Plasma kallikrein binding protein

Abstract

To provide a plasma kallikrein binding protein and use method thereof.SOLUTION: A plasma kallikrein binding protein binds to or competes for binding with a protein (for example, epi-Kal2) and / or a same epitope as a low molecule (AEBSF, for example), and does not bind to a pre-plasma kallikrein. The plasma kallikrein binding protein may have a full length (for example, IgG, IgM, IgA, IgD and IgE) or may include only an antigen binding fragment (for example, Fab, F(ab')2 or scFv fragment). The binding protein may include two heavy chain immunoglobulins and two light chain immunoglobulins or may have a single chain antibody.SELECTED DRAWING: None

Description

[Background technology]

[0001] Plasma kallikrein is a type of serine protease. Prekallikrein is the precursor of plasma kallikrein. Summary of the Invention

[0002] Plasma kallikrein is a serine protease component of the contact system and a potential drug target for various inflammatory, cardiovascular, infectious (sepsis) and neoplastic diseases (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). The contact system is activated by factor XIIa upon contact with foreign or negatively charged surfaces or by prolylcarboxypeptidase on the endothelial cell surface (Figure 1) (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 potentiates inflammation through the production of the inflammatory nonapeptide bradykinin. As the major kininogenase in the circulation, plasma kallikrein is primarily responsible for the generation of bradykinin in the vasculature. Hereditary angioedema (HAE) is caused by a genetic deficiency in the C1 inhibitor protein (C1-INH), the major natural plasma kallikrein inhibitor. Patients with HAE suffer from acute attacks of painful edema, often precipitated by unknown triggers (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). Drug use and genetic studies in animal models suggest that the plasma kallikrein-kinin system (plasma KKS) is involved in various diseases.

[0003] Plasma kallikrein binding proteins (e.g., antibodies, e.g., inhibitory antibodies) are useful as therapeutic agents for various diseases and conditions, e.g., diseases and conditions involving plasma kallikrein activity, due to their high potency and specificity and long blood residence time. High potency translates to high efficacy and low drug dose, and high specificity means that side effects can be reduced by inhibiting related off-target serine proteases. In general, the specificity of small molecule serine proteases is not as high as that of antibody inhibitors. Long blood residence time allows for less frequent dosing.

[0004] In some aspects, the disclosure features an isolated protein (e.g., an antibody, e.g., a human antibody) that binds to active plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein) and does not bind, for example, to pre-plasma kallikrein (e.g., human pre-plasma kallikrein and / or mouse pre-plasma kallikrein).

[0005] In some embodiments, the plasma kallikrein binding protein binds to or competes for binding with the same epitope as the kallikrein binding proteins described herein, hi some embodiments, the plasma kallikrein binding protein binds to or competes for binding with the same epitope as the proteins (e.g., epi-Kal2) and / or small molecules (e.g., AEBSF) described herein, and does not bind to pre-plasma kallikrein.

[0006] In some embodiments, the protein described herein is selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0007] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as X81-B01 and does not bind, for example, to pre-plasma kallikrein.

[0008] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as X67-D03 and does not bind, for example, to pre-plasma kallikrein.

[0009] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same site as X101-A01 and does not bind, for example, to pre-plasma kallikrein.

[0010] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same site as M162-A04 and does not bind, for example, to pre-plasma kallikrein.

[0011] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same site as X63-G06 and does not bind, for example, to pre-plasma kallikrein.

[0012] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0013] In certain embodiments, the protein binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope that overlaps with the active site of plasma kallikrein, and does not bind, for example, to pre-plasma kallikrein.

[0014] In some embodiments, the protein binds to one or more of the amino acids that form the catalytic triad of plasma kallikrein: His434, Asp483 and / or Ser578 (numbering based on the human sequence), and does not bind, for example, to pre-plasma kallikrein.

[0015] In some embodiments, the protein binds to one or more of the amino acids: Ser479, Tyr563 and / or Asp585 (numbering based on the human sequence) and does not bind to, for example, pre-plasma kallikrein.

[0016] In some embodiments, the plasma kallikrein binding protein binds to one or more of the amino acids: Arg551, Gln553, Tyr555, Thr558, and / or Arg560 (numbering based on the sequence of human kallikrein). In other embodiments, the plasma kallikrein binding protein binds to two, three, four, or five (i.e., all) of the amino acids: Arg551, Gln553, Tyr555, Thr558, and / or Arg560 (numbering based on the human sequence), and does not bind, for example, to pre-plasma kallikrein.

[0017] In some embodiments, the plasma kallikrein binding protein binds to one or more of the following amino acids: S478, N481, S525, and K526 (numbering based on the sequence of human kallikrein). In other embodiments, the plasma kallikrein binding protein binds to two, three, or four (i.e., all) of the following amino acids: S478, N481, S525, and K526 (numbering based on the sequence of human kallikrein).

[0018] In some embodiments, the plasma kallikrein binding protein reduces factor XIIa and / or bradykinin production by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% compared to a standard, e.g., factor XIIa and / or bradykinin production under the same conditions in the absence of the protein.

[0019] In some embodiments, the plasma kallikrein binding protein has an apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 10, 1, 0.5 or 0.2 nM.

[0020] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain.

[0021] In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the plasma kallikrein binding protein is an IgG, for example, IgG1, IgG2, IgG3 or IgG4. The plasma kallikrein binding protein can be a soluble Fab (sFab).

[0022] In some embodiments, the plasma kallikrein binding protein has a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2, IgG3, or IgG4, with a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human.

[0023] In some embodiments, the plasma kallikrein binding protein is physically associated with a moiety that enhances blood residence time, such as a moiety described herein.

[0024] In other embodiments, the plasma kallikrein binding protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen binding site of one of the binding proteins herein. The VH and VL regions of the Fab may be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0025] In one embodiment, the plasma kallikrein binding protein is a human or humanized antibody or is non-immunogenic in humans, e.g., the protein includes one or more human antibody framework regions, e.g., all human framework regions.

[0026] In one embodiment, the plasma kallikrein binding protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a human Fc domain.

[0027] In one embodiment, the plasma kallikrein binding protein is a primate or primatized antibody or is non-immunogenic in humans, e.g., the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

[0028] In one embodiment, the plasma kallikrein binding protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a primate Fc domain. "Primate" includes humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0029] In one embodiment, the plasma kallikrein binding protein comprises a human framework region or a framework region that is at least 95, 96, 97, 98, or 99% identical to a human framework region.

[0030] In certain embodiments, the plasma kallikrein binding protein does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0031] In certain embodiments, the plasma kallikrein binding protein is capable of binding to cells or tissues, eg, which express plasma kallikrein.

[0032] In one embodiment, plasma kallikrein binding proteins are physically associated with nanoparticles and can be used to target the nanoparticles to cells or tissues expressing plasma kallikrein.

[0033] In some aspects, the disclosure features isolated proteins (eg, antibodies, eg, human antibodies) that bind to or compete for binding to the same epitope as a kallikrein binding protein described herein.

[0034] In some embodiments, the protein binds to or competes for binding to the same epitope as a protein (eg, epi-Kal2) and / or small molecule (eg, AEBSF) described herein.

[0035] In some embodiments, the isolated protein comprises a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence: The heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or The light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0036] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from the heavy chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. and the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from the light chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04 (respectively).

[0037] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0038] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X81-B01, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X81-B01.

[0039] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X67-D03, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X67-D03.

[0040] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X63-G06, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X63-G06.

[0041] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from M162-A04, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from M162-A04.

[0042] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0043] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04; and / or the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04 (respectively).

[0044] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X81-B01 and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X81-B01.

[0045] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X67-D03 and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X67-D03.

[0046] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0047] In some embodiments, the protein comprises a heavy chain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. and / or a light chain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

[0048] In some embodiments, the protein comprises a heavy chain of X81-B01 and / or a light chain of X81-B01.

[0049] In some embodiments, the protein comprises a heavy chain of X67-D03 and / or a light chain of X67-D03.

[0050] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0051] In some embodiments, the plasma kallikrein binding protein reduces factor XIIa and / or bradykinin production by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% compared to a standard, e.g., factor XIIa and / or bradykinin production under the same conditions in the absence of the protein.

[0052] In some embodiments, the protein comprises one or more of the following features: (a) human CDRs or human framework regions; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the HC variable domains described herein; (c) the LC immunoglobulin variable domain sequence comprises at least one CDR that is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the LC variable domains described herein. (d) the LC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., in its entirety or in the framework regions or CDRs) to an LC variable domain described herein; (e) the HC immunoglobulin variable domain sequence is (f) the protein binds to an epitope bound by a protein described herein or competes for binding with a protein described herein; (g) a primate CDR or primate framework region; (h) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., entirely or in a framework region or CDR) to a HC variable domain described herein; (f) the protein binds to an epitope bound by a protein described herein or competes for binding with a protein described herein; (g) a primate CDR or primate framework region; (h) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to a HC variable domain described herein; (i) the HC immunoglobulin variable domain sequence comprises a CDR2 that differs from the CDR2 of the HC variable domain described herein by at least one, but not more than two, three, four, five, six, seven or eight amino acids; (j) the HC immunoglobulin variable domain sequence comprises a CDR3 that differs from the CDR3 described herein by at least one, but not more than two, three, four, five or six amino acids;(k) the LC immunoglobulin variable domain sequence comprises a CDR1 that differs from CDR1 of an LC variable domain described herein by at least one, but not more than two, three, four or five amino acids; (l) the LC immunoglobulin variable domain sequence comprises a CDR2 that differs from CDR2 of an LC variable domain described herein by at least one, but not more than two, three or four amino acids; (m) the LC immunoglobulin variable domain sequence comprises a CDR3 that differs from CDR3 of an LC variable domain described herein by at least one, but not more than two, three, four or five amino acids. (n) the LC immunoglobulin variable domain sequence differs from the LC variable domain described herein by at least one, but not by 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., in total or in the framework regions or CDRs); and (o) the HC immunoglobulin variable domain sequence differs from the HC variable domain described herein by at least one, but not by 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., in total or in the framework regions or CDRs).

[0053] In some embodiments, the protein has an apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 10, 1, 0.5 or 0.2 nM.

[0054] In some embodiments, the antibody does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0055] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain and a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0056] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0057] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0058] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy chain antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0059] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0060] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0061] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0062] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) light chain CDRs selected from the corresponding CDRs of the light chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0063] In a preferred embodiment, the protein comprises one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, as well as the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. and M35-G04.

[0064] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having X81-B01 light and heavy chains.

[0065] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain of X81-B01.

[0066] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain of X81-B01.

[0067] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chain antibody variable regions of an antibody selected from X81-B01.

[0068] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain antibody variable region of X81-B01.

[0069] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain antibody variable region of X81-B01.

[0070] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having one or more (eg, one, two or three) heavy chain CDRs from the corresponding CDRs of the X81-B01 heavy chain.

[0071] In a preferred embodiment, the protein is an antibody (eg, a human antibody) that has one or more (eg, one, two or three) light chain CDRs from the corresponding CDRs of the X81-B01 light chain.

[0072] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs from the X81-B01 heavy chain and one or more (e.g., one, two or three) light chain CDRs from the corresponding CDRs of the X81-B01 light chain.

[0073] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chains of X67-D03.

[0074] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain of X67-D03.

[0075] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain of X67-D03.

[0076] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chain antibody variable regions of an antibody selected from X67-D03.

