratope comprising 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, Gly 102, and Trp 103. In certain embodiments, the first antigen-binding region comprises a VH region comprising 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, Gly 102, and Trp 103. In certain embodiments, the first antigen-binding region comprises a VL region comprising a paratope comprising 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 first antigen-binding region comprises a VL region comprising a paratope comprising at least two, three, four, five, six, seven, eight, nine, or all ten 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 first antigen-binding region comprises a VL region comprising 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 first antigen-binding region comprises a VL region comprising 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 first antigen-binding region comprises a VL region comprising 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. In certain embodiments, the first antigen-binding region comprises a humanized antibody, a human antibody, or a chimeric antibody.

[0017] In certain embodiments, the first antigen-binding region comprises a humanized antibody. In certain embodiments, the first antigen-binding region comprises a human heavy chain 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 the 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. In certain embodiments, the Fc region comprises one or more amino acid substitutions, wherein the one or more substitutions result in increased 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.

[0018] In certain embodiments of the multispecific antigen-binding proteins described herein, the second antigen-binding region comprises a heavy chain comprising a second VH chain sequence comprising the three heavy chain CDR sequences CDR-H1, CDR-H2 and CDR-H3, and a light chain comprising a second VL chain sequence comprising the three light chain CDR sequences CDR-L1, CDR-L2 and CDR-L3.

[0019] In certain embodiments, the second antigen-binding region binds to one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D. In certain embodiments, the second antigen-binding region comprises a VH amino acid sequence that is 90% or more, 95% or more, or 99% or more identical to the VH amino acid sequence set forth in SEQ ID NO: 269. In certain embodiments, the second antigen-binding region comprises a VL amino acid sequence that is 90% or more, 95% or more, or 99% or more identical to the VL amino acid sequence set forth in SEQ ID NO: 268. In certain embodiments, the second antigen-binding region binds to an epitope of VEGF-A that includes at least one of amino acids 82-91 set forth in SEQ ID NO. 265. In certain embodiments, the second antigen-binding region comprises a sequence that is 90% or more, 95% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 266 or 267. In certain embodiments, the second antigen-binding region does not comprise an Fc domain.

[0020] In certain embodiments, the multispecific antigen-binding protein comprises a heterodimeric Fc domain. In certain embodiments, the multispecific antigen-binding protein comprises a bispecific antibody. In certain embodiments, the multispecific antigen-binding protein consists of a bispecific antibody. In certain embodiments, the multispecific antigen-binding protein comprises one or more additional antigen-binding domains that specifically bind to human fibrin or fibrinogen γC domain. In certain embodiments, the multispecific antigen-binding protein comprises one or more additional antigen-binding domains that specifically bind to VEGF.

[0021] In certain embodiments, the multispecific antigen-binding protein is used in the treatment of an ocular disorder or condition. In certain embodiments, the multispecific antigen-binding protein is formulated for administration to a subject by intravitreal injection.

[0022] In certain aspects, described herein is an isolated polynucleotide or set of polynucleotides encoding a first antigen-binding region, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof, optionally wherein the polynucleotide or set of polynucleotides comprises cDNA. In certain aspects, described herein is an isolated polynucleotide or set of polynucleotides encoding a second antigen-binding region, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof, optionally wherein the polynucleotide or set of polynucleotides comprises cDNA.

[0023] In certain aspects, described herein is a vector or set of vectors comprising a polynucleotide or set of polynucleotides described herein.

[0024] In certain aspects, described herein are host cells comprising a polynucleotide or set of polynucleotides, or a vector or set of vectors described herein.

[0025] In certain aspects, described herein are methods of producing a multispecific antigen binding protein described herein, the method comprising expressing the multispecific antigen binding protein, or antigen-binding region thereof, in a host cell described herein, and isolating the expressed multispecific antigen binding protein, or antigen-binding region thereof.

[0026] In certain aspects, described herein are pharmaceutical compositions comprising a multispecific antigen-binding protein described herein and a pharmaceutically acceptable excipient.

[0027] In certain aspects, described herein are kits comprising a multispecific antigen binding protein described herein or a pharmaceutical composition described herein and instructions for use.

[0028] In certain aspects, described herein are methods for treating an ocular disorder or condition, the methods comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen binding protein described herein or a pharmaceutical composition described herein. In certain embodiments, the ocular disorder or condition is selected from the group consisting of retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment.

[0029] In certain aspects, described herein are methods for treating a condition associated with Mac-1 binding to fibrin or fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen binding protein described herein or a pharmaceutical composition thereof described herein.

[0030] In certain aspects, described herein are methods of inhibiting microglial activation, the methods comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen binding protein described herein or a pharmaceutical composition thereof described herein.

[0031] In certain aspects, described herein are methods of producing a multispecific antigen binding protein, the method comprising expressing the multispecific antigen binding protein, or antigen-binding region thereof, in a host cell as described herein, and isolating the expressed multispecific antigen binding protein, or antigen-binding region thereof.

[0032] In certain aspects, described herein are methods of preventing an ocular disorder or condition, the method comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen binding protein described herein or a pharmaceutical composition thereof described herein.

[0033] In certain aspects, described herein are methods of treating an ocular disorder or condition associated with increased angiogenesis in a subject in need thereof, comprising administering to the subject a multispecific antigen binding protein described herein or a pharmaceutical composition thereof described herein.

[0034] A method of preventing an ocular disorder or condition associated with increased angiogenesis in a subject in need thereof, comprising administering to the subject a multispecific antigen binding protein as described herein, or a pharmaceutical composition thereof as described herein.

[0035] 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 explanation of the drawings]

[0036] [Figure 1] 1 is a graph depicting the uveitis clinical scores of rats administered intravitreally with an isotype control, murine antibody clone 60143 with an Fc-stabilizing LALA mutation (low dose = 10 μg / eye; high dose = 50 μg / eye), positive control FTY-720 (administered by oral gavage at a dose of 0.3 mg / kg), and naive mice without induced experimental autoimmune encephalomyelitis (EAE). [Figure 2] FIG. 1 is a graph showing vascular leakage by quantitative fluorescein angiography (qFA) in rats treated at the indicated concentrations with the anti-VEGF antigen-binding construct Eylea (aflibercept) and the murine anti-fibrin antibody clone 60143 with an Fc-stabilizing LALA mutation ("m60143-LALA"). [Figure 3] 1 is a graph showing choroidal neovascularization (CNV) lesion area in rats treated at the indicated concentrations with the anti-VEGF antigen-binding construct Eylea (aflibercept) and the murine anti-fibrin antibody clone 60143 with the Fc-stabilizing LALA mutation ("m60143-LALA"). DETAILED DESCRIPTION OF THE INVENTION

[0037] 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 methodology 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 the protocols and conditions defined by the manufacturer, unless otherwise specified.

[0038] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0039] It is understood that the aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0040] 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 includes the recited components and can include other components other than the explicitly recited components that do not substantially affect the condition being treated, but do not include any other components that substantially affect the condition being treated; or, if 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 concentrations or amounts sufficient to substantially affect the condition being treated. When a method is described as "consisting essentially of" recited steps, the method may include the recited steps and include other steps that do not substantially affect the condition being treated, but the method does not include any other steps other than the explicitly recited steps that substantially affect the condition being treated. As a non-limiting example, when a composition is described as "consisting essentially of" components, the composition may further include any amount of a pharmaceutically acceptable carrier, vehicle, or diluent, and such other components that do not substantially affect the condition being treated.

[0041] 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 integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0042] 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 cell and their derived progeny, respectively. Such antibodies may not be completely identical in nucleic acid content to the parent cell and may contain mutations. A "recombinant host cell" or "host cell" refers to a cell containing an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells.

[0043] 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.

[0044] As used herein, the term "prokaryote" refers to prokaryotic organisms. For example, non-eukaryotes may be from the phylogenetic domains Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.), or Archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, e.g., Haloferax volcanii, etc.). volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) may belong to the phylogenetic domain.

