Plasma kallikrein antibodies and uses thereof

Abstract

To provide antibodies that bind plasma kallikrein and such antibodies or antigen binding fragments thereof.SOLUTION: Provided herein are isolated antibodies that bind to OX40 or antigen binding fragments thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 142,748, filed January 28, 2021; U.S. Provisional Patent Application No. 63 / 159,323, filed March 10, 2021; U.S. Provisional Patent Application No. 63 / 220,194, filed July 9, 2021; and U.S. Provisional Patent Application No. 63 / 262,838, filed October 21, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The field of the invention is immunology, in particular therapeutic antibodies and the use of those antibodies to treat disease. [Background technology]

[0003] Plasma kallikrein is a serine protease and a potential drug target for various inflammatory, cardiovascular, infectious (sepsis), and tumor diseases (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies intrinsic coagulation through its feedback activation of factor XII and enhances inflammation through the production of the proinflammatory nonapeptide bradykinin. As the major kininogenase in the circulation, plasma kallikrein is primarily responsible for the production of bradykinin in the vasculature. Genetic deficiency of C1-inhibitor protein (C1-INH), the major natural inhibitor of plasma kallikrein, causes hereditary angioedema (HAE). Patients with HAE suffer from acute attacks of painful edema, often caused by unknown triggers (Zuraw BL et al., N. Engl J Med 359, 1027-1036, 2008). The plasma kallikrein-kinin system (plasma KKS) has been implicated in a variety of diseases through the use of pharmacological agents and genetic studies in animal models. Plasma kallikrein-binding proteins (e.g., antibodies, e.g., inhibitory antibodies) are useful therapeutic agents for a variety of diseases and conditions, including those related to the level of plasma kallikrein activity.

[0004] Despite the efforts made to date, there remains a need for therapeutic antibodies that target plasma kallikrein. Summary of the Invention

[0005] The inventions described herein are based, in part, on the discovery of high-affinity binding proteins, e.g., antibodies and antigen-binding fragments, that bind to plasma kallikrein, e.g., human plasma kallikrein. The antibodies have high specificity for plasma kallikrein, e.g., human plasma kallikrein. The antibodies have high selectivity for plasma kallikrein relative to prekallikrein and lack significant off-target binding to trypsin and other serine proteases. These binding proteins may also have an extended half-life, e.g., an extended half-life in the blood. Because of these and other characteristics, the antibodies may be useful in treating various disorders mediated by plasma kallikrein, e.g., hereditary angioedema and bradykinin-dependent edema, and may facilitate treatment of these disorders at lower doses and / or at longer dosing intervals compared to other antibodies.

[0006] In one aspect of the present invention, disclosed herein are antibodies, including antigen-binding fragments, that bind plasma kallikrein, which may be human plasma kallikrein, for example, human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO:32.

[0007] The antibodies and antigen-binding fragments disclosed herein comprise HCDR1, HCDR2, and H It may comprise a heavy chain variable region (VH) comprising CDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

[0008] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, 23, 29, or 43; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, 19, 24, 30, or 44; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, 25, or 31, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, 20, or 27; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, 18, 21, or 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, 22, or 28.

[0009] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, or 19; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0010] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21 or 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0011] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21 or 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0012] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 44; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0013] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, or 19; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0014] For example, the antibody or binding fragment thereof may comprise a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18; and The LCDR3 may have a VL comprising the amino acid sequence shown in SEQ ID NO:13.

[0015] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0016] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0017] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0018] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27; CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0019] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 44; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, and a VL in which LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0020] For example, the antibody or binding fragment thereof may have a VH in which HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 with up to 1, 2, or 3 amino acid substitutions relative to the amino acid sequence; CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, or 19 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions relative to the amino acid sequence; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions relative to the amino acid sequence; LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions relative to the amino acid sequence; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18 with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence; and a VL in which LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13 with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence. In some antibodies or binding fragments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain at most one amino acid substitution, while in other antibodies or binding fragments, at most one CDR selected from HCDR1, HCDR2, or HCDR3 contains at most one substitution, and / or at most one CDR selected from LCDR1, LCDR2, and / or LCDR3 contains at most one substitution.

[0021] The VL of an antibody or binding fragment disclosed herein may comprise the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6, for example, SEQ ID NO: 6. The VH of an antibody or binding fragment disclosed herein may comprise the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5, for example, SEQ ID NO: 5. The VH may comprise the amino acid sequence set forth in SEQ ID NO: 5, and the VL may comprise the amino acid sequence set forth in SEQ ID NO: 6. The VH may comprise the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5, and the VL may comprise the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6.

[0022] The light chain of an antibody or binding fragment disclosed herein may comprise the amino acid sequence set forth in SEQ ID NO: 8. The heavy chain of an antibody or binding fragment disclosed herein may comprise the amino acid sequence set forth in SEQ ID NO: 10, 9, or 7. For example, the light chain may comprise the amino acid sequence of SEQ ID NO: 8 and the heavy chain may comprise the amino acid sequence of SEQ ID NO: 7. For example, the light chain may comprise the amino acid sequence of SEQ ID NO: 8 and the heavy chain may comprise the amino acid sequence of SEQ ID NO: 9. For example, the light chain may comprise the amino acid sequence of SEQ ID NO: 8 and the heavy chain may comprise the amino acid sequence of SEQ ID NO: 10.

[0023] The antibody or antigen-binding fragment may have a VH region having an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:5, 3 or 1, and / or a VL region having an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:2, 4 or 6.

[0024] The antibody or antigen-binding fragment may have a heavy chain amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the sequence set forth in SEQ ID NO: 10, 9, or 7, and / or a light chain sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the sequence set forth in SEQ ID NO: 8.

[0025] The CDR sequences of the antibodies and binding fragments disclosed herein may be inserted between human or humanized framework sequences. The antibodies may be, for example, intact or full-length antibodies.

[0026] The antibodies or antigen-binding fragments disclosed herein may bind to at least one of the following residues of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO: 32: K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625. For example, they may bind to one or more of the following residues of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO: 32: K550, R551, K585, S597, Y617, or T625.

[0027] The antibodies or antigen-binding fragments disclosed herein have an affinity for human plasma kallikrein, e.g., human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO: 32, at about 1, 2, 3, 4, or 5 x 10 mAb, as measured by a surface plasmon resonance (SPR) assay. -9 K below M D They may bind at a pH of 6.0-7.4, which is essentially pH-independent. D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ , 1 mg / mL BSA, 0.02% Tween-20 as determined by SPR at 37°C in the same buffer, and / or a K between 0.1 nM and 5 nM for human plasma kallikrein D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ as determined by SPR at 37 °C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and / or a K between 0.1 nM and 5 nM for human plasma kallikrein D , and a K between 250 nM and 2,000 nM for human prekallikrein. D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+, as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4.

[0028] The present invention also covers antibodies and binding fragments thereof that compete with the antibodies or binding fragments disclosed herein for binding to plasma kallikrein. For example, such antibodies may bind to the same epitope on human plasma kallikrein as the antibodies disclosed herein. For example, such antibodies may bind to one or more of residues K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625 of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO:32. For example, such an antibody may bind to one or more of the following residues of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO: 32: K550, R551, K585, S597, Y617, or T625. Such an antibody may have the following characteristics: (i) a serum half-life of at least 20 days; (ii) a substantially pH-independent K value between pH 6.0 and 7.4; D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ (iii) a K between 0.1 nM and 5 nM for human plasma kallikrein, as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA and 0.02% Tween-20; D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ as determined by SPR at 37 °C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and / or (iv) a K between 0.1 nM and 5 nM for human plasma kallikrein D , and a K between 250 nM and 2,000 nM for human prekallikrein. D(e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).

[0029] The antibodies or antigen-binding fragments disclosed herein may be monoclonal antibodies. They may be humanized antibodies. They may have a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM. If the constant region is of class IgG, the subclass may be selected from IgG1, IgG2, IgG3, and IgG4. If the heavy chain constant region is IgG1, it may have M252Y, S254T, and T256E mutations, or M428L and N434S mutations, numbered according to the EU numbering catalog. The heavy chain variable region and the light chain variable region may or may not be linked by a linker. When they are linked by a linker, such as in scFv antibodies, the linker is (GGGGS). n (SEQ ID NO: 35); (GGGGA) n (SEQ ID NO: 36), or any combination thereof, where each n is independently 1 to 5.

[0030] In another aspect, the invention provides isolated nucleic acids comprising a nucleotide sequence encoding the heavy chain or heavy chain variable region (VH) of an antibody or antigen-binding fragment disclosed herein, as well as isolated nucleic acids encoding the light chain or light chain variable region (VL) of an antibody or antigen-binding fragment disclosed herein. The heavy and light chains may be encoded on the same nucleic acid sequence, or the heavy chain may be encoded on one nucleic acid and the light chain may be encoded separately by another nucleic acid. The VL and VH may be encoded on the same nucleic acid sequence, or the VH may be encoded on one nucleic acid and the VL may be encoded separately by another nucleic acid. An expression vector may contain nucleic acids encoding both the heavy and light chains, or an expression vector may contain nucleic acids encoding both the VL and VH chains. A host cell may contain an expression vector containing nucleic acids encoding both the heavy and light chains, or a host cell may contain an expression vector containing nucleic acid encoding the heavy chain and an expression vector containing nucleic acid encoding the light chain. A host cell may contain an expression vector containing nucleic acid encoding both the VH and VL, or a host cell may contain an expression vector containing nucleic acid encoding the VH and an expression vector containing nucleic acid encoding the VL. The host cells may be grown under conditions such that they express the heavy and light chains, or VH and VL, to produce the antibody or antigen-binding fragment of the invention.

[0031] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment disclosed herein, or a nucleic acid encoding same. The pharmaceutical composition may be, for example, injectable or intravenously administrable.

[0032] In yet another aspect, the present invention provides a method of treating a subject having a disease or disorder selected from hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, or burn injury, by administering a plasma kallikrein antibody or antigen-binding fragment thereof, as disclosed herein, or a pharmaceutical composition containing same, to the subject.

[0033] The disclosed antibodies or antigen-binding fragments may be used in methods of treating plasma kallikrein-mediated disorders in a subject, including hereditary angioedema or bradykinin-dependent edema. The disclosed antibodies or antigen-binding fragments thereof may also be used in the manufacture of a medicament for treating the aforementioned conditions, such as hereditary angioedema or bradykinin-dependent edema.

[0034] The disclosed antibodies or binding fragments thereof may be used to reduce bradykinin levels in a subject, to reduce or inhibit bradykinin production in a subject, and / or to reduce or inhibit plasma kallikrein activity in a subject. [Brief explanation of the drawings]

[0035] [Figure 1] 1 is a bar graph showing the selectivity of various hybridoma lines for human plasma kallikrein based on relative light units (RLU). [Figure 2] 1 is a graph showing the inhibitory activity of various antibodies against human plasma kallikrein. [Figure 3]1 provides graphs showing the inhibitory activity of various antibodies against human plasma kallikrein at pKal concentrations of 1 nM (top graph) and 10 nM (bottom graph). [Figure 4] 1 provides a graph showing the inhibitory activity of various antibodies against human plasma kallikrein at a pKal concentration of 1 nM. [Figure 5] 1 provides a graph showing that MAb4-YTE is more potent than DX-2930 in inhibiting bradykinin production in vitro. [Figure 6] 1 provides graphs of surface plasmon resonance (SPR) binding data comparing the affinity of DX-2930, MAb4-YTE, and MAb4-LS for pKal (top row of graphs) and preKal (bottom row of graphs) at pH 7.4 and pH 6.0. [Figure 7] 1 is a graph showing plasma kallikrein (pKal) activity plotted against the concentration of MAb4-YTE in a cross-species pKal binding assay as described in Example 2. As shown, MAb4-YTE is a potent inhibitor of pKal in humans as well as monkeys, rats, and rabbits. [Figure 8] 1 provides graphs of pharmacokinetic data showing concentrations of MAb4, MAb4-LS, and MAb4-YTE over time in cynomolgus monkeys. [Figure 9]

[0023] Figure 1 provides graphs showing that MAb4-YTE has a substantially extended plasma half-life in vivo in cynomolgus monkeys compared to DX-2930. Antibody concentration over time is shown for DX-2930 in the top graph and for MAb4-YTE in the middle graph, with the data from the top and middle graphs shown together in the bottom graph. [Figure 10] 1 is a graph showing the longer duration of action in vivo of MAb4-YTE compared to a comparative antibody. [Figure 11] 1 provides a graph showing the IC50 of gabexate mesylate, DX-2930, and MAb4-YTE in a trypsin inhibition assay. [Figure 12]Graphs showing competitive binding of MAb4-LS and MAb4-YTE antibodies to the DX-2930 surface, competitive binding of MAb4-YTE and DX-2930 antibodies to the MAb4-LS surface, and competitive binding of MAb4-LS and DX-2930 antibodies to the MAb4-YTE surface. [Figure 13] FIG. 1 shows the peptide interaction between human pKal and MAb4-YTE. [Figure 14] A-J show the interaction between human pKal and MAb4-YTE. A shows a ribbon / surface representation of the front view of pKal-MAb4-YTE epitope cross-link mapping. B shows a ribbon / surface representation of the back view of pKal-MAb4-YTE epitope cross-link mapping. C shows a ribbon / surface representation of the side view 1 of pKal-MAb4-YTE epitope cross-link mapping. D shows a ribbon / surface representation of the side view 2 of pKal-MAb4-YTE epitope cross-link mapping. E shows a ribbon / surface representation of the top view of pKal-MAb4-YTE epitope cross-link mapping. F shows a ribbon representation of the front view of pKal-MAb4-YTE epitope cross-link mapping. G shows a ribbon representation of the back view of pKal-MAb4-YTE epitope cross-link mapping. H shows a ribbon representation of side view 1 of pKal-MAb4-YTE epitope cross-linking mapping. I shows a ribbon representation of side view 2 of pKal-MAb4-YTE epitope cross-linking mapping. J shows a ribbon representation of top view of pKal-MAb4-YTE epitope cross-linking mapping. [Figure 15] FIG. 1 is a diagram of the peptide interaction between human pKal and DX-2930. [Figure 16]A-J show the interaction between human pKal and DX-2930. A shows a ribbon / surface representation of the front view of pKal-DX-2930 epitope cross-linking mapping. B shows a ribbon / surface representation of the back view of pKal-DX-2930 epitope cross-linking mapping. C shows a ribbon / surface representation of the side view 1 of pKal-DX-2930 epitope cross-linking mapping. D shows a ribbon / surface representation of the side view 2 of pKal-DX-2930 epitope cross-linking mapping. E shows a ribbon / surface representation of the top view of pKal-DX-2930 epitope cross-linking mapping. F shows a ribbon representation of the front view of pKal-DX-2930 epitope cross-linking mapping. G shows a ribbon representation of the back view of pKal-DX-2930 epitope cross-linking mapping. H shows a ribbon representation of side view 1 of pKal-DX-2930 epitope cross-link mapping. I shows a ribbon representation of side view 2 of pKal-DX-2930 epitope cross-link mapping. J shows a ribbon representation of top view of pKal-DX-2930 epitope cross-link mapping. [Figure 17] 1 is a map showing the residues of human pKal cross-linked by antibody DX-2930 compared to antibody MAb4-YTE, as described in Example 5 below, demonstrating that these antibodies bind to different epitopes on pKal. [Figure 18A] The nucleic acid sequence (top row; SEQ ID NO: 48) and amino acid sequence (bottom row; SEQ ID NO: 10) of the heavy chain of MAb4-YTE are shown, beginning in Figure 18A, continuing in Figure 18B, and ending in Figure 18C. [Figure 18B] The nucleic acid sequence (top row; SEQ ID NO: 48) and amino acid sequence (bottom row; SEQ ID NO: 10) of the heavy chain of MAb4-YTE are shown, beginning in Figure 18A, continuing in Figure 18B, and ending in Figure 18C. [Figure 18C] The nucleic acid sequence (top row; SEQ ID NO: 48) and amino acid sequence (bottom row; SEQ ID NO: 10) of the heavy chain of MAb4-YTE are shown, beginning in Figure 18A, continuing in Figure 18B, and ending in Figure 18C. [Figure 19A]The nucleic acid sequence (top row; SEQ ID NO: 49) and amino acid sequence (bottom row; SEQ ID NO: 8) of the light chain of MAb4-YTE are shown, beginning in Figure 19A and ending in Figure 19B. [Figure 19B] The nucleic acid sequence (top row; SEQ ID NO: 49) and amino acid sequence (bottom row; SEQ ID NO: 8) of the light chain of MAb4-YTE are shown, beginning in Figure 19A and ending in Figure 19B. DETAILED DESCRIPTION OF THE INVENTION

[0036] The inventions described herein are based, in part, on the discovery of high-affinity binding proteins, e.g., antibodies and antigen-binding fragments, that bind to plasma kallikrein, e.g., human plasma kallikrein. The antibodies have high specificity for plasma kallikrein, e.g., human plasma kallikrein. The antibodies have high selectivity for plasma kallikrein relative to prekallikrein and lack significant off-target binding to trypsin and other serine proteases. These binding proteins may also have an extended half-life, e.g., an extended half-life in the blood. Because of these and other characteristics, the antibodies may be useful in treating various disorders mediated by plasma kallikrein, e.g., hereditary angioedema and bradykinin-dependent edema, and may facilitate treatment of these disorders at lower doses and / or at longer dosing intervals compared to other antibodies.