[0077] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain antibody variable region of X67-D03.

[0078] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain antibody variable region of X67-D03.

[0079] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having one or more (eg, one, two or three) heavy chain CDRs from the corresponding CDRs of the X67-D03 heavy chain.

[0080] In a preferred embodiment, the protein is an antibody (eg, a human antibody) that has one or more (eg, one, two or three) light chain CDRs from the corresponding CDRs of the X67-D03 light chain.

[0081] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs from the X67-D03 heavy chain and one or more (e.g., one, two or three) light chain CDRs from the corresponding CDRs of the X67-D03 light chain.

[0082] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain.

[0083] In some embodiments, the plasma kallikrein binding protein has a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2, IgG3, or IgG4, with a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human.

[0084] In some embodiments, the plasma kallikrein binding protein is physically associated with a moiety that enhances blood residence time, such as a moiety described herein.

[0085] In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, such as IgG1, IgG2, IgG3 or IgG4. The protein can be a soluble Fab (sFab).

[0086] In other embodiments, the protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that contains the antigen binding site of one of the binding proteins herein. The VH and VL regions of the Fab may be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0087] In one embodiment, the protein is a human or humanized antibody, or is non-immunogenic in humans, e.g., the protein includes one or more human antibody framework regions, e.g., all human framework regions.

[0088] In one embodiment, the protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a human Fc domain.

[0089] In one embodiment, the protein is a primate or primatized antibody, or is non-immunogenic in humans, e.g., the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

[0090] In one embodiment, the protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0091] In one embodiment, the protein comprises a human framework region or a framework region that is at least 95, 96, 97, 98 or 99% identical to a human framework region.

[0092] In certain embodiments, the protein does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0093] In certain embodiments, the protein is capable of binding to cells or tissues that express, for example, plasma kallikrein.

[0094] In one embodiment, the protein is physically associated with the nanoparticles and can be used to target the nanoparticles to cells or tissues expressing plasma kallikrein.

[0095] In some aspects, the disclosure features a pharmaceutical composition comprising a kallikrein binding protein described herein, e.g., including a pharma- ceutically acceptable carrier. In some embodiments, the composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of other protein species. In one embodiment, the pharmaceutical composition does not bind to plasma kallikrein (e.g., human plasma kallikrein) or has a K of 5000 nM or greater. i,appThe antibody may be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99 or 99.9% free of fragments of binding proteins that bind to plasma kallikrein (e.g., human plasma kallikrein) at 100, ...

[0096] In some aspects, the disclosure features a method of treating or preventing a plasma kallikrein-associated disease in a subject, the method includes administering to the subject an isolated protein (e.g., an antibody, e.g., a human antibody) that binds to plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein) and does not bind, e.g., to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein).

[0097] In some embodiments, the protein binds to or competes for binding to the same epitope as a protein (eg, epi-Kal2) and / or small molecule (eg, AEBSF) described herein.

[0098] In some embodiments, the protein binds to the same epitope as, or competes for binding with, a kallikrein binding protein described herein.

[0099] In some embodiments, the plasma kallikrein-associated disease is selected from the group consisting of rheumatoid arthritis, gout, intestinal 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, clotting on a ventricular assist device or stent, clotting on a ventricular assist device or stent, 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 burn injury. In some embodiments, the plasma kallikrein binding protein reduces abnormal clotting associated with the contact activation system (i.e., the intrinsic activation system) by at least 10%, e.g., as measured by an APTT clotting assay. In other embodiments, the plasma kallikrein binding protein reduces abnormal clotting associated with the contact activation system by 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 100% (i.e., no abnormal clotting is detected).

[0100] In some embodiments, the plasma kallikrein binding protein is administered in combination with another treatment for the disease.

[0101] In some embodiments, the protein described herein is selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0102] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as X81-B01.

[0103] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as X67-D03.

[0104] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0105] In certain embodiments, the protein binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope that overlaps with the active site of plasma kallikrein.

[0106] In some embodiments, the protein binds to one or more of the amino acids that form the catalytic triad of plasma kallikrein: His434, Asp483, and / or Ser578 (numbering based on the human sequence).

[0107] In some embodiments, the protein binds to one or more amino acids at Ser479, Tyr563 and / or Asp585 (numbering based on the human sequence).

[0108] In other embodiments, the protein binds to one or more of the following amino acids: Arg551, Gln553, Tyr555, Thr558, and / or Arg560 (numbering based on the human sequence). In some embodiments, the plasma kallikrein binding protein binds to one or more of the following amino acids: S478, N481, S525, and K526 (numbering based on the human kallikrein sequence).

[0109] In some embodiments, the plasma kallikrein binding protein reduces factor XIIa and / or bradykinin production by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% compared to a standard, e.g., factor XIIa and / or bradykinin production under the same conditions in the absence of the protein.

[0110] In some embodiments, the plasma kallikrein binding protein has an apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.

[0111] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain.

[0112] In some embodiments, the plasma kallikrein binding protein has a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2, IgG3, or IgG4, with a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human.

[0113] In some embodiments, the plasma kallikrein binding protein is physically associated with a moiety that enhances blood residence time, such as a moiety described herein.

[0114] In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the plasma kallikrein binding protein is an IgG, for example, IgG1, IgG2, IgG3 or IgG4. The plasma kallikrein binding protein can be a soluble Fab (sFab).

[0115] In other embodiments, the plasma kallikrein binding protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen binding site of one of the binding proteins herein. The VH and VL regions of the Fab may be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0116] In one embodiment, the plasma kallikrein binding protein is a human or humanized antibody or is non-immunogenic in humans, e.g., the protein includes one or more human antibody framework regions, e.g., all human framework regions.

[0117] In one embodiment, the plasma kallikrein binding protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a human Fc domain.

[0118] In one embodiment, the plasma kallikrein binding protein is a primate or primatized antibody or is non-immunogenic in humans, e.g., the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

[0119] In one embodiment, the plasma kallikrein binding protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a primate Fc domain. "Primate" includes humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0120] In one embodiment, the plasma kallikrein binding protein comprises a human framework region or a framework region that is at least 95, 96, 97, 98, or 99% identical to a human framework region.

[0121] In certain embodiments, the plasma kallikrein binding protein does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0122] In certain embodiments, the plasma kallikrein binding protein is capable of binding to cells or tissues, eg, which express plasma kallikrein.

[0123] In one embodiment, plasma kallikrein binding proteins are physically associated with nanoparticles and can be used to target the nanoparticles to cells or tissues expressing plasma kallikrein.

[0124] A method of treating or preventing a plasma kallikrein-associated disorder in a subject is provided, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0125] In some embodiments, the plasma kallikrein-associated disease is selected from the group consisting of 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 clotting ventricular assist device or stent 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, and burn injury. In some embodiments, the plasma kallikrein binding protein reduces abnormal clotting 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 100% (i.e., no abnormal clotting is detected)), e.g., as measured by an APTT clotting assay.

[0126] In some embodiments, the protein is administered in combination with another treatment for the disease.

[0127] In some embodiments, the protein is administered in combination with a second agent selected from the group consisting of ecallantide, a C1 esterase inhibitor, aprotinin, a bradykinin B2 receptor inhibitor (eg, icatibant).

[0128] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from the heavy chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04; and / or The light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from the light chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

[0129] In some embodiments, the protein inhibits plasma kallikrein.

[0130] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X81-B01, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X81-B01.

[0131] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X67-D03, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X67-D03.

[0132] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0133] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. and / or the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0134] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X81-B01 and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X81-B01.

[0135] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X67-D03 and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X67-D03.

[0136] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or comprises a light chain of a protein described herein.

[0137] In some embodiments, the protein comprises the heavy chains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. and / or light chains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

[0138] In some embodiments, the protein comprises a heavy chain of X81-B01 and / or a light chain of X81-B01.

[0139] In some embodiments, the protein comprises a heavy chain of X67-D03 and / or a light chain of X67-D03.

[0140] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0141] In some embodiments, the plasma kallikrein binding protein reduces factor XIIa and / or bradykinin production by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% compared to a standard, e.g., factor XIIa and / or bradykinin production under the same conditions in the absence of the protein.

[0142] In some embodiments, the protein comprises one or more of the following features: (a) human CDRs or human framework regions; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the HC variable domains described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the LC variable domains described herein; (d) the LC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the LC variable domains described herein. , is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., in whole or in the framework regions or CDRs) to an LC variable domain described herein; (e) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., in whole or in the framework regions or CDRs) to an HC variable domain described herein; (f) the protein binds to an epitope bound by a protein described herein or competes for binding with a protein described herein; and (g) a primate CDR or primate framework region.

[0143] In some embodiments, the protein has an apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.

[0144] In some embodiments, the antibody does not bind to prekallikrein (eg, human prekallikrein) but does bind to the activated form of plasma kallikrein (eg, human plasma kallikrein).

[0145] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain and a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0146] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0147] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0148] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy chain antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0149] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0150] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0151] In a preferred embodiment, the protein is an antibody having one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0152] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) light chain CDRs selected from the corresponding CDRs of the light chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0153] In a preferred embodiment, the protein comprises one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, as well as the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. and M35-G04.

[0154] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having X81-B01 light and heavy chains.

[0155] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain of X81-B01.

[0156] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain of X81-B01.

[0157] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chain antibody variable regions of an antibody selected from X81-B01.

[0158] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain antibody variable region of X81-B01.

[0159] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain antibody variable region of X81-B01.

[0160] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having one or more (eg, one, two or three) heavy chain CDRs from the corresponding CDRs of the X81-B01 heavy chain.

[0161] In a preferred embodiment, the protein is an antibody (eg, a human antibody) that has one or more (eg, one, two or three) light chain CDRs from the corresponding CDRs of the X81-B01 light chain.

[0162] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs from the X81-B01 heavy chain and one or more (e.g., one, two or three) light chain CDRs from the corresponding CDRs of the X81-B01 light chain.

[0163] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chains of X67-D03.

[0164] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain of X67-D03.

[0165] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain of X67-D03.

[0166] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chain antibody variable regions of an antibody selected from X67-D03.

[0167] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain antibody variable region of X67-D03.

[0168] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain antibody variable region of X67-D03.

[0169] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having one or more (eg, one, two or three) heavy chain CDRs from the corresponding CDRs of the X67-D03 heavy chain.

[0170] In a preferred embodiment, the protein is an antibody (eg, a human antibody) that has one or more (eg, one, two or three) light chain CDRs from the corresponding CDRs of the X67-D03 light chain.

[0171] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs from the X67-D03 heavy chain and one or more (e.g., one, two or three) light chain CDRs from the corresponding CDRs of the X67-D03 light chain.

[0172] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain.

[0173] In some embodiments, the plasma kallikrein binding protein has a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2, IgG3, or IgG4, with a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human.

[0174] In some embodiments, the plasma kallikrein binding protein is physically associated with a moiety that enhances blood residence time, such as a moiety described herein.

[0175] In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, such as IgG1, IgG2, IgG3 or IgG4. The protein can be a soluble Fab (sFab).

[0176] In other embodiments, the protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that contains the antigen binding site of one of the binding proteins herein. The VH and VL regions of the Fab may be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0177] In one embodiment, the protein is a human or humanized antibody, or is non-immunogenic in humans, e.g., the protein includes one or more human antibody framework regions, e.g., all human framework regions.

[0178] In one embodiment, the protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a human Fc domain.