[0045] As used herein, "effective amount" or "therapeutically effective amount" refers to the 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, that is effective, alone or in combination with another therapy, to produce or contribute to the desired therapeutic effect. Examples of desired therapeutic effects are an enhanced immune response, slowing or delaying tumor development; disease stabilization; or amelioration of one or more symptoms. An effective amount can be given in one or more administrations.

[0046] 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 preventing disease recurrence, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or palliating the disease state, and remission or improved prognosis.

[0047] 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.

[0048] 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.

[0049] The term "in vitro" refers to processes performed within living cells that are grown apart from the organism, for example, grown in tissue culture.

[0050] The term "in vivo" refers to processes that take place within an organism.

[0051] The term "package insert" is used to refer to instructions customarily included in commercial packages (e.g., kits) of therapeutic or diagnostic agents that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic agent.

[0052] 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.

[0053] The terms "co-administration," "co-administering," and "in combination with" include the 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 before the administration of a second therapeutic agent.

[0054] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0055] 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.

[0056] 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.

[0057] The term "about" implies and encompasses the indicated value and a range above and below that value. In certain embodiments, the term "about" refers to the specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "about" refers to the specified value ± 1 standard deviation of that value.

[0058] 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 and agonizes a receptor.

[0059] 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.

[0060] For any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0061] The term "optionally," when used consecutively, means the inclusion of one to all of the listed combinations, and contemplates all subcombinations.

[0062] 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).

[0063] The term "affinity" refers to the strength of the sum of noncovalent 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 intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope).

[0064] 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.

[0065] As used herein, "k a " (M -1 ×sec -1 The term k ) refers to the binding rate constant for a particular antibody-antigen interaction. This value is k on Also called value.

[0066] 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 measured 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.

[0067] As used herein, "K A " (M -1 The term K ) refers to the binding equilibrium constant of a particular antibody-antigen interaction. A = k a / k d .

[0068] The term "multispecific antigen-binding protein" is used herein in its broadest sense and includes molecules comprising polypeptides that have the ability to bind to two or more different antigens. Multispecific antigen-binding proteins include multispecific antibodies and antibodies that comprise binders with affinity for one or more antigens.

[0069] The term "antigen-binding region" or "antigen-binding region" is used herein in its broadest sense and includes the region of a multispecific antigen-binding protein comprising a polypeptide that contains affinity for an antigen or epitope.

[0070] The term "fibrin antigen-binding region" refers to an antigen-binding region that has affinity for fibrin, and includes fibrin antibodies.

[0071] The term "VEGF antigen binding region" refers to an antigen binding region that has affinity for one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

[0072] The term "antibody" is used herein in its broadest sense and includes specific 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. Antibodies include any alternative antibody formats known in the art, including, but not limited to, single-domain antibodies, diabodies, knobs-into-hole antibodies, scFv, scFv dimers, BsFv, dsFv, a(dsFv)2, dsFv-dsFv', Fv fragments, Fab, Fab', F(ab')2, ds diabodies, minibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0073] 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 antibodies provided herein bind to an epitope of fibrin that is conserved between or within fibrin proteins from different species.

[0074] The term "epitope" refers to the part of an antigen that specifically binds to an antibody.

[0075] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops").

[0076] 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, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0077] 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 the 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.

[0078] 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 compared to the non-human species antibody.

[0079] The term "multispecific antibody" or "multi-specific antibody" refers to an antibody that comprises two or more different antigen-binding regions that collectively specifically bind to two or more different epitopes.

[0080] 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 in each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.

[0081] 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, excluding variations that may normally arise during the production of monoclonal antibodies. Such variations 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.

[0082] 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.

[0083] A "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.

[0084] 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 intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0085] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0086] A "single-chain Fv" or "sFv" or "scFv" comprises the VH and VL domains of an antibody, wherein 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 enables the scFv to form the desired structure for antigen binding. For a review of scFvs, 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.

[0087] 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.

[0088] 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 can be easily expressed 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).

[0089] 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 that of a naturally occurring antibody 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.

[0090] The term "antibody fragment" refers to an antibody that comprises 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.

[0091] The term "Fc domain" or "Fc region" is used herein to define the 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.

[0092] The term "substantially purified" refers to constructs described herein, or variants thereof, that may be substantially or essentially free from components that are 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.

[0093] 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 specified 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 needed 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., a functional domain, or over the entire length of the two sequences being compared.

[0094] In sequence comparison, usually, one sequence 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, and if necessary, partial sequence coordinate is designated, and sequence algorithm program parameter is designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence according to designated program parameter.

[0095] Optimal alignment of sequences for comparison can be achieved, 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 search for similarity 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).

[0096] Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. 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.

[0097] It should be noted 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.

[0098] Multispecific antigen-binding proteins The present application provides multispecific antigen-binding proteins comprising at least two different antigen-binding domains, wherein at least one antigen-binding domain binds to a human fibrin or fibrinogen γC domain and at least one antigen-binding domain binds to at least one isoform of VEGF.

[0099] The multispecific antigen-binding proteins of the present disclosure can comprise any antigen-binding domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, binding antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single-domain antibodies (sdAbs) such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camelid-derived nanobodies, as well as any antigen-binding domain that binds to an alternative scaffold known in the art to function as an antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., a single-chain TCR. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the multispecific antigen-binding protein will ultimately be used. For example, when used in humans, it may be beneficial for the antigen-binding domain of the multispecific antigen-binding protein to comprise human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

[0100] In some embodiments, the antigen-binding domain of the multispecific antigen-binding protein comprises an antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a chimeric antibody. In some embodiments, the antigen-binding domain comprises an antigen-binding fragment of an antibody. In some embodiments, the antigen-binding domain comprises an F(ab) fragment. In certain embodiments, the antigen-binding domain comprises an F(ab') fragment. In some embodiments, the antigen-binding domain comprises an scFv. In some embodiments, the antigen-binding domain comprises two single-chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a different epitope on the same antigen. In some embodiments, the antigen-binding domain comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to different epitopes on the same antigen. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In certain embodiments, the VH and VL are separated by a peptide linker. In certain embodiments, the scFv comprises a VH-L-VL or VL-L-VH structure, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0101] In some embodiments, each of the one or more scFvs comprises a VH-L-VL or VL-L-VH structure, where VH is the heavy chain variable domain, L is a peptide linker, and VL is the light chain variable domain. When two or more scFvs are linked, each scFv can be linked to the next scFv with a peptide linker. In some embodiments, each of the one or more scFvs is separated by a peptide linker. In some embodiments, each of the two scFvs binds to a different epitope on the same antigen.

[0102] In some embodiments, the antigen-binding domain comprises a single domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0103] In some embodiments, the multispecific antigen-binding proteins of the present disclosure may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds to the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different antigen. In some embodiments, the two or more antigen-binding domains provide logical gating, such as "or" logical gating, to the multispecific antigen-binding protein.

[0104] In some embodiments, the multispecific antigen-binding protein comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are linked to each other via a flexible linker. In some embodiments, each of the two antigen-binding domains can be independently selected from an antibody, an antigen-binding fragment of an antibody, an scFv, an sdAb, or a recombinant fibronectin domain.

[0105] In certain embodiments, the multispecific antigen-binding protein comprises antigen-binding domains comprising a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of the bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) has its VH (VH1) positioned upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) has its VL (VL2) positioned upstream of its VH (VH2), such that the entire bispecific antibody molecule has the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) has its VL (VL1) positioned upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) has its VH (VH2) positioned upstream of its VL (VL2), such that the overall bispecific antibody molecule has the configuration VL1VH1-VH2-VL2. In some embodiments, a linker is positioned between the two antibodies or antibody fragments (e.g., scFvs), e.g., between VL1 and VL2 when the construct is configured as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is configured as VL1-VH1-VH2-VL2. The linker can be a linker described herein, e.g., a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. Generally, the linker between two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is disposed between the VL and VH of a first scFv. In some embodiments, the linker is disposed between the VL and VH of a second scFv. In constructs with multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a multispecific antigen-binding protein comprises a VL, a VH, and may further comprise one or more linkers in an arrangement described herein.