[0037] The antibodies described herein have the following characteristics: (i) a serum half-life of at least 20 days; (ii) a substantially pH-independent K between pH 6.0 and 7.4; D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ (iii) a K between 0.1 nM and 5 nM for human plasma kallikrein, as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA and 0.02% Tween-20; D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+as determined by SPR at 37 °C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and / or (iv) a K between 0.1 nM and 5 nM for human plasma kallikrein D , and a K between 250 nM and 2,000 nM for human prekallikrein. D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).

[0038] These binding proteins can also be used in compositions, eg, pharmaceutical compositions, to treat various plasma kallikrein-mediated disorders such as hereditary angioedema and bradykinin-dependent edema.

[0039] I. Plasma kallikrein antibodies As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies. The antibodies described herein include antibodies containing two immunoglobulin heavy chains linked to two immunoglobulin light chains. Full-length or intact antibodies are included.

[0040] Antibody light chains typically contain one variable region (V L ) and one constant region (C L ) and a heavy chain usually consists of one variable region (V H) and at least three constant regions (CH1, CH2, and CH3) (more, depending on the isotype). The variable regions of each light / heavy chain pair, usually found in the amino-terminal portion of each chain, form the antibody-binding site, which is primarily responsible for antigen recognition and determines the binding specificity of the antibody. Thus, an intact or full-length antibody generally has two binding sites, i.e., is bivalent. The two binding sites may be the same and target the same antigen; or, as in bifunctional or bispecific antibodies, the two binding sites may be different, e.g., each binding site targets a different antigen or different epitopes of the same antigen.

[0041] The carboxy-terminal portion of an immunoglobulin heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Human heavy chains are further typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA, and IgE, respectively.

[0042] Typically, both the heavy and light chain variable domains of an antibody contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within four relatively conserved framework regions (FRs). The CDRs are usually lined by the framework regions, enabling binding to a specific epitope. Generally, from N- to C-terminus, both the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. As used herein, "HCDR1," "HCDR2," and "HCDR3" refer to CDR1, 2, or 3, respectively, of the heavy chain variable region, and "LCDR1," "LCDR2," and "LCDR3" refer to CDR1, 2, or 3, respectively, of the light chain variable region.

[0043] The assignment of amino acids to each CDR is generally based on the Kabat definition (e.g., Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616), or according to the definition of Chothia (see, e.g., Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883), but may also follow, for example, IMGT (see, e.g., Lefranc (2005), Nucl. Acids Res., 33, D593-D597; Lefranc et al., (2003), Dev. Comp. Immunol., 27, 55-77; Lefranc et al. al., (2005), Dev. Comp. Immunol., 29, 185-203 (2005); Lefranc et al., (2005), Dev. Comp. Immunol., 2005, 29, 917-938) or AbM (see, e.g., Martin, et al., (1989) Proc. Natl. Acad. Sci. USA, 86, 9268-9272; Pedersen et al., (1992), Immunomethods, 1, 126; Rees et al., (1996) In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172).

[0044] As used herein, the term "parent antibody" is used to refer to an antibody obtained by exposing the immune system to an antigen prior to modifying the antibody for its intended use, such as humanizing the antibody for use as a human therapeutic antibody.

[0045] As used herein, unless otherwise indicated, the term "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length or intact antibody, e.g., a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments; single-domain antibodies; recombinant heavy-chain-only antibodies (VHH); and shark heavy-chain-only antibodies (VNAR).

[0046] As used herein, the term "Fab fragment" refers to a fragment of one light chain and one heavy chain C H It is understood to mean an antibody fragment comprising the antibody fragment 1 and the variable region.

[0047] As used herein, the term "Fc" region refers to at least the C region of an antibody, H 2 and C H It refers to an antibody fragment containing two heavy chain fragments containing three domains.

[0048] As used herein, the term "Fab' fragment" refers to a fragment comprising one light chain and one V H Domain and C H 1 domain and even C H 1 and C H An antibody fragment containing a portion or fragment of one heavy chain containing the region between the two domains is understood to mean an antibody fragment containing a portion or fragment of one heavy chain containing the region between the two domains. The interchain disulfide bond formed between the two heavy chains of two Fab' fragments can form an F(ab')2 fragment.

[0049] As used herein, the term "Fv region" refers to the region of an antibody that comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0050] The term "single-chain Fv" or "scFv" refers to the VH and V L scFv refers to an antibody fragment containing V domains, where these domains are present in a single polypeptide chain. Generally, scFv polypeptides are H Domains and V L The scFv further comprises a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun (1994), THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0051] The term "monoclonal antibody," as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences in the variable domains, particularly the CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al. (1975), Nature, 256:495, or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., (1991) Nature, 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597. See also Presta, (2005) J. Allergy Clin. Immunol. 116:731.

[0052] As used herein, the term "chimeric antibody" is understood to mean an antibody having variable domains derived from a first antibody and constant domains derived from a second antibody, wherein the first and second antibodies are derived from different species; see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0053] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences derived from both human and non-human (e.g., murine, rat) antibodies. For example, a humanized antibody can include a variable region in which the CDRs or portions thereof are derived from or correspond to non-human immunoglobulin sequences and all or substantially all or portions of the FRs are derived from or correspond to human immunoglobulin sequences. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (e.g., a human light chain constant region and / or a human heavy chain constant region, e.g., a human Fc, or human CH1, CH2, and / or CH3 domains). The CDR sequences described herein may be inserted between human or humanized framework sequences.

[0054] Humanization can be performed to reduce the immunogenicity of antibodies, such as non-human antibodies, when administered to humans. One humanization approach creates chimeric proteins in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, for example, Morrison et al., 1984, PROC.NAT.ACAD.SCI.81:6851-6855; Neuberger et al., 1984, NATURE 312:604-608; U.S. Patent No. 6,893,625 (Robinson); U.S. Patent No. 5,500,362 (Robinson); and U.S. Patent No. 4,816,567 (Cabilly).

[0055] In an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted onto the framework of another species. For example, mouse CDRs can be grafted onto human FRs. In some embodiments, the CDRs of the light and heavy chain variable regions of an antibody are grafted onto human FRs or consensus human FRs. To create a consensus human FR, FRs derived from the amino acid sequences of several human heavy or light chains are aligned to identify a consensus amino acid sequence. CDR porting is U.S. Pat. ; 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 5,530,101 (Queen); Jones et al.(1986)NATURE 321:522-525;Riechmann et al.(1988)NATURE 332:323-327;Verhoeyen et al.(1988)SCIENCE 239:1534-1536; and Winter (1998) FEBS LETT 430:92-94.

[0056] In an approach called "SUPERHUMANIZATION™," human CDR sequences are selected from human germline genes based on the structural similarity of the human CDRs to the mouse antibody being humanized. See, e.g., U.S. Pat. No. 6,881,557 (Foote) and See Tan et al., 2002, J. IMMUNOL. 169:1119-1125.

[0057] Other methods for reducing immunogenicity include "reshaping," "hyperchimerization," and "veneering / resurfacing." See, for example, Vaswami et al., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al., 1996, PROT. ENGINEER, 9:895-904; and U.S. Patent No. 6,072,035 (Hardman). In the veneering / resurfacing approach, surface-accessible amino acid residues in mouse antibodies are replaced with amino acid residues more frequently found at the same positions in human antibodies. This type of antibody resurfacing is described, for example, in U.S. Patent No. 5,639,641 (Pedersen).

[0058] Another approach to converting murine antibodies into a form suitable for human medical use is known as ACTIVMAB™ technology (Vaccinex, Inc., Rochester, NY), which involves the use of vaccinia virus-based vectors to express antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains can be generated. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer). Another approach to converting murine antibodies into a form suitable for human use is the technology commercially implemented by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to create an "epitope-focused" library for antibody selection. Another approach to modifying murine antibodies into a form suitable for human medical use is the HUMAN ENGINEERING™ technology, commercially practiced by XOMA (US) LLC. See, e.g., International (PCT) Publication No. WO 93 / 11794 and U.S. Patent Nos. 5,766,886 (Studnicka); 5,770,196 (Studnicka); 5,821,123 (Studnicka); and 5,869,619 (Studnicka).

[0059] The antibodies disclosed herein can also be converted to a different type, such as a human IgG. By converting an antibody to a human antibody, the human subject will no longer recognize the antibody as foreign. The conversion of non-human IgG antibodies to human IgG antibodies is well known and can be performed routinely once the native sequence is known. As discussed herein, antibodies can be modified according to known methods. Such methods are described, for example, in Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy." Nature 332(6162):332-323; Tsurushita N, Park M, Pakabunto K, Ong K, Avdalovic A, Fu H, Jia A, Vasquez M, Kumar S. (2004), the entire contents of which are incorporated by reference. Thus, the antibodies of the present invention can be humanized.

[0060] As used herein, the term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell.

[0061] As used herein, the terms "specific binding," "immunospecific binding," "immunospecifically binds," or "specifically binds" refer to an antibody that binds to a predetermined antigen (e.g., plasma kallikrein) or an epitope present on that antigen. In this state, the antibody -7 The dissociation constant (K D ) to bind a predetermined antigen (e.g., plasma kallikrein) and / or its K for binding to a nonspecific antigen other than the predetermined antigen (e.g., BSA, casein, or another nonspecific polypeptide). D At least two times lower than Dand binds to a predetermined antigen with an affinity that is at least 2-fold greater, at least 10-fold greater, at least 20-fold greater, or at least 100-fold greater than its affinity for other antigens. The phrases "antibody that recognizes plasma kallikrein" and "antibody specific for plasma kallikrein" are used interchangeably herein with the term "antibody that immunospecifically binds to plasma kallikrein." In some embodiments, the antibody specifically or preferentially binds to plasma kallikrein over other proteins, such as, but not limited to, human prekallikrein. In some embodiments, the antibody, or antigen-binding fragment of the antibody, binds to its antigen (plasma kallikrein) with an affinity that is at least 2-fold greater, at least 10-fold greater, at least 20-fold greater, or at least 100-fold greater than its affinity for other antigens.

[0062] Methods for determining the specificity and affinity of mAbs by competitive inhibition are described in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589 601 (1983), which references are incorporated herein by reference in their entireties.

[0063] As used herein, the term "homologue" refers to a protein sequence having at least 40% but less than 100% sequence homology or identity to a reference sequence. The percent identity between two peptide chains can be determined by pairwise sequence comparison using the default settings of the AlignX module in Vector NTI v.9.0.0 (Invitrogen Corp., Carlsbad, Calif.) or other suitable sequence comparison software, such as BLAST. In some embodiments, an antibody or antigen-binding fragment thereof has at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology or identity to a sequence described herein. In some embodiments, the antibody has conservative substitutions compared to the sequences described herein. Exemplary conservative substitutions are shown in Table 1 and are encompassed within the scope of the disclosed subject matter. Conservative substitutions may be made in the framework region or antigen-binding site, as long as they do not adversely affect the properties of the antibody. Substitutions may be made to improve antibody properties, such as stability and affinity. Conservative substitutions will produce molecules with similar functional and chemical characteristics to the molecule in which they are modified. Exemplary amino acid substitutions are shown in Table 1 below. [Table 1]

[0064] In some embodiments, variants of the proteins and peptides provided herein are provided. In some embodiments, the variants comprise substitutions, deletions, or insertions. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) substitutions. As described herein, substitutions can be conservative. In some embodiments, the substitutions are non-conservative. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) deletions. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) insertions. In some embodiments, the substitutions, deletions, or insertions are present in a CDR provided herein. In some embodiments, the substitutions, deletions, or insertions are not present in a CDR provided herein.

[0065] Typically, the variant antibodies provided herein, or antigen-binding fragments thereof, retain at least 10% of their plasma kallikrein binding activity when the activity is expressed on a molar basis (compared to the unmodified antibody or reference antibody, which can be a parent antibody). In some embodiments, the variant antibodies (or antigen-binding fragments thereof) or antigen-binding fragments of the antibodies provided herein retain at least 20%, 50%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or more of the plasma kallikrein binding affinity as the unmodified antibody or reference antibody, which can be a parent antibody. As described herein, the antibodies or antigen-binding fragments of the invention It is also intended that the antibody may include conservative or non-conservative amino acid substitutions, which may also be referred to as "conservative variants" or "function-conservative variants" of the antibody, that do not substantially alter its biological activity.

[0066] As used herein, the term "epitope" refers to a portion of any molecule capable of being recognized and bound by an antibody at one or more of the antigen-binding regions of the Ab. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and possess specific charge characteristics as well as specific three-dimensional structural features. Examples of epitopes include, but are not limited to, the residues to which the antibodies provided herein bind. An epitope may be a linear or contiguous epitope, i.e., a linear sequence of amino acids in the primary structure of an antigen, e.g., human plasma kallikrein (SEQ ID NO: 32). Alternatively, in other embodiments, an epitope may be a conformational epitope, which has a specific three-dimensional shape when the antigen assumes its secondary structure. For example, a conformational epitope may include nonlinear, i.e., noncontiguous, amino acids of an antigen.

[0067] The antibodies disclosed herein may be, for example, inhibitory antibodies with high potency, specificity, and long serum retention, where high potency translates into high efficacy at low drug doses and high specificity reduces side effects due to inhibition of related off-target serine proteases.

[0068] An antibody or antigen-binding fragment thereof that binds to plasma kallikrein, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence or variant thereof as provided herein, has the following characteristics: a serum half-life of at least 20 days; a substantially pH-independent K between pH 6.0 and 7.4; D K between 0.1 nM and 5 nM for human plasma kallikrein (e.g., as determined by SPR at 37 °C in a buffer containing 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca, 1 mg / mL BSA, 0.02% Tween-20). D(e.g., as determined by SPR at 37°C in a buffer containing 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca, 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and / or a KD between 0.1 nM and 5 nM for human plasma kallikrein and a KD between 250 nM and 2,000 nM for human prekallikrein. D (e.g., as determined by SPR at 37°C in a buffer containing 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca2+, 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).