[0179] In one embodiment, the protein is a primate or primatized antibody, or is non-immunogenic in humans, e.g., the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

[0180] In one embodiment, the protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0181] In one embodiment, the protein comprises a human framework region or a framework region that is at least 95, 96, 97, 98 or 99% identical to a human framework region.

[0182] In certain embodiments, the protein does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0183] In certain embodiments, the protein is capable of binding to cells or tissues that express, for example, plasma kallikrein.

[0184] In one embodiment, the protein is physically associated with the nanoparticles and can be used to target the nanoparticles to cells or tissues expressing plasma kallikrein.

[0185] In some aspects, the disclosure features a method of promoting wound healing in a subject, the method includes administering to the subject an isolated protein (e.g., an antibody, e.g., a human antibody) that binds to plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein) and does not bind, e.g., to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein).

[0186] In some embodiments, the protein binds to or competes for binding with the same epitope as the kallikrein binding proteins described herein, In some embodiments, the protein binds to or competes for binding with the same epitope as the proteins (e.g., epi-Kal2) and / or small molecules (e.g., AEBSF) described herein.

[0187] In some embodiments, the plasma kallikrein binding protein is administered in combination with another treatment for wound healing.

[0188] In some embodiments, the protein described herein is selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0189] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as X81-B01.

[0190] In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as X67-D03.

[0191] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (eg, human prekallikrein), but does bind to the activated form of plasma kallikrein (eg, human plasma kallikrein).

[0192] In certain embodiments, the protein binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope that overlaps with the active site of plasma kallikrein.

[0193] In some embodiments, the protein binds to one or more of the amino acids His434, Asp483, and / or Ser578 (numbering based on the human sequence) that form the catalytic triad of plasma kallikrein. In other embodiments, the protein binds to one or more of the amino acids Arg551, Gln553, Tyr555, Thr558, and / or Arg560 (numbering based on the human sequence) that form the region for substrate recognition. In some embodiments, the plasma kallikrein binding protein binds to one or more of the amino acids S478, N481, S525, and K526 (numbering based on the human kallikrein sequence).

[0194] In some embodiments, the protein binds to one or more of the following amino acids: Ser479, Tyr563 and / or Asp585 (numbering based on the human sequence).

[0195] In some embodiments, the plasma kallikrein binding protein reduces factor XIIa and / or bradykinin production by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% compared to a standard, e.g., factor XIIa and / or bradykinin production under the same conditions in the absence of the protein.

[0196] In some embodiments, the plasma kallikrein binding protein has an apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.

[0197] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain.

[0198] In some embodiments, the plasma kallikrein binding protein has a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2, IgG3, or IgG4, with a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human.

[0199] In some embodiments, the plasma kallikrein binding protein is physically associated with a moiety that enhances blood residence time, such as a moiety described herein.

[0200] In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the plasma kallikrein binding protein is an IgG, for example, IgG1, IgG2, IgG3 or IgG4. The plasma kallikrein binding protein can be a soluble Fab (sFab).

[0201] In other embodiments, the plasma kallikrein binding protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen binding site of one of the binding proteins herein. The VH and VL regions of the Fab may be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0202] In one embodiment, the plasma kallikrein binding protein is a human or humanized antibody or is non-immunogenic in humans, e.g., the protein includes one or more human antibody framework regions, e.g., all human framework regions.

[0203] In one embodiment, the plasma kallikrein binding protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a human Fc domain.

[0204] In one embodiment, the plasma kallikrein binding protein is a primate or primatized antibody or is non-immunogenic in humans, e.g., the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

[0205] In one embodiment, the plasma kallikrein binding protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a primate Fc domain. "Primate" includes humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0206] In one embodiment, the plasma kallikrein binding protein comprises a human framework region or a framework region that is at least 95, 96, 97, 98, or 99% identical to a human framework region.

[0207] In certain embodiments, the plasma kallikrein binding protein does not include sequences derived from mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0208] In certain embodiments, the protein is capable of binding to cells or tissues that express, for example, plasma kallikrein.

[0209] In one embodiment, plasma kallikrein binding proteins are physically associated with nanoparticles and can be used to target the nanoparticles to cells or tissues expressing plasma kallikrein.

[0210] In some aspects, the disclosure features a method of promoting wound healing in a subject, the method including: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0211] In some embodiments, the protein is administered in combination with another treatment for wound healing.

[0212] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from the heavy chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04; and / or The light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from the light chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

[0213] In some embodiments, the protein inhibits plasma kallikrein.

[0214] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X81-B01, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X81-B01.

[0215] In some embodiments, one, two or three (e.g., three) CDR regions from the heavy chain variable domain are derived from X67-D03, and / or one, two or three (e.g., three) CDR regions from the light chain variable domain are derived from X67-D03.

[0216] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0217] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04; and / or the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04 (respectively).

[0218] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X81-B01 and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X81-B01.

[0219] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X67-D03 and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X67-D03.

[0220] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0221] In some embodiments, the protein comprises the heavy chains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. and / or light chains of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

[0222] In some embodiments, the protein comprises a heavy chain of X81-B01 and / or a light chain of X81-B01.

[0223] In some embodiments, the protein comprises a heavy chain of X67-D03 and / or a light chain of X67-D03.

[0224] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to the active form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0225] In some embodiments, the plasma kallikrein binding protein reduces factor XIIa and / or bradykinin production by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% compared to a standard, e.g., factor XIIa and / or bradykinin production under the same conditions in the absence of the protein.

[0226] In some embodiments, the protein comprises one or more of the following features: (a) human CDRs or human framework regions; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the HC variable domains described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the LC variable domains described herein; (d) the LC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the LC variable domains described herein. is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., in whole or in the framework regions or CDRs) to the LC variable domain described herein; (e) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., in whole or in the framework regions or CDRs) to the HC variable domain described herein; (f) the protein binds to an epitope bound by a protein described herein or competes for binding with a protein described herein; (g) a primate CDR or primate framework region.

[0227] In some embodiments, the protein has an apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 5, 1, 0.5 or 0.2 nM.

[0228] In some embodiments, the antibody does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0229] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain and a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0230] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0231] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0232] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy chain antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0233] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0234] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0235] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0236] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) light chain CDRs selected from the corresponding CDRs of the light chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0237] In a preferred embodiment, the protein comprises one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, as well as the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. and M35-G04.

[0238] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having X81-B01 light and heavy chains.

[0239] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain of X81-B01.

[0240] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain of X81-B01.

[0241] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chain antibody variable regions of an antibody selected from X81-B01.

[0242] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain antibody variable region of X81-B01.

[0243] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain antibody variable region of X81-B01.

[0244] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having one or more (eg, one, two or three) heavy chain CDRs from the corresponding CDRs of the X81-B01 heavy chain.

[0245] In a preferred embodiment, the protein is an antibody (eg, a human antibody) that has one or more (eg, one, two or three) light chain CDRs from the corresponding CDRs of the X81-B01 light chain.

[0246] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs from the X81-B01 heavy chain and one or more (e.g., one, two or three) light chain CDRs from the corresponding CDRs of the X81-B01 light chain.

[0247] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chains of X67-D03.

[0248] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain of X67-D03.

[0249] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain of X67-D03.

[0250] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having light and heavy chain antibody variable regions of an antibody selected from X67-D03.

[0251] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a heavy chain antibody variable region of X67-D03.

[0252] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having a light chain antibody variable region of X67-D03.

[0253] In a preferred embodiment, the protein is an antibody (eg, a human antibody) having one or more (eg, one, two or three) heavy chain CDRs from the corresponding CDRs of the X67-D03 heavy chain.

[0254] In a preferred embodiment, the protein is an antibody (eg, a human antibody) that has one or more (eg, one, two or three) light chain CDRs from the corresponding CDRs of the X67-D03 light chain.

[0255] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs from the X67-D03 heavy chain and one or more (e.g., one, two or three) light chain CDRs from the corresponding CDRs of the X67-D03 light chain.

[0256] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain.

[0257] In some embodiments, the plasma kallikrein binding protein has a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2, IgG3, or IgG4, with a blood residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in a human.

[0258] In some embodiments, the plasma kallikrein binding protein is physically associated with a moiety that enhances blood residence time, such as a moiety described herein.

[0259] In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, such as IgG1, IgG2, IgG3 or IgG4. The protein can be a soluble Fab (sFab).

[0260] In other embodiments, the protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that contains the antigen binding site of one of the binding proteins herein. The VH and VL regions of the Fab may be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0261] In one embodiment, the protein is a human or humanized antibody, or is non-immunogenic in humans, e.g., the protein includes one or more human antibody framework regions, e.g., all human framework regions.

[0262] In one embodiment, the protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a human Fc domain.

[0263] In one embodiment, the protein is a primate or primatized antibody, or is non-immunogenic in humans, e.g., the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

[0264] In one embodiment, the protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98 or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0265] In one embodiment, the protein comprises a human framework region or a framework region that is at least 95, 96, 97, 98 or 99% identical to a human framework region.

[0266] In certain embodiments, the protein does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0267] In certain embodiments, the protein is capable of binding to cells or tissues that express, for example, plasma kallikrein.

[0268] In one embodiment, the protein is physically associated with the nanoparticles and can be used to target the nanoparticles to cells or tissues expressing plasma kallikrein.

[0269] In some aspects, the disclosure features a method of treating or preventing rheumatoid arthritis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0270] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0271] In some embodiments, the protein is administered in combination with another treatment for rheumatoid arthritis.

[0272] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0273] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0274] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0275] In some aspects, the disclosure features a method of treating or preventing gout in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0276] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0277] In some embodiments, the protein is administered in combination with another treatment for gout.

[0278] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0279] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0280] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0281] In some aspects, the disclosure features a method of treating or preventing a bowel disease in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0282] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0283] In some embodiments, the protein is administered in combination with another treatment for the intestinal disease.

[0284] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0285] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0286] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0287] In some aspects, the disclosure features a method of treating or preventing oral mucositis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0288] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0289] In some embodiments, the protein is administered in combination with another therapy.

[0290] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0291] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0292] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0293] In some aspects, the disclosure features a method of treating or preventing neuropathic pain in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0294] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0295] In some embodiments, the protein is administered in combination with another treatment for neuropathic pain.

[0296] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0297] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0298] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0299] In some aspects, the disclosure features a method of treating or preventing inflammatory pain in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0300] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0301] In some embodiments, the protein is administered in combination with another treatment for inflammatory pain.

[0302] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0303] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0304] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0305] In some aspects, the disclosure features a method of treating or preventing spinal stenosis-degenerative spinal disease in a subject, the method including: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0306] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0307] In some embodiments, the protein is administered in combination with another treatment for spinal stenosis-degenerative spinal disease.

[0308] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0309] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0310] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0311] In some aspects, the disclosure features a method of treating or preventing arterial or venous thrombosis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0312] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0313] In some embodiments, the protein is administered in combination with another treatment for arterial or venous thrombosis.

[0314] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0315] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0316] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0317] In some aspects, the disclosure features a method of treating or preventing post-operative ileus in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0318] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0319] In some embodiments, the protein is administered in combination with another treatment for post-operative ileus.

[0320] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0321] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0322] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0323] In some aspects, the disclosure features a method of treating or preventing an aortic aneurysm in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0324] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0325] In some embodiments, the protein is administered in combination with another treatment for the aortic aneurysm.

