Multi-specific antigen-binding proteins which bind human fibrin yc or fibrinogen yc domain and vascular endothelial growth factor and methods of use

Abstract

Described herein are multi-specific antigen-binding proteins that bind human fibrin γC or fibrinogen γC domain and vascular endothelial growth factor, and methods of use thereof. In some embodiments, described herein are pharmaceutical compositions comprising the multi-specific antigen-binding proteins that bind fibrin γC or fibrinogen γC domain and vascular endothelial growth factor. In some embodiments, the multi-specific antigen-binding proteins and methods described herein are used for treatment of disorders or conditions of the eye.

Description

Multi-Specific Antigen-Binding Proteins Which Bind Human Fibrin yC or Fibrinogen yC Domain and Vascular Endothelial Growth Factor and Methods of UseCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 573,364, filed on April 2, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. The XML copy of the Sequence Listing, created on April 1, 2025, is named THB-01 IWO SL.xml and is 336,139 bytes in size.BACKGROUND

[0003] 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 etiologies, they are all characterized by the loss of photoreceptors or retinal ganglion cells (RGCs), resulting in deterioration of vision and, in some cases, blindness. Activation of microglia contributes to both neuronal and oligodendrocyte death via release of cytokines and nitric oxide. It is possible that interventions targeting activated microglia could alleviate photoreceptor and RGC death, thereby helping patients preserve their sight.

[0004] The y377-395 epitope of the fibrin yC or fibrinogen yC domain is the binding epitope of fibrin to CDl lb / CD18 (Mac-1) and CDl lc / CD18. The fibrin y377-395 peptide functions as an inhibitor of microglia activation by blocking fibrin binding to CD1 lb / CD18 (Mac-1) and CD1 lc / CD18. Because fibrin mediates blood coagulation by binding via a distinct epitope to the platelet integrin aubPs receptor, therapeutic agents (including antibodies), that block the CD1 lb / CD18 (Mac-1) and CD1 lc / CD18 binding epitope to fibrin can reduce the damaging effects of fibrin in the nervous system without affecting its beneficial effects in blood coagulation. Angiogenic ocular conditions such as retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration represent the leading cause 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, effective therapeutics that inhibit fibrin-induced microglial activation without affecting its beneficial effects in blood coagulation, and that also block VEGF, are needed for treatment of disorders and conditions of the eye.SUMMARY

[0006] Described herein, in some embodiments, are multi-specific antigen-binding proteins comprising: (a) a first antigen-binding region that specifically binds human fibrin yC or fibrinogen yC domain and comprising: a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR- H3; and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, respectively, wherein: (i) CDR-H1 comprises the sequence set forth in any one of SEQ ID NOs: 157, 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, 229, and 258; (ii) CDR-H2 comprises the sequence set forth in any one of SEQ ID NOs: 158, 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, 230 and 259; (iii) CDR-H3 comprises the sequence set forth in any one of SEQ ID NOs: 159, 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, 231, and 260; (iv) CDR-L1 comprises the sequence set forth in any one of SEQ ID NOs: 160, 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, 232 and 262; (v) CDR-L2 comprises the sequence set forth in any one of SEQ ID NOs: 161, 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 173, 185, 197, 209, 221, 233 and 263; and (vi) CDR-L3 comprises the sequence set forth in any one of SEQ ID NOs: 162, 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 174, 186, 198, 210, 222, 234 and 264; (b) a second antigen-binding region that specifically binds Vascular Endothelial Growth Factor (VEGF) comprising: (i) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 281-295 and 318; (ii) a heavy chain comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 269, 274, 277, 278, or 296-297, and a light chain comprising the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 268, 273, 279, 280, and 298-299, respectively; or (iii) an antigen-binding region comprising the sequence of SEQ ID NO: 267; c) optionally, a third antigen-binding domain that specifically binds human fibrin yC or fibrinogen yC domain; and d) optionally, a fourth antigen-binding domain that specifically binds VEGF.

[0007] In some embodiments, the first antigen-binding region comprises a VH sequence selected from a sequence set forth in any one of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254.

[0008] In some embodiments, the first antigen-binding region comprises a VL sequence selected from a sequence set forth in any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255.

[0009] In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in SEQ ID NO: 166. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 7 and a VL sequence set forth in SEQ ID NO: 10. In some 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 some 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 some 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 some 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 some 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 some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 79 and a VL sequence set forth in SEQ ID NO: 82. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 91 and a VL sequence set forth in SEQ ID NO: 94. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 103 and a VL sequence set forth in SEQ ID NO: 106. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 115 and a VL sequence set forth in SEQ ID NO: 118. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 127 and a VL sequence set forth in SEQ ID NO: 130. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 139 and a VL sequence set forth in SEQ ID NO: 142. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 151 and a VL sequence set forth in SEQ ID NO: 154. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 175 and a VL sequence set forth in SEQ ID NO: 178. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 187 and a VL sequence set forth in SEQ ID NO: 190. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 199 and a VL sequence set forth in SEQ ID NO: 202. Insome embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 211 and a VL sequence set forth in SEQ ID NO: 214. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 223 and a VL sequence set forth in SEQ ID NO: 226. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 235 and a VL sequence set forth in SEQ ID NO: 238. In some embodiments, the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 256 and a VL sequence set forth in SEQ ID NO: 257.

[0010] In some embodiments, the first antigen-binding region comprises a humanized, human, or chimeric first antigen-binding region.

[0011] In some embodiments, the first antigen-binding region comprises a humanized antibody or antigen-binding region.

[0012] In some embodiments, the first antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

[0013] In some embodiments, the first antigen-binding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

[0014] In some embodiments, the human Fc region comprises wild-type, human IgGl Fc.

[0015] In some embodiments, the human Fc domain comprises a sequence set forth in any one of SEQ ID NOs: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236.

[0016] In some embodiments, the heavy chain comprises a constant heavy chain sequence set forth in any one of SEQ ID NOs: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152,176, 188, 200, 212, 224, and 236.

[0017] In some embodiments, the light chain comprises a constant light chain sequence set forth in any one of SEQ ID NOs: 165, 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153,177, 189, 201, 213, 225, and 237.

[0018] In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 7, the VL sequence set forth in SEQ ID NO: 10, and the first antigenbinding region comprises a human Fc region, wherein the human Fc region comprises wildtype, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19, the VL sequence set forth in SEQ ID NO: 22, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding regioncomprises the VH sequence set forth in SEQ ID NO: 31, the VL sequence set forth in SEQ ID NO: 34, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43, the VL sequence set forth in SEQ ID NO: 46, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 55, the VL sequence set forth in SEQ ID NO: 58, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67, the VL sequence set forth in SEQ ID NO: 70, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79, the VL sequence set forth in SEQ ID NO: 82, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91, the VL sequence set forth in SEQ ID NO: 94, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103, the VL sequence set forth in SEQ ID NO: 106, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115, the VL sequence set forth in SEQ ID NO: 118, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127, the VL sequence set forth in SEQ ID NO: 130, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139, the VL sequence set forth in SEQ ID NO: 142, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth inSEQ ID NO: 151, the VL sequence set forth in SEQ ID NO: 154, and the first antigenbinding region comprises a human Fc region, wherein the human Fc region comprises wildtype, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163, the VL sequence set forth in SEQ ID NO: 166, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175, the VL sequence set forth in SEQ ID NO: 178, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187, the VL sequence set forth in SEQ ID NO: 190, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199, the VL sequence set forth in SEQ ID NO: 202, and the first antigenbinding region comprises a human Fc region, wherein the human Fc region comprises wildtype, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211, the VL sequence set forth in SEQ ID NO: 214, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223, the VL sequence set forth in SEQ ID NO: 226, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 235, the VL sequence set forth in SEQ ID NO: 238, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

[0019] In some 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 with the Fc without the one or more substitutions.

[0020] In some embodiments, the Fc region binds an Fey receptor selected from the group consisting of: FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.

[0021] In some embodiments, the first antigen-binding region comprises a monoclonal first antigen-binding region.

[0022] In some embodiments, the first antigen-binding region binds an y377-395 epitope of the fibrin yC or fibrinogen yC domain.

[0023] In some embodiments, the first antigen-binding region binds to peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241, and 249-253 with a KD of less than or equal to about 1, 2, 3, 4, 5, 6, 7, or 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

[0024] In some embodiments, the first antigen-binding region binds to a peptide comprising the sequence of the y377 -395 epitope of the human fibrin yC or fibrinogen yC domain with a KD of less than or equal to about 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

[0025] In some embodiments, the first antigen-binding region inhibits CD1 lb / CD18 (Mac-1) and CD1 lc / CD18 binding to fibrin yC or fibrinogen yC domain.

[0026] In some embodiments, the first antigen-binding region exhibits inhibition of microglial adhesion to the fibrin yC or fibrinogen yC domain.

[0027] In some embodiments, the first antigen-binding region binds human fibrin at any one of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

[0028] The In some embodiments, the first antigen-binding region binds human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten of amino acid residues Lys 411, lie 412, lie 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

[0029] In some embodiments, the first antigen-binding region comprises a VH region comprising a paratope that 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0030] In some embodiments, the first antigen-binding region comprises a VH region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all seventeen 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0031] In some embodiments, the first antigen-binding region comprises a VH region comprising a paratope that comprises amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0032] In some embodiments, the first antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0033] In some embodiments, the first antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0034] In some embodiments, the first antigen-binding region comprises a VL region comprising a paratope that comprises any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0035] In some embodiments, the first antigen-binding region comprises a VL region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine or all ten amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0036] In some embodiments, the first antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0037] In some embodiments, the first antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0038] In some embodiments, the first antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0039] In some embodiments, the second antigen-binding region binds one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

[0040] In some embodiments, the second antigen-binding region comprises a variable heavy chain sequence set forth in any one of SEQ ID NOs: 281-297 and 318.

[0041] In some embodiments, the second antigen-binding region comprises a variable heavy chain sequence set forth in SEQ ID NO: 296 or 297 and a variable light chain sequence set forth in SEQ ID NO: 298 or 299, respectively.

[0042] In some embodiments, the second antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NO: 274 and a light chain sequence set forth in SEQ ID NO: 273.

[0043] In some embodiments, the second antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NOs: 277 and 278 and a light chain sequence set forth in SEQ ID NO: 279 and 280.

[0044] In some embodiments, the second antigen-binding region comprises a humanized, human, or chimeric second antigen-binding region.

[0045] In some embodiments, the second antigen-binding region comprises a humanized antibody or antigen-binding region.

[0046] In some embodiments, the second antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

[0047] In some embodiments, the second antigen-binding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

[0048] In some embodiments, the second antigen-binding region comprises a monoclonal second antigen-binding region.

[0049] In some 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 any one of SEQ ID NOs: 269, 274, 277, 278, 281-297, 318, 330, and 331.

[0050] In some 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 any one of SEQ ID NOs: 268, 273, 279, 280, 298-299, 322, and 325.

[0051] In some embodiments, the second antigen -binding region binds an epitope of VEGF- A comprising at least one of amino acids 82-91 set forth in SEQ ID NO: 265.

[0052] In some 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 any one of SEQ ID NOs: 266, 267, and 332.

[0053] In some embodiments, the second antigen-binding region does not comprise an Fc domain.

[0054] In some embodiments, the multi-specific antigen-binding protein comprises the third antigen-binding region that specifically binds human fibrin yC or fibrinogen yC domain, wherein the third 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, respectively, wherein: (a) CDR-H1 comprises the sequence set forth in any one of SEQ ID NOs: 157, 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, 229, and 258; (b) CDR-H2 comprises the sequence set forth in any one of SEQ ID NOs: 158, 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, 230 and 259; (c) CDR-H3 comprises the sequence set forth in any one of SEQ ID NOs: 159, 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, 231, and 260; (d) CDR-L1 comprises the sequence set forth in any one of SEQ ID NOs: 160, 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, 232 and 262; (e) CDR-L2 comprises the sequence set forth in any one of SEQ ID NOs: 161, 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 173, 185, 197, 209, 221, 233 and 263; and (f) CDR-L3 comprises the sequence set forth in any one of SEQ ID NOs: 162, 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 174, 186, 198, 210, 222, 234 and 264.

[0055] In some embodiments, the third antigen-binding region comprises a VH sequence selected from a sequence set forth in any one of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79,91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254.

[0056] In some embodiments, the third antigen-binding region comprises a VL sequence selected from a sequence set forth in any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82,94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255.

[0057] In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in SEQ ID NO: 166. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 7 and a VL sequence set forth in SEQ ID NO: 10. In some embodiments, the thirdantigen-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 some embodiments, the third 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 some embodiments, the third 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 some embodiments, the third 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 some embodiments, the third 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 some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 79 and a VL sequence set forth in SEQ ID NO: 82. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 91 and a VL sequence set forth in SEQ ID NO: 94. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 103 and a VL sequence set forth in SEQ ID NO: 106. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 115 and a VL sequence set forth in SEQ ID NO: 118. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 127 and a VL sequence set forth in SEQ ID NO: 130. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 139 and a VL sequence set forth in SEQ ID NO: 142. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 151 and a VL sequence set forth in SEQ ID NO: 154. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 175 and a VL sequence set forth in SEQ ID NO: 178. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 187 and a VL sequence set forth in SEQ ID NO: 190. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 199 and a VL sequence set forth in SEQ ID NO: 202. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 211 and a VL sequence set forth in SEQ ID NO: 214. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 223 and a VL sequence set forth in SEQ ID NO: 226. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 235 and a VL sequence set forth in SEQ ID NO: 238. In some embodiments, the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 256 and a VL sequence set forth in SEQ ID NO: 257.

[0058] In some embodiments, the third antigen-binding region comprises a humanized, human, or chimeric third antigen-binding region.

[0059] In some embodiments, the third antigen-binding region comprises a humanized antibody or antigen-binding region.

[0060] In some embodiments, the third antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

[0061] In some embodiments, the third antigen-binding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

[0062] In some embodiments, the human Fc region comprises wild-type, human IgGl Fc.

[0063] In some embodiments, the human Fc domain comprises a sequence set forth in any one of SEQ ID NOs: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236.

[0064] In some embodiments, the heavy chain comprises a constant heavy chain sequence set forth in any one of SEQ ID NOs: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152,176, 188, 200, 212, 224, and 236.

[0065] In some embodiments, the light chain comprises a constant light chain sequence set forth in any one of SEQ ID NOs: 165, 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153,177, 189, 201, 213, 225, and 237.

[0066] In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 7, the VL sequence set forth in SEQ ID NO: 10, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19, the VL sequence set forth in SEQ ID NO: 22, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 31, the VL sequence set forth in SEQ ID NO: 34, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43, the VL sequence set forth in SEQ ID NO: 46, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VHsequence set forth in SEQ ID NO: 55, the VL sequence set forth in SEQ ID NO: 58, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67, the VL sequence set forth in SEQ ID NO: 70, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79, the VL sequence set forth in SEQ ID NO: 82, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91, the VL sequence set forth in SEQ ID NO: 94, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103, the VL sequence set forth in SEQ ID NO: 106, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115, the VL sequence set forth in SEQ ID NO: 118, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127, the VL sequence set forth in SEQ ID NO: 130, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139, the VL sequence set forth in SEQ ID NO: 142, and the third antigenbinding region comprises a human Fc region, wherein the human Fc region comprises wildtype, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151, the VL sequence set forth in SEQ ID NO: 154, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163, the VL sequence set forth in SEQ ID NO: 166, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175, the VLsequence set forth in SEQ ID NO: 178, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187, the VL sequence set forth in SEQ ID NO: 190, and the third antigenbinding region comprises a human Fc region, wherein the human Fc region comprises wildtype, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199, the VL sequence set forth in SEQ ID NO: 202, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211, the VL sequence set forth in SEQ ID NO: 214, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223, the VL sequence set forth in SEQ ID NO: 226, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc. In some embodiments, the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 235, the VL sequence set forth in SEQ ID NO: 238, and the third antigenbinding region comprises a human Fc region, wherein the human Fc region comprises wildtype, human IgGl Fc.

[0067] In some 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 with the Fc without the one or more substitutions.

[0068] In some embodiments, the Fc region binds an Fey receptor selected from the group consisting of: FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.

[0069] In some embodiments, the third antigen-binding region comprises a monoclonal third antigen-binding region.

[0070] In some embodiments, the third antigen-binding region binds an y377-395 epitope of the fibrin yC or fibrinogen yC domain.

[0071] In some embodiments, the third antigen-binding region binds to peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241, and 249-253 with a KD of less than or equal to about 1, 2, 3, 4, 5, 6, 7, or 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

[0072] In some embodiments, the third antigen-binding region binds to a peptide comprising the sequence of the y377 -395 epitope of the human fibrin yC or fibrinogen yC domain with a KD of less than or equal to about 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

[0073] In some embodiments, the third antigen-binding region inhibits CD1 lb / CD18 (Mac- 1) and CD1 lc / CD18 binding to fibrin yC or fibrinogen yC domain.

[0074] In some embodiments, the third antigen-binding region exhibits inhibition of microglial adhesion to the fibrin yC or fibrinogen yC domain.

[0075] In some embodiments, the third antigen-binding region binds human fibrin at any one of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

[0076] In some embodiments, the third antigen-binding region binds human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten of amino acid residues Lys 411, He 412, lie 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

[0077] In some embodiments, the third antigen-binding region comprises a VH region comprising a paratope that 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0078] In some embodiments, the third antigen-binding region comprises a VH region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all seventeen 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0079] In some embodiments, the third antigen-binding region comprises a VH region comprising a paratope that comprises amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0080] In some embodiments, the third antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0081] In some embodiments, the third antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0082] In some embodiments, the third antigen-binding region comprises a VL region comprising a paratope that comprises any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0083] In some embodiments, the third antigen-binding region comprises a VL region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine or all ten amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0084] In some embodiments, the third antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0085] In some embodiments, the third antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0086] In some embodiments, the third antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0087] In some embodiments, the multi-specific antigen-binding protein comprises the fourth antigen-binding region that specifically binds VEGF, wherein the fourth antigen-binding region comprises: (a) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 281-295 and 318; (b) a heavy chain comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 269, 274, 277, 278, or 296-297, and a light chain comprising the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs:268, 273, 279, 280, and 298-299, respectively; or (c) an antigen-binding region comprising the sequence of SEQ ID NO: 267.

[0088] In some embodiments, the fourth antigen-binding region binds one or more of VEGF- A, VEGF-B, VEGF-C, and VEGF-D.

[0089] In some embodiments, the fourth antigen-binding region comprises a variable heavy chain sequence set forth in any one of SEQ ID NOs: 281-297 and 318.

[0090] In some embodiments, the fourth antigen-binding region comprises a variable heavy chain sequence set forth in SEQ ID NO: 296 or 297 and a variable light chain sequence set forth in SEQ ID NO: 298 or 299, respectively.

[0091] In some embodiments, the fourth antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NO: 274 and a light chain sequence set forth in SEQ ID NO: 273.

[0092] In some embodiments, the fourth antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NOs: 277 and 278 and a light chain sequence set forth in SEQ ID NO: 279 and 280.

[0093] In some embodiments, the fourth antigen-binding region comprises a humanized, human, or chimeric fourth antigen-binding region.

[0094] In some embodiments, the fourth antigen-binding region comprises a humanized antibody or antigen-binding region.

[0095] In some embodiments, the fourth antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

[0096] In some embodiments, the fourth antigen-binding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

[0097] In some embodiments, the fourth antigen-binding region comprises a monoclonal fourth antigen-binding region.