[0069] In some embodiments, the antibody is a monoclonal antibody that binds to plasma kallikrein. In some embodiments, the antibody binds to amino acids of an epitope of plasma kallikrein. In some embodiments, plasma kallikrein has the following amino acid sequence: MILFKQATYF ISLFATVSCG CLTQLYENAF FRGGDVASMY TPNAQYCQMR CTFHPRCLF SFLPASSIND MEKRFGCFLK DSVTGTLPKV HRTGAVSGHS LKQCGHQISA CHRDIYKGVD MRGVNFNVSK VSSVEECQKR CTNNIRCQFF SYATQTFHKA EYRNNCLLKY SPGGTPTAIK VLSNVESGFS LKPCALSEIG CHMNIFQHLA FSDVDVARVL TPDAFVCRTI CTYHPNCLFF TFYTNVWKIE SQRNVCLLKT SESGTPSSST PQENTISGYS LLTCKRTLPE PCHSKIYPGV DFGGEELNVT FVKGVNVCQE TCTKMIRCQF FTYSLLPEDC KEEKCKCFLR LSMDGSPTRI AYGTQGSSGY SLRLCNTGDN SVCTTKTSTR IVGGTNSSWG EWPWQVSLQV KLTA QRHLCG GSLIGHQWVL TAAHCFDGLP LQDVWRIYSG ILNLSDITKD TPFSQIKEII IHQNYKVSEG NHDIALIKLQ APLNYTEFQK PICLPSKGDT STIYTNCWVT GWGFSKEKGE IQNILQKVNI PLVTNEECQK RYQDYKITQR Human plasma kallikrein (UNIPROT P03952, KLKB1) having the sequence MVCAGYKEGG KDACKGDSGG PLVCKHNGMW RLVGITSWGE GCARREQPGV YTKVAEYMDW ILEKTQSSDG KAQMQSPA (SEQ ID NO: 32).

[0070] In some embodiments, the plasma kallikrein is cynomolgus monkey plasma kallikrein (UNIPROT A0A2K5VTJ9-1, SEQ ID NO: 45), rabbit plasma kallikrein (UNIPROT G1T127, SEQ ID NO: 46), or rat plasma kallikrein (UNIPROT P14272, SEQ ID NO: 47). For example, the antibody or antigen-binding fragment thereof may bind to rabbit, rat, or monkey plasma kallikrein.

[0071] The sequence of human plasma kallikrein, as shown above, includes a signal peptide sequence at amino acid residues 1-19, which can be cleaved during post-translational processing. In some embodiments, the antibody binds to human plasma kallikrein lacking the signal peptide sequence, e.g., mature human plasma kallikrein.

[0072] In some embodiments, the antibody comprises an amino acid sequence provided herein.

[0073] The antibody sequence can be modified to produce a human IgG antibody. The sequence conversions provided herein can be modified to produce other types of antibodies. The CDRs can also be attached to other antibodies, proteins, or molecules to create antibody fragments that bind to plasma kallikrein, which can also be in the form of antibody-drug conjugates ("ADCs") or multispecific molecules. The sequences can also be made into chimeric antibodies as described herein.

[0074] In some embodiments, the antibody comprises an amino acid sequence comprising a sequence provided herein, or a fragment thereof. In some embodiments, the antibody comprises one or more amino acid sequences provided herein, an antigen-binding fragment thereof, or a human IgG variant thereof. A "human IgG variant thereof" refers to an antibody that has been modified to become a human IgG, if the starting antibody is not a human IgG antibody.

[0075] The antibody can also be modified into a chimeric antibody or a human antibody. The antibody can also be used in an injectable pharmaceutical composition. Also, as described herein, the antibody can be an isolated antibody or an engineered antibody.

[0076] In some embodiments, "derivatives" of antibodies, fragments, regions, or derivatives thereof are provided, which term includes those proteins encoded by genes that have been truncated or modified to result in molecular species that are functionally similar to immunoglobulin fragments. Modifications include, but are not limited to, the addition of genetic sequences encoding cytotoxic proteins, such as plant or bacterial toxins. Modifications can also include reporter proteins, such as fluorescent or chemiluminescent tags. Fragments and derivatives can be produced by any method.

[0077] Identification of these antigen-binding regions and / or epitopes recognized by the Abs described herein provides the information necessary to generate additional monoclonal antibodies with similar binding characteristics and therapeutic or diagnostic utility comparable to the embodiments of the present application.

[0078] The variable regions described herein can be combined with any type of constant region, including human or murine constant regions. Human genes encoding antibody constant (C) regions, fragments and regions can be derived from human fetal liver libraries by known methods. Human C region genes can be derived from any human cell, including cells that express and produce human immunoglobulins. Human C regions can be derived from any human cell, including cells that express and produce human immunoglobulins. H The regions can be derived from any of the known classes or isotypes of human heavy chains, including gamma, mu, alpha, delta, or epsilon, and their subtypes, such as G1, G2, G3, and G4. The heavy chain isotype is responsible for the various effector functions of the antibody, and therefore the C H The choice of region will be guided by the desired effector function, such as complement fixation or activity in antibody-dependent cellular cytotoxicity (ADCC). H The regions are derived from gamma 1 (IgG1), gamma 3 (IgG3), gamma 4 (IgG4), or μ (IgM). L The region can be derived from either human light chain isotype, kappa or lambda.

[0079] In some embodiments, the antibody comprises an Fc domain. In some embodiments, the Fc domain comprises mutations to extend the half-life of the antibody. In some embodiments, the Fc domain comprises mutations such as those described in U.S. Patent No. 7,670,600, which is incorporated herein by reference in its entirety. In some embodiments, the constant region comprises a mutation at amino acid residue 428 relative to a wild-type human IgG constant domain, numbered according to the EU numbering system of Kabat. Without being bound by any particular theory, antibodies comprising a mutation corresponding to residue 428 may have a longer half-life compared to the half-life of an IgG having a wild-type human IgG constant domain. In some embodiments, the mutation is a substitution of the native residue with threonine, leucine, phenylalanine, or serine. In some embodiments, the antibody further comprises one or more amino acid substitutions relative to the corresponding wild-type human IgG constant domain at one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 429-436, numbered according to the EU numbering system of Kabat. Specific mutations or substitutions at these positions are described in US Pat. No. 7,670,600, which is incorporated herein by reference in its entirety.

[0080] Other mutations can be used in the Fc domain, such as those provided in U.S. Patent No. 8,394,925, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region is a variant Fc region comprising amino acid substitutions at positions 428 and 434, where the amino acid substitutions are a non-wild-type leucine at position 428 and a non-wild-type serine at position 434, where the polypeptide is an antibody and numbering is according to the EU Catalogue of Kabat et al. In some embodiments, the Fc region comprises an S228P, L235E, M428L, or N434S substitution. In some embodiments, the Fc region comprises an M428L substitution. In some embodiments, the Fc region comprises an N434S substitution. In some embodiments, the Fc region comprises an M428L and N434S substitution, where numbering is according to the EU Catalogue of Kabat et al. In some embodiments, the Fc region comprises M252Y, S254T, and / or T256E substitutions, or the Fc region comprises M252Y, S254T, and T256E substitutions, where the numbering is according to the EU catalogue of Kabat et al.

[0081] In some embodiments, the antibodies described herein are used to detect the presence of an antigen. The antibodies can be used in any device or method for detecting the presence of an antigen.

[0082] CDR sequence of IA antibody The antibodies or antigen-binding fragments disclosed herein may be defined by their CDR sequences. For example, the antibodies or antigen-binding fragments may be defined by Kabat numbering. The antibody or antigen-binding fragment may comprise one or more of the amino acid sequences provided in Table 2, which are the CDRs of such antibodies or antigen-binding fragments. [Table 2]

[0083] The antibody or antigen-binding fragment may comprise one or more of the amino acid sequences provided in Table 3, which are the CDRs of the antibody or antigen-binding fragment as defined by Chothia numbering. [Table 3]

[0084] The antibody or antigen-binding fragment may comprise one or more of the amino acid sequences provided in Table 4, which are the CDRs of the antibody or antigen-binding fragment as defined by the IMGT numbering. [Table 4]

[0085] The antibody or antigen-binding fragment may comprise one or more of the amino acid sequences provided in Table 5, which are the CDRs of the antibody or antigen-binding fragment as defined by AbM numbering. [Table 5]

[0086] An antibody or antigen-binding fragment thereof may comprise a heavy or light chain CDR having the sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31. For example, the light chain CDR of the antibody, or antigen-binding fragment thereof, may have the sequence of SEQ ID NO: 11, 12, 13, 18, 20, 21, 22, 26, 27, or 28. For example, the heavy chain CDR of the antibody, or antigen-binding fragment thereof, may have the sequence of SEQ ID NO: 14, 15, 16, 17, 19, 23, 24, 25, 29, 30, or 31.xx.

[0087] The CDRs described herein can be interchanged with CDRs characterized by different definitions, such as Chothia and IMGT, as shown in the table above. For example, an LCDR1 defined by Kabat can be interchanged with an LCDR1 defined by Chothia, IMGT, or AbM; an LCDR2 defined by Kabat can be interchanged with an LCDR2 defined by Chothia, IMGT, or AbM, etc. For example, in some embodiments, an antibody or antigen-binding fragment thereof comprises a light chain variable region having an LCDR1, an LCDR2, and an LCDR3. and LCDR3 has the sequence of SEQ ID NO: 13, 22, or 28, while in some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 14, 23, 29, or 43; HCDR2 has the sequence of SEQ ID NO: 15, 17, 19, 24, 30, or 44; and HCDR3 has the sequence of SEQ ID NO: 16, 25, or 31.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 11, LCDR2 has the sequence of SEQ ID NO: 12, and LCDR3 has the sequence of SEQ ID NO: 13.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 11, LCDR2 has the sequence of SEQ ID NO: 18, and LCDR3 has the sequence of SEQ ID NO: 13.

[0090] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 20, LCDR2 has the sequence of SEQ ID NO: 21, and LCDR3 has the sequence of SEQ ID NO: 22.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 20, LCDR2 has the sequence of SEQ ID NO: 26, and LCDR3 has the sequence of SEQ ID NO: 22.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 27, LCDR2 has the sequence of SEQ ID NO: 21, and LCDR3 has the sequence of SEQ ID NO: 28.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 27, LCDR2 has the sequence of SEQ ID NO: 26, and LCDR3 has the sequence of SEQ ID NO: 28.

[0094] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 11, LCDR2 has the sequence of SEQ ID NO: 12, and LCDR3 has the sequence of SEQ ID NO: 28.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID NO: 11, LCDR2 has the sequence of SEQ ID NO: 18, and LCDR3 has the sequence of SEQ ID NO: 28.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 14, HCDR2 has the sequence of SEQ ID NO: 15, and HCDR3 has the sequence of SEQ ID NO: 16.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 14, HCDR2 has the sequence of SEQ ID NO: 17, and HCDR3 has the sequence of SEQ ID NO: 16.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 14, and CDR2 has the sequence of SEQ ID NO:19 and HCDR3 has the sequence of SEQ ID NO:16.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 23, HCDR2 has the sequence of SEQ ID NO: 24, and HCDR3 has the sequence of SEQ ID NO: 25.

[0100] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 29, HCDR2 has the sequence of SEQ ID NO: 30, and HCDR3 has the sequence of SEQ ID NO: 31.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 43, HCDR2 has the sequence of SEQ ID NO: 44, and HCDR3 has the sequence of SEQ ID NO: 25.

[0102] Different CDR motifs can be combined in any combination, including those not shown in the table above, for example, the following embodiments are provided as non-limiting examples of such combinations.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 12 or 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO: 14; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 15, 17, or 19; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 12; and LCDR3 has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 14; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 15; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 12; and LCDR3 has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 14; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 17; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 12; and LCDR3 has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 14; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 19; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 18; and LCDR3 has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 14; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 15; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 18; and LCDR3 has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 14; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 17; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 18; and LCDR3 has the amino acid sequence of SEQ ID NO: 13, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 14; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 19; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 16; or a variant of any of the foregoing.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 20; LCDR2 has the amino acid sequence of SEQ ID NO: 21 or 26; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO: 22, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO: 23; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 24; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 25; or a variant of any of the foregoing.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 20; LCDR2 has the amino acid sequence of SEQ ID NO: 21; and LCDR3 has the amino acid sequence of SEQ ID NO: 22, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 23; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 24; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 25; or a variant of any of the foregoing.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 20; LCDR2 has the amino acid sequence of SEQ ID NO: 26; and LCDR3 has the amino acid sequence of SEQ ID NO: 22, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 23; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 24; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 25; or a variant of any of the foregoing.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) LCDR1, LCDR the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 30; the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 31; or a variant of any of the foregoing.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 27; LCDR2 has the amino acid sequence of SEQ ID NO: 21; and LCDR3 has the amino acid sequence of SEQ ID NO: 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 29; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 30; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 31; or a variant of any of the foregoing.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 27; LCDR2 has the amino acid sequence of SEQ ID NO: 26; and LCDR3 has the amino acid sequence of SEQ ID NO: 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 29; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 30; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 31; or a variant of any of the foregoing.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 12 or 18; and the LCDR3 sequence has the amino acid sequence of SEQ ID NO: 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 sequence has the amino acid sequence of SEQ ID NO: 43; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 44; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 25; or a variant of any of the foregoing.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 12; and LCDR3 has the amino acid sequence of SEQ ID NO: 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 43; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 44; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 25; or a variant of any of the foregoing.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 11; LCDR2 has the amino acid sequence of SEQ ID NO: 18; and LCDR3 has the amino acid sequence of SEQ ID NO: 28, and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 43; heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 44; and heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 25; or a variant of any of the foregoing.

[0119] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 with up to 1, 2, 3, or 4 amino acid substitutions relative to that amino acid sequence; HCDR2 comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions relative to that amino acid sequence. and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, with up to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions relative to the amino acid sequence. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions relative to the amino acid sequence; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18, with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence. The amino acid substitutions may be conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." These variants may be derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, these variants may not be derived from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0120] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, 23, 29 or 43 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions relative to said amino acid sequence; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, 19, 24, 30 or 44 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions relative to said amino acid sequence; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, 25 or 31 with up to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions relative to said amino acid sequence. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, 20, or 27, with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions relative to the amino acid sequence; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, 18, 21, or 26, with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, 22, or 28, with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to the amino acid sequence. The amino acid substitutions may be conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." These variants may be derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, these variants may not be derived from the sequences provided herein, but may be de novo isolated, for example, according to the methods provided herein for obtaining antibodies.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has a sequence selected from SEQ ID NO: 11, 20, or 27, and wherein LCDR1 comprises at most one conservative amino acid substitution; LCDR2 has a sequence selected from SEQ ID NO: 12, 18, 21, and 26, and wherein LCDR2 comprises at most one conservative amino acid substitution; and LCDR3 has a sequence selected from SEQ ID NO: 13, 22, and 28, and wherein LCDR3 comprises at most one conservative amino acid substitution. and (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has a sequence selected from SEQ ID NO: 14, 23, or 29, and HCDR1 comprises at most one conservative amino acid substitution, HCDR2 has a sequence selected from SEQ ID NO: 15, 19, 24, or 30, and HCDR2 comprises at most one conservative amino acid substitution, and HCDR3 has a sequence selected from SEQ ID NO: 16, 25, or 31, and HCDR3 comprises at most one conservative amino acid substitution.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) LCDR1, LCDR and (ii) a light chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID NO: 11, and LCDR1 comprises at most one conservative amino acid substitution, LCDR2 has the sequence of SEQ ID NO: 18, and LCDR2 comprises at most one conservative amino acid substitution, and LCDR3 has the sequence of SEQ ID NO: 13, and LCDR3 comprises at most one conservative amino acid substitution; and

[0123] In some embodiments, the light chain variable region CDR1 is replaced with any of the other light chain CDR1 sequences disclosed herein. In some embodiments, the light chain variable region CDR2 is replaced with any of the other light chain CDR2 sequences disclosed herein. In some embodiments, the light chain variable region CDR3 is replaced with any of the other light chain CDR3 sequences disclosed herein. In some embodiments, the heavy chain variable region CDR1 is replaced with any of the other heavy chain CDR1 sequences disclosed herein. In some embodiments, the heavy chain variable region CDR2 is replaced with any of the other heavy chain CDR2 sequences disclosed herein. In some embodiments, the heavy chain variable region CDR3 is replaced with any of the other heavy chain CDR3 sequences disclosed herein.