[0326] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0327] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0328] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0329] In some aspects, the disclosure features a method of treating or preventing osteoarthritis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0330] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0331] In some embodiments, the protein is administered in combination with another treatment for osteoarthritis.

[0332] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0333] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0334] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0335] In some aspects, the disclosure features a method of treating or preventing vasculitis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0336] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0337] In some embodiments, the protein is administered in combination with another treatment for vasculitis.

[0338] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0339] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0340] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0341] In some aspects, the disclosure features a method of treating or preventing head trauma or peritumor cerebral edema in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0342] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0343] In some embodiments, the protein is administered in combination with another treatment for head trauma or peritumoral cerebral edema.

[0344] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0345] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0346] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0347] In some aspects, the disclosure features a method of treating or preventing sepsis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0348] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0349] In some embodiments, the protein is administered in combination with another treatment for sepsis.

[0350] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0351] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0352] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0353] In some aspects, the disclosure features a method of treating or preventing an acute middle cerebral artery (MCA) ischemic event (stroke) in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0354] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0355] In some embodiments, the protein is administered in combination with another treatment for an acute middle cerebral artery (MCA) ischemic event (stroke).

[0356] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0357] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0358] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0359] In some aspects, the disclosure features a method of treating or preventing restenosis (e.g., following angioplasty) in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0360] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0361] In some embodiments, the protein is administered in combination with another treatment for restenosis (eg, after angioplasty).

[0362] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0363] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0364] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0365] In some aspects, the disclosure features a method of treating or preventing systemic lupus erythematosus nephritis in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0366] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0367] In some embodiments, the protein is administered in combination with another treatment for systemic lupus erythematosus nephritis.

[0368] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0369] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0370] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0371] In some aspects, the disclosure features a method of treating or preventing a burn in a subject, the method comprising: administering to a subject an isolated protein (e.g., an antibody, e.g., a human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence; the heavy chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein; and / or the light chain immunoglobulin variable domain sequence comprises one, two or three (e.g., three) CDR regions from a light chain variable domain of a protein described herein; This protein binds to plasma kallikrein.

[0372] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0373] In some embodiments, the protein is administered in combination with another treatment for burn injury.

[0374] In some embodiments, the protein inhibits plasma kallikrein (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0375] In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein, and / or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of a protein described herein.

[0376] In some embodiments, the protein comprises a heavy chain of a protein described herein and / or a light chain of a protein described herein.

[0377] In some aspects, the disclosure features a method for detecting plasma kallikrein in a sample, the method including contacting the sample with a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) and detecting an interaction, if any, between the protein and plasma kallikrein.

[0378] In some embodiments, the protein comprises a detectable label.

[0379] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein). In some embodiments, the plasma kallikrein binding protein binds to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein and the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0380] In some aspects, the disclosure features a method of detecting plasma kallikrein in a subject, the method includes administering to the subject a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) and detecting an interaction, if any, between the protein and plasma kallikrein in the subject. For example, the detecting includes imaging the subject.

[0381] In some embodiments, the protein further comprises a detectable label.

[0382] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein). In some embodiments, the plasma kallikrein binding protein binds to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein and the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0383] In some aspects, the disclosure features a method of modulating plasma kallikrein activity, e.g., in a method of treating or preventing a plasma kallikrein-associated disease, the method includes modulating plasma kallikrein activity by contacting (e.g., in a human subject) plasma kallikrein with a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein).

[0384] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0385] In some embodiments, the plasma kallikrein-associated disease is selected from the group consisting of 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 peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis / vasculitis, and burn injury.

[0386] In some embodiments, the plasma kallikrein binding protein reduces aberrant clotting associated with the contact activation system (i.e., the intrinsic activation system) by at least 10%, e.g., as measured by an APTT clotting assay. In other embodiments, the plasma kallikrein binding protein reduces aberrant clotting associated with the contact activation system by 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 100% (i.e., no aberrant clotting is detected).

[0387] In some aspects, the disclosure features a method of treating a plasma kallikrein-associated disorder, the method including administering to a subject a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) in an amount sufficient to treat the plasma kallikrein-associated disorder in the subject. The method can further include providing to the subject a second therapy, e.g., a therapy for the plasma kallikrein-associated disorder described herein.

[0388] In some embodiments, the plasma kallikrein-associated disease is selected from the group consisting of 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 peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis / vasculitis, and burn injury.

[0389] Some aspects feature a method of imaging a subject, the method including administering to the subject a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) and detecting, e.g., an interaction between the protein and plasma kallikrein in the subject, if any.

[0390] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein). In some embodiments, the plasma kallikrein binding protein binds to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein and the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0391] In some embodiments, the protein does not inhibit plasma kallikrein activity.

[0392] In some embodiments, the protein inhibits plasma kallikrein activity (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0393] In some embodiments, the plasma kallikrein binding protein can include a detectable label (eg, a radionuclide or an MRI-detectable label).

[0394] In some embodiments, the subject has or is suspected of having a plasma kallikrein-related disorder. The method is useful, for example, for diagnosing a plasma kallikrein-related disorder.

[0395] In some embodiments, the plasma kallikrein-associated disease is selected from the group consisting of 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 peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, and burn injury.

[0396] In some embodiments, the plasma kallikrein binding protein reduces abnormal clotting 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 100% (i.e., no abnormal clotting is detected)), e.g., as measured by an APTT clotting assay.

[0397] In some aspects, the disclosure features a method for imaging plasma kallikrein, e.g., in a subject or sample (e.g., a biopsy sample), comprising administering a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) to, e.g., the subject or sample, and detecting an interaction, if any, between the protein and plasma kallikrein.

[0398] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein). In some embodiments, the plasma kallikrein binding protein binds to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein and the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0399] In some embodiments, the protein does not inhibit plasma kallikrein activity.

[0400] In some embodiments, the protein inhibits plasma kallikrein activity (eg, human plasma kallikrein and / or mouse plasma kallikrein).

[0401] In some embodiments, the plasma kallikrein binding protein can include a detectable label (eg, a radionuclide or an MRI-detectable label).

[0402] In some embodiments, the subject has or is suspected of having a plasma kallikrein-related disorder. The method is useful, for example, for diagnosing a plasma kallikrein-related disorder.

[0403] In some embodiments, the plasma kallikrein-associated disease is selected from the group consisting of 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 peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, and burn injury.

[0404] In some embodiments, the plasma kallikrein binding protein reduces abnormal clotting 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 100% (i.e., no abnormal clotting is detected)), e.g., as measured by an APTT clotting assay.

[0405] In one aspect, the disclosure features the use of a plasma kallikrein binding protein as described herein for the treatment of a disease as described herein, e.g., rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative 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 to promote wound healing.

[0406] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein). In some embodiments, the plasma kallikrein binding protein binds to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein and the activated form of plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0407] In one aspect, the disclosure features the use of a plasma kallikrein binding protein as described herein in the manufacture of a medicament for the treatment of a disease as described herein, e.g., rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, post-operative 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.

[0408] In some embodiments, the plasma kallikrein binding protein reduces abnormal clotting 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 100% (i.e., no abnormal clotting is detected)), e.g., as measured by an APTT clotting assay.

[0409] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein).

[0410] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0411] The contents of all cited references, including literature references, issued patents, published or unpublished patent applications, and those listed below, cited in this application are expressly incorporated herein by reference in their entirety. In the case of conflict, the present application, including definitions, will control. [Brief description of the drawings]

[0412] [Figure 1] 1 is a schematic depicting the role of plasma kallikrein (pKal) in the intrinsic coagulation pathway and inflammation. [Diagram 2] 1 shows the effect of M162-A04 on carrageenan-induced rat paw edema. Paw swelling was measured by the water displacement method. [Diagram 3] Figure 13: Effect of M162-A04 on carrageenan-induced thermal hyperalgesia. Pain latency was measured by the Hargreaves method after carrageenan injection. [Figure 4] FIG. 1 depicts an alignment of the light chain DNA sequences of non-germlined (X63-G06) and germlined, codon-optimized (X81-B01) versions of the same antibody found using ROLIC affinity maturation. Positions marked with an asterisk (*) are conserved, and spaces correspond to bases changed in X81-B01 by codon optimization or germlining. [Diagram 5]Figure 1 shows an alignment of the light chain amino acid sequences of a non-germlined (X63-G06) and a germlined, codon-optimized (X81-B01) version of the same antibody found using ROLIC affinity maturation. Positions marked with an asterisk (*) are conserved, and spaces correspond to amino acids changed in X81-B01 by germlining. A total of 11 amino acids are different between the non-germlined (X63-G06) and the germlined, codon-optimized (X81-B01) antibody. [Figure 6] FIG. 1 depicts an alignment of the heavy chain DNA sequences of a non-germlined (X63-G06) and a germlined, codon-optimized (X81-B01) version of the same antibody discovered using ROLIC affinity maturation. Positions marked with an asterisk (*) are conserved, and spaces correspond to bases changed in X81-B01 by codon optimization. [Figure 7] FIG. 1 shows an alignment of the heavy chain amino acid sequences of a non-germlined (X63-G06) and a germlined, codon-optimized (X81-B01) version of the same antibody found using ROLIC affinity maturation. Positions marked with an asterisk (*) are conserved. The two antibodies have the same heavy chain amino acid sequence. [Figure 8A] Figure 1 shows EPI-KAL2 competition for binding of X81-B01 to pKal. X81-B01 (IgG) was captured on the anti-human Fc fragment-specific surface of a CM5 BIACORE® chip. pKal (100 nM) was flowed over the surface in the presence (lower sensorgram) or absence (upper sensorgram) of 1 μM EPI-KAL2. [Figure 8B] Figure 1 shows EPI-KAL2 competition for binding of X67-D03 to pKal. X67-D03 (IgG) was captured on the anti-human Fc fragment specific surface of a CM5 Biacore chip. pKal (100 nM) was flowed over the surface in the presence (lower sensorgram) or absence (upper sensorgram) of 1 μM EPI-KAL2. [Figure 9]FIG. 13 depicts the results of CLIPS epitope mapping for the antibodies listed in Table 12. [Figure 10A] Figure 11 depicts a ClustalW alignment of pKal sequences from various species. Positions marked with "*" are conserved among species, while positions marked with ":" represent conservative substitutions among species. Positions marked with "." have non-conservative substitutions in some species. Stretches of amino acids marked with "@" symbols were shown to be highly solvent exposed by solvent accessible surface area calculations. Stretches of amino acids marked with "+" were identified as potential epitopes for the antibodies listed in Table 12. Amino acids highlighted in grey were found to be buried by solvent accessible surface area calculations when complexed with Kunitz domain active site inhibitors. Underlined positions are the amino acids that form the catalytic triad (His434, Asp483 and Ser578, numbering based on human sequence). [Figure 10B] Figure 11 depicts a ClustalW alignment of pKal sequences from various species. Positions marked with "*" are conserved among species, while positions marked with ":" represent conservative substitutions among species. Positions marked with "." have non-conservative substitutions in some species. Stretches of amino acids marked with "@" symbols were shown to be highly solvent exposed by solvent accessible surface area calculations. Stretches of amino acids marked with "+" were identified as potential epitopes for the antibodies listed in Table 12. Amino acids highlighted in grey were found to be buried by solvent accessible surface area calculations when complexed with Kunitz domain active site inhibitors. Underlined positions are the amino acids that form the catalytic triad (His434, Asp483 and Ser578, numbering based on human sequence). [Figure 10C]Figure 11 depicts a ClustalW alignment of pKal sequences from various species. Positions marked with "*" are conserved among species, while positions marked with ":" represent conservative substitutions among species. Positions marked with "." have non-conservative substitutions in some species. Stretches of amino acids marked with "@" symbols were shown to be highly solvent exposed by solvent accessible surface area calculations. Stretches of amino acids marked with "+" were identified as potential epitopes for the antibodies listed in Table 12. Amino acids highlighted in grey were found to be buried by solvent accessible surface area calculations when complexed with Kunitz domain active site inhibitors. Underlined positions are the amino acids that form the catalytic triad (His434, Asp483 and Ser578, numbering based on human sequence). [Figure 11A] FIG. 13 depicts a Biacore competition analysis of epi-kal2 against (i) DX-2922 antibody and (ii) M6-D09 antibody, as described in Example 12 herein. [Figure 11B] FIG. 13 depicts a Biacore competition analysis of epi-kal2 against (i) DX-2922 antibody and (ii) M6-D09 antibody, as described in Example 12 herein. [Figure 12] FIG. 13 depicts a Biacore competition analysis of AEBSF against (i) DX-2911 antibody and (ii) M6-D09 antibody, as described in Example 12 herein. [Figure 13] FIG. 13 depicts Biocore analysis showing that DX-2922 binds to plasma kallikrein bound to high molecular weight kininogen (HMWK). [Figure 14] FIG. 1 is a graph showing dose-dependent inhibition of edema by X101-A01 in carrageenan-induced paw edema (CPE) in rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0413] definition For convenience, certain terms used in the specification, examples and appended claims are defined here before further describing the invention. Other terms are defined as they are used herein.