[0106] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous 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 can 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.

[0107] An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kD) and one "heavy chain" (approximately 50-70 kD). The N-terminal domain of each chain defines a variable region of approximately 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. An IgG1 heavy chain is composed of VH, CH1, CH2, and CH3 domains, respectively, from the N-terminus to the C-terminus. The light chain is composed of VL and CL domains, from the N-terminus to the C-terminus. An IgG1 heavy chain includes a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are derived from or derived from immunoglobulin-based constructs such as diabodies or nanobodies. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain derived from a heavy chain antibody, such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain derived from a mammalian antibody, such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody, or any chimeric antibody.

[0108] In some embodiments, the multispecific antigen-binding proteins provided herein comprise one or more heavy chains. 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.

[0109] In some embodiments, the multispecific antigen-binding protein comprises an IgG1 antibody. In some embodiments, the multispecific antigen-binding protein comprises an IgG3 antibody. In some embodiments, the multispecific antigen-binding protein comprises an IgG2 antibody. In some embodiments, the multispecific antigen-binding protein comprises an IgG4 antibody.

[0110] Typically, a native four-chain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or complementarity-determining regions (CDRs), the latter of which exhibit the greatest sequence variability and / or are involved in antigen recognition. With the exception of CDR1 in the VH, CDRs generally contain amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein to refer to the portions of the variable regions that form the antigen-binding region. This particular region is 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 this definition includes overlapping or subsets of amino acid residues relative 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 encompassing 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 amino acid sequence of the variable region of an antibody.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] (Table A) Residues in the CDRs according to the Kabat and Chothia numbering scheme. TIFF2025533065000002.tif42133 * The C-terminus of CDR-H1, numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the CDR.

[0115] 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 specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0116] 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. The antigen-binding domain can comprise CDRs 1, 2, and 3 from the heavy chain, in that order; and CDRs 1, 2, and 3 from the light chain, in that order.

[0117] Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural and 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. Epitopes may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for determining epitopes, such as testing the binding of the antibody to fibrin variants with different point mutations or chimeric fibrin variants.

[0118] To screen for antibodies that bind to the epitope on the target antigen (e.g., fibrin) to which the 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.

[0119] A chimeric antibody has a portion of the heavy and / or light chain derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0120] 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 (e.g., obtained from a human source or designed de novo) that utilizes the human antibody repertoire or human antibody coding sequences. Human antibodies specifically exclude humanized antibodies.

[0121] 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, specific amino acids in the framework and constant domains of the heavy and / or light chains of a 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 because the altered amino acid residues are not important for immunospecific binding of the antibody to its antigen, or because the changes made to the amino acid sequence are conservative changes such that binding of the humanized antibody to the antigen is not significantly worse than 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.

[0122] 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" or "bi-specific antibody"). In some aspects, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody").

[0123] The antigen binding domain of the multispecific antigen binding protein may comprise an antibody or variable domain described herein, such as the clones set forth 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.

[0124] In some embodiments, the multispecific antigen-binding protein comprises a monoclonal antibody.

[0125] In some embodiments, the multispecific antigen-binding protein or antigen-binding region thereof is produced by a hybridoma, hi other embodiments, the antibody is produced by a recombinant cell engineered to express the desired variable and constant domains.

[0126] In some embodiments, the multispecific antigen binding protein comprises one or more single chain antibodies or other antibody derivatives or variants thereof that retain the antigen specificity and lower hinge region.

[0127] In some embodiments, the multispecific antigen-binding protein may be a multifunctional 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 an scFv.

[0128] In some embodiments, the antibody can form an immune complex, for example, the immune complex can be a tumor cell coated with the antibody.

[0129] In sequence comparison, usually, one sequence 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, and if necessary, partial sequence coordinate is designated, and sequence algorithm program parameter is designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence according to designated program parameter.

[0130] Optimal alignment of sequences for comparison can be achieved, 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 search for similarity 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).

[0131] 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 / ).

[0132] fibrin antigen-binding region V H domain In some embodiments, the fibrin antigen-binding region provided herein is 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 sequence V of SEQ ID NO: 13. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 14. H In some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 15.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.

[0133] In some embodiments, the fibrin antigen-binding regions provided herein are selected from the group consisting of exemplary V and VF domains provided in SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. 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, 20, 21, 22, 23, 24, or 25 amino acid substitutions. HIn 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 by, for example, 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, but may be de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0134] V L domain In some embodiments, the fibrin antigen-binding region provided herein comprises a VL sequence selected from SEQ ID NO:21.

[0135] In some embodiments, the antibodies provided herein comprise a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to 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 by, for example, 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] VH-VL combinations In some embodiments, the fibrin antigen-binding region provided herein is 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.

[0137] In some embodiments, the antibodies provided herein comprise a 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 sequence 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 sequence V of SEQ ID NO: 14. H Sequence and V of SEQ ID NO: 21 LIn some embodiments, the antibodies provided herein comprise the sequence V of SEQ ID NO: 15. 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.

[0138] 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.

[0139] In some embodiments, the fibrin antigen-binding regions provided herein are selected from the group consisting of exemplary V and VF domains provided in SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. 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 NOs: 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 by, for example, 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, but may be isolated de novo, for example, by the methods provided herein for obtaining antibodies.

[0140] CDR In some embodiments, the fibrin antigen-binding region provided herein is 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 two to three 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.

[0141] In some embodiments, the CDR is a CDR having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to 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 the 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 the 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 fibrin antigen-binding regions 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, but may be isolated de novo, e.g., by the methods provided herein for obtaining antibodies.

[0142] In some embodiments, the fibrin antigen-binding region provided herein comprises 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.

[0143] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 the 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 the 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.

[0145] In some embodiments, the fibrin antigen-binding region provided herein comprises a selected CDR-H3 of SEQ ID NOs: 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 to the CDR-H3 of SEQ ID NOs: 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the CDR-H3 is a selected CDR-H3 of SEQ ID NOs: 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 fibrin antigen-binding regions 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, but may be isolated de novo, e.g., by the methods provided herein for obtaining antibodies.

[0146] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises the 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 to the CDR-H1 of SEQ ID NO: 1. In some embodiments, the CDR-H1 is the 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 fibrin antigen-binding region provided herein comprises 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 to 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0156] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0157] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0158] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0159] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0160] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0161] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0162] In some embodiments, the fibrin antigen-binding region provided herein comprises 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 the 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 the 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 the 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0163] In some embodiments, the fibrin antigen-binding region provided herein comprises the 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 to the CDR-L3 of SEQ ID NO: 6. In some embodiments, the CDR-L3 is the 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0164] In some embodiments, the fibrin antigen-binding region provided herein comprises the 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 to the CDR-L2 of SEQ ID NO: 5. In some embodiments, the CDR-L2 is the 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0165] In some embodiments, the fibrin antigen-binding region provided herein comprises the 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 to the CDR-L1 of SEQ ID NO: 4. In some embodiments, the CDR-L1 is the 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 fibrin antigen-binding region described in this paragraph is referred to herein as a "variant." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0166] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-L3 of SEQ ID NO: 6 and CDR-L2 of SEQ ID NO: 5. In some embodiments, the antibodies provided herein comprise CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, CDR-L3 is CDR-L3 of SEQ ID NO: 6 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with up to 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0167] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0168] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 25, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0169] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0170] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 27, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0171] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 28, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0172] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 29, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0173] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H3 of SEQ ID NO: 30, CDR-H2 of SEQ ID NO: 2, CDR-H1 of SEQ ID NO: 1, CDR-L3 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 5, and 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, 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; 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; CDR-H1 is CDR-H1 of SEQ ID NO: 1 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 6 with a maximum of 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 5 with a maximum of 1, 2, 3, or 4 amino acid substitutions; and 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 fibrin antigen-binding regions described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, 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 de novo isolated, for example, by the methods provided herein for obtaining antibodies.