[0124] The antibody or binding fragment thereof of the present invention may have HCDR1, HCDR2, and / or HCDR3 with sequences corresponding to those of the heavy chain variable region sequence of SEQ ID NO: 1, 3, or 5, and / or may have LCDR1, LCDR2, and / or LCDR3 with sequences corresponding to those of the light chain variable region sequence of SEQ ID NO: 2, 4, or 6. For example, the antibody or binding fragment thereof of the present invention may have HCDR1, HCDR2, and HCDR3 with sequences corresponding to those of the heavy chain variable region sequence of SEQ ID NO: 3 or 5; and may have LCDR1, LCDR2, and LCDR3 with sequences corresponding to those of the light chain variable region sequence of SEQ ID NO: 4 or 6. For example, an antibody or binding fragment thereof of the present invention may have HCDR1, HCDR2, and HCDR3 with sequences corresponding to the HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region sequence of SEQ ID NO: 5; and may have LCDR1, LCDR2, and LCDR3 with sequences corresponding to the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region sequence of SEQ ID NO: 6. CDRs may be defined by Kabat, Chothia, IMGT, or AbM. For example, the CDRs are as defined by Kabat. For example, the CDRs are as defined by Chothia.

[0125] Variable and full-length sequences of IB antibodies In some embodiments, the antibody comprises one or more peptides having the following sequences provided in Table 6, or variants thereof: [Table 6-1] [Table 6-2]

[0126] In some embodiments, the antibody or antigen-binding fragment comprises a sequence selected from one or more of the following sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or variants thereof.

[0127] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, 3, or 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, 4, or 6.

[0128] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, 4, or 6.

[0129] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, 4, or 6.

[0130] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, 4, or 6.

[0131] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2.

[0132] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4, or the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:3 and a light chain comprising the amino acid sequence of SEQ ID NO:4.

[0133] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.

[0134] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5, or the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

[0135] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, or the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:6.

[0136] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.

[0137] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7, 9, or 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0138] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:8.

[0139] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:8.

[0140] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0141] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5.

[0142] The antibodies or antigen-binding fragments provided herein may comprise a heavy chain variable region comprising the amino acid sequence provided in SEQ ID NO: 1, 3, or 5 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. For example, one or more substitutions may be found in only the CDR regions, only the framework regions, or in both the CDR and framework regions. For example, the heavy chain variable region may comprise the amino acid sequence provided in SEQ ID NO: 1, 3, or 5. The antibody may have at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1, 3, or 5, wherein none of the mutations in SEQ ID NO: 1, 3, or 5 occur within HCDR1, HCDR2, or HCDR3. For example, mutations are found in one or more framework regions. In some examples, the amino acid substitution(s) may be (a) conservative amino acid substitution(s). The antibodies described in this paragraph are referred to herein as "variants." Such variants may be derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, such variants may not be derived from the sequences provided herein and may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0143] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6.

[0144] The antibodies or antigen-binding fragments provided herein may comprise a light chain variable region comprising the amino acid sequence provided in SEQ ID NO: 2, 4, or 6 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. For example, one or more substitutions may be found only in the CDR regions, only in the framework regions, or in both the CDR and framework regions. For example, the light chain variable region may have at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, 4, or 6, wherein any mutations in SEQ ID NO: 2, 4, or 6 do not occur within LCDR1, LCDR2, or LCDR3. For example, mutations are found in one or more framework regions. In some examples, the amino acid substitution(s) may be (a) conservative amino acid substitution(s). The antibodies described in this paragraph are referred to herein as "variants." These variants may be derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. Alternatively, these variants are not derived from the sequences provided herein and may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0145] The heavy chain variable region (VH) and light chain variable region (VL) sequences can be of any type, including, but not limited to, an scFv type in which the VH and VL regions are linked by a peptide linker. An example of a peptide linker that can be used to link the various peptides provided herein is (GGGGS) n (SEQ ID NO: 35); (GGGGA) n (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1 to 5. In some embodiments, the variable regions, e.g., heavy chain variable region and light chain variable region, are not linked by a linker, e.g., a peptide linker, while in other embodiments, the variable regions, e.g., heavy chain variable region and light chain variable region, are linked by a linker, e.g., a peptide linker.

[0146] In some embodiments, the antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM, hi some embodiments, the antibody comprises a heavy chain constant region of an IgG class and subclass selected from IgG1, IgG2, IgG3, and IgG4.

[0147] In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region comprises a wild-type (WT) sequence. In some embodiments, the IgG1 heavy chain constant region comprises M252Y, S254T, and T256E mutations, or M428L and N434S mutations, when numbered according to the EU numbering system. In some embodiments, the IgG1 heavy chain constant region comprises M252Y, S254T, and T256E mutations, when numbered according to the EU numbering system of Kabat. In the heavy chain amino acid sequence disclosed herein as SEQ ID NO: 9, which contains the M428L and N434S mutations, the M428L mutation occurs at residue 432 of the heavy chain sequence and the N434S mutation occurs at residue 438 of the heavy chain sequence. In the heavy chain amino acid sequence disclosed herein as SEQ ID NO: 10, which contains the M252Y, S254T and T256E mutations, the M252Y mutation occurs at residue 256 of the heavy chain sequence, the S254T mutation occurs at residue 258 of the heavy chain sequence, and the T256E mutation occurs at residue 260 of the heavy chain sequence.

[0148] In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:10, 9, or 7.

[0149] In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:10.

[0150] In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:9.

[0151] In some embodiments, the antibody or antigen-binding fragment thereof comprises a peptide having a sequence set forth in any of SEQ ID NOs: 1-10 and 11-19.

[0152] In some embodiments, the antibody or antigen-binding fragment thereof comprises any of the foregoing sequences or variants thereof.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10, or a variant of any of the foregoing. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or a variant of any of the foregoing.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof has a V amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or any combination thereof. L Contains peptides. L The peptides can include variants of any of these sequences as provided herein.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof has a V having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10, or any combination thereof. H Contains peptides. H The peptides can include variants of any of these sequences provided herein.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10, and V L The peptide comprises the sequence of SEQ ID NO:2, 4, 6, or 8.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptides may have a sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10. Contains columns and V LThe peptides include sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 2, 4, 6, or 8.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10, and L The peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 2, 4, 6, or 8, with the proviso that the V L The peptide comprises a light chain CDR having the sequence of SEQ ID NO: 11, 12, 13, 18, 20, 21, 22, 26, 27, or 28, and H There is a proviso that the peptide comprises a heavy chain CDR having the sequence of SEQ ID NO: 14, 15, 16, 17, 19, 23, 24, 25, 29, 30, 31, 43, or 44. In some embodiments, H Chain or V L The CDRs in the chains are as shown in the combinations provided herein.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof is H Peptides and V L peptide, wherein V H The peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10, and LThe peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 2, 4, 6, or 8, with the proviso that the V L The peptide comprises an LCDR1 having a sequence selected from SEQ ID NOs: 11, 20, or 27; an LCDR2 having a sequence selected from SEQ ID NOs: 12, 18, 21, and 26; and an LCDR3 having a sequence selected from SEQ ID NOs: 13, 22, and 28; and H There is a proviso that the peptide comprises an HCDR1 having a sequence selected from SEQ ID NO: 14, 23, 29, or 43; an HCDR2 having a sequence selected from SEQ ID NO: 15, 17, 19, 24, 30, or 44; and an HCDR3 having a sequence selected from SEQ ID NO: 16, 25, or 31.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10, and L The peptide comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 2, 4, 6, or 8, with the proviso that the V L The peptide comprises an LCDR1 selected from SEQ ID NOs: 11, 20, or 27, wherein LCDR1 comprises at most one conservative amino acid substitution; an LCDR2 having a sequence selected from SEQ ID NOs: 12, 18, 21, and 26, wherein LCDR2 comprises at most one conservative amino acid substitution; and an LCDR3 having a sequence selected from SEQ ID NOs: 13, 22, and 28, wherein LCDR3 comprises at most one conservative amino acid substitution; and HThere is a proviso that the peptide comprises HCDR1 having a sequence selected from SEQ ID NO: 14, 23, 29, or 43, wherein HCDR1 comprises at most one conservative amino acid substitution; HCDR2 having a sequence selected from SEQ ID NO: 15, 17, 19, 24, 30, or 44, wherein HCDR2 comprises at most one conservative amino acid substitution; and HCDR3 having a sequence selected from SEQ ID NO: 16, 25, or 31, wherein HCDR3 comprises at most one conservative amino acid substitution.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof is H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 1 and V L The peptide comprises the sequence of SEQ ID NO:2.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 1 and V L The peptide comprises the sequence of SEQ ID NO:4, 6, or 8.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 3, and V L The peptide comprises the sequence of SEQ ID NO:4.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 3, and V L The peptide comprises the sequence of SEQ ID NO:2, 6, or 8.

[0165] In some embodiments, the antibody or antigen-binding fragment thereof is VH Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 5, and V L The peptide comprises the sequence of SEQ ID NO:6.

[0166] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 5, and V L The peptide comprises the sequence of SEQ ID NO:2, 4, or 8.

[0167] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 7, and L The peptide comprises the sequence of SEQ ID NO:6.

[0168] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 7, and L The peptide comprises the sequence of SEQ ID NO:8.

[0169] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 7, and L The peptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 9, and L The peptide comprises the sequence of SEQ ID NO:8.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 9, and L The peptide comprises the sequence of SEQ ID NO:6.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 9, and L The peptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 10, and L The peptide sequence Contains sequence number 8.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 10, and L The peptide comprises the sequence of SEQ ID NO:6.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof is V H Peptides and V L peptide, wherein V H The peptide comprises the sequence of SEQ ID NO: 10, and L The peptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0176] In addition to these specific combinations, the V HAny of the peptides disclosed herein may be L It can be combined with any of the peptides.

[0177] As provided herein, the different peptides described herein (V H or V L ) can be linked by a peptide linker, or can be linked for contiguous sequences instead without a peptide linker. In some embodiments, the peptide linker is (GGGGS) n (SEQ ID NO: 35); (GGGGA) n (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1 to 5. The linked peptide form is V H -ZV L or V L -ZV H wherein Z is a peptide linker. In some embodiments, Z is (GGGGS) n (SEQ ID NO: 35); (GGGGA) n (SEQ ID NO: 36), or any combination thereof, where each n is independently 1 to 5.

[0178] II. Antibody Epitope Binding In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues K550 or R551 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues K585 or S597 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, or S597 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues Y617 or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues Y617, M618, D619, W620, 1621, L622, E623, K624, or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues K550, R551, K585, S597, Y617, or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody binds to an epitope on human plasma kallikrein that includes at least one of residues K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625 of wild-type human plasma kallikrein (SEQ ID NO: 32).

[0179] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least one of the following residues of human plasma kallikrein listed in SEQ ID NO:32: K550, R551, K585, S597, Y617, or T625.

[0180] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least two of the following residues of human plasma kallikrein listed in SEQ ID NO:32: K550, R551, K585, S597, Y617, or T625.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least three of the following residues of human plasma kallikrein listed in SEQ ID NO:32: K550, R551, K585, S597, Y617, or T625.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least four of the following residues of human plasma kallikrein listed in SEQ ID NO:32: K550, R551, K585, S597, Y617, or T625.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least five of the following residues of human plasma kallikrein listed in SEQ ID NO:32: K550, R551, K585, S597, Y617, or T625.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof binds to the following residues of human plasma kallikrein listed in SEQ ID NO:32: K550, R551, K585, S597, Y617, or T625.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least one of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least two of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least three of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof cleaves the amino acid sequence of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622 , E623, K624, or T625.

[0190] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least five of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0191] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least six of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0192] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least seven of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0193] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least eight of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0194] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least nine of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0195] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least ten of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0196] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least eleven of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0197] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 12 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 13 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0199] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 14 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0200] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 15 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0201] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 16 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0202] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 17 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 18 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 19 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0205] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 20 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0206] In some embodiments, the antibody or antigen-binding fragment thereof binds to the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587 , G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0207] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 22 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0208] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 23 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0209] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least 24 of the following residues of wild-type human plasma kallikrein (SEQ ID NO: 32): K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

[0210] In some embodiments, the antibody or antigen-binding fragment thereof binds to at least K550 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least R551 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least K585 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least H586 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least N587 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least G588 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least M589 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least W590 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least R591 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least L592 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least V593 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least G594 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least I595 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least T596 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least S597 of wild-type human plasma kallikrein (SEQ ID NO: 32).In some embodiments, the antibody or antigen-binding fragment thereof binds to at least Y617 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least M618 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least D619 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least W620 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least I621 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least L622 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least E623 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least K624 of wild-type human plasma kallikrein (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof binds to at least T625 of wild-type human plasma kallikrein (SEQ ID NO: 32).

[0211] In some embodiments, the antibody or antigen-binding fragment thereof binds to a linear epitope of human plasma kallikrein. For example, the linear epitope has the amino acid sequence of SEQ ID NO: 37, 38, or 39. The antibody or antigen-binding fragment thereof may bind to one, two, or three of these linear epitopes. In further embodiments, the antibody or antigen-binding fragment does not bind to a linear epitope having the sequence of SEQ ID NO: 40, 41, and / or 42, and / or any amino acid residues in the amino acid sequences of SEQ ID NO: 40, 41, and / or 42.

[0212] III. Antibody complex The antibodies provided herein may be conjugated to a chemical moiety. The chemical moiety may be, among others, a polymer, a radionuclide, or a cytotoxic agent. The chemical moiety may be an MRI-detectable label. In some embodiments, this may be referred to as an antibody-drug conjugate. In some embodiments, the chemical moiety is a polymer that extends the half-life of the antibody molecule in the subject's body. Suitable polymers include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG-conjugated single-chain antibodies. Wen, et al. (2001) (Bioconj. Chem. 12:545-553) discloses conjugating antibodies with PEG linked to a radiometal chelator (diethylenetriaminepentaacetic acid (DTPA)). Examples of chemical moieties include, but are not limited to, antimitotic agents such as calicheamicins (e.g., ozogamicin), monomethyl auristatin E, mertansine, and the like. Other examples include, but are not limited to, biologically active antimicrotubule agents, alkylating agents, and DNA minor groove binders. Chemical moieties can be linked to antibodies via linking groups (maleimides), cleavable linkers such as cathepsin-cleavable linkers (valine-citrulline), and, in some embodiments, one or more spacers (e.g., para-aminobenzylcarbamate).

[0213] The antibodies and antibody fragments of the invention can also be used, e.g., 99 Tc, 90 Y, 111 In, 32 P, 14 C. 125 I, 3 H, 131 I, 11 C. 15 O. 13 N, 18 F, 35 S, 51 Cr,57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 EU, 67 CU, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th, and 40 K. 157 Gd, 55 Mn, 52 Tr and 56 It may be conjugated to a label such as Fe.

[0214] Antibodies and antibody fragments may also be used in combination with rare earth chelators, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 152 Fluorescent or chemiluminescent labels may be conjugated, including fluorophores such as Eu, dansyl, umbelliferone, luciferin, luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, aequorin labels, 2,3-dihydrophthalazinedione, biotin / avidin, spin labels, and stable free radicals.

[0215] The antibody molecule may also be conjugated to a cytotoxic agent such as, for example, diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), diansin protein, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogenin, restrictocin, phenomycin, and enomycin.

[0216] Any method known in the art for conjugating the antibody molecules of the present invention to various moieties may be used, including those described in Hunter, et al., (1962) Nature 144:945;David,et al.,(1974)Biochemistry 13:1014;Pain,et al.,(1981)J.Immunol.Meth.40:219;and Nygren,J.,(1982)Histochem.and Cytochem. 30: 407. Methods for conjugating antibodies are conventional and very well known in the art.

[0217] In some embodiments, provided herein are antibodies (e.g., anti-plasma kallikrein antibodies). In some embodiments, the antibody is a recombinant antibody that binds to plasma kallikrein. In some embodiments, the plasma kallikrein protein is a human plasma kallikrein protein. In some embodiments, the antibody does not specifically bind to prekallikrein protein. As used herein, the term "recombinant antibody" refers to an antibody that is not naturally occurring. In some embodiments, the term "recombinant antibody" refers to an antibody that is not isolated from a human subject.

[0218] IV. Nucleic Acids and Their Production Methods The present invention also contemplates nucleic acids encoding each of the antibodies or antigen-binding fragments disclosed herein, including nucleic acids encoding the heavy or light chains, or variable regions of the heavy or light chains, of the antibodies as disclosed herein. Thus, in some embodiments, nucleic acid molecules encoding the antibodies or fragments disclosed herein are provided. In some embodiments, the nucleic acid encodes the amino acid sequence of an antibody or antigen-binding fragment thereof, or a heavy or light chain, or a variable region of a heavy or light chain, as provided herein.