[0414] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0415] The term "agonist" as used herein is intended to refer to an agent that mimics or upregulates (e.g., enhances or supports) the biological activity of a protein. An agonist can be a wild-type protein or a derivative thereof that has at least one biological activity of the wild-type protein. An agonist can also be a compound that increases at least one biological activity of a protein. An agonist can also be a compound that increases the interaction of a polypeptide with another molecule, such as a target peptide or a target nucleic acid.

[0416] As used herein, "antagonist" is intended to refer to an agent that downregulates (e.g., suppresses or inhibits) at least one biological activity of a protein.Antagonists can be compounds that inhibit or reduce the interaction between a protein and another molecule, such as a target peptide or an enzyme substrate.Antagonists can also be compounds that reduce the amount of expressed protein present.

[0417] The term "antibody" refers to a protein that includes at least one immunoglobulin variable domain (variable region) or immunoglobulin variable domain (variable region) sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH or HV) and a light (L) chain variable region (abbreviated herein as VL or LV). In another example, an antibody can include two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" refers to complete antibodies as well as antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')). 2Also encompassed are IgA, Fd fragments, Fv fragments, scFv and domain antibody (dAb) fragments (de Wildt et al., Eur J Immunol. 1996;26(3):629-39). Antibodies may have structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes). Antibodies may be from any origin, although primate (human and non-human primate) and primatized antibodies are preferred.

[0418] The VH and VL regions can be further subdivided into regions of hypervariability called "complementarity determining regions" ("CDRs"), interspersed with regions of high conservation called "framework regions" ("FRs"). The extent of the framework regions and CDRs have been defined (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The Kabat definitions are used herein. Typically, each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0419] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming an immunoglobulin variable domain structure such that one or more CDR regions are arranged in a conformation suitable for an antigen binding site. For example, the sequence may include all or a portion of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may be missing one, two or more N-terminal or C-terminal amino acids, internal amino acids, may include one or more inserted or additional terminal amino acids, or may include other modifications. In one embodiment, a polypeptide comprising an immunoglobulin variable domain sequence may be associated with another immunoglobulin variable domain sequence to form an antigen binding site, e.g., a structure that preferentially interacts with plasma kallikrein.

[0420] The VH or VL chain of the antibody may further comprise all or a portion of a heavy or light chain constant region to form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, with the heavy and light immunoglobulin chains linked together, for example, by disulfide bonds. In IgG, the heavy chain constant region comprises three immunoglobulin domains, CH1, CH2 and CH3. The light chain constant region comprises a CL domain. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. The constant region of the antibody typically mediates the binding of the antibody to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system. The light chain of the immunoglobulin may be of type kappa or lambda. In one embodiment, the antibody is glycosylated. The antibody may function in antibody-dependent cellular cytotoxicity and / or complement-mediated cytotoxicity.

[0421] One or more regions of the antibody can be human or substantially human. For example, one or more variable regions can be human or substantially human. For example, one or more CDRs can be human, such as HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and / or LC CDR3. Each of the light chain (LC) and / or heavy chain (HC) CDRs can be human. The HC CDR3 can be human. One or more framework regions can be human, such as FR1, FR2, FR3 and / or FR4 of the HC and / or LC. For example, the Fc region can be human. In one embodiment, all framework regions are human, such as from a human somatic cell (e.g., hematopoietic or non-hematopoietic cell that produces immunoglobulins). In one embodiment, the human sequences are germline sequences, such as encoded by germline nucleic acid. In one embodiment, the framework (FR) residues of the selected Fab can be converted to the amino acid type of the corresponding residue in the germline genes of most similar primates, particularly human germline genes. One or more constant regions can be human or substantially human. For example, at least 70, 75, 80, 85, 90, 92, 95, 98 or 100% of the immunoglobulin variable domains, constant regions, constant domains (CH1, CH2, CH3 and / or CL1) or the complete antibody can be human or substantially human.

[0422] An antibody may be encoded in whole or in part by an immunoglobulin gene or a segment thereof. Examples of human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as a number of immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KDa or about 214 amino acids) are encoded at the NH2-terminus (about 110 amino acids) by a variable region gene and at the COOH-terminus by a kappa or lambda constant region gene. Full-length immunoglobulin "heavy chains" (about 50 KDa or about 446 amino acids) are similarly encoded by a variable region gene (about 116 amino acids) and the other of the constant region genes, e.g., gamma (encoding about 330 amino acids). The length of the human HC is highly variable, with the HC CDR3 varying from about 3 amino acid residues to more than 35 amino acid residues.

[0423] The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of functional binding fragments encompassed by the "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab') fragment, which is a bivalent fragment containing two Fab fragments linked at the hinge region by a disulfide bridge; 2(iii) Fd fragments consisting of the VH and CH1 domains; (iV) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (V) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (Vi) isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that can be engineered using recombinant techniques to produce a single protein chain in which the VL and VH regions pair to form a monovalent molecule known as a single chain Fv (scFv). See, e.g., U.S. Patent Nos. 5,260,203, 4,946,778, and 4,881,175; Birds et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0424] Antibody fragments can be obtained using any suitable technique, including conventional techniques known to those of skill in the art. The term "monospecific antibody" refers to an antibody that exhibits a single specificity and affinity for a particular target, e.g., epitope. This term encompasses "monoclonal antibodies" or "monoclonal antibody compositions," as used herein, and refers to a preparation of antibodies or fragments thereof of single molecular composition, regardless of the method by which the antibody is made.

[0425] An antibody can be "germlined" by replacing one or more non-germline amino acids in the framework regions with the corresponding germline amino acid of the antibody, so long as binding characteristics are substantially retained.

[0426] The inhibition constant (Ki) is a measure of the potency of an inhibitor. It is the concentration of inhibitor required to reduce the enzyme activity by half and is independent of the concentration of the enzyme or substrate. The apparent Ki (Ki,app ) is obtained. That is, the apparent Ki value is estimated by fitting the change in the pseudo-first-order rate constant as a function of inhibitor concentration to the Morrison equation (Equation 1). K i,app The y-intercept from linear regression analysis of the plot gives Ki.

number

[0427] As used herein, "binding affinity" refers to the apparent binding constant, or K A Refers to. A is the dissociation constant (K D ) is the reciprocal of the . A binding protein is, for example, a protein that has a binding affinity for a particular target molecule, such as plasma kallikrein, of at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 and 10 11 M -1 A binding protein can bind to a first target with higher affinity than to a second target by determining the K A (or K D (value) is the K A (or K D In such cases, the binding protein is more specific for a first target (e.g., a protein or mimetic thereof in a first conformation) than a second target (e.g., the same protein or a mimetic thereof in a second conformation; or a second protein). The difference in binding affinity (e.g., with respect to specificity or other comparison) may be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 It could be double.

[0428] Binding affinity can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectrometry (e.g., using a fluorescence assay). An example of a condition for evaluating binding affinity is in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). Using the above techniques, the concentration of bound and free binding protein can be measured as a function of binding protein (or target) concentration. The concentration of bound binding protein ([bound]) is related to the concentration of free binding protein ([free]) and the concentration of binding sites for the binding protein on the target by the following equation, where (N) is the number of binding sites per target molecule:

number

[0429] But always K A It is not necessary to precisely determine K, as it may be sufficient to obtain a quantitative measure of affinity, as determined, for example, using methods such as ELISA or FACS analysis. A Since affinity is proportional to the affinity of a given antibody, it can be used, for example, to obtain a qualitative measure of affinity or to estimate affinity, such as by comparison with activity in a functional assay (e.g., an in vitro or in vivo assay), e.g., determining whether a high affinity is, for example, 2-fold higher.

[0430] The term "binding protein" refers to a protein capable of interacting with a target molecule. This term is used interchangeably with "ligand." "Plasma kallikrein binding protein" refers to a protein capable of interacting with (e.g., binding to) plasma kallikrein, and specifically includes proteins that preferentially or specifically interact with and / or inhibit plasma kallikrein. A protein inhibits plasma kallikrein if it reduces the activity of plasma kallikrein compared to the activity of plasma kallikrein under the same conditions in the absence of the protein. In some embodiments, the plasma kallikrein binding protein is an antibody.

[0431] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0432] One or more framework and / or CDR amino acid residues of the binding protein may comprise one or more mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions or deletions) compared to the proteins described herein. The plasma kallikrein binding protein may have mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions or deletions) compared to the proteins described herein (e.g., at least 1, 2, 3 or 4 mutations and / or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations), e.g., mutations that do not substantially affect protein function. The mutations may be in the framework regions, CDRs and / or constant regions. In some embodiments, the mutations are in the framework regions. In some embodiments, the mutations are in the CDRs. In some embodiments, the mutations are in the constant regions. Whether a particular substitution will be tolerated, i.e., will not adversely affect biological properties such as binding activity, can be predicted, for example, by assessing whether the mutation is conservative or by the method of Bowie et al. (1990) Science 247:1306-1310.

[0433] Motif sequences of biopolymers may include positions at which various amino acids are allowed. For example, the symbol "X" in such a context generally represents any amino acid (e.g., any of the 20 naturally occurring amino acids) unless otherwise specified (e.g., represents any non-cysteine ​​amino acid). Other allowed amino acids can also be represented, for example, using parentheses and a slash. For example, "(A / W / F / N / Q)" means that alanine, tryptophan, phenylalanine, asparagine, and glutamine are allowed at that particular position.

[0434] A "substantially human" immunoglobulin variable region is one that contains a sufficient number of human framework amino acid positions so as not to elicit an immunogenic response in normal humans. A "substantially human" antibody is one that contains a sufficient number of human amino acid positions so as not to elicit an immunogenic response in normal humans.

[0435] "Epitope" refers to a site on a target compound to which a binding protein (e.g., an antibody such as a Fab or full-length antibody) binds. When the target compound is a protein, this site can be composed entirely of amino acid components, entirely of chemical modifications of the protein's amino acids (e.g., glycosyl), or a combination thereof. Overlapping epitopes contain at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular structure.