[0174] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 24, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0175] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 25, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0176] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 26, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0177] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 27, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0178] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 28, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0179] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 29, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0180] In some embodiments, the fibrin antigen-binding region provided herein comprises CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, CDR-H3 of SEQ ID NO: 30, CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0181] epitope In certain embodiments, described herein are fibrin antigen-binding regions that bind to human fibrin or fibrinogen γC domain, wherein the fibrin antigen-binding region 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, the fibrin antigen-binding region binds to human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten 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 fibrin antigen-binding region 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.

[0182] Paratope In certain embodiments, the fibrin antigen-binding region described herein comprises a VH region comprising 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, Gly 102, and Trp 103. In certain embodiments, the fibrin antigen-binding region comprises a VH region comprising a paratope comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the following 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 comprising a paratope comprising amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103. In certain embodiments, the fibrin antigen-binding region comprises a VH region comprising a paratope comprising 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, Gly 102, and Trp 103. In certain embodiments, the fibrin antigen-binding region comprises a VH region comprising 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, Gly 102, and Trp 103.

[0183] In certain embodiments, the fibrin antigen-binding region comprises a VL region comprising a paratope comprising 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 comprising a paratope comprising at least two, three, four, five, six, seven, eight, nine, or all ten 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 comprising 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 comprising 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 comprising 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.

[0184] In certain embodiments, the paratope of the fibrin antigen-binding region 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.

[0185] VEGF antigen binding region In certain aspects, the multispecific antigen-binding proteins described herein comprise at least one antigen-binding region that binds to VEGF. In certain aspects, the antigen-binding region that binds to VEGF binds one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D. In certain embodiments, the antigen-binding region that binds to VEGF comprises a VH amino acid sequence that is 90% or more, 95% or more, or 99% or more identical to the VH amino acid sequence set forth in SEQ ID NO: 269. In certain embodiments, the VEGF antigen-binding region comprises a VL amino acid sequence that is 90% or more, 95% or more, or 99% or more identical to the VL amino acid sequence set forth in SEQ ID NO: 268. In certain embodiments, the VEGF antigen-binding region binds to an epitope of VEGF-A comprising at least one of amino acids 82-91 of SEQ ID NO. 265. In certain embodiments, the VEGF antigen-binding region binds to an epitope of VEGF-A comprising amino acids 82-91 of SEQ ID NO. 265. In certain embodiments, the VEGF antigen-binding region comprises a sequence that is 90% or more, 95% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 266 or 267.

[0186] In certain embodiments, the VEGF antigen-binding region is a single domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, minibody, nanobody, domain antibody, or bivalent domain antibody. In certain embodiments, the VEGF antigen-binding region is not an antibody. In certain embodiments, the VEGF antigen-binding region is an antibody without an Fc region. In certain embodiments, the VEGF antigen-binding region comprises an Fc region of class IgA, IgD, IgE, IgG, or IgM. In certain embodiments, the VEGF antigen-binding region comprises one or more modifications known in the art to stabilize the Fc region and improve in vivo circulatory half-life. In certain embodiments, the VEGF antigen-binding region comprises one or more modifications to promote selective binding to an Fc-gamma receptor and / or C1q.

[0187] In certain embodiments, the multispecific antigen binding proteins described herein comprise multiple VEGF antigen binding regions. In certain embodiments, the multispecific antigen binding proteins comprise two, three, four, five, or more VEGF antigen binding regions. In certain embodiments, the multispecific antigen binding proteins comprise two or more different VEGF antigen binding regions.

[0188] Fc area The structures of various immunoglobulin Fc regions 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 can be a naturally occurring Fc region or an Fc region that has been modified as described in the art or elsewhere in this disclosure.

[0189] 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 known as 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 forming the dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc polypeptide of a dimeric IgG Fc comprises the IgG CH2 and IgG CH3 constant domain sequences. The Fc may be of the IgA, IgD, IgE, IgG, or IgM class, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0190] 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 (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes can also be bound by a particular FcR (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. Inhibiting 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)).

[0191] Modification of CH2 domain can affect FcR binding to Fc. Some amino acid modifications in the Fc region are known in the art to selectively alter 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.

[0192] 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.

[0193] In some embodiments, the antibodies described herein contain modifications to improve their ability to mediate effector function. Such modifications are known in the art and include defucosylation or engineering the affinity of Fc for activating receptors, primarily FCGR3a in the case of ADCC and C1q in the case of CDC. Table B below summarizes various designs reported in the literature for engineering effector function.

[0194] 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 encoding an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the antibody-producing cells from adding the sugar "fucose" to N-linked antibody carbohydrate moieties. (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 antibody production are selected for their ability to produce lower levels of antibody fucosylation; the antibodies can be completely defucosylated (meaning they contain no detectable fucose), or they can be partially defucosylated, meaning that the isolated antibody contains less than 95%, 85%, 75%, 65%, 55%, 45%, 35%, 25%, 15%, or 5% of the amount of fucose typically found for a similar antibody produced in a mammalian expression system.

[0195] 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 antibody can be defucosylated to improve effector function.

[0196] (Table B) CH2 domain and effector function engineering TIFF2025533065000003.tif78136

[0197] Fc modifications that reduce FcgR and / or complement binding and / or effector function are known in the art. Recent publications describe strategies 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 an IgG2 / IgG4 scaffold, or introducing mutations in the hinge or CH2 region 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 FcgR or complement binding to Fc.

[0198] Specific non-limiting examples of known amino acid modifications that reduce FcgR or complement binding to Fc include those identified in Table C below.

[0199] (Table C) Modifications that reduce FcgR or complement binding to Fc TIFF2025533065000004.tif105128

[0200] 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 encoding an enzyme that deflects the cellular pathway of fucose biosynthesis into cells used for antibody production. This prevents the antibody-producing cells from adding the sugar "fucose" to N-linked antibody carbohydrate moieties. (von Horsten et al. (2010) Glycobiology. 2010 Dec; 20 (12):1607-18.) Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 cells 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 antibody-producing cell lines for their ability to produce lower levels of antibody fucosylation.

[0201] 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).

[0202] The antibodies can be completely defucosylated (meaning 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 found in a similar antibody produced in a mammalian expression system.

[0203] In some aspects, the antibodies provided herein comprise an IgG1 domain with a 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.

[0204] 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 positions 298, 333, and 334 of the Fc region. 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.

[0205] Other exemplary glycosylation variants that can be incorporated into the antibodies provided herein are described in, e.g., 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; and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of which is incorporated by reference in its entirety.

[0206] 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.

[0207] 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.

[0208] join The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ) 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®).

[0209] With respect to antibody binding to a target molecule, the terms "bind," "specific binding," "specifically binds," "specific for," "selectively binds," and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to the non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated if antibody binding to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 50% of its affinity for fibrin. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 40% of its affinity for fibrin. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 30% of its affinity for fibrin. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 20% of its affinity for fibrin. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 10% of its affinity for fibrin. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 1% of its affinity for fibrin. In some embodiments, the affinity of the fibrin antibody for the non-target molecule is less than about 0.1% of its affinity for fibrin.

[0210] As used herein in the context of two or more antibodies, the terms "compete with" or "cross-compete with" indicate 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 "compete 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, one 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. One 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 one 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.

[0211] 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 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 an adjacent epitope 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 a first antibody described herein for binding to fibrin. In some cases, the second antibody can block or inhibit 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 more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0212] In some embodiments, the anti-fibrin antibody does not substantially bind to myeloid cells present outside the cancer tissue, hi some embodiments, the anti-fibrin antibody does not substantially bind to stimulatory myeloid cells present within the cancer tissue.