[0219] For example, in one embodiment, the nucleic acid encodes the heavy chain variable region of any one of SEQ ID NOs: 1, 3, or 5, while in another embodiment, the nucleic acid encodes the light chain variable region of any one of SEQ ID NOs: 2, 4, or 6. In a specific embodiment, the nucleic acid encodes the heavy chain sequence of SEQ ID NO: 7, 9, or 10, while in another embodiment, the nucleic acid encodes the light chain sequence of SEQ ID NO: 8.

[0220] In one embodiment, the nucleic acid encodes a heavy chain or heavy chain variable region comprising: an HCDR1 having the amino acid sequence of SEQ ID NO: 14, 23, 29, or 43; an HCDR2 having the amino acid sequence of SEQ ID NO: 15, 17, 19, 24, 30, or 44; and an HCDR3 having the amino acid sequence of SEQ ID NO: 16, 25, or 31.

[0221] In another embodiment, the nucleic acid encodes a light chain or light chain variable region comprising: an LCDR1 having the amino acid sequence of SEQ ID NO: 11, 20, or 27; an LCDR2 having the amino acid sequence of SEQ ID NO: 12, 18, 21, or 26; and an LCDR3 having the amino acid sequence of SEQ ID NO: 13, 22, or 28.

[0222] In another embodiment, an antibody of the invention comprises a heavy chain variable region encoded by the nucleic acid sequence of SEQ ID NO: 33 and a light chain variable region encoded by the nucleic acid sequence of SEQ ID NO: 34. An antibody of the invention may also comprise a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 48 and a light chain encoded by the nucleic acid sequence of SEQ ID NO: 49.

[0223] As described herein, production of antibodies with known sequences is routine and can be done by any method. Methods for generating antibodies such as those disclosed herein are known in the art. Once the sequence is known, the antibody can be generated according to known methods, including by following the examples provided herein.

[0224] For example, DNA molecules encoding the light chain variable region and / or heavy chain variable region can be chemically synthesized using the sequence information provided herein. The synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, for example, constant region-encoding sequences and expression control sequences, to generate conventional gene expression constructs encoding the desired antibodies. The generation of defined gene constructs is within the routine skill of those in the art. Alternatively, the sequences provided herein can be cloned from hybridomas by conventional hybridization or polymerase chain reaction (PCR) techniques using synthetic nucleic acid probes whose sequences are based on the sequence information provided herein or prior art sequence information for the heavy and light chain-encoding genes of murine antibodies in hybridoma cells.

[0225] Nucleic acids encoding the desired antibodies can be incorporated (linked) into expression vectors, which can be introduced into host cells via conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2), and myeloma cells, which do not otherwise produce IgG proteins. Transformed host cells can be grown under conditions that allow the host cells to express genes encoding the variable regions of immunoglobulin light and / or heavy chains. Thus, the present invention provides vectors containing nucleic acids encoding antibodies or fragments thereof of the present invention, as well as cells containing such nucleic acids or vectors.

[0226] Specific expression and purification conditions vary depending on the expression system used. For example, if a gene is expressed in E. coli, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies and can be recovered after cell disruption by French press or sonication. The refractile bodies are then solubilized, and the protein is refolded and cleaved by methods known in the art.

[0227] When the engineered gene is to be expressed in eukaryotic host cells, such as CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, polyA sequence, and stop codon. Optionally, the vector or gene construct may contain an enhancer and an intron. Optionally, the expression vector may contain a gene encoding all or part of the constant region. The gene constructs can be introduced into eukaryotic host cells using conventional techniques. The host cells can be constructed using the V L or V H Fragment of V L -V H Heterodimer, V H -V L or V L -V HThe host cells may express single polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be linked to a moiety having another function (e.g., cytotoxicity). In some embodiments, host cells are transfected with a single vector expressing polypeptides that express all or part of a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In some embodiments, host cells are transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In some embodiments, host cells are co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising all or part of a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide comprising all or part of a light chain or light chain variable region).

[0228] Polypeptides comprising immunoglobulin heavy or light chain variable regions can be produced by growing (culturing) host cells transfected with expression vectors encoding such variable regions under conditions that allow expression of the polypeptide. After expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, for example, affinity tags such as glutathione-S-transferase (GST) or histidine tags.

[0229] Monoclonal antibodies that bind plasma kallikrein, or antigen-binding fragments of such antibodies, can be produced by growing (culturing) host cells transfected with (a) an expression vector encoding a complete or partial immunoglobulin heavy chain and another expression vector encoding a complete or partial immunoglobulin light chain; or (b) a single expression vector encoding both chains (e.g., complete or partial heavy and light chains) under conditions that allow expression of both chains. Intact antibodies (or antigen-binding fragments) can be recovered and purified or isolated using techniques known in the art, for example, affinity tags such as protein A, protein G, glutathione-S-transferase (GST), or histidine tags. It is within the ordinary skill in the art to express heavy and light chains from a single expression vector or from two separate expression vectors.

[0230] Nucleic acid sequences encoding the antibodies described herein can be genomic or cDNA, or RNA (e.g., mRNA) encoding at least one of the variable regions described herein. A convenient alternative to using chromosomal gene fragments as a source of DNA encoding V region antigen-binding segments is the use of cDNA for the construction of chimeric immunoglobulin genes, as reported, for example, by Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987), the references of which are incorporated herein by reference in their entirety. The use of cDNA requires that the gene be combined with gene expression elements appropriate for the host cell to achieve synthesis of the desired protein. The use of cDNA sequences has an advantage over genomic sequences (which contain introns) in that cDNA sequences can be expressed in bacteria or other hosts lacking appropriate RNA splicing systems.

[0231] For example, cDNAs encoding V-region antigen-binding segments capable of detecting, binding to, or neutralizing plasma kallikrein can be provided using known methods based on the use of the amino acid sequences provided herein. Because the genetic code is degenerate, it is possible to generate cDNAs encoding specific V-region antigen-binding segments capable of detecting, binding to, or neutralizing plasma kallikrein. More than one codon can be used to encode an amino acid (Watson, et al., infra). Using the genetic code, one or more different oligonucleotides can be identified, each capable of encoding an amino acid. The probability that a particular oligonucleotide will actually constitute an actual XXX-encoding sequence can be estimated by considering unusual base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in the eukaryotic or prokaryotic cell expressing the antibody or fragment. Such "codon usage rules" are disclosed by Lathe, et al., J. Molec. Biol. 183:112 (1985). Using Lathe's "codon usage rules," a single oligonucleotide or set of oligonucleotides is identified that contains the theoretically "most likely" nucleotide sequence capable of encoding an antibody variable or constant region sequence.

[0232] V. Pharmaceutical Compositions and Administration The plasma kallikrein binding proteins disclosed herein, including antibodies and antigen-binding fragments thereof, and including the nucleic acid molecules encoding them, can be formulated, for example, as pharmaceutical compositions for administration to a subject.

[0233] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent or drug (e.g., an antibody or antigen-binding fragment described herein) with a carrier (inert or active) suitable for in vivo or in vitro diagnostic or therapeutic use.

[0234] Pharmaceutical compositions containing antibodies, fragments thereof, or nucleic acids encoding such antibodies as disclosed herein can be presented in unit dosage form and can be prepared by any suitable method. In some embodiments, to prepare pharmaceutical or sterile compositions of the anti-plasma kallikrein antibodies or other proteins provided herein, the antibodies or antigen-binding fragments thereof or other proteins provided herein are mixed with a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Useful formulations can be prepared by methods known in the pharmaceutical arts. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0235] The phrases "pharmaceutically acceptable" and "pharmacologically acceptable," as used herein, refer to compounds, molecular entities, compositions, materials, and / or dosage forms that do not produce adverse, allergic, or other undesired reactions when administered to animals or humans, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biologics standards. "Pharmaceutically acceptable" and "pharmacologically acceptable" can mean approved or licensed by a federal or state regulatory agency or a corresponding agency in a country other than the United States, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0236] As used herein, "carrier" refers to a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a pharmaceutical agent, such as an antibody or binding fragment thereof, from one organ or part of the body to another organ or part of the body.

[0237] As used herein, a "pharmaceutically acceptable excipient" refers to a substance that aids in the administration of and / or absorption by a subject of an active agent, e.g., an antibody or binding fragment thereof, and can be included in a composition of the invention without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, e.g., phosphate buffered saline, emulsions (e.g., oil / water or water / oil emulsions), lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, dextrose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and pigments. Such preparations may be sterilized and, if desired, mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or flavoring substances, which do not deleteriously react with the compositions of the present invention. For examples of excipients, see Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0238] Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, and phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose.

[0239] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), phosphate buffered saline (PBS), or dextrose solution. The carrier must be stable under the conditions of manufacture and storage and must be preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0240] Pharmaceutical preparations are preferably sterile.Sterilization can be achieved, for example, by filtration through sterile filtration membranes.If the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0241] Formulations of the antibodies provided herein may be prepared, for example, by mixing with an acceptable carrier, excipient, or stabilizer in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In some embodiments, the antibody is diluted to the appropriate concentration with sodium acetate solution, pH 5-6, and NaCl or sucrose is added for osmolality. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0242] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined, for example, by the LD 50 (50% lethal dose of the population) and ED 50 The therapeutic index (LD) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the dose (therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD). 50 / ED 50 In certain embodiments, antibodies that exhibit large therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within the ED range with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form and route of administration employed.

[0243] The antibody compositions of the present invention may be administered to a subject in accordance with, for example, Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

[0244] The method of administration can be varied, and suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, by inhalation, insufflation, topical, dermal, transdermal, or intra-arterial.

[0245] In some embodiments, the antibody or antigen-binding fragment thereof can be administered by an invasive route, such as injection. In some embodiments, the antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intraarticularly (e.g., into an arthritic joint), intratumorally, or by inhalation or aerosol delivery. Administration by non-invasive routes (e.g., orally; e.g., pill, capsule, or tablet) is also within the scope of embodiments of the present invention. Administration by intravenous infusion is one method by which an antibody may be administered to a subject.

[0246] In some embodiments, the anti-plasma kallikrein antibody or antigen-binding fragment thereof is administered in combination with at least one additional therapeutic agent, such as, but not limited to, any therapeutic agent used to treat a disorder provided herein. In some embodiments, the antibody is administered in combination with another treatment for a disorder provided herein. For example, the anti-plasma kallikrein antibodies of the invention may be administered with one or more additional therapies used to treat HAE, such as BERINERT® (C1 esterase inhibitor), FIRAZYR® (icatibant injection), KALBITOR® (ecallantide), RUCONEST® (C1 esterase inhibitor, contest alfa), CINRYZE® (C1 esterase inhibitor), HAEGARDA® (C1 esterase inhibitor subcutaneous), TAKHZYRO® (lanadelumab-flyo), ORLADEYO™ (berotralstat), galadacinab, donidalorsen, synthetic 17-α-alkylated androgens such as danazol and stanozolol, and / or antifibrinolytic agents such as aminocaproic acid. The anti-plasma kallikrein antibodies may be administered simultaneously with one or more of the aforementioned additional therapies, e.g., they may be co-administered. The anti-plasma kallikrein antibody may be administered sequentially before or after one of the aforementioned therapies, for example, on different days or weeks or even months than any of the aforementioned additional therapies.

[0247] The compositions can be administered by medical devices known in the art. The pharmaceutical compositions of the invention can be administered by injection with a hypodermic needle, including a pre-filled syringe or an autoinjector.

[0248] The pharmaceutical composition may be administered by a needleless hypodermic injection device, such as those disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0249] Pharmaceutical compositions may be administered by infusion. Examples of well-known implants and modular forms for administering pharmaceutical compositions include U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Pat. No. 4,447,233, which discloses an infusion pump for delivering drugs at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; and U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art. Infusion may also be performed by intravenously delivering an antibody in a pharmaceutically acceptable carrier, such as saline, over a defined period of time. Intravenous delivery of pharmaceutical compositions may occur through a port implanted in a subject's vein or artery.

[0250] Alternatively, the antibody may be administered in a local rather than systemic manner, for example, via injection or in a depot or sustained release formulation. Furthermore, the antibody may be administered in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antibody.

[0251] The dosing regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells within the biological matrix. Preferably, the dosing regimen delivers enough therapeutic antibody to achieve improvement in the target disease state while simultaneously minimizing undesirable side effects. Thus, the amount of biologic delivered will depend in part on the particular therapeutic antibody and the severity of the condition being treated. Guidelines for selecting appropriate doses of therapeutic antibodies are available (e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.Ltd,Oxfordshire,UK;Kresina(ed.)(1991)Monoclonal Antibodies,Cytokines and Arthritis,Marcel Dekker,New York,NY;Bach(ed.)(1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker,New York,NY;Baert,et al.(2003)New Engl.J.Med.348:601-608;Milgrom et al.(1999)New Engl.J.Med.341:1966-1973;Slamon et al.(2001)New Engl.J.Med.344:783-792;Beniaminovitz et al.(2000)New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

[0252] Determination of appropriate dosages can be made by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, dosages are initiated at amounts slightly less than the optimal dose and thereafter increased as desired or necessary for any negative side effects. The dose is gradually increased until an optimal effect is achieved. Important diagnostic criteria include, for example, evaluation of symptoms of inflammation and levels of inflammatory cytokine production. Generally, it is desirable that the biologic used be derived from the same species as the animal being treated, thereby minimizing immune response to the reagent. For human subjects, for example, chimeric antibodies, humanized antibodies, and fully human antibodies may be desirable.

[0253] The antibody or antigen-binding fragment thereof can be provided by continuous infusion, or by doses administered, for example, daily, 1 to 7 times per week, weekly, biweekly, monthly, bimonthly, or quarterly. In some embodiments, the total weekly dose is generally at least 0.05 μg / kg body weight, more typically at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg, or more (e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (2003) Cancer (See Immunol. Immunother. 52:133-144). Doses may also be provided to achieve a predetermined target concentration of antibody in the subject's serum, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, antibodies are administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, 1500, 2000, or 2500 mg / subject on a weekly, biweekly, "every four weeks," monthly, bimonthly, or quarterly basis.

[0254] VI. Therapeutic Methods and Medical Uses The plasma kallikrein binding proteins disclosed herein, including antibodies and antigen-binding fragments thereof, can be used to treat diseases in a subject.

[0255] As used herein, the term "treating" or "treatment" includes inhibiting or delaying the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such a disorder. The term further includes ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.

[0256] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody or antigen-binding fragment thereof that, when administered to a cell, tissue, or subject, alone or in combination with an additional therapeutic agent, is effective to cause a measurable improvement in one or more symptoms of a disease or condition, or in the progression of such a disease or condition, e.g., HAE or a condition for which plasma kallikrein is known to cause the observed pathology. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to cause at least partial improvement of a symptom, e.g., treatment, cure, prevention, or amelioration of a related medical condition, e.g., HAE or a condition for which plasma kallikrein is known to cause the observed pathology, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent reduces a diagnostic criterion or parameter by at least 10%, usually at least 20%, and preferably at least about 30%. More preferably, it will result in an improvement of at least 40%, and most preferably at least 50%. When a subjective scale is used to assess disease severity, an effective amount can also result in an improvement in the subjective scale. In some embodiments, an amount is a therapeutically effective amount if it is an amount that can be used to treat or prevent the symptoms of HAE or a condition in which plasma kallikrein is known to cause the observed pathology. For example, a therapeutically effective amount of an antibody or binding fragment thereof of the present invention may reduce the frequency of HAE attacks or the severity of HAE attacks, e.g., the level of edema experienced by a subject during an attack.

[0257] The term "subject" as used herein refers to any living organism, such as a mammal (e.g., rat, mouse, dog, cat, rabbit) and an animal, including, for example, a human. In one embodiment, the subject is a human. A subject may also be referred to as a patient. In some embodiments, the subject is a subject in need thereof. A subject "in need thereof" refers to a subject who has been identified as needing treatment for the condition to be treated and who is being treated with the specific intent of treating such condition. The condition can be, for example, any of the conditions described herein. "Subject" is used interchangeably with "patient."