[0098] In some embodiments, the fourth 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 any one of SEQ ID NOs: 269, 274, 277, 278, 281-297, 318, 330, and 331.

[0099] In some embodiments, the fourth 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 any one of SEQ ID NOs: 268, 273, 279, 280, 298-299, 322, and 325.

[0100] In some embodiments, the fourth antigen-binding region binds an epitope of VEGF-A comprising at least one of amino acids 82-91 set forth in SEQ ID NO: 265.

[0101] In some embodiments, the fourth 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 any one of SEQ ID NOs: 266, 267, and 332.

[0102] In some embodiments, the fourth antigen-binding region does not comprise an Fc domain.

[0103] In some embodiments, the multi-specific antigen-binding proteins comprise a heterodimeric Fc domain.

[0104] In some embodiments, the multi-specific antigen-binding protein comprises a bispecific IgG, a bi-specific or a tetrabody (e.g., heavy chain and scFv fusion, heavy chain and scdsFv fusion, heavy chain and sdAb fusion).

[0105] In some embodiments, the multi-specific antigen-binding protein is a bi-specific IgG.

[0106] In some embodiments, the multi-specific antigen-binding protein is a bi-specific tetrabody.

[0107] In some embodiments, the bi-specific tetrabody comprises a heavy chain and scFv fusion, a heavy chain and scdsFv fusion, or a heavy chain and sdAb fusion.

[0108] Further described herein, in certain embodiments, are multi-specific antigenbinding proteins of any one of the above embodiments, for use in the treatment of a disorder or condition of eye.

[0109] In some embodiments, the multi-specific antigen-binding protein is formulated for administration to a subject by intravitreal injection.

[0110] Further described herein, in certain embodiments, are isolated polynucleotides or a set of polynucleotides encoding the first antigen-binding regions, a VH thereof, a VL thereof, a light chain thereof, a heavy chain thereof, or an antigen-binding portion thereof; optionally wherein the polynucleotide or set of polynucleotides comprises cDNA.

[0111] Further described herein, in certain embodiments, are isolated polynucleotides or set of polynucleotides encoding the second antigen-binding regions, a VH thereof, a VL thereof, a light chain thereof, a heavy chain thereof, or an antigen-binding portion thereof; optionally wherein the polynucleotide or set of polynucleotides comprises cDNA.

[0112] Further described herein, in certain embodiments, are vectors or set of vectors comprising the polynucleotide or set of polynucleotides.

[0113] Further described herein, in certain embodiments, are host cells comprising the polynucleotide or set of polynucleotides or the vector or set of vectors.

[0114] Further described herein, in certain embodiments, are pharmaceutical compositions comprising the multi-specific antigen-binding proteins and a pharmaceutically acceptable excipient.

[0115] Further described herein, in certain embodiments, are kits comprising the multispecific antigen-binding proteins or pharmaceutical compositions and instructions for use.

[0116] Further described herein, in certain embodiments, are methods for treating a disorder or condition of the eye, the method comprising administering to a mammalian subject a therapeutically effective amount a multi-specific antigen-binding protein or a pharmaceutical composition.

[0117] In some embodiments, the disorder or condition of the eye is selected from the group consisting of retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment.

[0118] Further described herein, in certain embodiments, are methods for treating a pathology associated with CD1 lb / CD18 (Mac-1) binding to fibrin or CD1 lb / CD18 (Mac-1) binding with fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount a multi-specific antigen-binding protein or a pharmaceutical composition.

[0119] Further described herein, in certain embodiments, are methods of inhibiting microglia activation, the method comprising administering to a mammalian subject a therapeutically effective amount of a multi-specific antigen-binding protein or a pharmaceutical composition.

[0120] Further described herein, in certain embodiments, are methods of producing a multi-specific antigen-binding protein, the method comprising expressing a multi-specific antigen-binding protein or antigen-binding region thereof in a host cell and isolating the expressed multi-specific antigen-binding protein or antigen-binding region thereof.

[0121] Further described herein, in certain embodiments, are methods of preventing a disorder or condition of the eye, the method comprising administering to a mammalian subject a therapeutically effective amount a multi-specific antigen-binding protein or a pharmaceutical composition.

[0122] Further described herein, in certain embodiments, are methods of treating a disorder or condition of the eye associated with increased vascularization in a subject in needthereof, comprising administering to the subject a multi-specific antigen-binding protein or a pharmaceutical composition.

[0123] Further described herein, in certain embodiments, are methods of preventing a disorder or condition of the eye associated with increased vascularization in a subject in need thereof, comprising administering to the subject a multi-specific antigen-binding protein or a pharmaceutical composition.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0124] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings, where:

[0125] FIG. 1 is a graph depicting the uveitis clinical score of rats administered intravitreally isotype control, murinized antibody clone 60143-with Fc stabilization LALA mutations (low dose = 10 pg / eye; high dose = 50 pg / eye), positive control FTY-720 (administered by oral gavage at a dose of 0.3 mg / kg), and naive mice with no experimental autoimmune encephalomyelitis (EAE) induction.

[0126] FIG. 2 is a graph showing vascular leakage via quantitative fluorescein angiography (qFA) in rats treated with the anti-VEGF antigen-binding construct Eylea (aflibercept) and murinized anti-fibrin antibody clone 60143-with Fc stabilization LALA mutations (“m60143 -LALA’) at the indicated concentrations.

[0127] FIG. 3 is a graph showing choroidal neovascularization (CNV) lesion area in rats treated with the anti-VEGF antigen-binding construct Eylea (aflibercept) and murinized antifibrin antibody clone 60143-with Fc stabilization LALA mutations (“m60143 -LALA’) at the indicated concentrations.

[0128] FIG. 4 is a graph showing vascular leakage via qFA in rats treated with anti-fibrin antibody clone 60143-LALA, an anti-VEGF antibody (bevacizumab), exemplary bi-specific 60143 / bevacizumab binding protein of the disclosure, isotype control (anti-RSV, which is not present in mice), or a bi-specific 60143 / anti-RSV antigen-binding protein. qFA images were recorded at 4 minutes and 6 minutes post-fluorescein injection and differences in the integrated density (IntDen Diff) between the two times post-injection were calculated as a readout of vascular leakage. Results shown are individual eyes, depicting the mean ± 95% confidence interval, and analyzed by one-way ANOVA, with Dunnett’s multiple comparison to the isotype control.

[0129] FIG. 5 is a heatmap showing the differences in levels of expression and p values thereof of cytokines in rats treated anti-RSV isotype control (“Iso”) versus bevacizumab (“Avastin”) or anti-fibrin antibody clone 60143-LALA (“60143 -LAL A”) versus anti-RSV isotype control (“Iso”). Results shown were analyzed by one-way ANOVA, with Dunnett’s multiple comparison to the isotype control.

[0130] FIG. 6 is a set of graphs showing the concentration of cytokines TNFa and IL- 17a, respectively, in rats, 14 days following treatment with anti-fibrin antibody clone 60143- LALA, bevacizumab, a bi-specific 60143 / anti-RSV antigen-binding protein, or an anti-RSV isotype control, as described in FIG. 4. Results shown depict the mean ± 95% confidence interval, and analyzed by one-way ANOVA, with Dunnett’s multiple comparison to the isotype control.

[0131] FIG. 7 is a set of graphs showing the concentration of cytokines VEGF and fractalkine (CX3CL1), respectively, in rats, 14 days following treatment with anti-fibrin antibody clone 60143-LALA, bevacizumab, a bi-specific 60143 / anti-RSV antigen-binding protein, or an anti-RSV isotype control, as described in FIG. 4. Results shown depict the mean ± 95% confidence interval, and analyzed by one-way ANOVA, with Dunnett’s multiple comparison to the isotype.

[0132] FIG. 8 is a set of graphs showing the concentration of cytokines MIP-la, IL- lb, IP10, and leptin, respectively, in rats, 14 days following treatment with anti-fibrin antibody clone 60143, bevacizumab, a bi-specific 60143 / anti-RSV antigen-binding protein, or an anti- RSV isotype control, as described in FIG. 4. Results shown depict the mean ± 95% confidence interval, and analyzed by one-way ANOVA, with Dunnett’s multiple comparison to the isotype.

[0133] FIG. 9 is a schematic (left panel) and graph (right panel), respectively, showing a biolayer interferometry (BLI) experiment to demonstrate binding of an exemplary bi-specific antigen-binding protein (e.g., a bi-specific tetrabody (e.g., heavy chain and scFv fusion, heavy chain and scdsFv fusion, and heavy chain and VHH fusion)) of the disclosure with the fibrin / fibrinogen yC-P2 peptide and with VEGF.

[0134] FIG. 10 is a schematic showing exemplary bi-specific tetrabodies (immunoglobulin G (IgG) fused to two variable domains (VHH)) of the disclosure with an anti-fibrin / fibrinogen yC-P2 peptide IgG and an anti-VEGF VHH fused to the CH3 domain (left panel) or constant light chain (CL) domain (right panel) of the IgG, respectively.

[0135] FIG. 11 is a schematic showing exemplary bi-specific tetrabodies (IgG and Fab fusions) of the disclosure with an anti-fibrin / fibrinogen yC-P2 peptide IgG fused to two anti- VEGF Fabs. Two different HC-LC pairings are indicated.

[0136] FIG. 12 is a schematic showing exemplary bi-specific antigen-binding proteins (IgG-fusion protein fusions) of the disclosure with an anti-fibrin / fibrinogen yC-P2 peptide IgG fused to Aflibercept, which is a recombinant fusion protein consisting of the extracellular domains of human VEGF receptor 1 and 2 (domains 2 and 3) fused to the Fc portion of human IgGl .DETAILED DESCRIPTIONDefinitions

[0137] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0138] As used herein, the singular form “a,” “an,” and “the” includes plural references unless indicated otherwise.

[0139] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0140] For all compositions described herein, and all methods using a composition described herein, the compositions can either comprise the listed components or steps or can “consist essentially of’ the listed components or steps. When a composition is described as “consisting essentially of’ the listed components, the composition contains the components listed, and may contain other components which do not substantially affect the condition being treated, but do not contain any other components which substantially affect thecondition being treated other than those components expressly listed; or, if the composition does contain extra components other than those listed which substantially affect the condition being treated, the composition does not contain a sufficient concentration or amount of the extra components to substantially affect the condition being treated. When a method is described as “consisting essentially of’ the listed steps, the method contains the steps listed, and may contain other steps that do not substantially affect the condition being treated, but the method does not contain any other steps which substantially affect the condition being treated other than those steps expressly listed. As a non-limiting specific example, when a composition is described as ‘consisting essentially of a component, the composition may additionally contain any amount of pharmaceutically acceptable carriers, vehicles, or diluents and other such components which do not substantially affect the condition being treated.

[0141] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0142] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), which each include the primary transformed or transfected cell and progeny derived therefrom. Such progeny may not be completely identical in nucleic acid content to a parent cell and may contain mutations. A “recombinant host cell” or “host cell” refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells.

[0143] An “effective amount” or “therapeutically effective amount” as used herein refers to an amount of a therapeutic compound, such as a multi-specific antigen-binding protein, administered to an individual, either as a single dose or as part of a series of doses, which is effective to produce or contribute to a desired therapeutic effect, either alone or in combination with another therapeutic modality. Examples of a desired therapeutic effect is enhancing an immune response, slowing, or delaying tumor development; stabilization ofdisease; and amelioration of one or more symptoms. An effective amount may be given in one or more dosages.

[0144] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed during the course of clinical pathology. Desirable effects of treatment include preventing recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. None of these terms require the supervision of medical personnel.

[0145] The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate an immune response in a subject.

[0146] As used herein, the terms “subject” or “individual” mean a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep.

[0147] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic or diagnostic products.

[0148] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0149] The terms “co-administration,” “co-administer,” and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently, or sequentially within no specific time limits.

[0150] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[0151] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0152] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0153] The term “about” indicates and encompasses an indicated value and a range above and below that value. For example, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. Where applicable, the term “about” indicates the designated value(s) ± one standard deviation of that value(s).

[0154] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor.

[0155] The term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. An “antagonist” is an entity that binds to and antagonizes a receptor.

[0156] For any of the structural and functional characteristics described herein, methods of determining these characteristics are known in the art.

[0157] The term “optionally” is meant, when used sequentially, to include from one to all of the enumerated combinations and contemplates all sub-combinations.

[0158] The term “amino acid” refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (He; 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 (Vai; V).

[0159] The term “affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a multi-specific antigen-binding protein) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (c.g, multi-specific antigen-binding protein and antigen or epitope).

[0160] The term “ka” (sec-1), as used herein, refers to the dissociation rate constant of a particular antigen-binding region-antigen interaction. This value is also referred to as the koir value.

[0161] The term “ka” (M^xsec-1), as used herein, refers to the association rate constant of a particular antigen-binding region-antigen interaction. This value is also referred to as the konvalue.

[0162] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antigen-binding region-antigen interaction. KD = kd / ka. In some embodiments, the affinity of an antigen-binding region is described in terms of the KD for an interaction between such antigen-binding region and its antigen. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.

[0163] The term “KA” (M'1), as used herein, refers to the association equilibrium constant of a particular antigen-binding region-antigen interaction. KA = ka / kd.

[0164] The term “multi-specific antigen-binding protein” is used herein in its broadest sense to include molecules comprising polypeptides that have the capability of binding to two or more distinct antigens. Multi-specific antigen-binding proteins include multi-specific antibodies and antibodies comprising binding agents that have affinity to one or more antigens.

[0165] The term “antigen -binding region” is used interchangeable herein in their broadest sense to include a first or a second region of a multi-specific antigen-binding protein comprising a polypeptide comprising affinity to an antigen or epitope.

[0166] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding regions that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antigen -binding proteins. Antibodies include any alternative antibody format 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 a bivalent domain antibody.

[0167] A “fibrin antigen-binding region,” “anti-fibrin antigen-binding region,” or “fibrinspecific antigen-binding region” is an antigen-binding region of a multi-specific antigenbinding protein, as provided herein, which specifically binds to the antigen fibrin. In some embodiments, the fibrin antigen-binding region binds the extracellular domain of fibrin. In some embodiments, a fibrin antigen-binding region provided herein binds to an epitope of fibrin that is conserved between or among fibrin proteins from different species.

[0168] A “VEGF antigen-binding region,” “anti-VEGF antigen-binding region,” or “VEGF-specific antigen-binding region” is an antigen-binding region of a multi-specific antigen-binding protein, as provided herein, which specifically binds to one or more VEGF antigens (E.G., VEGF-A, VEGF-B, VEGF-C, and VEGF-D).

[0169] The term “epitope” means a portion of an antigen that specifically binds to an antigen-binding region.

[0170] The term “hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”).

[0171] The terms “chimeric antibody” and “chimeric antigen-binding region” refer to an antibody or antigen-binding region, respectively, 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.

[0172] The terms “human antibody” and “human antigen-binding region” refer to an antibody or antigen-binding region which possess an amino acid sequence corresponding to that of an antibody or antigen-binding region produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody / antigen -binding region repertoire or human antibody / antigen -binding region-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies and antigen-binding regions specifically exclude humanized antibodies and antigen-binding regions.

[0173] The terms “humanized antibody” and “humanized antigen-binding regions” refer to a protein having a sequence that differs from the sequence of an antibody or antigenbinding region derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody or antigen-binding region is less likely to induce an immune response, and / or induces a less severe immune response, as compared to the non-human species antibody or antigen-binding region, when it is administered to a human subject.

[0174] 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 two or more different epitopes.

[0175] A “monospecific antigen-binding region” is an antigen-binding region that comprises one or more binding sites that specifically bind to a single epitope. An example ofa monospecific antigen-binding region is a scFv which recognizes an epitope. The binding specificity may be present in any suitable valency.

[0176] The term “monoclonal antigen-binding region” refers to an antigen-binding region from a population of substantially homogeneous antigen-binding regions. A population of substantially homogeneous antigen-binding regions comprises antigen-binding regions that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antigen-binding region. Such variants are generally present in only minor amounts. A monoclonal antigen-binding region is typically obtained by a process that includes the selection of a single antigen-binding region from a plurality of antigen-binding region. For example, the selection process is the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antigen-binding region, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0177] The term “single-chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In some embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule. As described in more detail herein, an scFv has a variable domain of light chain (VL) connected from its C-terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternately the scFv comprises of polypeptide chain where in the C- terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain.

[0178] The “Fab fragment” (also referred to interchangeably herein as “fragment antigen binding” or “Fab”) contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains, respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are 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 CHI domain including one or more cysteines from the antibody hinge region.

[0179] “F(ab')2” fragments contain two Fab' fragments joined, near the hinge region, by disulfide bonds. F(ab')2 fragments may be generated, for example, by recombinant methodsor by pepsin digestion of an intact antibody. The F(ab') fragments can be dissociated, for example, by treatment with B-mercaptoethanol.

[0180] “Fv” fragments comprise a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain.

[0181] “Single-chain Fv” or “sFv” or “scFv” includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, 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 scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. In some embodiments, a multi-specific antigen-binding protein herein is a bispecific antibody (e.g., a bi-specific IgG or bi-specific tetrabody (e.g., heavy chain and scFv fusion, heavy chain and scdsFv fusion, and heavy chain and VHH fusion)).

[0182] “ scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, anFc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL- VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0183] 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 the other variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. sdAbs are fairly stable and easy to express as fusion partner 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).

[0184] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region. For example, when used to refer to an IgG molecule, a “full length antibody” is an antibody that comprises two heavy chains and two light chains.

[0185] 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 an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0186] The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0187] The term “substantially purified” refers to a construct described herein, or variant thereof that may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., a native cell, or host cell in the case of recombinantly produced heteromultimer that in some embodiments, is substantially free of cellular material includes 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.

[0188] The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer 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 measured 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 persons of skill) or by visual inspection. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0189] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0190] Optimal alignment of sequences for comparison can be conducted, e.g., 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. 45:443 (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).

[0191] Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range 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.

[0192] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.Multi-Specific Antigen-Binding Proteins

[0193] The present application provides multi-specific antigen-binding proteins which comprises at least two distinct antigen-binding regions; wherein at least one antigen-binding region binds human fibrin yC or fibrinogen yC domain, and at least one antigen-binding region binds at least one isoform of VEGF.

[0194] Without wishing to be bound by theory, multi-specific antigen-binding proteins of the disclosure, by way of fibrin antigen-binding regions, antagonize and block CD1 lb / CD18 (Mac-1) receptor binding to fibrin and block binding of the al-domain, which is present on the cell surface of integrin receptors CDl lb / CD18 (Mac-1) and CDl lc / CD18on microglia, macrophages, and dendritic cells. Binding of the al-domain activates multiple signal transduction pathways which trigger an inflammatory response that leads to the secretion of cytokines that indirectly and directly damage nerves, leading to neuroinflammation.Meanwhile, by way of VEGF antigen-binding regions, the multi-specific antigen-binding proteins of the disclosure neutralize VEGF and inhibit angiogenesis, such that the multispecific antigen-binding proteins are readily suitable for use in methods of inhibiting microglial activation; and treating and preventing pathologies associated with CD1 lb / CD18(Mac-1) binding to fibrin or CD1 lb / CD18 (Mac-1) binding with fibrinogen, and disorders and conditions of the eye associated with increased vascularization.