[0213] 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.

[0214] In some embodiments, the antibodies provided herein have a nucleotide sequence 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 × 10, as measured by Biacore assay. -6 In some embodiments, the antibodies provided herein have a cytotoxicity of about 1 x 10 -5 M, 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, or 1 x 10 -9 K below M D It binds to human fibrin.

[0215] In some embodiments, the antibodies provided herein have a nucleotide sequence 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 to 3, 4 to 2, 3 to 1, 1.9 to 1.8, 1.8 to 1.7, 1.7 to 1.6, 1.6 to 1.5, 1.9 to 1.5, 1.5 to 1, 1 to 0.8, 1 to 0.5, 0.9 to 0.6, 0.7 to 0.4, 0.6 to 0.2, 0.5 to 0.3, 0.3 to 0.2, 0.2 to 0.1, 0.1 to 0.01, 0.01 to 0.001, or 0.001 to 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 a concentration of 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 even 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 x 10 as measured by a Biacore assay. 5 (1 / Ms) or greater a In some embodiments, the antibodies provided herein bind to human fibrin at a concentration of 4 to 7, 4 to 4.5, 4.5 to 5, 5 to 5.5, 5.5 to 6, 6 to 6.5, or 6.5 to 7, 7 to 8, 8 to 9, or 9 to 10 x 10 as measured by a Biacore assay. 5 (1 / Ms)K a It binds to human fibrin.

[0216] function "Effector function" refers to a biological activity mediated by the Fc region of an antibody, and this activity can 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.

[0217] 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 pharmaceutically acceptable excipients. In some embodiments, the compositions are sterile. Pharmaceutical compositions typically contain an effective amount of an antibody.

[0218] These compositions may contain, in addition to one or more of the antibodies disclosed herein, pharmaceutically 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 precise nature of the carrier or other material may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal.

[0219] Pharmaceutical compositions for oral administration can be in the form of tablets, capsules, powders, or liquids. 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 solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0220] 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 sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary.

[0221] 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 prophylaxis 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, e.g., dosage, etc., 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 practitioners. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0222] The compositions may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the condition being treated.

[0223] method Preparation method The antibodies described herein can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or VH of the antibody (e.g., the light and / or heavy chain of the 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 nucleic acids are provided. In one embodiment, the nucleic acid is provided in a multicistronic vector. In a further embodiment, a host cell comprising such 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 the 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 the antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the 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., a Y0, NS0, Sp20 cell). In one embodiment, a method of producing an antibody is provided, the method comprising culturing a host cell containing nucleic acid encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0224] For recombinant production of antibodies, for example, nucleic acids encoding the antibodies as described above are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0225] 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% or less of the dry weight of the cells. When the heteromultimer or variant thereof is recombinantly produced by a host cell, the protein is present in the culture 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 or less of the dry weight of the cells. In certain embodiments, the "substantially purified" heteromultimer produced by the methods described herein has 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.

[0226] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0227] 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 producing recombinant host cells. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or may be integrated into the host genome. Host cells may include CHO, derivatives of CHO, NS0, Sp20, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0228] For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., 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, the antibody may be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0229] 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).

[0230] 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. Numerous baculovirus strains have been identified that can be used to transfect insect cells, particularly Spodoptera frugiperda cells.

[0231] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0232] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the 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).

[0233] 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 antibody 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 proportion of antibody in the expression product.

[0234] 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 compared to monomeric heavy or light chain polypeptides or other antibodies.

[0235] In some embodiments, there are methods for producing glycosylated antibodies in stable mammalian cells as described herein, the methods comprising identifying and purifying a desired glycosylated antibody, in some embodiments, the identification is by one or both of liquid chromatography and mass spectrometry.

[0236] If necessary, antibodies can be purified or isolated after expression. Proteins can be isolated or purified by a variety of methods known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed 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 combined with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins can find use in the present invention for antibody purification. 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 enabled by specific fusion partners. For example, glutathione resins are used when GST fusions are utilized, and Ni-based resins are used when His-tags are utilized. +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.

[0237] 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.

[0238] 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 derivatives.

[0239] Furthermore, the antibodies described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. 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. Furthermore, amino acids can be D (dextrorotatory) or L (levorotatory).

[0240] How to use In one aspect, the present application provides a method of contacting fibrin and VEGF with a multispecific antigen binding agent described herein, resulting in inhibition of microglial adhesion to fibrin or fibrinogen γC domain and neutralization of VEGF.

[0241] In one aspect, the application provides a method of using a multispecific antigen binding protein as described herein for the treatment of a condition associated with Mac-1 binding to fibrin or fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen binding protein as described herein or a pharmaceutical composition comprising a multispecific antigen binding protein.

[0242] In certain aspects, described herein are methods of inhibiting microglial activation, the methods comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen binding protein or a pharmaceutical composition comprising a multispecific antigen binding protein described herein.

[0243] In certain aspects, described herein are methods of treating or preventing an ocular disorder or condition comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen-binding protein or a pharmaceutical composition comprising a multispecific antigen-binding protein described herein. In certain embodiments, the ocular disorder or condition is wet age-related macular degeneration ("WAMD"), age-related macular degeneration ("AMD"), diabetic retinopathy, diabetic macular edema (DME), central retinal vein occlusion (RVO), pathologic myopia, polypoidal choroidal vasculopathy, retinitis pigmentosa, glaucoma, uveitis, or retinal detachment.

[0244] Administration method In some embodiments, the methods provided herein are useful for treating an ocular disorder or condition in an individual. In one embodiment, the individual is a human.

[0245] In some embodiments, the multispecific antigen-binding protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intravitreally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. An effective amount of the multispecific antigen-binding protein can be administered for the treatment of an ocular disorder or condition. The appropriate dosage of the multispecific antigen-binding protein can be determined based on the type of ocular condition being treated, the type of multispecific antigen-binding protein, the severity and course of the ocular condition, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0246] In some embodiments, the multispecific antigen-binding proteins provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic or immunotherapeutic agent may be administered with the antibodies provided herein. Additional therapeutic agents include agents used to treat or prevent ocular disorders or conditions selected from the group consisting of: wet age-related macular degeneration ("WAMD"), age-related macular degeneration ("AMD"), diabetic retinopathy, diabetic macular edema (DME), central retinal vein occlusion (RVO), pathological myopia, polypoidal choroidal vasculopathy, retinitis pigmentosa, glaucoma, uveitis, or retinal detachment.

[0247] The additional therapeutic agent may be administered by any suitable means. In some embodiments, the multispecific antigen-binding protein provided herein and the additional therapeutic agent are comprised in the same pharmaceutical composition. In some embodiments, the multispecific antigen-binding protein provided herein and the additional therapeutic agent are comprised in different pharmaceutical compositions.

[0248] In embodiments where the multispecific antigen-binding protein provided herein and the additional therapeutic agent are comprised in different pharmaceutical compositions, administration of the multispecific antigen-binding protein can occur prior to, concurrently with, and / or after administration of the additional therapeutic agent. In some embodiments, administration of the multispecific antigen-binding protein provided herein and the additional therapeutic agent occurs within about one month of each other. In some embodiments, administration of the multispecific antigen-binding protein provided herein and the additional therapeutic agent occurs within about one week of each other. In some embodiments, administration of the multispecific antigen-binding protein provided herein and the additional therapeutic agent occurs within about one day of each other. In some embodiments, administration of the multispecific antigen-binding protein provided herein and the additional therapeutic agent occurs within about 12 hours of each other. In some embodiments, administration of the multispecific antigen-binding protein provided herein and the additional therapeutic agent occurs within about one hour of each other.