[0258] In some embodiments, the methods involve administering a therapeutically or prophylactically effective amount of one or more antibodies or antigen-binding fragments of antibodies described herein, or a pharmaceutical composition containing a therapeutically effective amount, to a susceptible subject or a subject exhibiting a condition in which plasma kallikrein is known to have caused the observed pathology. Any active form of an antibody can be administered, including, but not limited to, scFv, Fab, and F(ab')2 fragments, and other forms of antibodies provided herein.

[0259] As used herein, "plasma kallikrein-associated pathology" refers to a condition caused by the function or abnormal expression of plasma kallikrein. These conditions include, but are not limited to, hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, and burns. In some embodiments, the pathology is hereditary angioedema. In some embodiments, the condition is bradykinin-dependent edema.

[0260] In some embodiments, the antibodies provided herein are used to treat a subject having or suspected of having a plasma kallikrein-associated disorder, which in some embodiments is selected from the group consisting of hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumor cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, and burn injury.

[0261] In some embodiments, the plasma kallikrein binding protein reduces abnormal coagulation associated with the contact activation system (i.e., the intrinsic activation system) by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., no detectable abnormal coagulation)), as measured, for example, by an APTT coagulation assay.

[0262] In some embodiments, the antibodies provided herein can be used to treat a disorder described herein, e.g., hereditary angioedema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced clotting, head trauma or peritumor cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn, or to promote wound healing. In some embodiments, the disorder is hereditary angioedema or bradykinin-dependent edema. In some embodiments, the disorder is hereditary angioedema.

[0263] In some embodiments, the antibodies provided herein are used in the manufacture of a medicament for the treatment of a disorder described herein, e.g., hereditary angioedema, diabetic macular edema, retinal edema, Factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced clotting, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn, or in the manufacture of a medicament for wound healing.

[0264] Treatment of an individual may include administering a therapeutically effective amount of an antibody or antigen-binding fragment described herein, or a pharmaceutical composition containing such an antibody or fragment. The antibody may be provided in a kit such as those provided herein. The antibody may be used or administered alone or in combination with another therapeutic, analgesic, or diagnostic agent such as those provided herein. When providing a patient with an antibody or fragment thereof capable of binding to plasma kallikrein or an antibody capable of protecting against plasma kallikrein in a recipient patient, the dosage of the administered agent will vary depending on factors such as the patient's age, weight, height, sex, general condition, medical history, etc.

[0265] Antibodies capable of treating conditions associated with plasma kallikrein activity, or antibodies used in the treatment of plasma kallikrein-associated pathologies, are intended to be provided to a subject in an amount sufficient to affect the reduction, elimination, or amelioration of plasma kallikrein-associated symptoms or pathologies. Such conditions include, but are not limited to, hereditary angioedema, bradykinin-dependent edema, hereditary angioedema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, and burns. For example, the condition is hereditary angioedema (HAE).

[0266] Thus, in some embodiments, methods are provided for treating a subject with a plasma kallikrein-mediated disorder. In some embodiments, the method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof, as provided herein. In some embodiments, the disorder is hereditary angioedema, bradykinin-dependent edema, hereditary angioedema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (F ... In some embodiments, the disorder is hereditary angioedema. In some embodiments, the disorder is bradykinin-dependent edema. In some embodiments, the disorder is bradykinin-dependent edema.

[0267] Also disclosed herein are methods for reducing or inhibiting plasma kallikrein activity in a subject in need thereof. For example, the antibodies or binding fragments thereof disclosed herein can be administered to a subject in a therapeutically effective amount to reduce or inhibit the activity of plasma kallikrein, such as human plasma kallikrein, in the subject. The subject may have a plasma kallikrein-related disorder disclosed herein, such as HAE. In some embodiments, plasma kallikrein activity may be reduced or inhibited in an amount sufficient to prevent HAE attacks in the subject for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0268] Also disclosed herein are methods for reducing or inhibiting bradykinin production or activity in a subject in need thereof. For example, the antibodies or binding fragments disclosed herein can be administered to a subject in a therapeutically effective amount to reduce bradykinin levels or activity in the subject. The subject may have a plasma kallikrein-related disorder disclosed herein, such as HAE. In some embodiments, the bradykinin production or activity may be reduced or inhibited in an amount sufficient to prevent HAE attacks in the subject for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0269] As provided herein, the antibodies or antigen-binding fragments thereof can be administered in conjunction with other therapeutic agents, which may include, for example, one or more additional therapeutic agents used to treat HAE, such as BERINERT® (C1 esterase inhibitor), FIRAZYR® (icatibant injection), KALBITOR® (ecallantide), RUCONEST® (C1 esterase inhibitor, contest alfa), CINRYZE® (C1 esterase inhibitor), HAEGARDA® (C1 esterase inhibitor subcutaneous), TAKHZYRO® (lanadelumab-flyo), ORLADEYO™ (berotralstat), galadacimab, donidalorsen, synthetic 17-alpha-alkylated androgens such as danazol and stanozolol, and / or antifibrinolytic agents such as aminocaproic acid. The additional therapeutic agent(s) can be administered simultaneously or sequentially with the antibodies disclosed herein.

[0270] Kits useful for practicing the embodiments described herein are also provided. The kits of the present invention can include a first container containing or packaged in association with an antibody described above. The kits can also include another container containing or packaged in association with a solution necessary or convenient for practicing the embodiments. The container can be made of glass, plastic, or foil and can be a vial, bottle, pouch, tube, bag, or the like. The kits can also contain written information, such as instructions for practicing the embodiments or analytical information, such as the amounts of reagents contained in the first container means. The container, along with the written information, can be placed within another container device, such as a box or bag.

[0271] In some embodiments, an antibody that binds to a plasma kallikrein protein is provided. In some embodiments, the antibody is isolated. In some embodiments, the antibody binds to a plasma kallikrein protein. It specifically binds to klein.

[0272] In some embodiments, the antibody inhibits or neutralizes the function of a plasma kallikrein protein, e.g., human plasma kallikrein. As used herein, the term "neutralize" means that the activity or function of a protein is inhibited. The inhibition can be complete or partial. In some embodiments, the activity or function of the protein is inhibited by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99%. The percent inhibition can be based on the function or activity of the protein in the absence of the antibody. In some embodiments, the antibody inhibits coagulation functions promoted by plasma kallikrein. Thus, the present invention provides methods for modulating plasma kallikrein activity, e.g., by contacting plasma kallikrein with a plasma kallikrein antibody disclosed herein or a pharmaceutical composition comprising the antibody, to modulate the activity of plasma kallikrein, such as human plasma kallikrein. In some embodiments, the method comprises administering to a subject an antibody provided herein, or a pharmaceutical composition comprising the antibody, to modulate plasma kallikrein activity in the subject.

[0273] The articles "a" and "an" are used in this disclosure to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, "an element" means one element or more than one element.

[0274] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated. The expression "and / or" in connection with more than two listed objects should be understood to have the same meaning unless otherwise understood from the context.

[0275] The phrase "at least one" should be understood to include each of the listed items following the phrase individually, and various combinations of two or more of the listed items, unless otherwise understood from context and application.

[0276] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood as open-ended and non-limiting, e.g., not excluding additional, unrecited elements or steps, unless otherwise specified or understood from context.

[0277] When the term "about" is used before a quantitative value, the present invention also includes that particular quantitative value itself unless otherwise specifically stated. As used herein, the term "about" refers to a ±5% variation from the nominal value unless otherwise indicated or inferred from the context.

[0278] As used herein, the term "in combination with" means that the agents being described can be administered to an animal or subject together as a mixture, simultaneously as a single agent, or sequentially as a single agent in any order.

[0279] For example, when molecular weights of polymers are provided rather than absolute values, the molecular weight should be understood to be average molecular weight unless otherwise stated or understood from the context.

[0280] Any and all examples or exemplary phrases herein, such as "such as" The use of "comprising" or "comprising" is merely intended to better describe the invention and does not limit the scope of the invention unless recited in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0281] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0282] Throughout this description, when compositions are described as having, including, or comprising particular ingredients, or when processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of, or consist of, the recited ingredients, and that there are processes and methods of the invention that consist essentially of, or consist of, the recited processing steps.

[0283] In this application, when an element or component is said to be included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or that the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0284] Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether expressly or implicitly stated herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, when a particular compound is referenced, unless otherwise understood from the context, that compound can be used in various embodiments of the compositions of the invention and / or methods of the invention. In other words, although embodiments have been described and illustrated within this application in a manner that permits clear and concise writing and drawing of the application, it is intended and understood that embodiments may be variously combined or separated without departing from the present teachings and invention(s). For example, it will be understood that all features described and illustrated herein may be applicable to all aspects of the invention(s) described and illustrated herein.

[0285] The subject matter will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the claims should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce essentially similar results. [Example]

[0286] Example 1: Generation of novel plasma kallikrein antibodies Five mice with prekallikrein (prekal) knockout were immunized with activated human plasma kallikrein (pkal; Enzyme Research Labs, South Bend, IN) via the heel joint route. When serum titers indicated a strong immune response, draining lymph nodes were harvested, and lymphocytes were fused with a myeloma line using electrofusion. The resulting hybridomas were seeded into thirty 384-well plates and screened by ELISA for binding to human pkal and prekal. Hybridoma lines that preferentially bound pkal were subcloned and expanded.

[0287] The monoclonal IgG was again tested for binding to pkal and prekal by ELISA and for inhibition of pkal (1 nM enzyme) enzyme activity in a fluorogenic peptide substrate assay. The hybridoma was sequenced to determine the heavy and light chain variable domains (SEQ ID NO: 1 and SEQ ID NO: 2; MAb1). As shown in Figure 3, recombinantly expressed MAb1 retained the inhibitory activity of hybridoma-derived IgG. The nucleic acid sequence of the heavy chain variable domain of MAb1 is provided as SEQ ID NO: 33, and the nucleic acid sequence of the light chain variable domain of MAb1 is provided as SEQ ID NO: 34.

[0288] The human germline sequences closest to the mouse variable domains were identified by comparison with the human germline sequences listed in IMGT (imgt.org). To expand the mouse sequences toward the human germline, nested sets of framework modifications were designed for both the heavy and light chains, expressed in pairs, and tested for binding affinity to pkal and potency in inhibiting pkal enzymatic activity. The mutant VH5_VL4 (MAb2) maintained both binding affinity and inhibitory potency and was optimally humanized (SEQ ID NO: 3, SEQ ID NO: 4). Additionally, the structurally responsible NG residue in the heavy chain CDR2 was successfully modified to NA to reduce the risk of deamidation (SEQ ID NO: 5).

[0289] The MAb1 light chain CDR sequences were diversified to construct a Fab phage display library. Using sequential selection with pkal and deselection with prekal, pkal-selective clones were enriched. Selected clones were expressed as IgG and tested for inhibition of pkal enzyme activity (Figure 4). The light chain CDR2 mutant clone VL4_213T.05 (MAb3-VL; SEQ ID NO: 6) was identified as a selective mutant with high potency for pkal inhibition. This light chain CDR2 grafted onto a humanized VL4 mutant (SEQ ID NO: 6, MAb3-VL) maintained selectivity and activity when expressed with a humanized responsible fixed heavy chain variable region (SEQ ID NO: 5, MAb3-VH). A humanized, optimized construct of MAb3 VL and VH sequences, when expressed as a full-length human IgG1 kappa format, was designated MAb4 (heavy chain: SEQ ID NO: 7, light chain: SEQ ID NO: 8).

[0290] To further optimize the therapeutic potential of MAb4, the heavy chain constant domain was modified by introducing LS (corresponding to M428L and N434S according to EU numbering) or YTE mutations (corresponding to M252Y, S254T, and T256E according to EU numbering) (SEQ ID NO: 9 and SEQ ID NO: 10, respectively). When expressed together with the light chain SEQ ID NO: 8, these humanized IgG constructs were designated MAb4-LS and MAb4-YTE, respectively.

[0291] Example 2: In vitro characterization of plasma kallikrein antibodies The in vitro potency of MAb4 and its Fc variants, MAb4-YTE and MAb4-LS, was examined in a physiologically relevant functional assay, compared with that of the commercially available monoclonal antibody DX-2930, known to inhibit plasma kallikrein and the subject of clinical trials for the treatment of HAE. Specifically, the ability of each antibody to inhibit pKal cleavage of its natural substrate, high-molecular-weight kininogen (HMWK), was examined. In these experiments, 10 or 30 nM pKal was preincubated with the inhibitor antibody for 1 h at room temperature, followed by the addition of 600 nM HMWK for 20 min. The reaction was stopped by the addition of 0.5 μM AEBSF. The concentration of bradykinin (BK) catalyzed by pKal and released from HMWK was measured using a 1 / 100 dilution of the reaction product using a commercially available ELISA kit (Enzo, Farmingdale, NY). The newly generated BK in the pKal inhibition assay was significantly higher than that in the control group (Figure 1). A comparison of the antibodies tested with the reference antibody DX-2930 is shown in Figure 5. The Mab4, Mab4-YTE, and Mab4-LS constructs exhibited more potent pKal inhibition and steeper Hill slopes than DX-2930. In this context, MAb4 and its Fc variants were potent inhibitors of HMWK cleavage by pKal, exhibiting Hill slopes of approximately 2 (see, e.g., Figure 5), consistent with the hypothesis that MAb4 and its Fc variants may be allosteric inhibitors of pKal. Furthermore, the introduction of YTE or LS modifications into the Mab4 construct did not substantially affect the potency of the construct in the inhibition assay.

[0292] The HMWK assay parameters were physiologically relevant because the high molecular weight kininogen concentrations used were those circulating in humans, and the plasma kallikrein concentrations used were within the range of those estimated in plasma from patients with hereditary angioedema (HAE) during an attack. Both Mab4-YTE and DX-2930 demonstrated dose-dependent inhibition of bradykinin production, indicating a decrease in plasma kallikrein activity, and as shown in Figure 5, Mab4-YTE was more potent than DX-2930 in inhibiting plasma kallikrein activity and bradykinin release.

[0293] IC of antibodies to provide an indication of relative potency 90 The IC90 values ​​were also determined. In HAE, the therapeutically relevant level of inhibition to prevent HAE attacks is thought to be approximately 90% inhibition of plasma kallikrein. This corresponds to a measurement called IC90, which is the concentration of a drug that produces a 90% inhibitory effect. In the HMWK functional assay, the IC90 values ​​were determined as shown in Figure 5. 90 The IC value is the concentration of antibody that results in 90% inhibition of kallikrein activity, which is considered to be a therapeutically relevant level of inhibition. 90 The IC value of MAb4 and its Fc variants was 300 nM, whereas the IC 90 The value is 30 nM. Therefore, this in vitro HMWK functional assay demonstrated that MAb4-YTE has approximately 10-fold higher potency than DX-2930. Clinical trials with DX-2930 have shown that steady-state plasma levels of 200-300 nM DX-2930 are required to optimally reduce HAE attack rates and maximize attack-free duration, so the results of this assay are consistent with the clinical efficacy observed with DX-2930.