[0195] Multi-specific antigen-binding proteins of the present disclosure can comprise any antigen-binding region that binds to the antigen including, without limitation, a fusion protein, monoclonal antigen-binding region, a polyclonal antigen-binding region, a recombinant antigen-binding region, a bispecific antibody, a conjugated antibody, a human antibody or antigen-binding region thereof, a humanized antibody or antigen-binding region thereof, and a functional fragment thereof, including but not limited to a single-domain antibody (sdAb) also referred to interchangeably herein as a variable domain (VHH) of camelid derived nanobody, a heavy chain variable domain (VH), a light chain variable domain (VL), and an alternative scaffold known in the art to function as antigen-binding region, such as a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., single chain TCR, and the like. In some embodiments, it is beneficial for the antigen-binding region to be derived from the same species in which the multi-specific antigen-binding protein will ultimately be used in. For example, for use in humans, it is beneficial for the antigen-binding region of the multispecific antigen-binding protein to comprise human or humanized residues for the antigenbinding region of an antibody or antibody fragment.

[0196] In some embodiments, the antigen-binding region of the multi-specific antigenbinding protein comprises an antibody (e.g., an IgG). In some embodiments, a multi-specific antigen-binding protein of the disclosure comprises a bi-specific antibody (e.g., a bi-specific IgG). In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric antibody.

[0197] In some embodiments, the antigen-binding region comprises an antigen-binding fragment of an antibody. In some embodiments, the antigen-binding region comprises a F(ab) fragment. In some embodiments, the antigen-binding region comprises a F(ab') fragment. In some embodiments, the antigen-binding region comprises an scFv (e.g., scFv or scdsFv). In some embodiments, the antigen-binding region comprises two single chain variable fragments (scFvs or scdsFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding region comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind different antigens. In some embodiments, the scFv is a human scFv. In some embodiments,the scFv is a humanized scFv. In some embodiments, the scFv is a chimeric scFv. In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0198] In some embodiments, a multi-specific antigen-binding protein of the disclosure is a tetrabody comprising a heavy chain and scFv (e.g., scFv or scdsFv) fusion. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When there are two or more scFv linked together, each scFv can be linked to the next scFv with a peptide linked. 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 distinct epitope on the same antigen.

[0199] In some embodiments, the antigen-binding region comprises a single-domain antibody (sdAb; also known as VHH). In some embodiments, the sdAb is a humanized sdAb. In some embodiments, the sdAb is a chimeric sdAb.

[0200] In some embodiments, a multi-specific antigen-binding protein of the disclosure is a tetrabody comprising a heavy chain and sdAb fusion.

[0201] In some embodiments, a multi-specific antigen-binding protein of the present disclosure 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 antigenbinding domains. In some embodiments, each of the two or more antigen-binding domains binds the same antigen. In some embodiments, each of the two or more antigen-binding domains binds a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds a different antigen. In some embodiments, the two or more antigen-binding domains provide the multi-specific antigen-binding protein with logic gating, such as ‘or’ logic gating.

[0202] In some embodiments, the multi-specific antigen-binding protein comprises two or more (e.g., four) antigen-binding regions. In some embodiments, two or more antigenbinding regions are attached to one another via a viable flexible, stable linker. Glycine-serine-rich linkers of 2 to 80 amino acids or 6 to 30 ammo acids are, in some embodiments, used because the short side chain provides conformational flexibility and minimal immunogenicity, as well as because serine also improves solubility. Any suitable linker known in the art or described herein can be used, such as e.g., a (GS)n linker (SEQ ID NO: 336), a (GGS)n ((G2S)n) linker (SEQ ID NO: 337), a (GGGS)n ((G3S)n) linker (SEQ ID NO: 333), a (GGGGS)n ((G4S)n) linker (SEQ ID NO: 317), and a (GGGGGS)n ((G5S)n) linker (SEQ ID NO: 334), wherein n is 1 , 2, 3, 4, 5, or 6; or a GGSGGSGGS (G2S)3linker (SEQ ID NO: 335), a GGGSGGGSGGGS (G3S)3linker (SEQ ID NO: 328), a GGGGSGGGGSGGGGS (G4S)3linker (SEQ ID NO: 327), and a GGGGGSGGGGS (G5S)2linker (SEQ ID NO: 329). In some embodiments, antigen-binding regions are selected from an antibody (e.g., an IgG), an antigen-binding fragment of an antibody, an scFv (e.g., scFv or scdsFv), a Fab, a sdAb (VHH), or a recombinant fibronectin or VEGF domain. In some embodiments, one or two of the two or more antigen-binding regions are an IgG antibody. In some embodiments, one or two of the two or more antigen-binding regions are a scFv (e.g., scFv or scdsFv). In some embodiments, one or two of the two or more antigen-binding regions are a Fab. In some embodiments, one or two of the two or more antigen-binding regions are a sdAb.

[0203] In some embodiments, the multi-specific antigen-binding protein comprises an antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv or scdsFv) of a bi-specific antigen-binding protein, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VHi) upstream of its VL (VLi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall multi-specific antigen-binding protein has the arrangement VHi-VLi-VL2-VH2. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VLi) upstream of its VH (VHi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall multi-specific antigen-binding protein has the arrangement VLi VHi-VH2-VL2. In some embodiments, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VLi and VL2if the construct is arranged as VHi-VLi-VL2-VH2, or between VHi and VH2if the construct is arranged as VLi-VHi-VH2-VL2. The linker, in some embodiments, is a linker as described herein, e.g., a (G4S)n linker (SEQ ID NO: 317), wherein n is 1, 2, 3, 4, 5, or 6. In someembodiments, the linker is GS. In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, a linker is disposed between the VL and VH of the first scFv. In some embodiments, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers can be the same or different. Accordingly, in some embodiments, a multi-specific antigen-binding protein comprises VLs, VHs, and, in some embodiments, further comprises one or more linkers in an arrangement as described herein.

[0204] The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), e.g., IgGl, IgG?, IgGs, IgG4, IgAl, and IgA?. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.

[0205] An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The IgGl heavy chain comprises of the VH, CHI, CH2 and CH3 domains, respectively, from the N to C-terminus. The light chain comprises of the VL and CL domains from N to C terminus. The IgGl heavy chain comprises a hinge between the CHI and CH2 domains. In some embodiments, the immunoglobulin constructs comprise at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to a therapeutic polypeptide. In some embodiments, the immunoglobulin domain found in a multi-specific antigen-binding protein provided herein is from or derived from an immunoglobulin-based construct such as a diabody or a nanobody. In some embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody such as a camelid antibody. In some embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody, or any chimeric antibody.

[0206] In some embodiments, the multi-specific antigen-binding proteins provided herein comprise one or more heavy chains. In some embodiments, the heavy chain is an IgA. In some embodiments, the heavy chain is an IgD. In some embodiments, the heavy chain is an IgE. In some embodiments, the heavy chain is an IgG. In some embodiments, the heavy chain is an IgM. In some embodiments, the heavy chain is an IgGl. In some embodiments, the heavy chain is an IgG2. In some embodiments, the heavy chain is an IgG3. In some embodiments, the heavy chain is an IgG4. In some embodiments, the heavy chain is an IgAl. In some embodiments, the heavy chain is an IgA2.

[0207] In some embodiments, the multi-specific antigen-binding protein comprises an IgGl antibody. In some embodiments, the multi-specific antigen-binding protein comprises an IgG3 antibody. In some embodiments, the multi-specific antigen-binding protein comprises an IgG2 antibody. In some embodiments, the multi-specific antigen-binding protein comprises an IgG4 antibody.

[0208] Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the complementarity determining regions (CDRs), the latter being of highest sequence variability and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the 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 herein interchangeably in reference to portions of the variable region that form the antigen-binding regions. This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0209] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol..273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732- 745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.

[0210] Table 1 provides 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.

[0211] CDRs may be assigned, for example, 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, incorporated by reference in its entirety.Table 1. Residues in CDRs according to Kabat and Chothia numbering schemes* The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the CDR.

[0212] The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0213] One example of an antigen-binding region is an antigen-binding region formed by a VH-VL dimer of an antibody. Another example of an antigen-binding region is an antigenbinding region formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin. An antigen-binding region can include CDRs 1, 2, and 3 from a heavy chain in that order; and CDRs 1, 2, and 3 from a light chain in that order.

[0214] Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope, in some embodiments, comprises amino acid residues thatare directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigen-binding region binds can be determined using known techniques for epitope determination such as, for example, testing for antigen-binding region binding to fibrin variants with different point-mutations, or to chimeric fibrin variants.

[0215] To screen for antigen-binding regions which bind to an epitope on a target antigen interest (e.g., fibrin or VEGF), a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, or additionally, epitope mapping can be performed by methods known in the art.

[0216] Chimeric antibodies are antibodies 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.

[0217] Human antibodies or antigen-binding regions thereof are antibodies which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0218] A humanized antibody or antigen-binding region thereof has a sequence that differs from the sequence 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, as compared to the non-human species antibody, when it is administered to a human subject. In some embodiments, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce the humanized antibody. In some embodiments, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. In some embodiments, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immuno-specific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen.Examples of how to make humanized antibodies are found in U.S. Pat. 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.

[0219] The two or more different epitopes are, in some embodiments, epitopes on the same antigen (e.g., a single fibrin molecule expressed by a cell) or on different antigens (e.g., different fibrin molecules expressed by the same cell, or a fibrin molecule and a VEGF molecule). In some embodiments, a multi-specific antigen-binding protein binds two different epitopes (i.e., a “bispecific antibody” or “bi-specific antibody”). In some embodiments, a multi-specific antibody binds three different epitopes (i.e., a “trispecific antibody”).

[0220] The antigen-binding regions of the multi-specific antigen-binding proteins can include an antigen-binding region or variable domain described herein such as the antigenbinding region of the clones set forth in the drawings and / or tables. In some embodiments, the multi-specific antigen-binding protein comprises an alternative scaffold. In some embodiments, the multi-specific antigen-binding protein consists of an alternative scaffold. In some embodiments, the multi-specific antigen-binding protein consists essentially of an alternative scaffold. In some embodiments, the multi-specific antigen-binding protein comprises two or more antibody fragments. In some embodiments, the multi-specific antigenbinding protein consists of two or more antibody fragments. In some embodiments, the multispecific antigen-binding protein consists essentially of two or more antibody fragments.

[0221] In some embodiments the multi-specific antigen-binding protein or antigenbinding region thereof is produced by hybridomas. In some embodiments, the antibodies are produced by recombinant cells engineered to express the desired variable and constant domains.

[0222] In some embodiments the multi-specific antigen-binding protein comprise one or more single chain antibodies or other antibody derivatives retaining the antigen specificity and the lower hinge region or a variant thereof.

[0223] In some embodiments the multi-specific antigen-binding protein is a polyfunctional antibody, recombinant antibody, human antibody, humanized antibody, or fragment or variant thereof. In some embodiments, the multi-specific antigen-binding protein comprises antibody fragments or a derivative thereof, which can be selected from a Fab fragment, a Fab'2 fragment, a CDR, and scFv (e.g., heavy chain and scFv fusions).

[0224] In some embodiments, the antibodies are capable of forming an immune complex. For example, an immune complex can be a tumor cell covered by antibodies.

[0225] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0226] Optimal alignment of sequences for comparison can be conducted, e.g., 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).

[0227] 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 / ).

[0228] In some embodiments, multi-specific antigen-binding proteins of the disclosure comprise amino acid substitutions to enable heterodimerization of two heavy chains, such as with a knob-into-hole approach, and / or CrossMAb or other constant domain interface technologies (See e.g., Surowka et al. MAbs 13, 1967714 (2021), Barlow etal. MAbs 17, 2479531 (2025), and U.S. Patent Publication No. US 2023 / 0265134, each of which is incorporated by reference in its entirely as it pertains to constant domain interface technologies) to enable light chain pairing yet prevent unspecific binding of the light chains, as exemplified in the amino acid sequences of SEQ ID NOs: 304-307.

[0229] In some embodiments, a multi-specific antigen-binding protein comprises a leader sequence, such as SEQ ID NO: 300.

[0230] In some embodiments, a multi-specific antigen-binding protein (e.g., anti-fibrin P2 / anti-VEGF antigen-binding protein (e.g., a bi-specific IgG)) of the disclosure comprises one or more (e.g., one, two, three, four, or more) of any one of the amino acid sequences ofSEQ ID NOs: 304-315, 318, 321, 322, 324, 325, and 330-332 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0231] Further, exemplary multi-specific antigen-binding proteins of the disclosure are provided, for example, as SEQ ID NOs: 301-303, 320, 323, and 326. In some embodiments, a multi-specific antigen-binding protein of the disclosure comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 301-303, 320, 323, and 326.Fibrin Antigen-Binding Regions

[0232] In some embodiments, the multi-specific antigen-binding proteins described herein comprise at least one (e.g., one or two e.g., a first antigen-binding region and a third antigen-binding region) antigen-binding region that binds human fibrin yC or fibrinogen yC domain.VH Domains

[0233] In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 163. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 7. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 19. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 31. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 43. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 55. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 67. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 79. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 91. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 103. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 115. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 127. In some embodiments, a multi-specific antigen-bindingprotein provided herein comprises a VH sequence of SEQ ID NO: 139. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 151. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 175. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 187. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 199. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 211. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 223. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 235. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VH sequence of SEQ ID NO: 254.

[0234] In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VH sequence provided in SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, a multispecific antigen-binding protein provided herein comprises a VH sequence provided in SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254, 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 embodiments, 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 a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.VL Domains

[0235] In some embodiments, a fibrin antigen-binding region provided herein comprises a VL sequence selected from any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ IDNO: 166. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 10. In some embodiments, a multi-specific antigenbinding protein provided herein comprises a VL sequence of SEQ ID NO: 22. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 34. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 46. In some embodiments, a multispecific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 58. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 70. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 82. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 94. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 106. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 118. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 130. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 142. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 154. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 178. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 190. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 202. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 214. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 226. In some embodiments, a multi-specific antigen -binding protein provided herein comprises a VL sequence of SEQ ID NO: 238. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence of SEQ ID NO: 255.

[0236] In some embodiments, a fibrin antigen-binding region provided herein comprises a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VL sequence provided in SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, a multi-specific antigen-binding protein provided herein comprises a VL sequence provided in SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255, 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 embodiments, 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 a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.VH-VL Combinations

[0237] In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in any one of SEQ ID NOs: 166, 321, or 324. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in SEQ ID NO: 166. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 7 and a VL sequence set forth in SEQ ID NO: 10. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 19 and a VL sequence set forth in SEQ ID NO: 22. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 31 and a VL sequence set forth in SEQ ID NO: 34. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 43 and a VL sequence set forth in SEQ ID NO: 46. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 55 and a VL sequence set forth in SEQ ID NO: 58. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 67 and a VL sequence set forth in SEQ ID NO: 70. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 79 and a VL sequence set forth in SEQ ID NO: 82. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 91 and a VL sequence set forth in SEQ ID NO: 94. In some embodiments, a fibrin antigen-binding region provided herein comprises a VHsequence set forth in SEQ ID NO: 103 and a VL sequence set forth in SEQ ID NO: 106. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 115 and a VL sequence set forth in SEQ ID NO: 118. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 127 and a VL sequence set forth in SEQ ID NO: 130. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 139 and a VL sequence set forth in SEQ ID NO: 142. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 151 and a VL sequence set forth in SEQ ID NO: 154. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 175 and a VL sequence set forth in SEQ ID NO: 178. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 187 and a VL sequence set forth in SEQ ID NO: 190. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 199 and a VL sequence set forth in SEQ ID NO: 202. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 211 and a VL sequence set forth in SEQ ID NO: 214. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 223 and a VL sequence set forth in SEQ ID NO: 226. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 235 and a VL sequence set forth in SEQ ID NO: 238. In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 256 and a VL sequence set forth in SEQ ID NO: 257.

[0238] In some embodiments, a fibrin antigen-binding region provided herein comprises a VH sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to an illustrative VH sequence provided in any one of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254; and a VL sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to a VL sequence provided in any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, a multi-specific antigenbinding protein provided herein comprises a VH sequence provided in any one of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 amino acid substitutions, and a VL sequence provided in any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255, 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 embodiments, 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 a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.CDRs

[0239] In some embodiments, a fibrin antigen-binding region provided herein comprises one to three CDRs of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, a multi-specific antigen-binding protein provided herein comprises two to three CDRs of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, a multi-specific antigenbinding protein provided herein comprises three CDRs of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, the CDRs are Exemplary CDRs. In some embodiments, the CDRs are Kabat CDRs. In some embodiments, the CDRs are Chothia CDRs. In some embodiments, the CDRs are AbM CDRs. In some embodiments, the CDRs are Contact CDRs. In some embodiments, the CDRs are IMGT CDRs.

[0240] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254. In some embodiments, the CDR-H1 is a CDR-H1 of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is a CDR-H2 of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 is a CDR-H3 of a VHdomain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, a fibrin antigen-binding region described in this paragraph are referred to herein as “variants.” In some embodiments, such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.

[0241] In some embodiments, a fibrin antigen-binding region provided herein comprises one to three CDRs of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, a multi-specific antigen-binding protein provided herein comprises two to three CDRs of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, a multi-specific antigen-binding protein provided herein comprises three CDRs of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, the CDRs are Exemplary CDRs. In some embodiments, the CDRs are Kabat CDRs. In some embodiments, the CDRs are Chothia CDRs. In some embodiments, the CDRs are AbM CDRs. In some embodiments, the CDRs are Contact CDRs. In some embodiments, the CDRs are IMGT CDRs.

[0242] In some embodiments, the CDRs are CDRs having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1, CDR-L2, or CDR-L3 of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, the CDR-L1 is a CDR-L1 of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-L2 is a CDR-L2 of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-L3 is a CDR- L3 of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255, with up to 1, 2, 3, 4, 5, 6, 7, or 8 aminoacid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, a fibrin antigen-binding region described in this paragraph are referred to herein as “variants.” In some embodiments, such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.

[0243] In some embodiments, a fibrin antigen-binding region provided herein comprises one to three CDRs of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254 and one to three CDRs of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, a multi-specific antigen-binding protein provided herein comprises two to three CDRs of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254 and two to three CDRs of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, a multi-specific antigen-binding protein provided herein comprises three CDRs of a VH domain selected from SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254 and three CDRs of a VL domain of any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255. In some embodiments, the CDRs are Exemplary CDRs. In some embodiments, the CDRs are Kabat CDRs. In some embodiments, the CDRs are Chothia CDRs. In some embodiments, the CDRs are AbM CDRs. In some embodiments, the CDRs are Contact CDRs. In some embodiments, the CDRs are IMGT CDRs.

[0244] In some embodiments, a fibrin antigen-binding region provided herein comprises a (a) CDR-H1 selected from the sequences set forth in any one of SEQ ID NOs: 157, 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, 229, and 258; (b) CDR-H2 selected from the sequences set forth in any one of SEQ ID NOs: 158, 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, 230 and 259; (c) CDR-H3 selected from the sequences set forth in any one of SEQ ID NOs: 159, 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, 231, and 260; (d) CDR-L1 selectedfrom the sequences set forth in any one of SEQ ID NOs: 160, 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, 232 and 262; (e) CDR-L2 selected from the sequences set forth in any one of SEQ ID NOs: 161, 5, 17, 29, 41, 53, 65, 77, 89, 101, 113,125, 137, 149, 173, 185, 197, 209, 221, 233 and 263; and (f) CDR-L3 selected from the sequences set forth in any one of SEQ ID NOs: 162, 6, 18, 30, 42, 54, 66, 78, 90, 102, 114,126, 138, 150, 174, 186, 198, 210, 222, 234 and 264, respectively, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, a fibrin antigen-binding region described in this paragraph are referred to herein as “variants.” In some embodiments, such variants are derived from a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.