[0249] Kits and Articles of Manufacture The present application provides a kit comprising any one or more of the multispecific antigen-binding proteins described herein. In some embodiments, the kit further comprises a component selected from a secondary antibody, an immunohistochemical analysis reagent, a pharmaceutically acceptable excipient, and an instruction manual, and any combination thereof. In one particular embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the multispecific antigen-binding proteins described herein together with one or more pharmaceutically acceptable excipients.

[0250] The present application also provides an article of manufacture comprising any one of the multispecific antigen-binding proteins or kits described herein. Examples of articles of manufacture include vials (including sealed vials). [Example]

[0251] Below are examples of specific modes 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 numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0252] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. 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 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0253] material and method Antigens were biotinylated using Pierce's EZ-Link Sulfo-NHS-Biotinylation Kit. Goat F(ab')2 anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE), and streptavidin-AF633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Goat anti-human IgG-PE (Human-PE) was obtained from Southern Biotech. Anti-mouse APC was obtained from Jackson ImmunoResearch.

[0254] Example 1: 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 induced in animals by immunization 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-matured antibodies after intravitreal administration of antifibrin antibodies in a rat EAU model.

[0255] 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 except Group 6 were immunized with IRBP emulsion in complete Freund's adjuvant (CFA) on Day 0. Similarly, animals in Groups 1–4 received a single intravitreal injection of the sponsor's test product on Day 0. Animals in Group 5 received a single oral dose of the positive control, FTY-720, daily. 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 track the extent of disease. Clinical observations were performed as follows: Frequency: Once each test day. Procedure: Groups were randomized prior to evaluation to keep examiners masked. Animals were observed under a dissecting microscope and scored on a scale of 0 to 4 based on anterior segment clinical disease. Photographs of the anterior chamber were taken during clinical evaluation. Clinical Observation Scoring: 0-0.5: No disease; the eye is translucent. Some blood vessels in the iris are dilated. 1: Congested blood vessels in the iris; abnormal pupil constriction (or dilation). 2: Slight haze in the anterior chamber. 3: Moderately opaque anterior chamber, but pupil still visible. 4: Opaque anterior chamber and unclear pupil.

[0256] All animals were euthanized on day 14, and whole eyes (OU) were harvested immediately after euthanasia. After 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. The eyes for cytokine analysis were hemisected and the retina harvested. Before wax embedding, each eye was carefully oriented for optimal microscopic examination. 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 performed blinded. Clinical scoring / uveitis grading was determined as follows: 0: No disease, normal retinal structure. 0.5: Trace amount. Less than 1 / 4: Mild inflammatory cell infiltration of the retina with or without photoreceptor damage. 1: ≥1 / 4 Mild inflammation and / or photoreceptor outer segment damage. 2: ≥1 / 4 mild to moderate inflammation and / or involvement of the outer nuclear layer. 3: ≥1 / 4 moderate to marked inflammation and / or involvement of the inner nuclear layer. 4: 1 / 4 or more severe inflammation and / or full-thickness retinal damage.

[0257] As shown in Figure 1, rats administered low or high doses of the murine ADI-60143-LALA Fc-stabilized antibody clone showed a significant reduction in 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.

[0258] Example 2: Laser-induced choroidal neovascularization study Laser-induced choroidal neovascularization (LCNV) was created in 6- to 8-week-old Brown Norway rats on experimental day 0. Animals were anesthetized with an intraperitoneal (IP) injection of ketamine / xylazine and topically administered 1% tropicamide for pupil dilation. Using a handheld cover slip as a contact lens and Gen Teal lubricating ophthalmic gel as a medium for contact between the cover slip and the corneal surface, six lesions were created equidistant from the optic nerve head in the mid-peripheral retina using a Nidek GYC-500 green laser photocoagulator connected to a Nidek SL-1800 slit lamp.

[0259] The laser parameters included: wavelength 532 nm, spot size 100 μm, duration 0.1 seconds, and 120 mW. The anti-fibrin antibody disclosed herein was administered by intravitreal (IVIT) injection on the same day as laser irradiation. Six days after laser irradiation, vascular leakage was assessed by quantitative fluorescein angiography (qFA). Seven days after laser irradiation, the animals were sacrificed for choroidal neovascularization (CNV) area analysis.

[0260] On day 1 of the experiment, treatments were administered bilaterally by IVIT injection according to Table 1 below.

[0261] (Table 1) TIFF2025533065000005.tif68140

[0262] Because qFA and optical coherence tomography (OCT) are non-terminal procedures (e.g., survival procedures), the rats used for these readings were also used for CNV area analysis, serum collection, and histological examination.

[0263] Animals were anesthetized with isoflurane and treated with topical proparacaine prior to injection to provide local analgesia. IVIT injections up to 2.0 μL in volume were performed under an operating microscope (Zeiss Microscopy) using a custom-made, short-barrel, 2 μL syringe with a detachable 33-gauge needle (Hamilton Co.). After injection, neomycin / polymyxin B / gramicidin eye drops were instilled to prevent infection. Treatments were randomized across animals and housing cages.

[0264] Lesion location images were captured by optical coherence tomography (OCT) using a Heidelberg Spectralis on experimental days 0, 3, and 7. Animals were anesthetized with an intraperitoneal (IP) injection of ketamine / xylazine. A-scans were used to identify the location of CNV lesions, and high-resolution B-scans were acquired for each lesion and 3D deconvolution was performed to determine lesion volume. Because OCT is a survival procedure, animals were also used for serum collection.

[0265] On day 6 of the experiment, vascular leakage was assessed by qFA (Figure 2). Animals were anesthetized with an IP injection of ketamine / xylazine. Sodium fluorescein was administered via tail vein injection at a dose of 500 mg / kg. Fluorescent fundus images of one eye of each animal were captured using a Micron IV imaging system 4 and 2 minutes after fluorescein injection. A masked observer quantified the fluorescence intensity of one lesion per eye using ImageJ software using the "integrated density" function. The difference in integrated density between the two post-injection measurements was recorded as a readout of vascular leakage. Because qFA is a survival procedure, animals were used for CNV area analysis.

[0266] On experimental day 7, animals were euthanized and their eyes were enucleated. The extent of CNV at the site of Bruch's membrane rupture was measured by a masked observer using computer-assisted image analysis of choroidal plane mounts stained with FITC-labeled isolectin B4. Because the anti-fibrin antibody was administered to both eyes of the same animal, the area of ​​all lesions in both eyes of each animal was averaged and reported as a single data point (Figure 3). Statistical analysis of area measurements was performed by ANOVA and parametric post-hoc tests. Retinas were paraffin-embedded and sectioned for further analysis.

[0267] 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 details can be made therein without departing from the spirit and scope of the invention.

[0268] 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.

[0269] Unofficial sequence listing TIFF2025533065000006.tif198150TIFF2025533065000007.tif221150TIFF2025533065000008.tif221150TIFF2025533065000009.tif221150TIFF2025533065000010.tif221150TIFF2025533065000011.tif221150TIFF2025533065000012.tif212150TIFF2025533065000013.tif220150TIFF2025533065000014.tif221150TIFF2025533065000015.tif221150TIFF2025533065000016.tif221150TIFF2025533065000017.tif221150TIFF2025533065000018.tif221150TIFF2025533065000019.tif221150TIFF2025533065000020.tif221150TIFF2025533065000021.tif221150TIFF2025533065000022.tif221150TIFF2025533065000023.tif221150TIFF2025533065000024.tif221150TIFF2025533065000025.tif221150TIFF2025533065000026.tif219150TIFF2025533065000027.tif219150TIFF2025533065000028.tif220150TIFF2025533065000029.tif221150TIFF2025533065000030.tif198150

Claims

1. A multispecific antigen-binding protein comprising at least two different antigen-binding regions, wherein a first antigen-binding region specifically binds to a human fibrin or fibrinogen γC domain and a second antigen-binding region specifically binds to vascular endothelial growth factor (VEGF).