[0294] The kinetic properties and selectivity of the lead pkal antibodies were estimated by SPR at 37°C, pH 7.4, or pH 6.0. MAb4-LS (SEQ ID NOS: 9 and 8) and MAb4-YTE (SEQ ID NOS: 10 and 8) exhibited low single-digit nanomolar affinity for pkal and substantial selectivity for prekal (Figure 6). Indeed, MAb4-LS and MAb4-YTE, as well as DX-2930, have over 1000-fold higher affinity for plasma kallikrein than for prekallikrein (Figure 6). The K of MAb4-YTE for human plasma kallikrein was D was 1.1 nM at pH 7.4 and 0.9 nM at pH 6.0, while the K DThe K values ​​were 2.2 nM at pH 7.4 and 1.2 nM at pH 6.0. These antibodies did not show significant pH dependence in target binding to human plasma kallikrein, particularly within the pH range of 6.0 to 7.4. In contrast, the reference antibody DX-2930 had K values ​​of 18 nM at pH 7.4 and 236 nM at pH 6.0. D (Figure 6). As a result, the Mab-YTE and Mab4-LS antibodies have stronger binding affinities (greater than 5-fold, e.g., 8-16-fold) for plasma kallikrein than the reference antibody DX-2930, which is consistent with the approximately 10-fold greater potency of Mab4-YTE compared to DX-2930 in functional plasma kallikrein inhibition assays. Furthermore, the DX-2930 antibody exhibited considerable pH sensitivity under the same conditions. For example, the DX-2930 antibody exhibited a K D Table 7 shows the K of MAb4-YTE and DX-2930 based on SPR binding as described above at pH 7.4 and 37°C. a , K. d and K. D Indicates the value of [Table 7]

[0295] In summary, the data presented in Figure 6 demonstrate that antibodies Mab4-YTE or Mab4-LS possess the following characteristics: (a) K is virtually pH independent between pH 6.0 and 7.4 D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA and 0.02% Tween-20); (b) K between 0.1 nM and 5 nM for human plasma kallikrein D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ , as determined by SPR at 37 °C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and (c) K between 0.1 nM and 5 nM for human plasma kallikrein D , and a K between 250 nM and 2,000 nM for human prekallikrein. D (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca 2+ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).

[0296] hFcRn binding of plasma kallikrein antibodies with Fc mutations (MAb4-LS and MAb4-YTE) was also evaluated. Two dilution series of MAb4-LS and MAb4-YTE were prepared: 2000 nM to 31.25 nM at pH 6.0 in PBS containing 0.05% P20 (polysorbate 20), and 2000 nM to 500 nM at pH 7.4. IgG1 was used as a control antibody. Antibody samples were evaluated for FcRn binding via SPR using a Biacore T200. The Biacore chip was CM5 (Sino Biological, catalog no. CT009-H08H) coupled to human FcRn. The running buffer used was PBS 0.05% P20, pH 6, or pH 7.4. hFcRn binding at pH 6.0 is shown in Table 8. Both MAb4-LS and MAb4-YTE showed a significant improvement in serotonin activity compared to unmodified MAb4 (2.29 × 10 1 K d compared to 3.10 x 10 2 and 2.77 × 10 2 K d ) and DX-2930 (1.67 x 10 1 K dThe pH-dependent hFcRn binding was enhanced by a decreased off-rate (10-fold slower) at pH 6.0 compared to MAb4 and DX-2930. At pH 7.4, there was no evidence of binding for all three antibodies, suggesting that FcRn binding of MAb4, MAb4-LS, and MAb4-YTE is pH-dependent (data not shown). Thus, without wishing to be bound by theory, both the MAb4-LS and MAb4-YTE antibodies may bind better to FcRn in endosomes (pH 6) and weaker to FcRn in the blood (pH 7.4) compared to the parent MAb4 antibody, allowing the antibodies to recirculate into the blood via the IgG-FcRn recycling pathway, thereby extending the circulating half-lives of MAb4-LS and MAb4-YTE by approximately 3-4 fold compared to MAb4 and DX-2930 (see Table 10 in Example 3). [Table 8]

[0297] Additionally, MAb4-YTE was shown to potently inhibit plasma kallikrein enzyme activity in humans as well as cynomolgus monkeys, rats, and rabbits, as assessed using Pro-Phe-Arg-AMC (PFR-AMC), a small fluorescent substrate of pKal. MAb4-YTE antibody was incubated with 1 nM pKal from each species at the indicated concentrations, as shown in the graph in Figure 7, in triplicate wells for 1 hour at 37°C. After addition of 10 μM fluorogenic reporter substrate PFR-AMC, pKal activity was measured for 60 minutes using a fluorescent plate reader in kinetic mode. Linear regression analysis of the first 40 minutes of substrate release data from each reaction well was performed to obtain the initial reaction rate (RFU / min) for each inhibitor concentration. Initial reaction rates were fitted (four-parameter fit) to obtain the IC 50 Values ​​were obtained and normalized to obtain the percentage of enzyme activity within the upper and lower limits of the fitted curve. IC 50The data are shown in Table 9. The level of pKal activity plotted against the concentration of MAb4-YTE in nM is shown in Figure 7. The data demonstrate that MAb4-YTE is a potent inhibitor of pKal not only in humans, but also in monkeys, rabbits, and rats. [Table 9]

[0298] Example 3: In vivo pharmacokinetic characterization of plasma kallikrein antibodies MAb4 (SEQ ID NOS: 7 and 8), MAb4-LS (SEQ ID NOS: 9 and 8), and MAb4-YTE (SEQ ID NOS: 10 and 8) were further characterized in pharmacokinetic studies in cynomolgus monkeys in parallel with another plasma kallikrein antibody, DX-2930. These antibodies were tested simultaneously but independently, rather than directly compared. N = 18 male cynomolgus monkeys weighing 3.0–4.0 kg, either LM-naive or naive, were purchased from Hainan Jingang Biotech Co., Ltd. Antibodies were formulated in 20 mM histidine, 150 mM NaCl, 0.01% Tween 80, pH 6.5. For these studies, all antibodies were administered to male cynomolgus monkeys (N = 3) at 5 mg / kg via IV infusion over 10 minutes. At each time point, blood was collected from the cephalic and saphenous veins into pre-chilled citrate tubes. Samples were collected weekly for 42 days (DX-2930 and MAb4) or 84 days (MAb4-YTE and MAb4-LS). Blood samples were placed on wet ice and centrifuged at 4°C to obtain plasma within 15 minutes of sample collection. Plasma samples were stored at approximately -70°C. The presence of each anti-plasma kallikrein antibody in the plasma samples was determined by ELISA using plates coated with anti-human antibodies as capture agents and human IgG heavy and light chain monkey adsorbent antibodies (Southern Biotech, Cat#: 2049-01 and Bethyl, Cat# A80-319P) as detection reagents. ELISA plates were read using a SpectraMax M2.

[0299] MAb4 and DX-2930 (MAb4 half-life shown in Figure 8 and DX-2930 half-life shown in Figure 9) had terminal half-lives of 10–12 days, consistent with those reported in the USFDA review document and publications for DX-2930 in nonhuman primates. In contrast, MAb4-LS exhibited a terminal half-life of approximately 23 days, and MAb4-YTE exhibited a terminal half-life of approximately 34 days (see Figure 8). The terminal half-life of MAb4-YTE is approximately 3–4 times longer than that observed for DX-2930 and MAb4. These results demonstrate the superiority of the MAb4 antibody with its extended half-life. This result was surprising, as the introduction of LS or YTE mutations into other plasma kallikrein antibodies was found not to increase half-life (data not shown). Additionally, the increased inhibitory potency and half-life of the newly generated antibodies indicate that effective doses of MAb4-YTE or MAb4-LS may be maintained in vivo for extended periods compared to DX-2930. Figure 9 shows the PK half-lives of MAb4-YTE (middle panel) and DX-2930 (top panel) compared to the IC90 of each antibody's pKal. (The data from the top and middle panels are overlaid on each other in the bottom panel to show the plasma levels of both MAb4-YTE and DX-2930.) While DX-2930 plasma levels fall below the predicted minimum therapeutic concentration (IC90) by approximately day 10, MAb4-YTE remains above the predicted minimum therapeutic concentration (IC90) for over 84 days. Table 10 provides a summary of PK data from cynomolgus monkey studies. Without being bound by theory, the increased pH-dependent FcRn binding of the Fc-modified antibodies described in Example 2 slowed clearance and extended half-life in cynomolgus monkeys. [Table 10]

[0300] Evaluation of the in vitro efficacy data and half-life results from the non-human primate studies suggests the potential duration of efficacy of the antibody. Specifically, the plasma concentration of the antibody measured at each time point from the cynomolgus monkey studies can be used to predict the expected level of plasma kallikrein inhibition at that time point based on the plasma kallikrein inhibition observed at that concentration of antibody in the in vitro efficacy assay.

[0301] Figure 10 provides a predictive model based on pKal inhibition measured in the in vitro functional assay and in vivo PK data comparing the in vivo percent pKal inhibition by Mab4-YTE and DX-2930 based on plasma concentrations and HMWK assay data from the cynomolgus monkey PK study. In the cynomolgus monkey study, DX-2930 plasma levels fell below the minimum therapeutic concentration or IC90 predicted by the in vitro potency assay by approximately day 10, and by day 20, the predicted in vitro potency assay reached a level resulting in approximately 50% inhibition of plasma kallikrein. In contrast, in the cynomolgus monkey study, plasma levels of MAb4-YTE remained above the IC90 predicted by the in vitro potency assay for the entire duration of the study, 84 days.

[0302] These preclinical data suggest that at equivalent doses, MAb4-YTE has a half-life of several months in humans, resulting in a significantly longer duration of action than DX-2930. This may potentially enable even lower doses of MAb4-YTE with a longer duration of action than DX-2930. Therefore, MAb4-YTE may be an effective preventative therapy for HAE patients by inhibiting the pathological activity of plasma kallikrein for a long period of time. In addition, the antibody may be administered less frequently while maintaining therapeutically effective plasma levels necessary to prevent HAE attacks. Therefore, antibodies such as MAb4-YTE or MAb4-LS may be administered at less frequent intervals, for example, every three months, or even longer. This represents a significant advantage over currently available HAE treatments such as DX-2930. Thus, MAb4-YTE may combine the benefits of less frequent administration, lower dosages, and long-term suppression of HAE attacks.

[0303] Example 4. In vitro characterization of off-target binding of plasma kallikrein antibodies A membrane proteome array assay was performed to identify nonspecific binding of MAb4, and no off-target binding was observed for MAb4 (data not shown).

[0304] The antibody was further examined to determine whether it contained effector function and was found to have no effector function (data not shown).

[0305] The specificity of the antibodies was also determined by assessing whether they bound to unrelated serine proteases. The antibodies were screened against a panel of serine proteases (approximately 20), and no significant cross-reactive binding was observed (data not shown).

[0306] To determine whether MAb4 exhibits cross-reactivity, a trypsin inhibition assay was developed and performed. 0.25 nM trypsin, human pancreas (Cat# 16-19-032000, Athens Research and Technology Inc.), and a dilution series of MAb4-YTE starting at 1 μM were incubated with PFR-AMC (fluorogenic substrate, Bachem, Cat# 4004023) in buffer for 2 hours. Fluorescence intensity was measured every 5 minutes. Gabexate mesylate was used as a positive control, with a dilution series starting at 1 μM. Each assay was performed in duplicate. The results are presented in Figure 11 and Table 11. MAb4-YTE did not inhibit trypsin activity, and an IC50 could not be calculated. In contrast, antibody DX-2930 inhibited trypsin activity with an IC50 (243 nM-376 nM), which is within the potency range of DX-2930, which inhibits the functional activity of plasma kallikrein (approximately 300 nM). Thus, Mab4-YTE exhibited a superior protease selectivity profile compared to DX-2930. [Table 11]

[0307] Additional serine protease inhibition assays were developed and implemented to determine whether MAb4 exhibited cross-reactivity. MAb4-YTE and DX-2930 were tested in duplicate in 10-dose assays with 3-fold serial dilutions starting at 1 μM against 16 proteases. Control compounds were tested in 10-dose assays with 3-fold serial dilutions starting at 10 μM or 100 μM (tPA, matriptase 2, kallikrein 5, kallikrein 2, kallikrein 1, granzyme B, and APC). Protease activity was monitored as a time course measurement of the increase in fluorescent signal from a fluorescently labeled peptide substrate, and the initial linear portion of the signal slope (signals / min) was analyzed. Percent enzyme activity for titration was calculated by considering the vehicle control in the presence of PBS buffer as 100% activity. IC50 values ​​for proteinase inhibition by MAb4-YTE and DX-2930 are summarized in Table 12 below. [Table 12]

[0308] Empty cells indicate no inhibitory effect or compound activity that could not be fitted with an IC50 curve. IC50 values ​​greater than 1 μM were estimated based on the best curve fit available.

[0309] The IC50 of MAb4-YTE against the serine proteases tested in this assay could not be calculated. IC50 values ​​of greater than 1 μM (1.00E-06) were estimated for DX-2930 against hepsin and thrombin A. The differences in the serine protease inhibition profiles were consistent with the increased protease selectivity of MAb4-YTE compared to DX-2930.

[0310] In summary, MAb4-YTE did not inhibit trypsin activity or the serine proteases tested, and therefore does not exhibit off-target inhibitory activity. In contrast, the DX-2930 plasma kallikrein antibody exhibited significant trypsin inhibitory activity as well as off-target inhibitory activity for hepsin and thrombin A, demonstrating the superior target specificity and selectivity of MAb4-YTE due to its lack of off-target activity.

[0311] Example 5: Characterization of the epitope of plasma kallikrein antibodies Competitive binding assay

[0312] SPR-based competition experiments (Biosensor Tools, Salt Lake City, UT) show that MAb4 binds to an epitope distinct from that of DX-2930, which has been shown to be a competitive inhibitor binding to the active site (Kenniston, 2014) (Figure 12). DX-2930, MAb4-LS, and MAb4-YTE were amine-coupled to a Xantec 30M sensor chip using standard NHS / ECD coupling with 10 mM NaAc, pH 5.0. For capture and stacking studies, the running buffer contained HBS (pH 7.4) containing 1.3 mM Ca++ with 1 mg / ml BSA and 0.02% p20. Human plasma kallikrein was captured on various antibody surfaces (times 0–180 s, 1 μM), and then each surface-bound complex was probed with 1 μM of each mAb.

[0313] When each antibody was tested against itself, no binding was observed (Figure 12). However, both MAb4-LS and MAb4-YTE bound to kallikrein captured by the DX-2930 antibody surface (Figure 12). In addition, DX-2930 bound to kallikrein captured by both the MAb4-LS and MAb4-YTE antibodies (Figure 12). These results indicate that MAb4-LS and MAb4-YTE bind to epitopes distinct from those of DX-2930.

[0314] Cross-linking and mass fingerprinting of pKal-antibody complexes

[0315] Human pKal was first characterized by peptide mass fingerprinting. Human plasma kallikrein protein was subjected to proteolysis with trypsin, chymotrypsin, Asp-N, elastase, and thermolysin, followed by nLC-QExactive+Orbitrap MS / MS analysis. An nLC Ultimate 3000-RSLC system coupled to a QExactive+ mass spectrometer (Thermo Scientific) was used. 7 μM human pKal was incubated with 1 μL of DSS d0 / d12 (2 mg / mL; DMF) and then incubated at room temperature for 180 minutes. A control sample was prepared without the addition of cross-linking reagent. The sample was incubated at room temperature for 180 minutes. After incubation, the cross-linking reaction was stopped by adding 1 μL of ammonium bicarbonate (final concentration 20 mM) and incubated at room temperature for 60 minutes. Next, both tubes were dried using a speed vac and then resuspended in 8 M urea (10 μL). After mixing, DTT (1 μL, 500 mM) was added to both tubes. The mixtures were then incubated at 37°C for 60 minutes. After incubation, iodoacetamide (1 μL, 1 M) was added to each tube, which was then incubated for an additional 60 minutes at room temperature in the dark. After incubation, 100 μL of proteolysis buffer was added to both tubes. Trypsin buffer contained 50 mM Ambic, pH 8.5, 5% acetonitrile; chymotrypsin buffer contained 100 mM Tris-HCl, 10 mM CaCl2, pH 7.8; ASP-N buffer contained 50 mM phosphate buffer, pH 7.8; elastase buffer contained 50 mM Tris-HCl, pH 8.0; and thermolysin buffer contained 50 mM Tris-HCl, 0.5 mM CaCl2, pH 9.0.

[0316] Trypsin proteolysis

[0317] 100 μL of reduced / alkylated human pKal was mixed with 1 μL of trypsin (Promega) at a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37°C.

[0318] Chymotrypsin proteolysis

[0319] 100 μL of reduced / alkylated human pKal was mixed with 0.5 μL of chymotrypsin (Promega) at a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 25°C.

[0320] ASP-N proteolysis

[0321] 100 μL of reduced / alkylated human pKal was mixed with 0.5 μL of ASP-N (Promega) at a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 37°C.