[0245] In some embodiments, the CDR-H3 is a CDR-H3 of any one of SEQ ID NOs: 159, 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, 231, and 260, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is a CDR-H2 of any one of SEQ ID NOs: 158, 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, 230 and 259, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H1 is a CDR-Hl of any one of SEQ ID NOs: 157, 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, 229, and 258, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-L3 is a CDR-L3 of any one of SEQ ID NOs: 162, 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 174, 186, 198, 210, 222, 234 and 264, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-L2 is a CDR-L2 of any one of SEQ ID NOs: 161, 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 173, 185, 197, 209, 221, 233 and 263, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-L1 is a CDR-L1 of any one of SEQ ID NOs: 160, 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, 232 and 262, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, 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 a sequence provided herein, for example, by affinity maturation, site directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from a sequence provided herein and are, for example, isolated de novo according to the methods provided herein for obtaining antibodies.

[0246] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, a CDR-H3 of SEQ ID NO: 3, a CDR-L1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0247] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 13, a CDR-H2 of SEQ ID NO: 14, a CDR-H3 of SEQ ID NO: 15, a CDR-L1 of SEQ ID NO: 16, a CDR-L2 of SEQ ID NO: 17, and a CDR-L3 of SEQ ID NO: 18.

[0248] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 26, a CDR-H3 of SEQ ID NO: 27, a CDR-L1 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 29, and a CDR-L3 of SEQ ID NO: 30.

[0249] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 37, a CDR-H2 of SEQ ID NO: 38, a CDR-H3 of SEQ ID NO: 39, a CDR-L1 of SEQ ID NO: 40, a CDR-L2 of SEQ ID NO: 41, and a CDR-L3 of SEQ ID NO: 42.

[0250] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 49, a CDR-H2 of SEQ ID NO: 50, a CDR-H3 of SEQ ID NO: 51, a CDR-L1 of SEQ ID NO: 52, a CDR-L2 of SEQ ID NO: 53, and a CDR-L3 of SEQ ID NO: 54.

[0251] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 61, a CDR-H2 of SEQ ID NO: 62, a CDR-H3 of SEQ ID NO: 63, a CDR-L1 of SEQ ID NO: 64, a CDR-L2 of SEQ ID NO: 65, and a CDR-L3 of SEQ ID NO: 66.

[0252] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 73, a CDR-H2 of SEQ ID NO: 74, a CDR-H3 of SEQ ID NO: 75, a CDR-L1 of SEQ ID NO: 76, a CDR-L2 of SEQ ID NO: 77, and a CDR-L3 of SEQ ID NO: 78.

[0253] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 85, a CDR-H2 of SEQ ID NO: 86, a CDR-H3 of SEQ ID NO: 87,a CDR-L1 of SEQ ID NO: 88, a CDR-L2 of SEQ ID NO: 89, and a CDR-L3 of SEQ ID NO: 90.

[0254] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 97, a CDR-H2 of SEQ ID NO: 98, a CDR-H3 of SEQ ID NO: 99, a CDR-L1 of SEQ ID NO: 100, a CDR-L2 of SEQ ID NO: 101, and a CDR-L3 of SEQ ID NO: 102.

[0255] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 109, a CDR-H2 of SEQ ID NO: 110, a CDR-H3 of SEQ ID NO: 111, a CDR-L1 of SEQ ID NO: 112, a CDR-L2 of SEQ ID NO: 113, and a CDR-L3 of SEQ ID NO: 114.

[0256] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 121, a CDR-H2 of SEQ ID NO: 122, a CDR-H3 of SEQ ID NO: 123, a CDR-L1 of SEQ ID NO: 124, a CDR-L2 of SEQ ID NO: 125, and a CDR-L3 of SEQ ID NO: 126.

[0257] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 133, a CDR-H2 of SEQ ID NO: 134, a CDR-H3 of SEQ ID NO: 135, a CDR-L1 of SEQ ID NO: 136, a CDR-L2 of SEQ ID NO: 137, and a CDR-L3 of SEQ ID NO: 138.

[0258] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 145, a CDR-H2 of SEQ ID NO: 146, a CDR-H3 of SEQ ID NO: 147, a CDR-L1 of SEQ ID NO: 148, a CDR-L2 of SEQ ID NO: 149, and a CDR-L3 of SEQ ID NO: 150.

[0259] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 157, a CDR-H2 of SEQ ID NO: 158, a CDR-H3 of SEQ ID NO: 159, a CDR-L1 of SEQ ID NO: 160, a CDR-L2 of SEQ ID NO: 161, and a CDR-L3 of SEQ ID NO: 162.

[0260] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 169, a CDR-H2 of SEQ ID NO: 170, a CDR-H3 of SEQ ID NO: 171, a CDR-L1 of SEQ ID NO: 172, a CDR-L2 of SEQ ID NO: 173, and a CDR-L3 of SEQ ID NO: 174.

[0261] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 181, a CDR-H2 of SEQ ID NO: 182, a CDR-H3 of SEQ ID NO:183, a CDR-L1 of SEQ ID NO: 184, a CDR-L2 of SEQ ID NO: 185, and a CDR-L3 of SEQ ID NO: 186.

[0262] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 193, a CDR-H2 of SEQ ID NO: 194, a CDR-H3 of SEQ ID NO: 195, a CDR-L1 of SEQ ID NO: 196, a CDR-L2 of SEQ ID NO: 197, and a CDR-L3 of SEQ ID NO: 198.

[0263] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 205, a CDR-H2 of SEQ ID NO: 206, a CDR-H3 of SEQ ID NO: 207, a CDR-L1 of SEQ ID NO: 208, a CDR-L2 of SEQ ID NO: 209, and a CDR-L3 of SEQ ID NO: 210.

[0264] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 217, a CDR-H2 of SEQ ID NO: 218, a CDR-H3 of SEQ ID NO: 219, a CDR-L1 of SEQ ID NO: 220, a CDR-L2 of SEQ ID NO: 221, and a CDR-L3 of SEQ ID NO: 222.

[0265] In some embodiments, a fibrin antigen-binding region provided herein comprises a CDR-H1 of SEQ ID NO: 229, a CDR-H2 of SEQ ID NO: 230, a CDR-H3 of SEQ ID NO: 231, a CDR-L1 of SEQ ID NO: 232, a CDR-L2 of SEQ ID NO: 233, and a CDR-L3 of SEQ ID NO: 234.Epitopes

[0266] In some embodiments, described herein are fibrin antigen-binding regions that bind human fibrin or the fibrinogen yC domain, wherein the fibrin antigen-binding region binds human fibrin at any one of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, He 420, and Gly 421 relative to SEQ ID NO: 265. In some embodiments, the fibrin antigen-binding region binds human fibrin at least two, three, four, five, six, seven, eight, nine, or all ten of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265. In some embodiments, the fibrin antigen-binding region binds human fibrin at amino acid residues Lys 411, lie 412, lie 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265. In some embodiments the amino acid residue of the human fibrin yC or fibrinogen yC domain epitope bind the paratope of the antigen-binding region with a distance of less than 5 Angstroms or less, 4 Angstroms or less, 3 Angstroms or less, or 2 Angstroms or less.Paratopes

[0267] In some embodiments, the fibrin antigen-binding regions described herein comprise a VH region comprising a paratope that binds human fibrin yC or fibrinogen yC 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 some embodiments, the fibrin antigen-binding region comprises a VH region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all seventeen 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 relative to the amino acid sequence set forth in SEQ ID NO: 163. In some embodiments, the antigen-binding regions comprises a VH region comprising a paratope that comprises amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103 relative to the amino acid sequence set forth in SEQ ID NO: 163. In some embodiments, the fibrin antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163. In some embodiments, the fibrin antigenbinding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

[0268] In some embodiments, the fibrin antigen-binding region comprises a VL region comprising a paratope that comprises any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the antigen-binding regions comprises a VL region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine or all ten amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the antigen-binding regions comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the antigen-binding regions comprises a VLregion comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the antigen-binding regions comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

[0269] In some embodiments the paratope of the fibrin antigen-binding region binds the amino acid residues of the human fibrin yC or fibrinogen yC domain epitope with a distance of less than 5 Angstroms or less, 4 Angstroms or less, 3 Angstroms or less, or 2 Angstroms or less.VEGF Antigen-Binding Regions

[0270] In some embodiments, the multi-specific antigen-binding proteins described herein comprise at least one (e.g., one or two e.g., a second antigen-binding region and a fourth antigen-binding region) antigen -binding region that binds VEGF. In some embodiments, the antigen-binding region that binds VEGF, binds one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D. In some embodiments, the VEGF antigen-binding region comprises a sequence set forth in SEQ ID NO: 270. In some embodiments, the VEGF antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NO: 272 and a light sequence set for in SEQ ID NO: 271. In some embodiments, the VEGF antigen-binding region comprises a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 281-295 and 318 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the VEGF antigen-binding region comprises a heavy chain comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 269, 274, 277, 278, or 296-297, and a light chain comprising the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 268, 273, 279, 280, and 298-299 respectively (e.g., the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 269 and the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 268; or the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 274 and the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 273). In some embodiments, the VEGF antigen-binding region comprises a heavy chain comprising an antigen-binding region comprising the sequence of SEQ ID NO: 267 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the VEGF antigen-binding region comprises a variable heavy chain sequence set forth in any one of SEQ ID NOs: 281-297 and 318 or a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the VEGF antigen-binding region comprises a variable heavy chain sequence set forth in SEQ ID NO: 296 or 297 and a variable light chain sequence set forth in SEQ ID NO: 298 or 299, respectively, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the VEGF antigenbinding region comprises a heavy chain sequence set forth in SEQ ID NO: 274 and a light chain sequence set forth in SEQ ID NO: 273 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the VEGF antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NOs: 277 and 278 and a light chain sequence set forth in SEQ ID NO: 279 and 280 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, respectively. In some embodiments, the antigen-binding region that binds 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 some 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 some embodiments, the VEGF antigen-binding region binds an epitope of VEGF -A comprising at least one of amino acids 82-91 of SEQ ID NO: 265. In some embodiments, the VEGF antigen-binding region binds an epitope of VEGF-A comprising amino acids 82-91 of SEQ ID NO: 265. In some 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 NOs: 266 or 267.

[0271] In some embodiments, the VEGF antigen-binding region comprises a heavy chain variable region of any one of SEQ ID NOs: 281-295 and 318 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0272] In some embodiments, the VEGF antigen-binding region comprises a heavy chain comprising the sequence of any one of SEQ ID NOs: 269, 274, 277, 278, or 296-297, and a light chain comprising the sequence of any one of SEQ ID NOs: 268, 273, 279, 280, and 298- 299, respectively, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, respectively.

[0273] In some embodiments, VEGF antigen-binding region comprises a heavy chain comprising the sequence of SEQ ID NO: 330 or 331 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0274] In some embodiments, VEGF antigen-binding region comprises a light chain comprising the sequence of SEQ ID NO: 321, 322, or 325 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0275] In some embodiments, the VEGF antigen-binding region comprises the sequence of SEQ ID NO: 267 or 332 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0276] In some embodiments, the VEGF antigen-binding region is a VEGF trap, composed of extracellular sequences from VEGF receptors. In some embodiments, the sequences are from the VEGF binding domain of human VEGFR1 and the third binding domain from human VEGFR2 as set forth in SEQ ID NO: 332 . This and any suitable VEGF trap known in the art may be used, such as those described in Holash et al., Proc Natl Acad Sci U SA 99, 11393-11398 (2002), Papadopoulos et al., Angiogenesis 15, 171-185 (2012), and Stewart et al. Br J Ophthalmol. 2008;92:667-66, the disclosures of each of which are incorporated herein in their entirety as they pertain to VEGF traps.

[0277] In some embodiments, a VEGF antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 269 and a VL sequence set forth in SEQ ID NO: 268.

[0278] In some embodiments, a VEGF antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 274 and a VL sequence set forth in SEQ ID NO: 273.

[0279] In some embodiments, a VEGF antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 277 and / or 278 and a VL sequence set forth in SEQ ID NO: 279 and / or 280.

[0280] In some embodiments, a VEGF antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 296 and a VL sequence set forth in SEQ ID NO:298.

[0281] In some embodiments, a VEGF antigen-binding region provided herein comprises a VH sequence set forth in SEQ ID NO: 297 and a VL sequence set forth in SEQ ID NO:299.

[0282] In some embodiments, the VEGF antigen-binding region is a single domain antibody, a diabody, an scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F(ab')2, a ds diabody, a minibody, a nanobody, a domain antibody, or a bivalent domain antibody. In some embodiments, the VEGF antigen-binding region isnot an antibody. In some embodiments, the VEGF antigen-binding region is an antibody that does not comprise and Fc region. In some embodiments, the VEGF antigen-binding region comprises an Fc region of the class IgA, IgD, IgE, IgG, or IgM. In some embodiments, the VEGF antigen-binding region comprises one or more modifications known in the art to stabilize the Fc region and improve circulation half-life in vivo. In some embodiments, the VEGF antigen-binding region comprises one or more modifications to promote selective binding of Fc-gamma receptors and / or Clq.

[0283] In some embodiments, the multi-specific antigen-binding proteins described herein comprise a plurality of VEGF antigen-binding regions. In some embodiments, the multi-specific antigen-binding proteins comprise 2, 3, 4, 5, or more VEGF antigen-binding regions. In some embodiments, the multi-specific antigen-binding proteins comprise more than one distinct VEGF antigen-binding region.Fc Region

[0284] The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, incorporated by reference in its entirety. The Fc region is, in some embodiments, a naturally occurring Fc region, or an Fc region modified as described in the art or elsewhere in this disclosure.

[0285] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. An “Fc polypeptide” of a dimeric Fc as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, an Fc polypeptide of a dimeric IgG Fc comprises an IgG CH2 and an IgG CH3 constant domain sequence. An Fc can be of the class IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgGs, IgG4, IgAi, and IgA?.

[0286] The terms “Fc receptor” and “FcR” are used to describe a receptor that binds to the Fc region of a multi-specific antigen-binding protein or an antibody. For example, an FcR is a native sequence human FcR. Generally, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses,including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Immunoglobulins of other isotypes can also be bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB 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 to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0287] Modifications in the CH2 domain can affect the binding of FcRs to the Fc. A number of amino acid modifications in the Fc region are known in the art for selectively altering the affinity of the Fc for different Fc gamma receptors. In some embodiments, the Fc comprises one or more modifications to promote selective binding of Fc-gamma receptors.

[0288] Exemplary mutations that alter the binding of FcRs to the 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(l-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 Sei. 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 U S 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, S 239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S2 39E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D and other mutations listed in WO2011 / 120134 and WO2011 / 120135, herein incorporated by reference. TherapeuticAntibody Engineering (by William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012) lists mutations on page 283.

[0289] In some embodiments a multi-specific antigen-binding protein described herein includes modifications to improve its ability to mediate effector function. Such modifications are known in the art and include afucosylation, or engineering of the affinity of the Fc towards an activating receptor, mainly FCGR3a for ADCC, and towards Clq for CDC. The following Table 2 summarizes various designs reported in the literature for effector function engineering.

[0290] Methods of producing antigen-binding proteins with little or no fucose on the Fc glycosylation site (Asn 297 EU numbering) without altering the amino acid sequence are well known in the art. The GlymaX® technology (ProBioGen AG) is based on the introduction of a gene for an enzyme which deflects the cellular pathway of fucose biosynthesis into cells used for antigen-binding protein production. This prevents the addition of the sugar “fucose” to the N-linked antigen-binding protein carbohydrate part by antigen-binding proteinproducing cells (von Horsten et al. (2010) Glycobiology . 2010 Dec; 20 (12): 1607-18).Another approach to obtaining antigen-binding proteins with lowered levels of fucosylation can be found in U.S. Patent No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to yield lower levels of fucosylation on antibodies can be fully afucosylated (meaning they contain no detectable fucose) or they can be partially afucosylated, meaning that the isolated antigen-binding protein contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose normally detected for a similar antigen-binding protein produced by a mammalian expression system.

[0291] Thus, in some embodiments, a multi-specific antigen-binding protein described herein can include a dimeric Fc that comprises one or more amino acid modifications as noted in Table 2 that confer improved effector function. In some embodiments, the multispecific antigen-binding protein can be afucosylated to improve effector function.Table 2: CH2 domains and effector function engineering

[0292] Fc modifications reducing FcgR and / or complement binding and / or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curr Opin Biotech 20:685-691, and Strohl, WR and Strohl LM, “Antibody Fc engineering for optimal antibody performance” In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing (2012), pp 225-249). These strategies include reduction of effector function through modification of glycosylation, use of IgG2 / IgG4 scaffolds, or the introduction of mutations in the hinge or CH2 regions of the Fc. For example, US Patent Publication No. 2011 / 0212087 (Strohl), International Patent Publication No. WO 2006 / 105338 (Xencor), US Patent Publication No. 2012 / 0225058 (Xencor), US Patent Publication No. 2012 / 0251531 (Genentech), and Strop et al ((2012) J. Mol. Biol. 420: 204- 219) describe specific modifications to reduce FcgR or complement binding to the Fc.

[0293] Specific, non-limiting examples of known amino acid modifications to reduce FcgR or complement binding to the Fc include those identified in the following Table 3:Table 3: Modifications to reduce FcgR or complement binding to the Fc

[0294] Methods of producing multi-specific antigen-binding proteins with little or no fucose on the Fc glycosylation site (Asn 297 EU numbering) without altering the amino acid sequence are well known in the art. The GlymaxX® technology (ProBioGen AG) is based on the introduction of a gene for an enzyme which deflects the cellular pathway of fucose biosynthesis into cells used for multi-specific antigen-binding protein production. This prevents the addition of the sugar “fucose” to the N-linked multi-specific antigen-binding protein carbohydrate part by multi-specific antigen-binding protein-producing cells, (von Horsten et al. (2010) Glycobiology. 2010 Dec; 20 (12): 1607-18.) Examples of cell lines capable of producing defucosylated multi-specific antigen-binding proteins 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 Lecl3 CHO cells, which are deficient in protein fucosylation (see Ripka et al., Arch.Biochem. Biophys., 1986, 249:533-545; U.S. Pat. Pub. 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 (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 multi-specific antigen-binding proteins with lowered levels of fucosylation can be found in U.S. Patent No. 8,409,572, which teaches selecting cell lines for antibody production for their ability to yield lower levels of fucosylation on antibodies.

[0295] Examples of cell lines capable of producing defucosylated multi-specific antigenbinding proteins include CHO-DG44 with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD) (see Henning vonHorsten et al., Glycobiol 2010, 20: 1607-1618) or Lecl3 CHO cells, which are deficient in protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Pat. Pub. 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 (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).

[0296] Multi-specific antigen-binding proteins can be fully afucosylated (meaning they contain no detectable fucose) or they can be partially afucosylated, meaning that the isolated multi-specific antigen-binding protein contains less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose normally detected for a similar multi-specific antigenbinding protein produced by a mammalian expression system.

[0297] In some embodiments, a multi-specific antigen-binding protein provided herein comprises an IgGl domain with reduced fucose content at position Asn 297 compared to a naturally occurring IgGl domain. Such Fc domains are known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, incorporated by reference in its entirety. In some embodiments, such multi-specific antigen-binding proteins do not comprise any fucose at position Asn 297. The amount of fucose is, in some embodiments, determined using any suitable method, for example as described in WO 2008 / 077546, incorporated by reference in its entirety.

[0298] In some embodiments, a multi-specific antigen-binding protein provided herein comprises an Fc region with one or more amino acid substitutions which improve ADCC, such as a substitution at one or more of positions 298, 333, and 334 of the Fc region. In some embodiments, a multi-specific antigen-binding protein provided herein comprises 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, incorporated by reference in its entirety.