2. the first antigen-binding region comprises 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; a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, 229, or 258; b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, 230, or 259; c. 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, 231, or 260; d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 160, 172, 184, 196, 208, 220, 232, or 262; e. 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, 233, or 263; and f. 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, 234 or 264; 2. The multispecific antigen-binding protein of claim 1.

3. 2. The multispecific antigen-binding protein of claim 1, wherein the first antigen-binding region 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, 235, or 254.

4. 3. The multispecific antigen-binding protein of claim 1 or 2, wherein the first antigen-binding region comprises a VL sequence selected from the sequence set forth in SEQ ID NO: 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 166, 178, 190, 202, 214, 226, 238, or 255.

5. 10. The multispecific antigen-binding protein of claim 1, wherein the first antigen-binding region 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.

6. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19 and the VL sequence set forth in SEQ ID NO:

22.

7. 3. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 31 and the VL sequence set forth in SEQ ID NO:

34.

8. 4. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43 and the VL sequence set forth in SEQ ID NO:

46.

9. 5. The multispecific antigen-binding protein of claim 1 , wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 55 and the VL sequence set forth in SEQ ID NO:

58.

10. 6. The multispecific antigen-binding protein of claim 1 , wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67 and the VL sequence set forth in SEQ ID NO:

70.

11. 8. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79 and the VL sequence set forth in SEQ ID NO:

82.

12. 9. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91 and the VL sequence set forth in SEQ ID NO:

94.

13. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103 and the VL sequence set forth in SEQ ID NO:

106.

14. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115 and the VL sequence set forth in SEQ ID NO:

118.

15. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127 and the VL sequence set forth in SEQ ID NO:

130.

16. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139 and the VL sequence set forth in SEQ ID NO:

142.

17. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151 and the VL sequence set forth in SEQ ID NO:

154.

18. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163 and the VL sequence set forth in SEQ ID NO:

166.

19. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175 and the VL sequence set forth in SEQ ID NO:

178.

20. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187 and the VL sequence set forth in SEQ ID NO:

190.

21. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199 and the VL sequence set forth in SEQ ID NO:

202.

22. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211 and the VL sequence set forth in SEQ ID NO:

214.

23. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223 and the VL sequence set forth in SEQ ID NO:

226.

24. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 235 and the VL sequence set forth in SEQ ID NO:

238.

25. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 256 and the VL sequence set forth in SEQ ID NO:

257.

26. 10. The multispecific antigen-binding protein of claim 1 , wherein the first antigen-binding region comprises a humanized first antigen-binding region, a human first antigen-binding region, or a chimeric first antigen-binding region.

27. 27. The multispecific antigen-binding protein of claim 26, wherein the first antigen-binding region comprises a humanized antibody.

28. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

29. 29. The multispecific antigen-binding protein of claim 28, wherein the human Fc region comprises a human heavy chain constant region of class IgG and a subclass selected from IgG1, IgG2, IgG3 and IgG4.

30. 30. The multispecific antigen-binding protein of claim 29, wherein the human Fc region comprises wild-type human IgG1 Fc.

31. 31. The multispecific antigen-binding protein of claim 30, wherein the human Fc domain comprises the 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.

32. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the heavy chain comprises a constant heavy chain sequence as set forth 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.

33. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the light chain comprises a constant light chain sequence as set forth 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.

34. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 7 and the VL sequence set forth in SEQ ID NO: 10; and the human Fc region comprises a wild-type human IgG1 Fc.

35. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19 and the VL sequence set forth in SEQ ID NO: 22; and the human Fc region comprises a wild-type human IgG1 Fc.

36. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 31 and the VL sequence set forth in SEQ ID NO: 34; and the human Fc region comprises wild-type human IgG1 Fc.

37. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43 and the VL sequence set forth in SEQ ID NO: 46; and the human Fc region comprises wild-type human IgG1 Fc.

38. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 55 and the VL sequence set forth in SEQ ID NO: 58; and the human Fc region comprises wild-type human IgG1 Fc.

39. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67 and the VL sequence set forth in SEQ ID NO: 70; and the human Fc region comprises a wild-type human IgG1 Fc.

40. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79 and the VL sequence set forth in SEQ ID NO: 82; and the human Fc region comprises a wild-type human IgG1 Fc.

41. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91 and the VL sequence set forth in SEQ ID NO: 94; and the human Fc region comprises a wild-type human IgG1 Fc.

42. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103 and the VL sequence set forth in SEQ ID NO: 106; and the human Fc region comprises a wild-type human IgG1 Fc.

43. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115 and the VL sequence set forth in SEQ ID NO: 118; and the human Fc region comprises a wild-type human IgG1 Fc.

44. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127 and the VL sequence set forth in SEQ ID NO: 130; and the human Fc region comprises a wild-type human IgG1 Fc.

45. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139 and the VL sequence set forth in SEQ ID NO: 142; and the human Fc region comprises a wild-type human IgG1 Fc.

46. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151 and the VL sequence set forth in SEQ ID NO: 154; and the human Fc region comprises a wild-type human IgG1 Fc.

47. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163 and the VL sequence set forth in SEQ ID NO: 166; and the human Fc region comprises a wild-type human IgG1 Fc.

48. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175 and the VL sequence set forth in SEQ ID NO: 178; and the human Fc region comprises a wild-type human IgG1 Fc.

49. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187 and the VL sequence set forth in SEQ ID NO: 190; and the human Fc region comprises a wild-type human IgG1 Fc.

50. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199 and the VL sequence set forth in SEQ ID NO: 202; and the human Fc region comprises a wild-type human IgG1 Fc.

51. 10. The multispecific antigen-binding protein of claim 9, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211 and the VL sequence set forth in SEQ ID NO: 214; and the human Fc region comprises a wild-type human IgG1 Fc.

52. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223 and the VL sequence set forth in SEQ ID NO: 226; and the human Fc region comprises a wild-type human IgG1 Fc.

53. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region 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 a wild-type human IgG1 Fc.

54. 54. The multispecific antigen-binding protein of any one of claims 28-53, wherein the Fc region comprises one or more amino acid substitutions, which substitutions result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more substitutions.

55. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

56. 10. The multispecific antigen-binding protein of claim 1 , wherein the first antigen-binding region comprises a monoclonal first antigen-binding region.

57. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region binds to the γ377-395 epitope of the fibrin or fibrinogen γC domain.

58. Approximately 1, 2, 3, 4, 5, 6, 7, or 8 × 10 as measured by surface plasmon resonance (SPR) single-cycle kinetics (SCK) assay -5 K below M D 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region binds to a peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241 and 249-253.

59. Approximately 8 × 10 as measured by surface plasmon resonance (SPR) single-cycle kinetics (SCK) assay -5 K below M D 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region binds to a peptide comprising the sequence of the γ377-395 epitope of the human fibrin or fibrinogen γC domain.

60. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region inhibits Mac-1 binding to fibrin or fibrinogen γC domain.

61. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region exhibits inhibition of microglial adhesion to fibrin or fibrinogen γC domain.

62. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region 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.

63. 63. The multispecific antigen-binding protein of claim 62, wherein the first antigen-binding region binds to human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten of the following amino acid residues: Lys 411, Ile 412, Ile 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, Ile 420, and Gly 421.

64. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises a VH region comprising 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, Gly 102, and Trp 103.

65. 65. The multispecific antigen-binding protein of claim 64, wherein the first antigen-binding region comprises a VH region comprising a paratope comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of the following 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.

66. 65. The multispecific antigen-binding protein of claim 64, wherein the first antigen-binding region comprises a VH region comprising a paratope comprising amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103.

67. 65. The multispecific antigen-binding protein of claim 64, wherein the first antigen-binding region comprises a VH region comprising a paratope comprising 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, Gly 102, and Trp 103.

68. 65. The multispecific antigen-binding protein of claim 64, wherein the first antigen-binding region comprises a VH region comprising 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, Gly 102, and Trp 103.