[0322] Elastase proteolysis

[0323] 100 μL of reduced / alkylated human pKal was mixed with 1 μL of elastase (Promega) at a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37°C.

[0324] Thermolysin proteolysis

[0325] 100 μL of reduced / alkylated human pKal was mixed with 2 μL of thermolysin (Promega) at a ratio of 1 / 50. The proteolysis mixture was incubated at 70° C. overnight.

[0326] After digestion, a final 1% formic acid was added to the solution. After proteolysis, 1 μL of the peptide solution generated by proteolysis was loaded onto a nano-liquid chromatography system (Ultimate 3000-RSLC). Quadrupole-Orbitrap MS analysis was performed according to the manufacturer's instructions. Based on the proteolysis MS results, overlapping peptide mappings of trypsin, chymotrypsin, ASP-N, elastase, and thermolysin were obtained, covering 99.05% of the human pKal sequence.

[0327] Next, after incubation with deuterated cross-linkers, the epitopes of the human pKal / MAb4-YTE and human pKal / DX-2930 complexes were determined as described above. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-Quadrupole-Orbitrap MS), and the generated data were analyzed using XQuest and Stavrox software.

[0328] 20 μL of the prepared mixture of human pKal / MAb4-YTE (1.4 μM and 0.8 μM) and human pKal / DX-2930 (1.4 μM and 0.4 μM) was mixed with 2 μL of DSS d0 / d12 (2 mg / mL; DMF) and incubated at room temperature for 180 minutes. After incubation, the reaction was stopped by adding 1 μL of ammonium bicarbonate (final concentration 20 mM), followed by incubation at room temperature for 1 hour. The solution was then dried using a speed vac, after which 20 μL of an 8 M urea suspension in HO was added. After mixing, 2 μL of DTT (500 mM) was added to the solution. The mixture was then incubated at 37°C for 1 hour. After incubation, 2 μL of iodoacetamide (1 M) was added, followed by incubation at room temperature in the dark for 1 hour. After incubation, 80 μL of proteolysis buffer was added. The trypsin buffer contained 50 mM Ambic, pH 8.5, and 5% acetonitrile; the chymotrypsin buffer contained 100 mM Tris-HCl, 10 mM CaCl, pH 7.8; the ASP-N buffer contained 50 mM phosphate buffer, pH 7.8; the elastase buffer contained 50 mM Tris-HCl, pH 8.0; and the thermolysin buffer contained 50 mM Tris-HCl, 0.5 mM CaCl, pH 9.0. Proteolysis with trypsin, chymotrypsin, ASP-N, elastase, and thermolysin was carried out using the conditions described above for pKal proteins. Cross-linked peptides were analyzed using Xquest version 2.0 and Stavrox 3.6 software.

[0329] The molecular interface between human PKAL and MAb4-YTE was characterized using chemical cross-linking, high-mass MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry. After proteolysis of the protein complex human-PKAL / MAb4-YTE containing deuterated d0d12 with trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-orbitrap MS / MS analysis detected 10 cross-linked peptides between human-PKAL and MAb4-YTE.

[0330] Analysis of the cross-linking data indicated that the MAB4-YTE interaction involves the following amino acids on human pKal: 550, 551, 585, 591, 597, 617, and 625. Figure 13 provides a diagram of the peptide interaction between human pKal and MAB4-YTE. The human pKal sequence is shown below, with the cross-linking epitope of MAB4-YTE indicated at residues 550, 551, 585, 597, 617, and 625. Figures 14A-J show ribbon / surface and ribbon models of the antibody epitope site on human pKal. The epitope site of the MAb4-YTE antibody was modeled on PDB structure 6O1G, with UNIPROT P03952 as the reference sequence. The amino acids shown in dark color in the model correspond to residues 550-551 (KR (SEQ ID NO: 37)), residues 585-597 (KHNGMWRLVGITS (SEQ ID NO: 38)), and residues 617-625 (YMDWILEKT (SEQ ID NO: 39)) of the human pKal sequence.

[0331] Analysis of the cross-linking data indicated that the DX-2930 interaction involves the following amino acids on human pKal: 434, 446, 475, 482, 555, 558, and 560. Figure 15 provides a diagram of the peptide interaction between human pKal and DX-2930. The human pKal sequence is shown below, with the DX-2930 cross-linking epitope indicated at residues 434, 446, 475, 482, 555, 558, and 560. Figures 16A-J show ribbon / surface and ribbon models of the antibody epitope site on human pKal. The epitope site for the DX-2930 antibody was modeled after PDB structure 6O1G. The amino acids shown in dark color in the model correspond to residues 434-446 (HCFDGLPLQDVWR (SEQ ID NO: 40)), residues 475-482 (YKVSEGNH (SEQ ID NO: 41)), and residues 555-560 (YKITQR (SEQ ID NO: 42)) of the human pKal sequence. Figure 17 provides a map showing the residues of human pKal cross-linked by antibody DX-2930 compared to antibody MAb4-YTE.

[0332] Cross-linking mass spectrometry data confirm that MAB4-YTE binds to a different epitope on human pKal compared to DX-2930. Without wishing to be bound by theory, a different binding site on plasma kallikrein for Mab4-YTE compared to DX-2930 may explain the improved protease selectivity profile, lack of pH sensitivity of plasma kallikrein binding, and the overall superior profile of Mab4-YTE compared to DX-2930.

[0333] Incorporation by Reference The entire disclosure of each patent and scientific document referred to in this specification is hereby incorporated by reference for all purposes.

[0334] equivalent The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the embodiments and the appended claims.

[0335] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8]

Claims

1. An antibody or antigen-binding fragment thereof that binds to plasma kallikrein, comprising: a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and a light chain variable region (VL) sequence comprising LCDR1, LCDR2, and LCDR3; a. (i) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, 23, 29, or 43; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, 19, 24, 30, or 44; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, 25, or 31; and (ii) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, 20, or 27; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, 18, 21, or 26; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, 22, or 28; b. (i) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, or 19; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; and (ii) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; c. (i) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:23; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:24; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; and (ii) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21 or 26; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22; d. (i) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:29; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:30; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:31; and (ii) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:27; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:21 or 26; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:28; or e. (i) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:43; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:44; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; and (ii) the antibody or antigen-binding fragment thereof, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

28.

2. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, or 19; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; and The antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or 18; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

13.

3. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

13. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising an amino acid sequence.

4. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; and The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

13.

5. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; and The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

13.

6. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:23; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:24; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; and The antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

22.

7. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:29; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:30; and the HCDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO:31; and The antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27; the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

28.

8. (i) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 44; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25; and (ii) The antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

28.

9. An antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3; a. the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 with up to 1, 2, or 3 amino acid substitutions relative to the amino acid sequence; the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 17, or 19 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions relative to the amino acid sequence; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions relative to the amino acid sequence; and b. the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions relative to the amino acid sequence; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 18 with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13 or 14 with up to 1, 2, 3, or 4 amino acid substitutions relative to the amino acid sequence; The antibody or antigen-binding fragment thereof, comprising the amino acid sequence set forth in SEQ ID NO: 13 with 1, 2, 3, or 4 amino acid substitutions.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contains at most one amino acid substitution.

11. 11. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein at most one CDR selected from HCDR1, HCDR2, or HCDR3 contains one substitution.

12. The antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein at most one CDR selected from LCDR1, LCDR2, and / or LCDR3 comprises one substitution.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 4, or 6.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or 14, wherein the VL region comprises the amino acid sequence shown in SEQ ID NO:

6.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 1, 3 or 5.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO:

5.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 5, and the VL region comprises the amino acid sequence shown in SEQ ID NO:

6.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 5, and the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 4, or 6.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 4, or 6.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 3, and the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 4, or 6.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, and the VL region comprises the amino acid sequence shown in SEQ ID NO:

2.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, and the VL region comprises the amino acid sequence shown in SEQ ID NO:

4.

23. the VH region comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5, and the V The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the L region comprises the amino acid sequence shown in SEQ ID NO:

6.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO:

8.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or 24, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 7, 9 or 10.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 24 or 25, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:

10.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 24 or 25, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:

9.

28. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 24 or 25, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 10, 9 or 7, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

29. 29. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 24 to 26, or 28, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

30. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 24, 25, 27 or 28, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:9 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

31. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 24, 25 or 28, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VH region comprises a sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5.

33. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the VL region comprises a sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6.

34. 34. The antibody or antigen-binding fragment thereof of claim 32 or 33, wherein the VL region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identical to the sequence set forth in SEQ ID NO: 6, 4, or 2, and the VH region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identical to the sequence set forth in SEQ ID NO: 1, 3, or 5.

35. the heavy chain has at least 80%, 85%, or 90% identical sequence to the sequence set forth in SEQ ID NO: 7, 9, or 10 13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, comprising a sequence having a percent homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

36. 36. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or 35, wherein the light chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the sequence set forth in SEQ ID NO:

8.

37. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 36, wherein the CDR sequences are inserted between human or humanized framework sequences.

38. The antibody of any one of claims 1 to 37, wherein the antibody is an intact antibody or a full-length antibody.

39. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 38, wherein the plasma kallikrein is human plasma kallikrein having the amino acid sequence shown in SEQ ID NO:

32.

40. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, wherein the antibody or antigen-binding fragment thereof binds to at least one of the following residues of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO: 32: K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625.

41. The antibody or antigen-binding fragment thereof according to claims 1 to 40, wherein the antibody or antigen-binding fragment thereof binds to at least one, two, three, four, five or six of the following residues of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO: 32: K550, R551, K585, S597, Y617, or T625.

42. The antibody or antigen-binding fragment thereof has a denaturing activity of about 1, 2, 3, 4, or 5 x 10 as measured by a surface plasmon resonance (SPR) assay. －９ K below M Ｄ 42. The method of any one of claims 1 to 41, wherein the antibody binds to human plasma kallikrein (SEQ ID NO: 32) at the

43. The antibody has the following characteristics: a. K that is virtually pH independent between pH 6.0 and 7.4 Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween 20) b. K between 0.1 nM and 5 nM for human plasma kallikrein Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and c. K between 0.1 nM and 5 nM for human plasma kallikrein Ｄ and K between 250 nM and 2,000 nM for human prekallikrein Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.

4.

44. 44. An isolated antibody or binding fragment thereof that competes with the antibody of any one of claims 1 to 43 for binding to plasma kallikrein.

45. An isolated antibody or binding fragment thereof, which binds to the same epitope on human plasma kallikrein as the antibody of any one of claims 1 to 44.

46. A recombinant antibody or antigen-binding fragment thereof that binds to plasma kallikrein, said antibody or antigen-binding fragment optionally comprising: a. a serum half-life of at least 20 days; b. K that is virtually pH independent between pH 6.0 and 7.4 Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20); c. K between 0.1 nM and 5 nM for human plasma kallikrein Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and d. K between 0.1 nM and 5 nM for human plasma kallikrein Ｄ and K between 250 nM and 2,000 nM for human prekallikrein Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4).

47. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to one or more of residues K550, R551, K585, H586, N587, G588, M589, W590, R591, L592, V593, G594, I595, T596, S597, Y617, M618, D619, W620, I621, L622, E623, K624, or T625 of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO:

32.

48. 48. The antibody or antigen-binding fragment thereof of claim 47, which binds to one or more of residues K550, R551, K585, S597, Y617, or T625 of human plasma kallikrein having the amino acid sequence set forth in SEQ ID NO:

32.

49. The antibody or antigen-binding fragment thereof has the following characteristics: a. K that is virtually pH independent between pH 6.0 and 7.4 Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20); b. K between 0.1 nM and 5 nM for human plasma kallikrein Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ , as determined by SPR at 37°C in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4); and c. K between 0.1 nM and 5 nM for human plasma kallikrein Ｄ and K between 250 nM and 2,000 nM for human prekallikrein Ｄ (e.g., 50 mM HEPES, 150 mM NaCl, 0.1 or 1.3 mM Ca ２＋ 37 in a buffer containing 1 mg / mL BSA, 0.02% Tween-20, pH 6.0 or 7.4 49. The antibody or antigen-binding fragment thereof of claim 47 or 48, having one or more of the following:

50. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 49, wherein the antibody is a monoclonal antibody.

51. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 50, wherein the antibody is a humanized antibody.

52. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, wherein the antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE and IgM.

53. 53. The antibody or antigen-binding fragment thereof of any one of claims 1 to 52, wherein the antibody comprises a heavy chain constant region of the class IgG and a subclass selected from IgG1, IgG2, IgG3, and IgG4.

54. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 53, wherein the antibody comprises an IgG1 heavy chain constant region.

55. 55. The antibody or antigen-binding fragment thereof of claim 54, wherein the IgG1 heavy chain constant region comprises a wild-type (WT) sequence.

56. 55. The antibody or antigen-binding fragment thereof of claim 54, wherein the IgG1 heavy chain constant region comprises the mutations M252Y, S254T, and T256E, or the mutations M428L and N434S, numbered according to the EU numbering catalogue.

57. 55. The antibody or antigen-binding fragment thereof of claim 54, wherein the IgG1 heavy chain constant region comprises the following mutations: M252Y, S254T, and T256E, numbered according to the EU numbering catalogue.

58. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 57, wherein the heavy chain variable region and the light chain variable region are not linked by a linker.

59. 58. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 57, wherein the antibody-binding fragment is an scFv antibody.

60. 60. The antibody or antigen-binding fragment thereof of any one of claims 1 to 57 or 59, wherein the heavy chain variable region and the light chain variable region are linked by a peptide linker.

61. The peptide linker is (GGGGS) ｎ (SEQ ID NO: 35); (GGGGA) ｎ (SEQ ID NO: 36), or any combination thereof, wherein each n is independently 1 to 5.

62. 62. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain or a heavy chain variable region (VH) of any one of claims 1 to 61.

63. 62. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain or a light chain variable region (VL) of any one of claims 1 to 61.

64. 63. An expression vector comprising the nucleic acid of claim 62.

65. 64. An expression vector comprising the nucleic acid of claim 63.

66. 66. The expression vector of claim 65, further comprising the nucleic acid of claim 62.

67. 66. A host cell comprising the expression vector of claim 64 and the expression vector of claim 65.

68. 67. A host cell comprising the expression vector of claim 66.

69. 1. A method for producing an antibody or antigen-binding fragment thereof that binds to plasma kallikrein, said method comprising: (a) growing the host cell of claim 67 or 68 under conditions such that the host cell expresses a polypeptide or polypeptides comprising a heavy chain or heavy chain variable region and a light chain variable region or light chain, thereby producing the antibody or the antigen-binding fragment of the antibody; (b) purifying the antibody or the antigen-binding fragment of the antibody.

70. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 61, or the nucleic acid of any one of claims 62 to 63.

71. 71. The pharmaceutical composition of claim 70, wherein the pharmaceutical composition is an injectable pharmaceutical composition.

72. 62. A method of treating a subject having a disease or disorder selected from hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn injury, the method comprising administering an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 61, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof.

73. 62. A method of treating a plasma kallikrein-mediated disorder in a subject, the method comprising administering to the subject an effective amount of an antibody of any one of claims 1 to 61, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof.

74. 74. The method of claim 73, wherein the disorder is hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced clotting, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn injury.

75. Hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, 612. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 611, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, for use in the treatment of a disease or disorder selected from postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn injury.

76. 62. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 61, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for use in the manufacture of a medicament for the treatment of a disease or disorder selected from hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn.

77. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 61, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for the treatment of a disease or disorder selected from hereditary angioedema, bradykinin-dependent edema, diabetic macular edema, retinal edema, factor XII-associated cold autoinflammatory syndrome (FACAS), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis / degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device- or stent-induced coagulation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, or burn injury.

78. 62. A method of reducing bradykinin levels or inhibiting bradykinin production in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or binding fragment thereof of any one of claims 1 to 61, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

79. 62. A method of reducing or inhibiting plasma kallikrein activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or binding fragment thereof of any one of claims 1 to 61, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

80. 80. The method of any one of claims 72 to 79, wherein the disease or disorder is hereditary angioedema.

81. 80. The method of any one of claims 72 to 79, wherein the disease or disorder is bradykinin-dependent edema.

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