[0299] Other illustrative glycosylation variants which are, in some embodiments, incorporated into the multi-specific antigen-binding proteins provided herein are described, for example, in U.S. Pat. Pub. Nos. 2003 / 0157108, 2004 / 0093621, 2003 / 0157108, 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, 2004 / 0132140, 2004 / 0110704, 2004 / 0110282, 2004 / 0109865; International Pat. Pub. Nos. 2000 / 61739, 2001 / 29246, 2003 / 085119,2003 / 084570, 2005 / 035586, 2005 / 035778; 2005 / 053742, 2002 / 031140; Okazaki et al., J. Mol. Biol., 2004, 336: 1239-1249; and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of which is incorporated by reference in its entirety.

[0300] In some embodiments, a multi-specific antigen-binding protein provided herein comprises an Fc region with at least one galactose residue in the oligosaccharide attached to the Fc region. Such multi-specific antigen -binding proteins variants have, in some embodiments, improved CDC function. Examples of such variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764; each of which his incorporated by reference in its entirety.

[0301] In some embodiments, a multi-specific antigen-binding protein provided herein comprises one or more alterations that improves or diminishes Clq binding and / or CDC. See U.S. Pat. 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.

[0302] In certain embodiments, the Fc region comprises one or more amino acid substitutions, wherein the one or more substitutions result in a decrease in one or more of ADCC activity, ADCP activity, or CDC activity compared with the Fc without the one or more substitutions.

[0303] In certain embodiments, the one or more amino acid substitutions is selected from the group consisting of S228P (SP), M252Y, S254T, T256E, M260Y, S262T, T264E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, P329A (PA), P329G (PG), P331S (PS), Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, L242A, L243A, and N434W (direct numbering system). In certain embodiments, the one or more amino acid substitutions comprises a specific combination of amino acid substitutions selected from the group consisting of M428L / N434S (LS);M252Y / S254T / T256E (YTE); T250Q / M428L; T307A / E380A / N434A; T256D / T307Q (DQ); T256D / T307W (DW); M252Y / T256D (YD); T307Q / Q311V / A378V (QVV);T256D / H285D / T307R / Q311V / A378V (DDRVV); L309D / Q311H / N434S (DHS); S228P / L235E (SPLE); L234A / L235A (LALA); LALA / PA; LALA / PS; LALA / PG; LALA / LS; LALA / YTE; M428L / N434A; L234A / G237A (LAGA); L234A / L235A / G237A (LALAGA); L234A / L235A / P329G; D265A / YTE; LALA / YTE; LAGA / YTE;LALAGA / YTE; LALAPG / YTE; N297A / LS; D265A / LS; LALAGA / LS; LALAPG / LS; N297A / DHS; D265A / DHS; LALA / DHS; LAGA / DHS; LALAGA / DHS; LALAPG / DHS; SP / YTE; SPLE / YTE; SP / LS; SPLE / LS; SP / DHS; SPLE / DHS; N297A / LA; D265A / LA;LALA / LA; LAGA / LA; LALAGA / LA; LALAPG / LA; N297A / N434A; D265A / N434A; LALA / N434A; LAGA / N434A; LALAGA / N434A; LALAPG / N434A; N297A / N434W; D265A / N434W; LALA / N434W; LAGA / N434W; LALAGA / N434W; LALAPG / N434W; N297A / DQ; D265A / DQ; LALA / DQ; LAGA / DQ; LALAGA / DQ; LALAPG / DQ; N297A / DW; D265A / DW; LALA / DW; LAG A / D W; LALAGA / DW; LALAPG / DW N297A / YD; D265A / YD; LALA / YD; LAGA / YD; LALAGA / YD; LALAPG / YD; T307Q / Q311V / A378V (QVV); N297A / QVV; D265A / QVV; LALA / QVV; LAGA / QVV; LALAGA / QVV; LALAPG / QVV; DDRVV; N297A / DDRVV; D265A / DDRVV; LALA / DDRVV; LAGA / DDRVV; LALAGA / DDRVV; and LALAPG / DDRVV.

[0304] In certain embodiments the human Fc region comprises a human IgGl Fc with LALA mutations. For example, a human Fc region can comprise a human IgGl Fc with LALA mutations at L42A / L243 A (EU numbering system). In certain embodiments the human Fc region comprises a human IgGl Fc with LALA / YTE mutations. In certain embodiments the human Fc region comprises a human IgGl Fc with LALA / LS mutations. In certain embodiments the human Fc region comprises a human IgGl Fc with LALA / PA mutations. In certain embodiments the human Fc region comprises a human IgGl Fc with LALA / PG mutations. In certain embodiments the human Fc region comprises a human IgGl Fc with LALA / PS mutations.Binding

[0305] The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. 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®).

[0306] With regard to the binding of a multi-specific antigen-binding protein to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competitionwith a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antigen-binding region to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 50% of the affinity for fibrin. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 40% of the affinity for fibrin. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 30% of the affinity for fibrin. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 20% of the affinity for fibrin. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 10% of the affinity for fibrin. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 1% of the affinity for fibrin. In some embodiments, the affinity of a fibrin antigen-binding region for a non-target molecule is less than about 0.1% of the affinity for fibrin.

[0307] When used herein in the context of two or more antibodies or antigen-binding regions thereof, the term “competes with” or “cross-competes with” indicates that the 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, after which a second fibrin antibody is added. In another exemplary assay, a first fibrin antibody is coated on a surface and contacted with fibrin, and then a second fibrin antibody is added. If the presence of the first fibrin antibody reduces binding of the second fibrin antibody, in either assay, then the antibodies compete with each other. The term “competes with” also includes combinations of antibodies where one antibody reduces binding of another antibody, but where no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit binding of each other, regardless of the order in which they are added. In some embodiments, one antibody reduces binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured in a competitive binding assay. A skilled artisan can select the concentrations of the antibodies used in the competition assays based on the affinities of the antibodies for fibrin and the valency of the antibodies. The assays described in this definition are illustrative, and a skilled artisan can utilize any suitable assay to determine if antibodies compete with each other. Suitable assays are described, for example, in Cox et al., “Immunoassay Methods,” in AssayGuidance 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.

[0308] A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, lOx, 20x, or lOOx) inhibits or blocks binding of the reference antibody by, e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as measured in a competitive binding assay. Antibodies identified by competition assay (competing antibody) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. For example, a second, competing antibody is identified that competes for binding to fibrin with a first antibody described herein. In certain embodiments, the second antibody can block or inhibit binding of the first antibody by, e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as measured in a competitive binding assay. In certain embodiments, the second antibody can displace the first antibody by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0309] Conversion of the blood coagulation protein fibrinogen to fibrin by thrombin exposes a cryptic epitope y377-395, termed P2, which can bind CD1 lb / CD18 (Mac-1) and CD1 lc / CD18 on microglia, macrophages and dendritic cells and trigger an inflammatory response. In some embodiments, an anti-fibrin antigen-binding region binds to residues y377- 395 of the fibrin yC or fibrinogen yC domain (SEQ ID NO: 31) of human fibrin. The binding epitope includes the residues within the 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 end residue of each range (e.g., residues 378-395 of human fibrin), or any combination thereof. In some embodiments, an anti-fibrin antigen-binding region described herein blocks binding of the al-domain, which is present on the cell surface of integrin receptors CD1 lb / CD18 (Mac-1) and CDl lc / CD18 on microglia, macrophages and dendritic cells and activates multiple signal transduction pathways which trigger an inflammatory response leading to the secretion of cytokines that indirectly and directly damage nerves, leading to neuroinflammation and neurodegen erati on .

[0310] In some embodiments, a multi-specific antigen-binding protein provided herein binds human fibrin with a KD of less than or equal to 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 IO’7to IO’9nM, as measured by Biacore assay. In some embodiments, the KD of the multi-specific antigen-binding protein provided herein is between about 0.001-0.01, 0.01-0.1, 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-1, 0.25-0.75, 0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6- 1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-2, 1-5, 2-7, 3-8, 3-5, 4-6, 5-7, 6-8, 7-9, 7-10, or 5-10 x IO’7to 10'9nM, as measured by Biacore assay. In some embodiments, a multi-specific antigenbinding protein provided herein binds human fibrin with a KD of less than or equal to about 1 x 10’5nM, 1 x IO’6nM, 1 x IO’7nM, 1 x IO’8nM, or 1 x IO’9nM.

[0311] In some embodiments, the multi-specific antigen-binding protein provided herein binds human fibrin with a KD of less than or equal to 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 10'7to 10'9nM, or less, as measured by Biacore assay. In some embodiments, the multi-specific antigen-binding protein provided herein binds human fibrin with a KD between 5-3, 4-2, 3-1, 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, 1.9-1.5, 1.5-1, 1-0.8, 1-0.5, 0.9-0.6, 0.7-0.4, 0.6-0.2, 0.5-0.3, 0.3-0.2, 0.2-0.1, 0.1-0.01, 0.01-0.001, or 0.001- 0.0001 x 10'7to 10'9nM as measured by Biacore assay. In some embodiments, the multispecific antigen-binding protein provided herein binds human fibrin with a Kd of less than or equal to 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 10'4(1 / s), or less, as measured by Biacore assay. In some embodiments, the multi-specific antigen-binding protein provided herein binds human fibrin with a Kd between 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 IO’4(1 / s) as measured by Biacore assay. In some embodiments, the multi-specific antigen-binding protein provided herein binds human fibrin with a Kaof greater than or equal to 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 105(l / Ms), or more, as measured by Biacore assay. In some embodiments, the multi-specific antigenbinding protein provided herein binds human fibrin with a Kabetween 4-7, 4-4.5, 4.5-5, 5- 5.5, 5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10 x 105(1 / Ms) as measured by Biacore assay.Function

[0312] “Effector functions” refer to those biological activities mediated by the Fc region of a multi-specific antigen-binding protein, which activities vary depending on the antibody isotype. Examples of antibody effector functions include receptor ligand blocking, agonism,or antagonism, Clq 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 multi-specific antigen-binding protein described herein is antagonism and blocksCD1 lb / CD18 (Mac-1) receptor binding to fibrin. In some embodiments, the effector function of the multi-specific antigen-binding protein described herein is neutralization of VEGF.Pharmaceutical Compositions

[0313] The present application provides compositions comprising the multi-specific antigen-binding proteins including pharmaceutical compositions comprising any one or more of the multi-specific antigen-binding proteins described herein with one or more pharmaceutically acceptable excipients. In some embodiments the composition is sterile. The pharmaceutical compositions generally comprise an effective amount of a multi-specific antigen-binding protein.

[0314] These compositions can comprise, in addition to one or more of the multi-specific antigen-binding proteins disclosed herein, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0315] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder, or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0316] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, and Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included, as required.

[0317] The multi-specific antigen-binding protein that is to be given to an individual, administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above are found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0318] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.MethodsMethods of Preparation

[0319] Multi-specific antigen-binding proteins described herein can be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In some embodiments, an isolated nucleic acid encoding a multi-specific antigen-binding protein described herein is provided. Such a nucleic acid encodes an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the multi-specific antigen-binding protein (e.g., the light and / or heavy chains of the multi-specific antigenbinding protein) 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 acid are provided. In some embodiments, the nucleic acid is provided in a multi ci str onic vector. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the multi-specific antigen-binding protein and an amino acid sequence comprising the VH of the multi-specific antigen-binding protein, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the multi-specific antigenbinding protein and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the multi-specific antigen-binding protein. Alternatively, andas would be understood by one of the art, the vector, in some embodiments, comprises a nucleic acid that encodes a fusion protein, VH, a VL, a light chain, a heavy chain, a subunit, an A chain, a B chain, a C chain, a D chain, or an antigen-binding portion thereof of the described multi-specific antigen-binding proteins. In some embodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell, or human embryonic kidney (HEK) cell, or lymphoid cell (e.g., YO, NSO, Sp20 cell). In some embodiments, a method of making a multi-specific antigen-binding protein is provided, wherein the method comprises culturing a host cell comprising nucleic acid encoding the multi-specific antigen-binding protein, as provided above, under conditions suitable for expression of the multi-specific antigenbinding protein, and optionally recovering the multi-specific antigen-binding protein from the host cell (or host cell culture medium).

[0320] For recombinant production of the multi-specific antigen-binding protein, a nucleic acid encoding a multi-specific antigen-binding protein, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the multi-specific antigen-binding protein).

[0321] When a heteromultimer or variant thereof is recombinantly produced by the host cells, the protein in some embodiments is present 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 the host cells, the protein, in some embodiments, is present in the culture medium 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 some embodiments, “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, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0322] Suitable host cells for cloning or expression of multi-specific antigen-binding protein-encoding vectors include prokaryotic or eukaryotic cells described herein.

[0323] Recombinant host cells or host cells are cells that include an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. The exogenous polynucleotide can be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, can be integrated into the host genome. Host cells can include CHO, derivatives of CHO, NSO, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0324] For example, a multi-specific antigen-binding protein is produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coh.) After expression, the multi-specific antigen-binding protein is, in some embodiments, isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0325] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for multi-specific antigen-binding protein-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of a multi-specific antigen-binding protein with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0326] Suitable host cells for the expression of glycosylated multi-specific antigenbinding proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which, in some embodiments, is used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0327] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0328] Vertebrate cells are, in some embodiments, also used as hosts. For example, mammalian cell lines that are adapted to grow in suspension are useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., 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 liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. 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, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for multi-specific antigen-binding protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0329] For example, in preparing an scFV of the multi-specific antigen-binding proteins described herein, VH and VL are amplified from hybridomas or phage display and the VH / VL are sequenced. A plasmid of any suitable orientation can be generated (e.g., an orientation of VH-linker-VL). The linker can be a flexible peptide linker of, for example, 15- 20 amino acids long and made up of glycine and serine with dispersed hydrophilic residues for increased solubility. scFVs can be produced in bacteria, such as redox-mutant bacteria, with an oxidizing cytoplasm. Alternatively, expression of the plasmid in the presence of molecular chaperones to help fold the scFV or targeting the plasmid to the oxidizing periplasm of bacteria can be used in the method or preparation.

[0330] In preparing a Fab of the multi-specific antigen-binding proteins described herein, one of several readily available methods can be employed. In some embodiments, enzymatic / chemical cleavage of a whole antibody to form F(ab')2 fragments is followed by the reduction of those fragments to yield Fab fragments. Alternatively, through the recombinant synthesis of F(ab')2 antibody fragments, chemical reduction of these fragments can yield Fab units.

[0331] Nanobodies of the multi-specific antigen-binding proteins described herein can be produced from a VH (i.e., camelization) in three known steps: (1) converting hydrophobic residues of the VH at positions 37, 44, 45, and 47 on the framework 2 (FR2) region (numbering according to Kabat et al. 1992) to hydrophilic residues, such as G; (2) insertion of an extra loop formed by a CDR1-CDR3 disulfide linkage, which is frequently found inVHHs; and (3 a) reshaping CDR3s of the antigen -binding region into another species, such as a chicken antigen-binding region. Alternatively (3b) a synthetic library based on randomized CDR3 codons can be generated in order to camelize the CDR3 of the antigen-binding region. This step is often required because the CDR3 in camelelized antibodies is longer and has additional tertiary structure, sometimes folding over and having more disulfide bridges. The CDR3 in cam elids often have a higher cystine frequency as well.

[0332] As would be understood by one of the art, the methods of preparation described herein can be readily adapted to produce a portion of a multi-specific antigen-binding protein described herein (e.g., a first antigen-binding region thereof), which can be operably linked or fused to a second portion of a multi-specific antigen-binding protein described herein (e.g., a second antigen-binding region thereof) by methods known in the art. In some embodiments, the multi-specific antigen-binding proteins described herein are produced in stable mammalian cells, by a method comprising: transfecting at least one stable mammalian cell with: nucleic acid encoding the multi-specific antigen-binding protein, in a predetermined ratio; and expressing the nucleic acid in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acid is determined in transient transfection experiments to determine the relative ratio of input nucleic acids that results in the highest percentage of the multi-specific antigen-binding protein in the expressed product.

[0333] In some embodiments, is the method of producing a multi-specific antigenbinding protein in stable mammalian cells as described herein wherein the expression product of the at least one stable mammalian cell comprises a larger percentage of the desired glycosylated multi-specific antigen-binding protein as compared to the monomeric heavy or light chain polypeptides, or other antibodies or multi-specific antigen-binding proteins.

[0334] In some embodiments, is the method of producing a glycosylated multi-specific antigen-binding protein in stable mammalian cells described herein, said method comprising identifying and purifying the desired glycosylated multi-specific antigen-binding protein. In some embodiments, the said identification is by one or both of liquid chromatography and mass spectrometry.

[0335] If required, the multi-specific antibodies antigen-binding proteins can be purified or isolated after expression. Proteins are isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such asFPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins can find use in the present disclosure for purification of multi-specific antibodies antigen-binding proteins. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies. Purification can often be enabled by a particular fusion partner. For example, multi-specific antibodies antigen-binding proteins are purified using glutathione resin if a GST fusion is employed, Ni+2affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag-tag is used. For general guidance in suitable purification techniques, see, e.g., incorporated entirely by reference Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994, incorporated entirely by reference. The degree of purification necessary will vary depending on the use of the multi-specific antigen-binding proteins. In some embodiments, no purification is necessary.

[0336] In some embodiments, the multi-specific antibodies antigen-binding proteins are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toy opearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q, and DEAE columns.

[0337] In some embodiments, the multi-specific antigen-binding 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 comparables.

[0338] In addition, multi-specific antibodies antigen-binding proteins described herein can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y and Hunkapiller et al., Nature, 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide is 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 include, but are not limited to, to the D-isomers of the common amino acids, 2,4diaminobutyric acid, alpha-amino isobutyric acid, 4aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic 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 in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).Methods of Use

[0339] In some embodiments, the present application provides methods of contacting fibrin and VEGF with a multi-specific antigen-binding protein described herein which results in inhibition of microglial adhesion to the fibrin yC or fibrinogen yC domain and neutralization of VEGF.

[0340] In some embodiments, the present application provides methods of using the multi-specific antigen-binding proteins described herein for treatment of a pathology associated with CD1 lb / CD18 (Mac-1) binding to fibrin or CD1 lb / CD18 (Mac-1) binding with fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount of a multi-specific antigen-binding protein or a pharmaceutical composition comprising a multi-specific antigen-binding protein described herein.

[0341] In some embodiments, described herein are methods of inhibiting microglia activation, the method comprising administering to a mammalian subject a therapeutically effective amount a multi-specific antigen-binding protein or a pharmaceutical composition comprising a multi-specific antigen-binding protein described herein.

[0342] In some embodiments, described herein are methods of treating or preventing a disorder or condition of the eye comprising administering to a mammalian subject a therapeutically effective amount of a multi-specific antigen-binding protein or a pharmaceutical composition comprising a multi-specific antigen-binding protein described herein. In some embodiments, the disorder or condition of the eye 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.

[0343] In some embodiments, the subject is a human. In some embodiments the subject has a disease or condition that can be treated with a multi-specific antigen-binding protein provided herein.Methods of Administration

[0344] In some embodiments, the methods provided herein are useful for the treatment of a disorder or condition of the eye in an individual. In some embodiments, the individual is a human.

[0345] In some embodiments, the multi-specific antigen-binding protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intravitreally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of multi-specific antigen-binding protein is, in some embodiments, administered for the treatment of a disorder or condition of the eye. The appropriate dosage of the multi-specific antigen-binding protein is, in some embodiments, determined based on the type of eye condition to be treated, the type of the multi-specific antigen-binding protein, the severity and course of the eye condition, the clinical condition of the individual, the individual’s clinical history and response to the treatment, and the discretion of the attending physician.