69. 69. The multispecific antigen-binding protein of any one of claims 62 to 68, wherein the first antigen-binding region comprises a VL region comprising a paratope comprising 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.

70. 70. The multispecific antigen-binding protein of claim 69, wherein the first antigen-binding region comprises a VL region comprising a paratope comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of the following 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.

71. 70. The multispecific antigen-binding protein of claim 69, wherein the first antigen-binding region comprises a VL region comprising a paratope comprising amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gln 50, Leu 92, Leu 94, and Leu 96.

72. 70. The multispecific antigen-binding protein of claim 69, wherein the first antigen-binding region comprises a VL region comprising 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.

73. 70. The multispecific antigen-binding protein of claim 69, wherein the first antigen-binding region comprises a VL region comprising 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.

74. 10. The multispecific antigen-binding protein of claim 1, wherein the first antigen-binding region comprises a humanized antibody, a human antibody, or a chimeric antibody.

75. 75. The multispecific antigen-binding protein of claim 74, wherein the first antigen-binding region comprises a humanized antibody.

76. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region comprises a heavy chain human Fc of a class selected from IgG, IgA, IgD, IgE and IgM.

77. 77. The multispecific antigen-binding protein of any one of claims 28 to 76, wherein the human Fc region comprises a human heavy chain constant region of class IgG and a subclass selected from IgG1, IgG2, IgG3 and IgG4.

78. 78. The multispecific antigen-binding protein of claim 77, wherein the human Fc region comprises wild-type human IgG1 Fc.

79. 78. The multispecific antigen-binding protein of claim 77, wherein the human Fc domain comprises the 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.

80. 80. The multispecific antigen-binding protein of any of claims 28-79, wherein the Fc region comprises one or more amino acid substitutions, which substitutions result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to an Fc without the one or more substitutions.

81. 81. The multispecific antigen-binding protein of any one of claims 28 to 80, wherein the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.

82. 10. The multispecific antigen-binding protein of claim 1 , wherein the first antigen-binding region comprises a monoclonal antibody.

83. Approximately 1, 2, 3, 4, 5, 6, 7, or 8 × 10 as measured by surface plasmon resonance (SPR) single-cycle kinetics (SCK) assay -5 K below M D 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region binds to a peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241 and 249-253.

84. Approximately 8 × 10 as measured by surface plasmon resonance (SPR) single-cycle kinetics (SCK) assay -5 K below M D 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region binds to a peptide comprising the sequence of the γ377-395 epitope of the human fibrin or fibrinogen γC domain.

85. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region inhibits Mac-1 binding to fibrin or fibrinogen γC domain.

86. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the first antigen-binding region exhibits inhibition of microglial adhesion to fibrin or fibrinogen γC domain.

87. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the second antigen-binding region comprises a heavy chain comprising a second variable heavy (VH) chain sequence comprising the three heavy chain CDR sequences CDR-H1, CDR-H2 and CDR-H3, and a light chain comprising a second variable light (VL) chain sequence comprising the three light chain CDR sequences CDR-L1, CDR-L2 and CDR-L3.

88. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the second antigen-binding region binds to one or more of VEGF-A, VEGF-B, VEGF-C and VEGF-D.

89. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the second antigen-binding region comprises a VH amino acid sequence that is 90% or more, 95% or more, or 99% or more identical to the VH amino acid sequence set forth in SEQ ID NO:

269.

90. 10. The multispecific antigen-binding protein of any one of the preceding claims, wherein the second antigen-binding region comprises a VL amino acid sequence that is 90% or more, 95% or more, or 99% or more identical to the VL amino acid sequence set forth in SEQ ID NO:

268.

91. 20. The multispecific antigen-binding protein of any one of the preceding claims, wherein the second antigen-binding region binds to an epitope of VEGF-A comprising at least one of amino acids 82 to 91 set forth in SEQ ID NO.

265.

92. 92. The multispecific antigen-binding protein of any one of claims 1 to 91, wherein the second antigen-binding region comprises a sequence that is 90% or more, 95% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 266 or 267.

93. 10. The multispecific antigen-binding protein of any of the preceding claims, wherein the second antigen-binding region does not comprise an Fc domain.

94. 10. The multispecific antigen-binding protein of any of the preceding claims, comprising a heterodimeric Fc domain.

95. 10. The multispecific antigen-binding protein of any of the preceding claims, comprising a bispecific antibody.

96. 10. A multispecific antigen-binding protein according to any one of the preceding claims, which consists of a bispecific antibody.

97. 10. The multispecific antigen-binding protein of any one of the preceding claims, comprising one or more further antigen-binding domains that specifically bind to a human fibrin or fibrinogen γC domain.

98. 10. The multispecific antigen-binding protein of any of the preceding claims, comprising one or more additional antigen-binding domains that specifically bind to VEGF.

99. 10. The multispecific antigen-binding protein of any of the preceding claims for use in the treatment of an ocular disorder or condition.

100. 10. The multispecific antigen-binding protein of any one of the preceding claims, formulated for administration to a subject by intravitreal injection.

101. 10. An isolated polynucleotide or set of polynucleotides encoding the first antigen-binding region, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof according to any one of the preceding claims, optionally comprising cDNA.

102. 10. An isolated polynucleotide or set of polynucleotides encoding the second antigen-binding region, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof according to any one of the preceding claims, optionally comprising cDNA.

103. 103. A vector or set of vectors comprising the polynucleotide or set of polynucleotides of claim 101 or 102.

104. 104. A host cell comprising the polynucleotide or set of polynucleotides of claim 101 or 102 or the vector or set of vectors of claim 103.

105. 101. A pharmaceutical composition comprising the multispecific antigen-binding protein of any one of claims 1 to 100 and a pharmaceutically acceptable excipient.

106. 106. A kit comprising the multispecific antigen-binding protein of any one of claims 1 to 100 or the pharmaceutical composition of claim 105, and instructions for use.

107. 106. A method for treating an ocular disorder or condition, said method comprising administering to a mammalian subject a therapeutically effective amount of the multispecific antigen-binding protein of any one of claims 1 to 100 or the pharmaceutical composition of claim 105.

108. 108. The method of claim 107, wherein the ocular disorder or condition is selected from the group consisting of retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment.

109. 106. A method for treating a condition associated with Mac-1 binding to fibrin or fibrinogen, said method comprising administering to a mammalian subject a therapeutically effective amount of a multispecific antigen-binding protein of any one of claims 1 to 100 or a pharmaceutical composition of claim 105.

110. 106. A method of inhibiting microglial activation, said method comprising administering to a mammalian subject a therapeutically effective amount of the multispecific antigen-binding protein of any one of claims 1 to 100 or the pharmaceutical composition of claim 105.

111. 105. A method of producing a multispecific antigen-binding protein, the method comprising expressing the multispecific antigen-binding protein or antigen-binding region thereof in a host cell of claim 104, and isolating the expressed multispecific antigen-binding protein or antigen-binding region thereof.

112. 106. A method of preventing an ocular disorder or condition, said method comprising administering to a mammalian subject a therapeutically effective amount of the multispecific antigen-binding protein of any one of claims 1 to 100 or the pharmaceutical composition of claim 105.

113. 106. A method of treating an ocular disorder or condition associated with increased angiogenesis in a subject in need thereof, said method comprising administering to said subject a multispecific antigen-binding protein of any one of claims 1 to 100 or a pharmaceutical composition of claim 105.

114. 106. A method of preventing an ocular disorder or condition associated with increased angiogenesis in a subject in need thereof, said method comprising administering to said subject a multispecific antigen-binding protein of any one of claims 1 to 100 or a pharmaceutical composition of claim 105.

115. 101. The multispecific antigen-binding protein of any one of claims 1 to 100, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 256 and the VL sequence set forth in SEQ ID NO: 257.

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