[0436] A first binding protein (e.g., an antibody) "binds to the same epitope" as a second binding protein (e.g., an antibody) if the first binding protein binds to the same site on the target compound as the second binding protein or binds to a site that overlaps with the site bound by the second binding protein (e.g., 50%, 60%, 70%, 80%, 90% or 100% overlap in terms of amino acid sequence or other molecular structure (e.g., glycosyl, phosphate or sulfate groups)).

[0437] A first binding protein (e.g., an antibody) "competes for binding" with a second binding protein (e.g., an antibody) if binding of the first binding protein to its epitope reduces (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the amount of the second binding protein that binds to that epitope. Competition can be direct (e.g., the first binding protein binds to an epitope that is the same as or overlaps with an epitope bound by the second binding protein) or indirect (e.g., binding of the first binding protein to its epitope causes a conformational change in the target compound that reduces the ability of the second binding protein to bind to its epitope).

[0438] Calculation of "homology" or "sequence identity" between two sequences (these terms are used interchangeably herein) is performed as follows: The sequences are aligned for optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). Optimal alignment is determined as maximum score using the GAP program of the GCG software package with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5 with a Blossum 62 scoring matrix. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules will be identical at that position (amino acid or nucleic acid "identity" as used herein is the same as amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions common to the two sequences.

[0439] In preferred embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98% or 100% of the length of the reference sequence. For example, a reference sequence can be the length of an immunoglobulin variable domain sequence.

[0440] A "humanized" immunoglobulin variable region is an immunoglobulin variable region that has been modified to contain a sufficient number of human framework amino acid positions so as not to elicit an immunogenic response in normal humans. "Humanized" immunoglobulins are described, for example, in U.S. Patent Nos. 6,407,213 and 5,693,762.

[0441] As used herein, the term "hybridize under low stringency, medium stringency, high stringency or very high stringency conditions" describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in this reference, both of which may be used. Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions at about 45°C in 6x sodium chloride / sodium citrate (SSC), followed by two washes in 0.2x SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the wash temperature may be increased to 55°C); (2) medium stringency hybridization conditions at about 45°C in 6x SSC, followed by two washes in 0.2x SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the wash temperature may be increased to 55°C); (3) high stringency hybridization conditions at 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (4) very high stringency hybridization conditions at 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C. Unless otherwise specified, very high stringency condition (4) is the preferred condition and is the condition to be used. The present disclosure includes nucleic acids that hybridize with the nucleic acids described herein or their complements, such as nucleic acids encoding the binding proteins described herein, at low, medium, high, or very high stringency. The nucleic acids can be the same length as the reference nucleic acid or within 30, 20, or 10% of the length. The nucleic acid can correspond to a region encoding an immunoglobulin variable domain sequence described herein.

[0442] An "isolated composition" refers to a composition that is at least 90% removed from at least one component of the natural sample from which it is obtained. An artificially produced or naturally produced composition can be a "composition" of "at least" some degree of purity if the species or species population of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-by-weight basis.

[0443] An "isolated" protein refers to a protein that is removed from at least 90% of at least one component of the natural sample from which it is obtained. A protein can be "at least" somewhat pure if the species or species population of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-by-weight basis.

[0444] The term "modulator" refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species, etc. (natural or non-natural) that may be capable of causing modulation, or an extract made from biological material, such as bacteria, plants, fungi, or animal cells or tissues. Modulators may be assayed (directly or indirectly) to evaluate their potential activity as an inhibitor or activator (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, antibacterial agent, inhibitor of microbial infection or growth, etc.) of a functional property, biological activity or process, or a combination thereof. In such an assay, multiple modulators may be screened simultaneously. The activity of a modulator may be known, unknown, or partially known.

[0445] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a binding agent, e.g., an antibody, without abolishing, or more preferably without substantially altering, the biological activity, whereas an "essential" amino acid residue can be altered to substantially abolish the activity of the binding agent.

[0446] A "patient," "subject," or "host" (the terms are used interchangeably) treated by the methods of the present invention can mean a human or non-human animal.

[0447] The terms "prekallikrein" and "pre-plasma kallikrein" are used interchangeably herein and refer to the zymogen form of active plasma kallikrein, also known as prekallikrein.

[0448] The terms "preventing" or "preventing" a disease in a subject refer to administering a pharmaceutical treatment, e.g., a drug, to a subject such that at least one symptom of the disease is prevented, which occurs prior to the clinical onset of an undesirable condition (e.g., a disease or other undesirable condition in a host animal) such that the host is prevented from developing the undesirable condition. "Prevention" of a disease is also referred to as "prophylaxis" or "prophylactic treatment."

[0449] As used herein, the term "substantially identical" (or "substantially homologous") is used herein to refer to a first amino acid or nucleic acid sequence that contains a sufficient number of identical or equivalent amino acid residues or nucleotides (e.g., having similar side chains, e.g., conservative amino acid substitutions) as a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins having) a similar activity, e.g., binding activity, preferential binding or biological activity. In the case of antibodies, the second antibody has the same specificity and at least 50%, at least 25%, or at least 10% of the affinity relative to the same antigen.

[0450] Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the binding proteins described herein. In some embodiments, plasma kallikrein binding proteins can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the HC and / or LC framework regions (e.g., HC and / or LC FR1, 2, 3 and / or 4) with a binding protein described herein. In some embodiments, plasma kallikrein binding proteins can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the HC and / or LC CDRs (e.g., HC and / or LC CDR1, 2 and / or 3) with a binding protein described herein. In some embodiments, the plasma kallikrein binding protein has about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the constant region (e.g., CH1, CH2, CH3 and / or CL1) to a binding protein described herein.

[0451] Also, substantial identity exists when the nucleic acid segment hybridizes under selected hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0452] Statistical significance can be determined by any known method. Examples of statistical tests include Student's T test, Mann Whitney non-parametric U test and Wilcoxon non-parametric statistical test. Some statistically significant relationships have P values ​​less than 0.05 or 0.02. Certain binding proteins can show statistically significant differences, for example, in specificity or binding (e.g., P values ​​less than 0.05 or 0.02). For example, terms such as "induce", "inhibit", "enhance", "elevate", "increase", "reduce" and the like, which represent distinguishable qualitative or quantitative differences, can refer to the difference between two states, for example, the statistically significant difference between two states.

[0453] A "therapeutically effective amount" preferably modulates a measurable parameter, such as plasma kallikrein activity, by a statistically significant degree or at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated subjects. The ability of a compound to modulate a measurable parameter, such as a disease-related parameter, can be evaluated in an animal model system that is predictive of efficacy in human diseases and conditions, such as rheumatoid arthritis or oral mucositis. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate the parameter in vitro.

[0454] "Treating" a disease (or condition) in a subject or a subject having a disease refers to administering medical therapy, e.g., administering a drug, to a subject such that at least one symptom of the disease is cured, alleviated, or reduced.

[0455] The term "preventing" a disease in a subject refers to administering a pharmaceutical treatment, e.g., a drug, to a subject such that at least one symptom of the disease is prevented, which occurs prior to the clinical onset of an undesirable condition (e.g., a disease or other undesirable condition in a host animal) such that the host is prevented from developing the undesirable condition. "Preventing" a disease is also referred to as "prophylaxis" or "prophylactic treatment."

[0456] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic amount is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0457] As used herein, the term "DX-2922" is used interchangeably with the term "X101-A01." Other variants of this antibody are described below. [Table 1]

[0458] Plasma kallikrein-binding protein The plasma kallikrein binding protein can be full-length (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD and IgE) or can include only an antigen-binding fragment (e.g., Fab, F(ab')2 or scFv fragment). The binding protein can include two heavy and two light immunoglobulin chains or can be a single chain antibody. The plasma kallikrein binding protein can be a recombinant protein, such as a humanized, CDR-grafted, chimeric, deimmunized or in vitro generated antibody, and can optionally include a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein binding protein is a monoclonal antibody.

[0459] In one aspect, the disclosure features a protein (e.g., an isolated protein) that binds to plasma kallikrein (e.g., human plasma kallikrein and / or mouse kallikrein) and includes at least one immunoglobulin variable region. For example, the protein includes a heavy chain (HC) immunoglobulin variable domain sequence and / or a light chain (LC) immunoglobulin variable domain sequence. In one embodiment, the protein binds to and inhibits plasma kallikrein, e.g., human plasma kallikrein and / or mouse kallikrein.

[0460] The protein may comprise one or more of the following features: (a) human CDRs or human framework regions; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., one, two or three) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the HC variable domains described herein; (c) the LC immunoglobulin variable domain sequence comprises at least one CDR that is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the LC variable domains described herein. (d) the LC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., in whole or in framework regions or CDRs) to the LC variable domain described herein; (e) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the LC variable domain described herein; (f) the protein binds to an epitope bound by a protein described herein or competes for binding with a protein described herein; (g) a primate CDR or primate framework region; (h) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (e.g., entirely or in a framework region or CDR) to a HC variable domain described herein; (f) the protein binds to an epitope bound by a protein described herein or competes for binding with a protein described herein; (g) a primate CDR or primate framework region; (h) the HC immunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to a HC variable domain described herein; (i) the HC immunoglobulin variable domain sequence comprises a CDR2 that differs from the CDR2 of the HC variable domain described herein by at least one, but not more than two, three, four, five, six, seven or eight amino acids; (j) the HC immunoglobulin variable domain sequence comprises a CDR3 that differs from the CDR3 described herein by at least one, but not more than two, three, four, five or six amino acids;(k) the LC immunoglobulin variable domain sequence comprises a CDR1 that differs from CDR1 of an LC variable domain described herein by at least one, but not more than two, three, four or five amino acids; (l) the LC immunoglobulin variable domain sequence comprises a CDR2 that differs from CDR2 of an LC variable domain described herein by at least one, but not more than two, three or four amino acids; (m) the LC immunoglobulin variable domain sequence comprises a CDR3 that differs from CDR3 of an LC variable domain described herein by at least one, but not more than two, three, four or five amino acids. (n) the LC immunoglobulin variable domain sequence differs from the LC variable domain described herein by at least one, but not by 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., in total or in the framework regions or CDRs); and (o) the HC immunoglobulin variable domain sequence differs from the HC variable domain described herein by at least one, but not by 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., in total or in the framework regions or CDRs).

[0461] The plasma kallikrein binding protein can be an isolated protein (e.g., at least 70, 80, 90, 95, or 99% free of other proteins). In some embodiments, the plasma kallikrein binding protein or composition thereof is inactive or partially active (e.g., has a K of 5000 nM or greater) compared to the plasma kallikrein binding protein. i,app The plasma kallikrein binding protein is isolated from antibody cleavage fragments (e.g., cleaved DX-2922) that bind to plasma kallikrein at a specific concentration. For example, the plasma kallikrein binding protein is at least 70% free of such antibody cleavage fragments; in other embodiments, the binding protein is at least 80%, at least 90%, at least 95%, at least 99% or 100% free of inactive or partially active antibody cleavage fragments.

[0462] The plasma kallikrein binding protein may further inhibit plasma kallikrein, for example human plasma kallikrein.

[0463] In some embodiments, the plasma kallikrein binding protein does not bind to prekallikrein (e.g., human prekallikrein and / or mouse prekallikrein), but does bind to activated plasma kallikrein (e.g., human plasma kallikrein and / or mouse kallikrein).