[0346] In some embodiments, the multi-specific antigen-binding protein provided herein is administered with at least one additional therapeutic agent. Any suitable additional therapeutic or immunotherapeutic agent is to be administered with a multi-specific antigenbinding proteins provided herein. Additional therapeutic agents include agents that are used to treat or prevent a disorder or condition of the eye 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), pathologic myopia, polypoidal choroidal vasculopathy, retinitis pigmentosa, glaucoma, uveitis, or retinal detachment.

[0347] In some embodiments, the agents are present in the cell or in the subject’s body at the same time or exert their biological or therapeutic effect at the same time. In some embodiments, therapeutic agents are in the same composition or unit dosage form. In some embodiments, therapeutic agents are in separate compositions or unit dosage forms. In some embodiments, a first agent can be administered prior to the administration of a second therapeutic agent.

[0348] The additional therapeutic agent can be administered by any suitable means. In some embodiments, the multi-specific antigen-binding protein provided herein and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, the multi-specific antigen-binding protein provided herein and the additional therapeutic agent are included in different pharmaceutical compositions.

[0349] In embodiments where the multi-specific antigen-binding protein provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the multi-specific antigen-binding protein can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent. In some embodiments, administration of the multi-specific antigen-binding protein provided herein and the additional therapeutic agent occur within about one month of each other. In some embodiments, administration of the multi-specific antigen-binding protein provided herein and the additional therapeutic agent occur within about one week of each other. In some embodiments, administration of the multi-specific antigen-binding protein provided herein and the additional therapeutic agent occur within about one day of each other. In some embodiments, administration of the multi-specific antigen-binding protein provided herein and the additional therapeutic agent occur within about twelve hours of each other. In some embodiments, administration of the multi-specific antigen-binding protein provided herein and the additional therapeutic agent occur within about one hour of each other.Kits and Articles of Manufacture

[0350] The present application provides kits comprising any one or more of the multispecific antigen-binding proteins described herein. In some embodiments, the kits further contain a component selected from any of secondary antibodies, reagents for immunohistochemistry analysis, pharmaceutically acceptable excipients, an instruction manual, and any combination thereof. In some embodiments, the kit comprises a pharmaceutical composition comprising any one or more of the multi-specific antigenbinding proteins described herein, with one or more pharmaceutically acceptable excipients.

[0226] The present application also provides articles of manufacture comprising any one of the multi-specific antigen-binding proteins or kits described herein. Examples of an article of manufacture include vials (including sealed vials).EXAMPLES

[0351] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present disclosure 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.

[0352] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully 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 3rdEd. (Plenum Press) Vols A and B(1992).Materials and Methods

[0353] Antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit from Pierce. Goat F(ab')2 anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE), and Streptavidin-AF633 (SA-633) were 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.Example 1: Anti-Fibrin P2 Treatment Decreases Inflammation in Uveitis Model

[0354] Experimental autoimmune uveitis (EAU) is an organ-specific autoimmune disease that targets the neural retina. This autoimmune response is induced when animals are immunized with retinal antigens (interphotoreceptor retinoid-binding protein (IRBP)], in this case). In order to confirm therapeutic role of anti-fibrin treatment in inflammatory eye conditions or diseases, the efficacy of the anti-fibrin affinity matured antibodies were tested in a rat EAU model after intravitreal administration of the anti-fibrin antibodies.

[0355] In this study, 52 Lewis rats were divided into six groups, namely 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). Animals from all groups, except Group 6, were immunized with an emulsion of IRBP in Complete Freund’s Adjuvant (CFA)on Day 0. Similarly, animals from Group 1-4 received a single intravitreal injection of sponsors test article once on Day 0. Animals in Group 5 received once daily oral administration of positive control FTY-720. After a period of 8-10 days, immunized animals developed uveitis in each eye. Clinical evaluations were performed for all the animals at baseline, day 4, 7, 11, and 14 to follow the extent of diseases developed. Clinical observations were performed as follows:Frequency. Once on each study day.Procedure -. Groups were randomized ahead of evaluations to keep the examiner masked. Animals were observed under a dissection microscope and scored on a scale of 0-4 based on their anterior clinical disease. Photographs of the anterior chamber were taken at the time of clinical evaluations.Clinical observation scoring'.0-0.5: No disease; eye is translucent. Some blood vessels in the iris may be dilated.1 : Engorged blood vessels in iris; abnormal pupil contraction (or dilation).2: Slight haziness to the anterior chamber.3: Moderately opaque anterior chamber, but pupil still visible.4: Opaque anterior chamber and obscured pupil.

[0356] All animals were euthanized on Day 14 and immediately following euthanasia, whole eyes (OU) were collected, upon verification of death, both eyes of each animal were carefully removed. One eye was collected for histological analysis, and the other eye was collected for cytokine analysis. Eyes for cytokine analysis were hemisected and retina were collected. Each eye was carefully orientated for optimal microscopic examination prior to wax embedding. Sections (5 pm) were cut and stained with hematoxylin and eosin for histological examination and scoring according to the following scale summarized below and as described by Caspi, et al. (2012). Histological analysis was masked to the examiner.Clinical Scoring / Uveitis grading was determined as follows:0: No disease, normal retinal architecture.0.5: Trace. <1 / 4 Mild inflammatory cell infiltration of the retina with or without photoreceptor damage.1 : > 14 Mild inflammation and / or photoreceptor outer segment damage.2: > 14 Mild to moderate inflammation and / or lesion extending to the outer nuclear layer.3 : >lA Moderate to marked inflammation and or lesion extending to the inner nuclear layer.4: > 14 Severe inflammation and / or full-thickness retinal damage.

[0357] As shown in FIG. 1, rats administered a low or high dose of the murinized ADI- 60143 - LAL A Fc-stabilized antibody clone exhibited a significantly reduced clinical uveitis score on Day 14 of the study. These results confirm that the affinity matured anti-fibrin antibodies decreased inflammation in subjects with uveitis and are therapeutically effective in a pre-clinical model of eye conditions related to vascular defects of the eye, such as uveitis.Example 2: Laser-Induced Choroidal Neovascularization Study

[0358] Laser-induced choroidal neovascularization (LCNV) was generated in 6- to 8- week-old Brown Norway rats on experiment Day 0. Animals were anesthetized with an intraperitoneal (IP) injection of ketamine / xylazine, and topical administration of 1% tropicamide for pupillary dilation. Using a handheld cover slip as a contact lens and Gen Teal lubricating eye gel as a medium contacting the cover slip to the surface of the cornea, a Nidek GYC-500 green laser photocoagulator coupled to a Nidek SL-1800 slitlamp was used to create six lesions equidistant from the optic nerve head in the retinal mid periphery.

[0359] Laser parameters included: 532 nm wavelength, 100 pm spot size, 0.1 sec duration, and 120 mW. The anti-fibrin antibody 60143, disclosed herein, was administered via intravitreal (IVIT) injection on the same day as the laser. 6 days post-laser, vascular leakage was assessed via quantitative fluorescein angiography (qFA). After 7 days post-laser, animals were sacrificed for choroidal neovascularization (CNV) area analysis.

[0360] On experiment Day 1, treatments were administered bilaterally via IVIT injection according to Table 4 below.Table 4

[0361] As qFA and optical coherence tomography (OCT) are non-terminal (e.g., survival procedure) procedures, rats used for these readouts were also used for CNV area analysis, serum collection, and histology.

[0362] Animals were anesthetized with isoflurane and treated with topical proparacaine before injection to provide topical analgesia. IVIT injections of up to 2.0 pL volume were given using a short barrel, 2 pL syringe with a custom made, detachable 33 -gauge needle (Hamilton Co.) under an operating microscope (Zeiss Microscopy). After injection, neomycin / polymyxin B / gramicidin ophthalmic drops were applied to prevent infection. Treatments were randomized between animals and housing cages.

[0363] At experiment Day 0, Day 3, and Day 7, images of lesion placement were captured by OCT using the Heidelberg Spectralis. 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 captured for each lesion and 3D deconvolution was performed to determine lesion volume. As OCT is a survival procedure, animals were also used for serum collection.

[0364] At experiment Day 6, vascular leakage was assessed via qFA (FIG. 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 were captured for one eye of each animal with a Micron IV imaging system at four minutes postfluorescein injection, and again two minutes later. Imaged software was used by a masked observer to quantify the fluorescence intensity for one lesion per eye using the “integrated density” function. The difference in integrated density between the two times post-injection was recorded as a readout of vascular leakage. As qFA is a survival procedure, animals were used for CNV area.

[0365] At experiment Day 7, animals were euthanized and their eyes enucleated. The extent of CNV at the Bruch’s membrane rupture sites was measured by masked observers using computer-assisted image analysis of FITC-conjugated isolectin B4-stained choroidal flat-mounts. As the anti-fibrin antibody was administered to both eyes in the same animal, the areas for all lesions in both eyes of each animal was averaged and reported as a single data point (FIG. 3). Statistical analysis of area measurements were performed by ANOVA and a parametric post hoc test. Retinas were paraffin embedded and sectioned for further analysis.Example 3: Design of Bi-Specific Anti-Fibrin / Fibrinogen yC P2 and Anti-VEGF IgGMaterials & Methods

[0366] A bi-specific IgG including each of SEQ ID NOs: 304-307 was constructed using the Genscript Clone EZ method, and the designed bi-specific IgG included knob-into-holes and CrossMAb approaches to ensure appropriate heavy and light chain pairing. The anti- fibrin / fibrinogen yc P2 Fc CH2 domain contained the LAL A modification (antibody clone 60143 LALA) to eliminate effector function. It will be understood by one of skill in the art that any suitable anti-fibrin, anti-fibrinogen, anti-fibrin / fibrinogen, or anti-VEGF antibody or antigen-binding fragment or domain thereof can be used.

[0367] Plasmid cDNA was co-transected into the mammalian TurboCHO expression platform (Genscript), and the antibody was purified with MabSelect PrismA+ HiLoad 26 / 600 Superdex (Genscript). The final product was 97% pure, as determined by SEC HPLC and endotoxin free (<0.1 EU / mg). Binding activity for fibrin / fibrinogen yC P2 and VEGF were confirmed with Octet Biolayer Interferometry (BLI).

[0368] In brief, BLI experiments were conducted, and it was demonstrated that the constructed bi-specific IgG bound both fibrin yC or fibrinogen yC and VEGF antigens simultaneously. Individual binding was performed with a fibrin / fibrinogen yC-P2 peptide on the capture sensor and with VEGF on the capture sensor.Example 4: Design of Bi-Specific Anti-Fibrin / Fibrinogen yC P2 and Anti-VEGF Tetrabodies

[0369] Using similar methods as described in Example 3, four bi-specific anti- fibrin / fibrinogen yC P2 and anti-VEGF heavy chain scFv-fusion tetrabodies (VL-VH and VH-VL orientations, respectively, with or without a disulfide bind at VH44-VL100 to stabilize protein and reduce aggregation, respectively) having the below sequences were constructed. In each of the four bi-specific tetrabodies, the constant heavy chain, below, wasused, which included Leu234Ala and Leu235 Ala mutations and a Pro331 Ser mutation relative to huIgGl, and a GS linker (each bolded and underlined):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS (SEQ ID NO: 309).

[0370] Additionally, in each of the four 60143 LALA / anti-VEGF scFv bi-specific tetrabodies, the huIgG Ckappa constant light chain, below, was used:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9).

[0371] In each of the four 60143 LALA / anti-VEGF scFv bi-specific tetrabodies, the 60143 LALA VH and 60143 LAL A VL sequences, respectively, below, were used.

[0372] At the N-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the 60143 LALA VH sequence (with a leader sequence; bolded and underlined), below, was cloned in:MGWSCIILFLVATATGVHSEVOLVQSGAEVKKPGASVKVSCKASGYTFTSTWIHW VRQAPGQGLEWIGLIDPSDSYTNYNQKFRGRATLTVDTSTSTAYMELSSLRSEDTAV YYCASSKPTGGWGQGTTVTVSS (SEQ ID NO: 310).

[0373] At the huIgG Ckappa constant light chain, the 60143 LALA VL sequence (with a leader sequence, bolded and underlined), below, was cloned in:MGWSCIILFLVATATGVHSDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSSGITYLS WYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQ NLELPLTFGGGTKVEIK (SEQ ID NO: 312).

[0374] In the first tetrabody (VL-VH orientation, without disulfide bind) of the four 60143 LALA / anti-VEGF scFv bi-specific tetrabodies, at the C-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the anti-VEGF VL-VH scFv sequence (which included two linkers (each bolded and underlined)), below, was cloned in:GSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLE WVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPY YYGTSHWYFDVWGQGTLVTVSS (SEQ ID NO: 311).

[0375] In the second tetrabody (VH-VL orientation, without disulfide bind), at the C- terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the anti-VEGF VL-VH scFV sequence (which included two linkers (each bolded and underlined)), below, was cloned in: GSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWIN TYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHW YFDVWGOGTLVTVSSGGGGSGGGGSGGGGSDIOLTQSPSSLSASVGDRVTITCSAS QDISNYLNWYQQKPGKAPKVLIYFTS SLHSGVPSRF SGSGSGTDFTLTIS SLQPEDF AT YYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 313).

[0376] In the third tetrabody (VL-VH orientation, with disulfide bind), at the C-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the anti-VEGF VL-VH scFv sequence (which included two linkers and two single nucleotide substitutions to enable disulfide binding at VH44 and VL100) was cloned in.

[0377] In the fourth tetrabody (VH-VL orientation, with disulfide bind), at the C- terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the anti-VEGF VH-VL scFv sequence (which included two linkers and two single nucleotide substitutions to enable disulfide binding at VH44 and VL100 (each bolded and underlined)), below, was cloned in: GSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKCLEWVGWIN TYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHW YFDVWGOGTLVTVSSGGGGSGGGGSGGGGSDIOLTQSPSSLSASVGDRVTITCSAS QDISNYLNWYQQKPGKAPKVLIYFTS SLHSGVPSRF SGSGSGTDFTLTIS SLQPEDF AT YYCQQYSTVPWTFGCGTKVEIK (SEQ ID NO: 314).Example 5: Laser-Induced Choroidal Neovascularization Study with Bi-Specific Antigen-binding ProteinsMaterials & MethodsLaser-induced choroidal neovascularization

[0378] Laser-induced choroidal neovascularization (LCNV) is a well-established model of choroidal angiogenesis that is used to assess wet age-related macular degeneration (AMD), as pathological neovascularization from the choroidal capillaries is elicited by damaging theBruch's membrane with laser application. LCNV was generated in 6- to 8-week-old Brown Norway rats on experiment day 0 (DO). Rats were anesthetized with an IP injection of ketamine / xylazine, and 1% tropicamide was applied for pupil dilation. Using a hand-held coverslip as a contact lens and lubricating eye gel as a medium contacting the coverslip to the surface of the cornea, a Nidek GYC-500 green laser photocoagulator coupled to a Nidek SL- 1800 slit-lamp was used to create six lesions equidistant from the optic nerve head in the retinal midperiphery. Laser parameters included: 532 nm wavelength, 100 pm spot size, 0.1 sec duration, and 120 mW. On Day 1 (DI), treatments were administered via intravitreal injection. Rats were anesthetized with isoflurane and topical analgesia was provided with proparacaine. 2.0 pL injections were given using a short barrel, 5 pL syringe with a custom- made, detachable 33 -gauge needle under an operating microscope. After injection, neomycin / polymyxin B / gramicidin ophthalmic drops were applied to prevent infection.Quantitative fluorescein angiography

[0379] Lesion permeability was assessed via quantitative fluorescein angiography (qFA). Rats were anesthetized as described supra, and 10% sodium fluorescein was administered via intraperitoneal (IP) injection at a dose of 1 pL / g body weight. Fluorescent fundus images were captured for one eye of each rat with a Micron IV imaging system at four minutes postfluorescein injection and again two minutes later. Imaged software was used by a masked observer to quantify the fluorescence intensity for one lesion per eye using the integrated density function. The difference in integrated density between 4- and 6-minutes post-injection was recorded as a readout of vascular leakage (FIG. 4).CNV lesion area and histology

[0380] Rats were euthanized and their eyes were enucleated. The extent of CNV at the Bruch’s membrane rupture sites was measured by masked observers using computer-assisted image analysis of FITC-conjugated isolectin-B4-stained choroidal flat-mounts. As the treatments were administered bilaterally, the areas for all lesions in both eyes of each rat were averaged and reported as a single data point. Statistical analyses of qFA and CNV area measurements were performed by ANOVA and a parametric post hoc test.

[0381] Statistical analyses were conducted with GraphPad Prism version 10.0 for Windows, GraphPad Software, Boston, MA.Cytokine Analysis

[0382] Cytokine expression of various cytokines including IL-17-alpha, TNF-alpha, MIP- 1-alpha, and IL-l-beta was determined on Day 14 in rats treated anti-RSV isotype control (“Iso”) versus bevacizumab (“Avastin”) or anti-fibrin antibody clone 60143-LALA (“60143- LALA”) versus anti-RSV isotype control (“Iso”). Results shown are analyzed by one-way ANOVA, with Dunnett’s multiple comparison to the isotype control.Results

[0383] An exemplary bi-specific antigen-binding protein of the disclosure (bi-specific IgG i.e., anti-fibrin antibody clone 60143 / anti-VEGF antibody (bevacizumab)) of the disclosure showed significant efficacy in reducing the permeability of the laser-induced neovascular lesions compared to isotype control (anti-RSV; FIG. 4). Additionally, differences in the expression and / or concentration of several cytokines, including VEGF and fractalkine, between the anti-fibrin antibody clone 60143 vs. anti-VEGF antibody bevacizumab treatment groups (FIGs. 5-8) indicated differences in the mechanism of action of an anti-fibrin antibody as compared to an anti-VEGF antibody.Example 6: Design and Characterization of Bi-Specific Antigen-binding Proteins Materials and Methods

[0384] Exemplary bi-specific antigen-binding proteins of the disclosure, such as bi- specific tetrabodies are designed and have benefits, including increased avidity for fibrin / fibrinogen yC-P2 deposited in tissue. Unlike bi-specific IgGs, some bi-specific tetrabodies do not require knob-into-holes and CrossMAb approaches for successful construction. Additionally, bi-specific tetrabodies have the unique advantage of requiring fewer genes for transfection.

[0385] To construct the bi-specific tetrabodies, genes are synthesized and cloned. cDNA is extracted and the sequences are confirmed. Transfection takes place, for example, in mammalian ExpiCHO cells for up to 9 days (about 100 mL to about 200 mL).

[0386] Using such methods or the methods described in Example 3, for example, bi- specific anti-fibrin / fibrinogen yC P2 and anti-VEGF heavy chain VHH-fusion tetrabodies (e.g., VHH on CH3 or CL) having the below sequences are constructed.

[0387] In exemplary bi-specific 60143 LALA / anti-VEGF heavy chain VHH fusion tetrabodies, the constant heavy chain of SEQ ID NO: 309 is used, which includes Leu234Ala and Leu235Ala mutations and a Ser228Pro mutation relative to huIgGl and a GS linker.

[0388] Additionally, in exemplary bi-specific 60143 LALA / anti-VEGF heavy chain VHH fusion tetrabodies, the huIgG Ckappa constant light chain of SEQ ID NO: 9 is used.

[0389] At the N-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the 60143 LALA VH sequence (with a leader sequence) of SEQ ID NO: 310 is cloned in. At the huIgG Ckappa constant light chain, the 60143 LALA VL sequence (with a leader sequence) of SEQ ID NO: 312 is cloned in.