[0464] In certain embodiments, the protein binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope that overlaps with the active site of plasma kallikrein.

[0465] In some embodiments, the protein binds to the same epitope as a protein described herein or competes for binding with it.

[0466] In some embodiments, the protein competes with or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0467] In some embodiments, the protein binds to CLIP peptides C1, C2, C3, C4, C5, C6, or C7, or two or more of these peptides (e.g., their corresponding positions on plasma kallikrein), e.g., the protein binds to C5 and C6. CLIP peptides C1-C7 are peptides in plasma kallikrein identified by CLIP epitope mapping (see Figure 9 and Figures 10A-10C). C1 corresponds to positions 55-67 of the plasma kallikrein catalytic domain, C2 corresponds to positions 81-94 of plasma kallikrein, C3 corresponds to positions 101-108, C4 corresponds to positions 137-151, C5 corresponds to positions 162-178, C6 corresponds to positions 186-197, and C7 corresponds to positions 214-217.

[0468] In some embodiments, the protein binds to an epitope depicted in FIG.

[0469] In some embodiments, the protein binds to one or more of the amino acids that form the catalytic triad of plasma kallikrein: His434, Asp483, and / or Ser578 (numbering based on the human sequence).

[0470] In some embodiments, the protein binds to one or more of the following amino acids: Arg551, Gln553, Tyr555, Thr558, and / or Arg560 (numbering based on the human sequence). In some embodiments, the plasma kallikrein binding protein binds to one or more of the following amino acids: S478, N481, S525, and K526 (numbering based on the human kallikrein sequence).

[0471] In some embodiments, the protein binds to one or more of the following amino acids: Ser479, Tyr563 and / or Asp585 (numbering based on the human sequence).

[0472] The active site cleft of plasma kallikrein contains three amino acids (His434, Asp483, and Ser578) that form the catalytic triad and result in enzymatic hydrolysis of bound substrates (the catalytic triad is underlined in FIG. 10). The peptides selected for CLIPS epitope mapping analysis were determined to be surface accessible and either form the active site or surround the active site. Peptide C1 contains active site histidine 434. Peptide C3 contains active site aspartic acid 483. Peptide C6 contains active site serine 578. Antibodies can bind to multiple surface-exposed amino acids that are discontinuous in the amino acid sequence. For example, from CLIP analysis, X81-B01 appears to bind to peptides C2, C3, C5, and C6.

[0473] In some embodiments, the protein binds to an epitope comprising one or more amino acids from CLIPS peptide C1, peptide C2, peptide C3, peptide C4, peptide C5, peptide C6, or peptide C7.

[0474] In some embodiments, the protein binds to an epitope that includes amino acids from at least two different CLIPS peptides, for example, from at least two of peptide C1, peptide C2, peptide C3, peptide C4, peptide C5, peptide C6, or peptide C7.

[0475] This protein is at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 and 10 11 M -1 In one embodiment, the protein can bind to plasma kallikrein, e.g., human plasma kallikrein, with a binding affinity of 1×10 -3 , 5×10 -4 s -1 or 1×10 -4 s -1 Slower than Koff In one embodiment, the protein binds to human plasma kallikrein at a concentration of 1×10 2 , 1×10 3 or 5×10 3 M -1 s -1 Faster than K on In one embodiment, the protein binds to plasma kallikrein but not to tissue kallikrein and / or plasma prekallikrein relative to plasma kallikrein (e.g., the protein binds to tissue kallikrein and / or plasma prekallikrein less efficiently (e.g., 5, 10, 50, 100 or 1000 times less efficiently than a negative control, or not at all).

[0476] In one embodiment, the protein is -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 and 10 -10 This protein inhibits human plasma kallikrein activity with a Ki of less than IC 50 The affinity of the protein for human plasma kallikrein may be less than 100 nM, 10 nM, 1, 0.5 or 0.2 nM. For example, the protein may modulate plasma kallikrein activity as well as the production of factor XIIa (e.g., from factor XII) and / or bradykinin (e.g., from high molecular weight kininogen (HMWK)). The protein may inhibit plasma kallikrein activity and / or the production of factor XIIa (e.g., from factor XII) and / or bradykinin (e.g., from high molecular weight kininogen (HMWK)). The affinity of the protein for human plasma kallikrein may be less than 100 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM. DIn one embodiment, the protein inhibits plasma kallikrein but not tissue kallikrein relative to plasma kallikrein (e.g., the protein inhibits tissue kallikrein less efficiently (e.g., 5, 10, 50, 100, or 1000 times less efficiently than a negative control, or not at all)).

[0477] In some embodiments, the apparent inhibition constant (K i,app ) is less than 1000, 500, 100, 5, 1, 0.5 or 0.2 nM.

[0478] The plasma kallikrein binding protein can be an antibody. The plasma kallikrein binding antibody can contain its HC and LC variable domain sequences in a single polypeptide (e.g., scFv) or in different polypeptides (e.g., IgG or Fab).

[0479] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain and a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, DX-2922, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0480] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0481] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0482] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy chain antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0483] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0484] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0485] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0486] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) light chain CDRs selected from the corresponding CDRs of the light chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04.

[0487] In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., one, two or three) heavy chain CDRs and one or more (e.g., one, two or three) light chain CDRs selected from the corresponding CDRs of the light chain group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

[0488] In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another embodiment, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4. The protein can be a soluble Fab. In other embodiments, the protein comprises a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises an antigen-binding site of one of the binding proteins herein. The VH and VL regions of the Fab can be provided as an IgG, Fab, Fab2, Fab2', scFv, pegylated Fab, pegylated scFv, pegylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable construct.

[0489] In one embodiment, the protein is a human or humanized antibody, or is non-immunogenic in humans. For example, the protein comprises one or more human antibody framework regions, such as all human framework regions or framework regions that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to a human framework region. In one embodiment, the protein comprises a human Fc domain or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc domain.

[0490] In one embodiment, the protein is a primate or primatized antibody, or is non-immunogenic in humans. For example, the protein comprises one or more primate antibody framework regions, such as all primate framework regions or framework regions that are 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to primate framework regions. In one embodiment, the protein comprises a primate Fc domain or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobo)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

[0491] In some embodiments, the primate antibody has an affinity for human plasma kallikrein of less than 1000, 500, 100, 10, 5, 1, 0.5 nM, e.g., less than 10 nM, less than 1 nM, or less than 0.5 nM. D The present invention may be characterized by the following:

[0492] In certain embodiments, the protein does not include sequences derived from a mouse or rabbit (eg, is not a mouse or rabbit antibody).

[0493] In some aspects, the disclosure provides for the use of a protein (e.g., a binding protein, e.g., an antibody) (e.g., a protein described herein) that binds to plasma kallikrein (e.g., human plasma kallikrein) and comprises at least one immunoglobin variable region in a method for the treatment (or prevention) of a plasma kallikrein-associated disease or condition. For example, a plasma kallikrein binding protein comprises a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. Numerous examples of plasma kallikrein binding proteins are described herein.

[0494] The plasma kallikrein binding protein can be an isolated protein (eg, at least 70, 80, 90, 95, or 99% free from other proteins).

[0495] The plasma kallikrein binding protein may further inhibit plasma kallikrein, e.g., human plasma kallikrein and / or mouse plasma kallikrein. In some embodiments, it is preferred that the plasma kallikrein binding protein binds to both human and mouse plasma kallikrein, so that the efficacy of such antibodies can be tested in mouse models.

[0496] Plasma kallikrein Examples of plasma kallikrein sequences from which plasma kallikrein binding proteins can be generated include human, mouse or rat plasma kallikrein amino acid sequences, sequences that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the above sequences, or fragments thereof, such as fragments of the sequences set out below.

[0497] The sequence of human plasma kallikrein used in the selection and subsequent screening is shown below (accession number NP_000883.2). The human plasma kallikrein used (86 kDa) was purified from human plasma and activated with factor XIIa from a commercial supplier. Factor XIIa activates prekallikrein by cleaving the polypeptide sequence at a single site (between Arg371-Ile372, the cleavage site is indicated by " / " in the sequence below), generating active plasma kallikrein, which is composed of two disulfide-bonded polypeptides: a heavy chain of about 52 kDa and a catalytic domain of about 34 kDa [Colman and Schmaier, (1997) "Contact System: A Vascular Biology Modulator With Anticoagulant, Profibrinolytic, Antiadhesive, and Proinflammatory Attributes" Blood, 90, 3819-3843].

[0498] GCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGG TPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQ FFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTR / IVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHD IALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA

[0499] The human, mouse and rat prekallikrein amino acid sequences and the mRNA sequences encoding them are shown below. The sequence of prekallikrein is the same as plasma kallikrein, except that active plasma kallikrein (pkal) has a single polypeptide chain that is cleaved at a single position (indicated by " / ") to produce two chains. The sequence listed below is the complete sequence including the signal sequence. The signal sequence is expected to be removed upon secretion from expressing cells.

[0500] Human plasma kallikrein (Accession number: NP_000883.2) >gi|78191798|ref|NP_000883.2|Plasma kallikrein B1 precursor [Homo sapiens] MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSIND MEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKR CTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLA FSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYS LLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLR LSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCG GSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQ APLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQR MVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDG KAQMQSPA