[0390] At the C-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence or at the C-terminus of the pcDNA3.1 huIgG Ckappa constant light chain, exemplary anti-VEGF VHH that are cloned in include: SEQ ID NO: 282 and SEQ ID NO: 283 (with, for example, GS linkers).

[0391] Following construction, proteins are purified by Protein A and size exclusion chromatography to a purity of >95%. Quality is characterized by SEC-HPLC, pCE-SDS, Intact Mass analysis, and / or endotoxin analysis. Selected molecules undergo biophysical characterization to identify developability of the respective bi-specific tetrabody. Such biophysical characterization assays can include AC-SINS analysis for colloidal stability; Tm and Tagg determination with DLS; hydrophobicity determination by HPLC-HIC; polyreactivity analysis; a heparin column assay to assess non-specific cell-interaction; freeze / thaw cycles analyzed by SEC-HPLC; accelerated stability analyses; and / or testing at 37 °C on Day 0, 7, and 14. Additionally, BLI and / or SPR testing can be conducted to evaluate specific binding to both fibrin / fibrinogen yC-P2 and VEGF, as well as confirmation of bivalent binding.Biolayer Interferometry

[0392] To demonstrate that exemplary bi-specific antigen-binding proteins of the disclosure (e.g., bi-specific antibodies and bi-specific tetrabodies) bind both fibrin yC or fibrinogen yC and VEGF antigens simultaneously, biolayer interferometry (BLI) and / or surface plasmon resonance (SPR) experiments are performed both with the fibrin / fibrinogen yC-P2 peptide and with VEGF.

[0393] Individual binding is performed with a fibrin / fibrinogen yC-P2 peptide on the capture sensor and with VEGF on the capture sensor. Simultaneous binding of, for example,a tetrabody is demonstrated with an experiment as shown, in FIG. 9, in which a tetrabody is bound to the fibrin / fibrinogen yC-P2. Buffer wash can be initiated to determine the koff for anti-fibrin / fibrinogen yC-P2 specificity. VEGF is added to demonstrate simultaneous binding.Biophysical Characterization

[0394] Viscosity (as a function of protein concentration and shear stress) is evaluated to ensure syringeability and injectability for intravitreal injection. Low viscosity, preferably < 10 centipoise, at high concentration, > 50 mg / mL, preferably > 100 mg / mL is desired for the IVT injection to enable less frequent dosing.Example 7: Design and Characterization of Bi-Specific IgG-VHHi Tetrabodies

[0395] To construct exemplary bi-specific tetrabodies of the disclosure, genes were synthesized and cloned. cDNA was extracted and the sequences were confirmed. Transfection was performed in mammalian CHO cells for up to 7-14 days (about 100 mL to about 1200 mL) and bi-specific anti-fibrin / fibrinogen yC P2 and anti-VEGF IgG-VHH fusion tetrabodies (e.g., anti-VEGF VHH fused to anti-fibrin / fibrinogen yC P2 IgG CH3 or CL; FIG. 10) having the below sequences were constructed.

[0396] A first set of three exemplary bi-specific 60143 LALA / anti-VEGF IgG-VHH fusion tetrabodies had at the N-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the 60143 LALA VH sequence (with a leader sequence) of SEQ ID NO: 310 cloned, which includes Leu234Ala and Leu235Ala mutations and a Pro228Ser mutation relative to huIgGl and a GS linker. At the huIgG Ckappa constant light chain, the 60143 LALA VL sequence (with a leader sequence) of SEQ ID NO: 312 was cloned in. At the C- terminus of the pcDNA3.1 huIgGl Ckappa constant light chain, an anti-VEGF VHH with SEQ ID NO: 318 (with a (GGGGS)3(G4S)3linker (SEQ ID NO: 327), a GGGS3(G3S)3linker (SEQ ID NO: 328) or a GGSGGSGGS (G2S)3linker (SEQ ID NO: 335), respectively) cloned in (FIG. 10, right panel). The bi-specific tetrabody with the (G4S)3linker (SEQ ID NO: 327) is identified here as “007,” the bi-specific tetrabody with the (G3S)3linker (SEQ ID NO: 328) is identified herein as “007-GS3,” and the bi-specific tetrabody with the (G2S)3linker (SEQ ID NO: 335) is identified herein as “007-GS2.”

[0397] A second exemplary bi-specific 60143 LALA / anti-VEGF IgG-VHH fusion tetrabody had at the N-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the 60143 LALA VH sequence (with a leader sequence) of SEQ ID NO: 310 cloned in, whichincludes Leu234Ala and Leu235Ala mutations and a Pro228Ser mutation relative to huIgGl and a GS linker. At the C-terminus of the pcDNA3.1 huIgGl constant heavy chain sequence, the anti-VEGF VHH with SEQ ID NO: 318 (with a (G2S)3linker (SEQ ID NO: 335) or (G4S)3linker (SEQ ID NO: 327)) was cloned in, while at the pcDNA3.1 huIgG Ckappa constant light chain, the 60143 LALA VL sequence (with a leader sequence) of SEQ ID NO: 312 was cloned in (FIG. 10, left panel) and is identified herein as “006.”

[0398] Following construction, size exclusion chromatography, melting temperature (Tm), aggregation temperature (Tagg), hydrophobicity on an HIC column, and Surface Plasmon Resonance (SPR) of the 60143 LALA and anti-VEGF components or simultaneous binding, respectively, were measured relative to 60143 LALA. The results are provided in Table 5. It was observed that using a (G3S)3(SEQ ID NO: 328) or (G2S)3linker (SEQ ID NO: 335) improved the stability, as observed with higher Tm and Tagg values. Furthermore, high affinity binding to fibrin and VEGF, as well as simultaneous binding was observed, as measured with SPR.Table 5ND = not determinedExample 8: Design and Characterization of Bi-Specific IgG-Fab Tetrabodies

[0399] To construct exemplary bi-specific tetrabodies of the disclosure, heavy chain and light chain genes were synthesized and cloned. cDNA was extracted and the sequences were confirmed. Transfection was performed in mammalian CHO cells for up to 7-14 days (about 100 mL to about 1200 mL) and bi-specific anti-fibrin / fibrinogen yC P2 and anti-VEGF IgG- Fab fusion tetrabodies (e.g., anti-VEGF Fab fused to anti-fibrin / fibrinogen yC P2 IgG; FIG. 11) having the below sequences were constructed. As opposed to using cross-mAb techniques, two heavy chain / light chain CHI and CK variant pairs were used to promote correct light chain pairing of the anti-fibrin heavy chain with the anti-fibrin light chain and the anti-VEGF heavy chain with the anti-VEGF light chain.

[0400] The first constructed bi-specific 60143 LALA-PA / anti-VEGF IgG-Fab fusion tetrabody (FIG. 11, left panel) included the sequence of SEQ ID NO: 320, which includes the 60143 LALA-PA heavy chain (set 1) having Leu234Ala and Leu235Ala mutations (bold) and a Pro329Ala mutation (bold underlined) relative to huIgGl as well as three further mutations (italic bold underline) to promote preferential light chain pairing of the anti-fibrin heavy chain with the anti-fibrin light chain and the anti-VEGF heavy chain with the anti-VEGF light chain linked by a GGGGGSGGGGS (SEQ ID NO: 329) linker (underlined) to a modified ranibizumab heavy chain (SEQ ID NO: 330; set 2) to promote preferential pairing:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSTWIHWVRQAPGQGLEWIGLIDPSD SYTNYNQKFRGRATLTVDTSTSTAYMELSSLRSEDTAVYYCASSKPTGGWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYE LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMN WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCA KYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC (SEQ ID NO: 30).Said first bi-specific 60143 LALA-PA / anti-VEGF IgG-Fab fusion tetrabody also included (expressed as one sequence) a modified 60143 variable light chain of SEQ ID NO: 321 (set 1) having three mutations (italic bold underline) to promote preferential paring as well as amodified ranibizumab light chain of SEQ ID NO: 322 (set 2) having three mutations (italic bold underline) to promote preferential pairing:DIVMTQSPLSLPVTPGEPAS ISCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQMS NLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIKRTVAAPS VFI FPPSDEQLKSGRASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS SRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 321); andDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFI FP PSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 322).

[0401] The second constructed bi-specific 60143 LALA-PA / anti-VEGF IgG-Fab fusion tetrabody (FIG. 11, right panel) included the sequence of SEQ ID NO: 323, which includes the 60143 LALA-PA heavy chain (set 2) having Leu234Ala and Leu235Ala mutations (bold) and a Pro329Ala mutation (bold underlined) relative to huIgGl as well as two further mutations (italic bold underline) to promote preferential light chain pairing of the anti-fibrin heavy chain with the anti-fibrin light chain and the anti-VEGF heavy chain with the anti- VEGF light chain linked by a GGGGGSGGGGS (SEQ ID NO: 329) linker (underlined) to a modified ranibizumab heavy chain (SEQ ID NO: 331; set 1) to promote preferential pairing:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSTWIHWVRQAPGQGLEWIGLIDPSDSY TNYNQKFRGRATLTVDTSTSTAYMELSSLRSEDTAVYYCASSKPTGGWGQGTTVTVSSASTK GPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWV RQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKY PYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYELSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSC (SEQ ID NO: 323).Said second bi-specific 60143 LALA-PA / anti-VEGF IgG-Fab fusion tetrabody also included (expressed as one sequence) a modified 60143 variable light chain of SEQ ID NO: 324 (set 2) having three mutations (italic bold underline) to promote preferential paring as well as amodified ranibizumab light chain of SEQ ID NO: 325 (set 1) having three mutations (italic bold underline) to promote preferential pairing:DIVMTQSPLSLPVTPGEPAS ISCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQMS NLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIKRTVAAPS VFI FPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS SELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 324); andDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFI FP PSDEQLKSGRASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 325).

[0402] The first and second bi-specific 60143 LALA-PA / anti-VEGF IgG-Fab fusion tetrabodies described herein were produced in CHO cells at yield of 64 mg / L and 36 mg / L, respectively. After protein A affinity chromatography, the first and second bi-specific 60143 LALA-PA / anti-VEGF IgG-Fab fusion tetrabodies observed 89% and 83% monomer content, respectively.Example 9: Design and Characterization of Bi-Specific IgG-VEGF-Trap Fusion Protein Tribody

[0403] To construct an exemplary bi-specific IgG-VEGF-trap fusion protein tribody (bivalent for fibrin / fibrinogen yC P2 and monovalent for VEGF) of the disclosure, heavy chain, light chain, and VEGF trap fusion protein genes were synthesized and cloned. The molecule was designed to assemble like a monospecific IgG. It also used a VEGF trap structure with human extracellular VEGF receptor sequences of human VEGFR1 and VEGFR2 . Chain pairing constructs like knob-into-holes and CrossMAb approaches were not needed. cDNA was extracted and the sequences were confirmed. Transfection was performed in mammalian CHO cells for up to 7-14 days (about 100 mL to about 1200 mL) and bi- specific anti-fibrin / fibrinogen yC P2 and VEGF trap IgG-fusion protein tribody (e.g., VEGF trap fusion protein fused e.g., to CH3 of anti-fibrin / fibrinogen yC P2 IgG; FIG. 12) having the below sequences were constructed.

[0404] The constructed bi-specific 60143 LALA-PA / VEGF trap IgG-fusion protein tribody included the sequence of SEQ ID NO: 326, which includes the 60143 LALA-PA heavy chain having Leu234Ala and Leu235Ala mutations (bold) and a Pro329Ala mutation (bold underlined) relative to huIgGl linked by a GGGGGSGGGGS (SEQ ID NO: 329) linker (underlined) to VEGFR portion of the aflibercept sequence (SEQ ID NO: 332; italic bold):QVQLVQSGAEVKKPGASVKVSCKASGYTFTSTWIHWVRQAPGQGLEWIGLIDPSD SYTNYNQKFRGRATLTVDTSTSTAYMELSSLRSEDTAVYYCASSKPTGGWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNH YT QKS L S L S PGGGGGSGGGGS. SDTGPPFVEMYSEIPEIIHMTEGPELVIPCRVTSP NITVTLKKEPLDTLIPDGKRIIWDSPKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQ TNTIIDWLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQS GSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO: 326). Said specific 60143 LALA-PA / VEGF trap IgG-fusion protein tribody also included (expressed as one sequence) a 60143 light chain of SEQ ID NO: 84.

[0405] The 60143 LALA-PA / VEGF trap IgG-fusion protein tribody was produced in CHO cells with a yield of 185 mg / L. After protein A affinity chromatography, 90% monomer content was observed. This high yield can be attributed to IgG design, which did not necessitate knob-in-hold design.

[0406] While the disclosure has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure.

[0407] All references, issued patents, and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.INFORMAL SEQUENCE LISTINGIll* For SEQ ID NOs: 309-315 and 320: bold = leader sequence; bold and underlined = linker; and italics and bold = disulfide bond

Claims

CLAIMS1. A multi-specific antigen-binding protein comprising:(a) a first antigen-binding region that specifically binds human fibrin yC or fibrinogen yC domain and comprising: a heavy chain comprising a variable heavy (VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3; and a light chain comprising a variable light (VL) chain sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, respectively, wherein:(i) CDR-H1 comprises the sequence set forth in any one of SEQ ID NOs: 157, 1, 13,25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, 229, and 258;(ii) CDR-H2 comprises the sequence set forth in any one of SEQ ID NOs: 158, 2, 14,26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, 230 and 259;(iii) CDR-H3 comprises the sequence set forth in any one of SEQ ID NOs: 159, 3, 15,27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, 231, and 260;(iv) CDR-L1 comprises the sequence set forth in any one of SEQ ID NOs: 160, 4, 16,28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, 232 and 262;(v) CDR-L2 comprises the sequence set forth in any one of SEQ ID NOs: 161, 5, 17,29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 173, 185, 197, 209, 221, 233 and 263; and(vi) CDR-L3 comprises the sequence set forth in any one of SEQ ID NOs: 162, 6, 18,30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 174, 186, 198, 210, 222, 234 and 264; and(b) a second antigen-binding region that specifically binds Vascular Endothelial Growth Factor (VEGF) comprising:(i) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 281-295 and 318;(ii) a heavy chain comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 269, 274, 277, 278, or 296-297, and a light chain comprising the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 268, 273, 279, 280, and 298-299, respectively; or(iii) an antigen-binding region comprising the sequence of SEQ ID NO: 267; and c) optionally, a third antigen-binding domain that specifically binds human fibrin yC or fibrinogen yC domain; and d) optionally, a fourth antigen-binding domain that specifically binds VEGF.

2. The multi-specific antigen-binding protein of claim 1, wherein the first antigen -binding region comprises a VH sequence selected from a sequence set forth in any one of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254.

3. The multi-specific antigen-binding protein of claim 1 or 2, wherein the first antigenbinding region comprises a VL sequence selected from a sequence set forth in any one of SEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255.

4. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in SEQ ID NO: 166.

5. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 7 and a VL sequence set forth in SEQ ID NO: 10.

6. The multi-specific antigen-binding protein of any one of claims 1-3, wherein 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.

7. The multi-specific antigen-binding protein of any one of claims 1-3, wherein 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.

8. The multi-specific antigen-binding protein of any one of claims 1-3, wherein 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.

9. The multi-specific antigen-binding protein of any one of claims 1-3, wherein 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.

10. The multi-specific antigen-binding protein of any one of claims 1-3, wherein 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.

11. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 79 and a VL sequence set forth in SEQ ID NO: 82.

12. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 91 and a VL sequence set forth in SEQ ID NO: 94.

13. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 103 and a VL sequence set forth in SEQ ID NO: 106.

14. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 115 and a VL sequence set forth in SEQ ID NO: 118.

15. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 127 and a VL sequence set forth in SEQ ID NO: 130.

16. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 139 and a VL sequence set forth in SEQ ID NO: 142.

17. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 151 and a VL sequence set forth in SEQ ID NO: 154.

18. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 175 and a VL sequence set forth in SEQ ID NO: 178.

19. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 187 and a VL sequence set forth in SEQ ID NO: 190.

20. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 199 and a VL sequence set forth in SEQ ID NO: 202.

21. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 211 and a VL sequence set forth in SEQ ID NO: 214.

22. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 223 and a VL sequence set forth in SEQ ID NO: 226.

23. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 235 and a VL sequence set forth in SEQ ID NO: 238.

24. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 256 and a VL sequence set forth in SEQ ID NO: 257.

25. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region comprises a humanized, human, or chimeric first antigen-binding region.

26. The multi-specific antigen-binding protein of claim 25, wherein the first antigen-binding region comprises a humanized antibody or antigen-binding region.

27. The multi-specific antigen-binding protein of any one of the above 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.

28. The multi-specific antigen-binding protein of claim 27, wherein the first antigen-binding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

29. The multi-specific antigen-binding protein of claim 28, wherein the human Fc region comprises wild-type, human IgGl Fc.

30. The multi-specific antigen-binding protein of claim 29, wherein the human Fc domain comprises a sequence set forth in any one of SEQ ID NOs: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236.

31. The multi-specific antigen-binding protein of any one of the above claims, wherein the heavy chain comprises a constant heavy chain sequence set forth in any one of SEQ ID NOs:164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236.

32. The multi-specific antigen-binding protein of any one of the above claims, wherein the light chain comprises a constant light chain sequence set forth in any one of SEQ ID NOs:165, 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153, 177, 189, 201, 213, 225, and 237.

33. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 7, the VL sequence set forth in SEQ ID NO: 10, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

34. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19, the VL sequence set forth in SEQ ID NO: 22, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

35. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 31, the VL sequence set forth in SEQ ID NO: 34, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

36. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43, the VL sequence set forth in SEQ ID NO: 46, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

37. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 55, the VL sequence set forth in SEQ ID NO: 58, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

38. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67, the VL sequence set forth in SEQ ID NO: 70, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

39. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79, the VL sequence set forth in SEQ ID NO: 82, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

40. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91, the VL sequence set forth in SEQ ID NO: 94, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

41. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103, the VL sequence set forth in SEQ ID NO: 106, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

42. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115, the VL sequence set forth in SEQ ID NO: 118, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

43. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127, the VL sequence set forth in SEQ ID NO: 130, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

44. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139, the VL sequence set forth in SEQ ID NO: 142, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

45. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151, the VL sequence set forth in SEQ ID NO: 154, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

46. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163, the VLsequence set forth in SEQ ID NO: 166, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

47. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175, the VL sequence set forth in SEQ ID NO: 178, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

48. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187, the VL sequence set forth in SEQ ID NO: 190, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

49. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199, the VL sequence set forth in SEQ ID NO: 202, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

50. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211, the VL sequence set forth in SEQ ID NO: 214, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

51. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223, the VL sequence set forth in SEQ ID NO: 226, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

52. The multi-specific antigen-binding protein of any one of claims 1-3, wherein the first antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 235, the VL sequence set forth in SEQ ID NO: 238, and the first antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

53. The multi-specific antigen-binding protein of any one of claims 27-52, wherein 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 with the Fc without the one or more substitutions.

54. The multi-specific antigen-binding protein of any one of the above claims, wherein the Fc region binds an Fey receptor selected from the group consisting of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.

55. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region comprises a monoclonal first antigen-binding region.

56. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region binds an y377-395 epitope of the fibrin yC or fibrinogen yC domain.

57. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region binds to peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241, and 249-253 with a KD of less than or equal to about 1, 2, 3, 4, 5, 6, 7, or 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

58. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region binds to a peptide comprising the sequence of the y377-395 epitope of the human fibrin yC or fibrinogen yC domain with a KD of less than or equal to about 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

59. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region inhibits CD1 lb / CD18 (Mac-1) and CD1 lc / CD18 binding to fibrin yC or fibrinogen yC domain.

60. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region exhibits inhibition of microglial adhesion to the fibrin yC or fibrinogen yC domain.

61. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region binds human fibrin at any one of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

62. The multi-specific antigen-binding protein of claim 61, wherein the first antigen-binding region binds human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten ofamino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

63. The multi-specific antigen-binding protein of any one of the above claims, wherein the first antigen-binding region comprises a VH region comprising a paratope that 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

64. The multi-specific antigen-binding protein of claim 63, wherein the first antigen-binding region comprises a VH region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all seventeen 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

65. The multi-specific antigen-binding protein of claim 63, wherein the first antigen-binding region comprises a VH region comprising a paratope that comprises amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103 relative to the amino acid sequence set forth in SEQ ID NO: 163.

66. The multi-specific antigen-binding protein of claim 63, wherein the first antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

67. The multi-specific antigen-binding protein of claim 63, wherein the first antigen-binding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

68. The multi-specific antigen-binding protein of any one of claims 61-67, wherein the first antigen-binding region comprises a VL region comprising a paratope that comprises any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

69. The multi-specific antigen-binding protein of claim 68, wherein the first antigen-binding region comprises a VL region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine or all ten amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

70. The multi-specific antigen-binding protein of claim 68, wherein the first antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

71. The multi-specific antigen-binding protein of claim 68, wherein the first antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

72. The multi-specific antigen-binding protein of claim 68, wherein the first antigen-binding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

73. The multi-specific antigen-binding protein of any one of the above claims, wherein the second antigen-binding region binds one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

74. The multi-specific antigen-binding protein of any one of the above claims, wherein the second antigen-binding region comprises a variable heavy chain sequence set forth in any one of SEQ ID NOs: 281-297 and 318.

75. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a variable heavy chain sequence set forth in SEQ ID NO: 296 or 297 and a variable light chain sequence set forth in SEQ ID NO: 298 or 299, respectively.

76. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NO: 274 and a light chain sequence set forth in SEQ ID NO: 273.

77. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NOs: 277 and 278 and a light chain sequence set forth in SEQ ID NO: 279 and 280.

78. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a humanized, human, or chimeric second antigen-binding region.

79. The multi-specific antigen-binding protein of claim 78, wherein the second antigenbinding region comprises a humanized antibody or antigen-binding region.

80. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

81. The multi-specific antigen-binding protein of claim 80, wherein the second antigenbinding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

82. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a monoclonal second antigen-binding region.

83. The multi-specific antigen-binding protein of any one of claims 1-73, 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 any one of SEQ ID NOs: 269, 274, 277, 278, 281-297, 318, 330, and 331.

84. The multi-specific antigen-binding protein of any one of claims 1-73 and 83, 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 any one of SEQ ID NOs: 268, 273, 279, 280, 298-299, 322, and 325.

85. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region binds an epitope of VEGF-A comprising at least one of amino acids 82-91 set forth in SEQ ID NO: 265.

86. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region comprises a sequence that is 90% or more, 95% or more, or 99% ormore identical to the amino acid sequence set forth in any one of SEQ ID NOs: 266, 267, and 332.

87. The multi-specific antigen-binding protein of any one of claims 1-73, wherein the second antigen-binding region does not comprise an Fc domain.

88. The multi-specific antigen-binding protein of any one of the above claims, wherein the multi-specific antigen-binding protein comprises the third antigen-binding region that specifically binds human fibrin yC or fibrinogen yC domain, wherein the third antigenbinding 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, respectively, wherein:(a) CDR-H1 comprises the sequence set forth in any one of SEQ ID NOs: 157, 1, 13,25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 169, 181, 193, 205, 217, 229, and 258;(b) CDR-H2 comprises the sequence set forth in any one of SEQ ID NOs: 158, 2, 14,26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 170, 182, 194, 206, 218, 230 and 259;(c) CDR-H3 comprises the sequence set forth in any one of SEQ ID NOs: 159, 3, 15,27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 171, 183, 195, 207, 219, 231, and 260;(d) CDR-L1 comprises the sequence set forth in any one of SEQ ID NOs: 160, 4, 16,28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 172, 184, 196, 208, 220, 232 and 262;(e) CDR-L2 comprises the sequence set forth in any one of SEQ ID NOs: 161, 5, 17,29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 173, 185, 197, 209, 221, 233 and 263; and(f) CDR-L3 comprises the sequence set forth in any one of SEQ ID NOs: 162, 6, 18,30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 174, 186, 198, 210, 222, 234 and 264.

89. The multi-specific antigen-binding protein of claim 88, wherein the third antigen-binding region comprises a VH sequence selected from a sequence set forth in any one of SEQ ID NOs: 163, 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 175, 187, 199, 211, 223, 235, and 254.

90. The multi-specific antigen-binding protein of claim 88 or 89, wherein the third antigenbinding region comprises a VL sequence selected from a sequence set forth in any one ofSEQ ID NOs: 166, 10, 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 178, 190, 202, 214, 226, 238, and 255.

91. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 163 and a VL sequence set forth in SEQ ID NO: 166.

92. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 7 and a VL sequence set forth in SEQ ID NO: 10.

93. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third 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.

94. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third 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.

95. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third 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.

96. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third 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.

97. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third 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.

98. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 79 and a VL sequence set forth in SEQ ID NO: 82.

99. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 91 and a VL sequence set forth in SEQ ID NO: 94.

100. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 103 and a VL sequence set forth in SEQ ID NO: 106.

101. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 115 and a VL sequence set forth in SEQ ID NO: 118.

102. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 127 and a VL sequence set forth in SEQ ID NO: 130.

103. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 139 and a VL sequence set forth in SEQ ID NO: 142.

104. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 151 and a VL sequence set forth in SEQ ID NO: 154.

105. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 175 and a VL sequence set forth in SEQ ID NO: 178.

106. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 187 and a VL sequence set forth in SEQ ID NO: 190.

107. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 199 and a VL sequence set forth in SEQ ID NO: 202.

108. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 211 and a VL sequence set forth in SEQ ID NO: 214.

109. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 223 and a VL sequence set forth in SEQ ID NO: 226.

110. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 235 and a VL sequence set forth in SEQ ID NO: 238.

111. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises a VH sequence set forth in SEQ ID NO: 256 and a VL sequence set forth in SEQ ID NO: 257.

112. The multi-specific antigen-binding protein of any one of claims 88-111, wherein the third antigen-binding region comprises a humanized, human, or chimeric third antigenbinding region.

113. The multi-specific antigen -binding protein of claim 112, wherein the third antigenbinding region comprises a humanized antibody or antigen-binding region.

114. The multi-specific antigen-binding protein of any one of claims 88-113, wherein the third antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

115. The multi-specific antigen-binding protein of claim 114, wherein the third antigenbinding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

116. The multi-specific antigen-binding protein of claim 115, wherein the human Fc region comprises wild-type, human IgGl Fc.

117. The multi-specific antigen-binding protein of claim 116, wherein the human Fc domain comprises a sequence set forth in any one of SEQ ID NOs: 164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236.

118. The multi-specific antigen-binding protein of any one of claims 88-117, wherein the heavy chain comprises a constant heavy chain sequence set forth in any one of SEQ ID NOs:164, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 176, 188, 200, 212, 224, and 236.

119. The multi-specific antigen-binding protein of any one of claims 88-118, wherein the light chain comprises a constant light chain sequence set forth in any one of SEQ ID NOs:165, 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153, 177, 189, 201, 213, 225, and 237.

120. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 7, the VL sequence set forth in SEQ ID NO: 10, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

121. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 19, the VL sequence set forth in SEQ ID NO: 22, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

122. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 31, the VL sequence set forth in SEQ ID NO: 34, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

123. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 43, the VL sequence set forth in SEQ ID NO: 46, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

124. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 55, the VL sequence set forth in SEQ ID NO: 58, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

125. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 67, the VL sequence set forth in SEQ ID NO: 70, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

126. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 79, the VL sequence set forth in SEQ ID NO: 82, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

127. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 91, the VLsequence set forth in SEQ ID NO: 94, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

128. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 103, the VL sequence set forth in SEQ ID NO: 106, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

129. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 115, the VL sequence set forth in SEQ ID NO: 118, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

130. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 127, the VL sequence set forth in SEQ ID NO: 130, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

131. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 139, the VL sequence set forth in SEQ ID NO: 142, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

132. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 151, the VL sequence set forth in SEQ ID NO: 154, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

133. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 163, the VL sequence set forth in SEQ ID NO: 166, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

134. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 175, the VL sequence set forth in SEQ ID NO: 178, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

135. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 187, the VL sequence set forth in SEQ ID NO: 190, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

136. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 199, the VL sequence set forth in SEQ ID NO: 202, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

137. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 211, the VL sequence set forth in SEQ ID NO: 214, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

138. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 223, the VL sequence set forth in SEQ ID NO: 226, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

139. The multi-specific antigen-binding protein of any one of claims 88-90, wherein the third antigen-binding region comprises the VH sequence set forth in SEQ ID NO: 235, the VL sequence set forth in SEQ ID NO: 238, and the third antigen-binding region comprises a human Fc region, wherein the human Fc region comprises wild-type, human IgGl Fc.

140. The multi-specific antigen-binding protein of any one of claims 116-139, wherein 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 with the Fc without the one or more substitutions.

141. The multi-specific antigen-binding protein of any one of claims 88-140, wherein the Fc region binds an Fey receptor selected from the group consisting of: FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.

142. The multi-specific antigen-binding protein of any one of claims 88-141, wherein the third antigen-binding region comprises a monoclonal third antigen-binding region.

143. The multi-specific antigen-binding protein of any one of claims 88-142, wherein the third antigen-binding region binds an y377-395 epitope of the fibrin yC or fibrinogen yC domain.

144. The multi-specific antigen-binding protein of any one of claims 88-143, wherein the third antigen-binding region binds to peptide comprising an amino acid sequence set forth in at least one of SEQ ID NOs: 241, and 249-253 with a KD of less than or equal to about 1, 2, 3, 4, 5, 6, 7, or 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

145. The multi-specific antigen-binding protein of any one of claims 88-144, wherein the third antigen-binding region binds to a peptide comprising the sequence of the y377-395 epitope of the human fibrin yC or fibrinogen yC domain with a KD of less than or equal to about 8 x 10'7to 10'9nM, as measured by surface plasmon resonance (SPR) single cycle kinetics (SCK) assay.

146. The multi-specific antigen-binding protein of any one of claims 88-145, wherein the third antigen-binding region inhibits CD1 lb / CD18 (Mac-1) and CD1 lc / CD18 binding to fibrin yC or fibrinogen yC domain.

147. The multi-specific antigen-binding protein of any one of claims 88-146, wherein the third antigen-binding region exhibits inhibition of microglial adhesion to the fibrin yC or fibrinogen yC domain.

148. The multi-specific antigen-binding protein of any one of claims 88-147, wherein the third antigen-binding region binds human fibrin at any one of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

149. The multi-specific antigen-binding protein of claim 148, wherein the third antigenbinding region binds human fibrin at at least two, three, four, five, six, seven, eight, nine, or all ten of amino acid residues Lys 411, He 412, He 413, Phe 415, Asn 416, Arg 417, Leu 418, Thr 419, lie 420, and Gly 421 relative to SEQ ID NO: 265.

150. The multi-specific antigen-binding protein of any one of claims 88-149, wherein the third antigen-binding region comprises a VH region comprising a paratope that comprises any one of amino acid residues Ser 31, Tyr 32, Trp 33, His 35, Trp 47, Leu 50, Asp 52, Asp54, 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

151. The multi-specific antigen-binding protein of claim 150, wherein the third antigenbinding region comprises a VH region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all seventeen 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

152. The multi-specific antigen-binding protein of claim 150, wherein the third antigenbinding region comprises a VH region comprising a paratope that comprises amino acid residues Ser 31, Trp 33, His 35, Asp 52, Asp 54, Tyr 56, Ser 94, Gly 101, Gly 102, and Trp 103 relative to the amino acid sequence set forth in SEQ ID NO: 163.

153. The multi-specific antigen-binding protein of claim 150, wherein the third antigenbinding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

154. The multi-specific antigen-binding protein of claim 150, wherein the third antigenbinding region comprises a VH region comprising a paratope that comprises 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 relative to the amino acid sequence set forth in SEQ ID NO: 163.

155. The multi-specific antigen-binding protein of any one of claims 150-154, wherein the third antigen-binding region comprises a VL region comprising a paratope that comprises any one of amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

156. The multi-specific antigen-binding protein of claim 155, wherein the third antigenbinding region comprises a VL region comprising a paratope that comprises at least two, three, four, five, six, seven, eight, nine or all ten amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91 or Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

157. The multi-specific antigen-binding protein of claim 155, wherein the third antigenbinding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

158. The multi-specific antigen-binding protein of claim 155, wherein the third antigenbinding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Gin 50, Asn 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

159. The multi-specific antigen-binding protein of claim 155, wherein the third antigenbinding region comprises a VL region comprising a paratope that comprises the amino acid residues His 27, Tyr 32, Tyr 36, Leu 46, Tyr 49, Gin 50, Ala 91, Leu 92, Leu 94, and Leu 96 relative to the amino acid sequence set forth in SEQ ID NO: 166.

160. The multi-specific antigen-binding protein of any one of claims 88-159, comprising a heterodimeric Fc domain.

161. The multi-specific antigen-binding protein of any one of claims 88-160, wherein the multi-specific antigen-binding protein comprises a bi-specific IgG, a bi-specific tribody or a tetrabody (e.g., heavy chain and scFv fusion, heavy chain and scdsFv fusion, heavy chain and sdAb fusion).

162. The multi-specific antigen-binding protein of any one of the above claims, wherein the multi-specific antigen-binding protein comprises the fourth antigen-binding region that specifically binds VEGF, wherein the fourth antigen-binding region comprises:(a) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 281-295 and 318;(b) a heavy chain comprising the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 269, 274, 277, 278, or 296-297, and a light chain comprising the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 268, 273, 279, 280, and 298-299, respectively; or(c) an antigen-binding region comprising the sequence of SEQ ID NO: 267.

163. The multi-specific antigen-binding protein of claim 162, wherein the fourth antigenbinding region binds one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

164. The multi-specific antigen-binding protein of claim 162 or 163, wherein the fourth antigen-binding region comprises a variable heavy chain sequence set forth in any one of SEQ ID NOs: 281-297 and 318.

165. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region comprises a variable heavy chain sequence set forth in SEQ ID NO: 296 or 297 and a variable light chain sequence set forth in SEQ ID NO: 298 or 299, respectively.

166. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NO: 274 and a light chain sequence set forth in SEQ ID NO: 273.

167. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region comprises a heavy chain sequence set forth in SEQ ID NOs: 277 and 278 and a light chain sequence set forth in SEQ ID NO: 279 and 280.

168. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region comprises a humanized, human, or chimeric fourth antigenbinding region.

169. The multi-specific antigen-binding protein of claim 168, wherein the fourth antigenbinding region comprises a humanized antibody or antigen-binding region.

170. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region comprises a heavy chain human Fc region of a class selected from IgG, IgA, IgD, IgE, and IgM.

171. The multi-specific antigen-binding protein of claim 170, wherein the fourth antigenbinding region comprises a human Fc region, and wherein the human Fc region comprises a human heavy chain constant region of the class IgG and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

172. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region comprises a monoclonal fourth antigen-binding region.

173. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth 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 any one of SEQ ID NOs: 269, 274, 277, 278, 281-297, 318, 330, and 331.

174. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth 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 any one of SEQ ID NOs: 268, 273, 279, 280, 298-299, 322, and 325.

175. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth antigen-binding region binds an epitope of VEGF-A comprising at least one of amino acids 82-91 set forth in SEQ ID NO: 265.

176. The multi-specific antigen-binding protein of any one of claims 162-164, wherein the fourth 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 any one of SEQ ID NOs: 266, 267, and 332.

177. The multi-specific antigen-binding protein of any one of the above claims, wherein the multi-specific antigen-binding protein is a bi-specific IgG.

178. The multi-specific antigen-binding protein of any one of the above claims, wherein the multi-specific antigen-binding protein is a bi-specific tribody or tetrabody.

179. The multi-specific antigen-binding protein of claim 178, wherein the bi-specific tetrabody comprises a heavy chain and scFv fusion, a heavy chain and scdsFv fusion, or a heavy chain and sdAb fusion.

180. The multi-specific antigen-binding protein of any one of the above claims for use in the treatment of a disorder or condition of eye.

181. The multi-specific antigen-binding protein of any one of the above claims formulated for administration to a subject by intravitreal injection.

182. An isolated polynucleotide or set of polynucleotides encoding the first antigenbinding region of any of the above claims, a VH thereof, a VL thereof, a light chain thereof, a heavy chain thereof, or an antigen-binding portion thereof, optionally wherein the polynucleotide or set of polynucleotides comprises cDNA.

183. An isolated polynucleotide or set of polynucleotides encoding the second antigenbinding region of any of the above claims, a VH thereof, a VL thereof, a light chain thereof, aheavy chain thereof, or an antigen-binding portion thereof, optionally wherein the polynucleotide or set of polynucleotides comprises cDNA.

184. A vector or set of vectors comprising the polynucleotide or set of polynucleotides of claim 182 or 183.

185. A host cell comprising the polynucleotide or set of polynucleotides of claim 182 or 183 or the vector or set of vectors of claim 184.

186. A pharmaceutical composition comprising the multi-specific antigen-binding protein of any one of claims 1-179 and a pharmaceutically acceptable excipient.

187. A kit comprising the multi-specific antigen-binding protein of any one of claims 1- 179 or a pharmaceutical composition of claim 186 and instructions for use.

188. A method for treating a disorder or condition of the eye, the method comprising administering to a mammalian subject a therapeutically effective amount the multi-specific antigen-binding protein of any one of claims 1-179 or the pharmaceutical composition of claim 186.

189. The method of claim 188, wherein the disorder or condition of the eye is selected from the group consisting of: retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment.

190. A method for treating a pathology associated with CD1 lb / CD18 (Mac-1) binding to fibrin or CD1 lb / CD18 (Mac-1) binding with fibrinogen, the method comprising administering to a mammalian subject a therapeutically effective amount the multi-specific antigen-binding protein of any one of claims 1-179 or the pharmaceutical composition of claim 186.

191. A method of inhibiting microglia activation, the method comprising administering to a mammalian subject a therapeutically effective amount of the multi-specific antigen -binding protein of any one of claims 1-179 or the pharmaceutical composition of claim 186.

192. A method of producing a multi-specific antigen-binding protein, the method comprising expressing a multi-specific antigen-binding protein or antigen-binding region thereof in the host cell of claim 185 and isolating the expressed multi-specific antigenbinding protein or antigen-binding region thereof.

193. A method of preventing a disorder or condition of the eye, the method comprising administering to a mammalian subject a therapeutically effective amount the multi-specific antigen-binding protein of any one of claims 1-179 or the pharmaceutical composition of claim 185.

194. A method of treating a disorder or condition of the eye associated with increased vascularization in a subject in need thereof, comprising administering to the subject the multispecific antigen-binding protein of any one of claims 1-179 or the pharmaceutical composition of claim 185.

195. A method of preventing a disorder or condition of the eye associated with increased vascularization in a subject in need thereof, comprising administering to the subject the multispecific antigen-binding protein of any one of claims 1-179 or the pharmaceutical composition of claim 185.

Images