[0501] Human plasma kallikrein mRNA (Accession number: NM_000892) >gi|78191797|ref|NM_000892.3|Homo sapiens kallikrein B, plasma (Fletcher factor) 1 (KLKB1), mRNA AGAACAGCTTGAAGACCGTTCATTTTTAAGTGACAAGAGACTCACCTCCAAGAAGCAATTGTGTTTTCAG AATGATTTTATTCAAGCAAGCAACTTTATTCATTTCCTTGTTTGCTACAGTTTCCTGTGGATGTCTGACT CAACTCTATGAAAACGCCTTCTTCAGAGGTGGGGATGTAGCTTCCATGTACACCCCAAATGCCCAATACT GCCAGATGAGGTGCACATTCCACCCAAGGTGTTTGCTATTCAGTTTTCTTCCAGCAAGTTCAATCAATGA CATGGAGAAAAGGTTTGGTTGCTTCTTGAAAGATAGTGTTACAGGAACCCTGCCAAAGTACATCGAACA GGTGCAGTTTCTGGACATTCCTTGAAGCAATGTGGTCATCAAATAAGTGCTTGCCATCGAGACATTTATA AAGGAGTTGATATGAGAGGAGTCAATTTTAATGTGTCTAAGGTTAGCAGTGTTGAAGAATGCCAAAAAG GTGCACCAGTAACATTCGCTGCCAGTTTTTTCATATGCCACGCAAACATTTCCAAGGCAGAGTACCGG AACAATTGCCTATTAAAGTACAGCTCCCGGAGGAACACCTACCGCTATAAAGGTGCTGAGTAACGTGGAAT CTGGATTCTCACTGAAGCCCTGTGCCCTTTCAGAAATTGGTTGCCACATGAACATCTTCCAGCATCTTGC GTTCTCAGATGTGGATGTTGCCAGGGTTCTCACTCCAGATGCTTTTTGTGTGTCGGACCATCTGCACCTAT CACCCCAACTGCCTCTTCTTTACATTCTATACAAATGTATGGAAAATCGAGTCACAAAAGAAATGTTTGTC TTCTTAAAACATCTGAAAGTGGCACACCAAGTTCCTCTACTCCTCAAGAAAACACCATATCTGGATATAG CCTTTTAACCTGCAAAAGAACTTTACCTGAACCCTGCCATTCTAAAATTTACCCGGGAGTTGACTTTGGA GGAGAAGAATTGAATGTGACTTTTGTTAAAGGAGTGAATGTTTGCCAAGAGACTTGCACAAAGATGATTC GCTGTCAGTTTTTCACTTATTCTTTACTCCCAGAAGACTGTAAGGAAGAGAAGTGTAAGTGTTTCTTAAG ATTATCTATGGATGGTTCTCCAACTAGGATTGCGTATGGGACACAAGGGAGCTCTGGTTACTCTTTGAGA TTGTGTAACACTGGGGACAACTCTGTCTGCACAACAAAAACAAGCACACGCATTGTTGGAGGAACAAACT CTTCTTGGGGAGAGTGGCCCTGGCAGGTGAGCCTGCAGGTGAAGCTGACAGCTCAGAGGCACCTGTGTGG AGGGTCACTCATAGGACACCAGTGGGTCCTCACTGCTGCCCACTGCTTTGATGGGCTTCCCCTGCAGGAT GTTTGGCGCATCTATAGTGGCATTTTAAATCTGTCAGACATTACAAAAGATACACCTTTCTCACAAATAA AAGAGATTATTATTCACCAAAACTATAAAGTCTCAGAAGGGAATCATGATATCGCCTTGATAAAACTCCA GGCTCCTTTGAATTACACTGAATTCCAAAAACCAATATGCCTACCTTCCAAAGGTGACACAAGCACAATT TATACCAACTGTTGGGTAACCGGATGGGGCTTCTCGAAGGAGAAAGGTGAAATCCAAAATATTCTACAAA AGGTAAATATTCCTTTGGTAACAAATGAAGAATGCCAGAAAAGATATCAAGATTATAAAATAACCCAACG GATGGTCTGTGCTGGCTATAAAGAAGGGGGAAAAGATGCTTGTAAGGGAGATTCAGGTGGTCCCTTAGTT TGCAAACACAATGGAATGTGGCGTTTGGTGGGCATCACCAGCTGGGGTGAAGGCTGTGCCCGCAGGGAGC AACCTGGTGTCTACACCAAAGTCGCTGAGTACATGGACTGGATTTTAGAGAAAACACAGAGCAGTGATGG AAAAGCTCAGATGCAGTCACCAGCATGAGAAGCAGTCCAGAGTCTAGGCAATTTTTACAACCTGAGTTCA AGTCAAATTCTGAGCCTGGGGGGTCCTCATCTGCAAAGCATGGAGAGTGGCATCTTCTTTGCATCCTAAG GACGAAAAACACAGTGCACTCAGAGCTGCTGAGGACAATGTCTGGCTGAAGCCCGCTTTCAGCACGCCGT AACCAGGGGCTGACAATGCGAGGTCGCAACTGAGATCTCCATGACTGTGTGTTGTGAAATAAAATGGTGA AAGATCAAAAAA

[0502] Mouse plasma kallikrein (Accession number: NP_032481.1) >gi|6680584|ref|NP_032481.1| Kallikrein B, plasma 1 [Mus musculus] MILFNRVGYFVSLFATVSCGCMTQLYKNTFFRGGDLAAIYTPDAQYCQKMCTFHPRCLLFSFLAVTPPKE TNKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQISACHRDIYKGLDMRGSNFNISKTDNIEECQKL CTNNFHCQFFTYATSAFYRPEYRKKCLLKHSASGTPTSIKSADNLVSGFSLKSCALSEIGCPMDIFQHSA FADLNVSQVITPDAFVCRTICTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIPQENAISGYS LLTCRKTRPEPCHSKIYSGVDFEGEELNVTFVQGADVCQETCTKTIRCQFFIYSLLPQDCKEEGCKCSLR LSTDGSPTRITYGMQGSSGYSLRLCKLVDSPDCTTKINARIVGGTNASLGEWPWQVSLQVKLVSQTHLCG GSIIGRQWVLTAAHCFDGIPYPDVWRIYGGILSLSEITKETPSSRIKELIIHQEYKVSEGNYDIALIKLQ TPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKEQGETQNILQKATIPLVPNEECQKKYRDYVINKQ MICAGYKEGGTDACKGDSGGPLVCKHSGRWQLVGITSWGEGCGRKDQPGVYTKVSEYMDWILEKTQSSDV RALETSSA

[0503] Mouse plasma kallikrein mRNA (Accession number: NM_008455.2) >gi|236465804|ref|NM_008455.2|Mus musculus kallikrein B, plasma 1 (Klkb1), mRNA AGACCGCCCTCGGTGCCATATTCAGAGGGCTTGAAGACCATCTTCATGTGAAGACTCCCTCTCCTCCAGA ACCACAACGTGACCATCCTTCCAGGATGATTTTATTCAACCGAGTGGGTTATTTTGTTTCCTTGTTTGCT ACCGTCTCCTGTGGGTGTATGACTCAACTGTATAAAAATACCTTCTTCAGAGGTGGGGATCTAGCTGCCA TCTACACCCCAGATGCCCAGTACTGTCAGAAGATGTGCACTTTTCACCCCAGGTGCCTGCTGTTCAGCTT TCTCGCCGTGACTCCACCCAAAGAGACAAATAAACGGTTTGGTTGCTTCATGAAAGAGAGCATTACAGGG ACTTTGCCAAGAATACACCGGACAGGGGCCATTTCTGGTCATTCTTTAAAGCAGTGTGGCCATCAAATAA GTGCTTGCCACCGAGACATATACAAAGGACTTGATATGAGAGGGTCCAACTTTAATATCTCTAAGACCGA CAATATTGAAGAATGCCAGAAACTGTGCACAAATAATTTTCACTGCCAATTTTTCACATATGCTACAAGT GCATTTTACAGACCAGAGTACCGGAAGAAGTGCCTGCTGAAGCACAGTGCAAGCGGAACACCCACCAGCA TAAAGTCAGCGGACAACCTGGTGTCTGGATTCTCACTGAAGTCCTGTGCGCTTTCGGAGATAGGTTGCCC CATGGATATTTTCCAGCACTCTGCCTTTGCAGACCTGAATGTAAGCCAGGTCATCACCCCCGATGCCTTT GTGTGTCGCACCATCTGCACCTTCCATCCCAACTGCCTTTTCTTCACGTTCTACACGAATGAATGGGAGA CAGAATCACAGAGAAATGTTTGTTTTCTTAAGACGTCTAAAAGTGGAAGACCAAGTCCCCCTATTCCTCA AGAAAACGCTATATCTGGATATAGTCTCCTCACCTGCAGAAAAACTCGCCCTGAACCCTGCCATTCCAAA ATTTACTCTGGAGTTGACTTTGAAGGGGAAGAACTGAATGTGACCTTCGTGCAAGGAGCAGATGTCTGCC AAGAGACTTGTACAAAGACAATCCGCTGCCAGTTTTTTATTTACTCCTTACTCCCCCAAGACTGCAAGGA GGAGGGGTGTAAATGTTCCTTAAGGTTATCCACAGATGGCTCCCCAACTAGGATCACCTATGGCATGCAG GGGAGCTCCGGTTATTCTCTGAGATTGTGTAAACTTGTGGACAGCCCTGACTGTACAACAAAAAATAATG CACGTATTGTGGGAGGAAAAACGCTTCTTTTAGGGGAGTGGCCATGGCAGGTCAGCCTGCAAGTGAAGCT GGTATCTCAGACCCATTTGTGTGGAGGGTCCATCATTGGTCGCCAATGGGTACTGACAGCTGCCCATTGC TTTGATGGAATTCCCTATCCAGATGTGTGGCGTATATATGGCGGAATTCTTAGTCTGTCCGAGATTACGA AAGAAACGCCCTTCCTCGAGAATAAAGGAGCTTATTATTCATCAGGAATACAAAGTCTCAGAAGGCAATTA TGATATTGCCTTAATAAAGCTTCAGACGCCCCCTGAATTATACTGAATTCCAAAAACCAATATGCCTGCCT TCCAAAGCTGACACAAATACAATTTATACCAACTGTTGGGTGACTGGATGGGGCTACACGAAGGAACAAG GTGAAACGCAAAATTTCTACAAAAGGCTACTATTCCTTTGGTACCAAATGAAGAATGCCAGAAAAAAAATA CAGAGATTATGTTTATAAACAAGCAGATGATCTGTGCTGGCTACAAAAGAGGCGGAACAGACGCTTGTAAG GGAGATTCCGGTGGCCCCTTAGTCTGTAAACACAGTGGACGGTGGCAGTTGGTGGGTATCACCAGCTGGG GTGAAGGCTGCGCCCGCAAGGACCAACCAGGAGTCTACACCAAAGTTTCTGAGTACATGGACTGGATATT GGAGAAGACACAGAGCAGTGATGTAAGAGCTCTGGAGACATCTTCAGCCTGAGGAGGCTGGGTACCAAGG AGGAAGAACCCAGCTGGCTTTACCACCTGCCCTCAAGGCAAACTAGAGCTCCAGGATTCTCGGCTGTAAA ATGTTGATAATGGTGTCTACCTCACATCCGTATCATTGGATTGAAAATTCAAGTGTAGATATAGTTGCTG AAGACAGCGTTTTGCTCAAGTGTGTTTCCTGCCTTGAGTCACAGGAGCTCCAATGGGAGCATTACAAAGA TCACCAAGCTTGTTAGGAAAGAGAATGATCAAAGGGTTTTATTAGGTAATGAAATGTCTAGATGTGATGC AATTGAAAAAAAGACCCCAGATTCTAGCACAGTCCTTGGGACCATTCTCATGTAACTGTTGACTCTGGAC CTCAGCAGATCTCAGAGTTACCTGTCCACTTCTGACATTTGTTTATTAGAGCCTGATGCTATTCTTTCAA GTGGAGCAAAAAAAAAAAAAAA

[0504] Rat plasma kallikrein (Accession number: NP_036857.2) >gi|162138905|ref|NP_036857.2| Kallikrein B, plasma 1 [Rattus norvegicus] MILFKQVGYFVSLFATVSCGCLSQLYANTFFRGGDLAAIYTPDAQHCQKMCTFHPRCLLFSFLAVSPTKE TDKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQLSACHQDIYEGLDMRGSNFNISKTDSIEECQKL CTNNIHCQFFTYATKAFHRPEYRKSCLLKRSSSGTPTSIKPVDNLVSGFSLKSCALSEIGCPMDIFQHFA FADLNVSHVVTPDAFVCRTVCTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIIQENAVSGYS LFTCRKARPEPCHFKIYSGVAFEGEELNATFVQGADACQETCTKTIRCQFFTYSLLPQDCKAEGCKCSLR LSTDGSPTRITYEAQGSSGYSLRLCKVVESSDCTTKINARIVGGTNSSLGEWPWQVSLQVKLVSQNHMCG GSIIGRQWILTAAHCFDGIPYPDVWRIYGGILNLSEITNKTPFSSIKELIIHQKYKMSEGSYDIALIKLQ TPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKERGETQNILQKATIPLVPNEECQKKYRDYVITKQ MICAGYKEGGIDACKGDSGGPLVCKHSGRWQLVGITSWGEGCARKEQPGVYTKVAEYIDWILEKIQSSKE RALETSPA

[0505] Rat plasma kallikrein mRNA (Accession number: NM_012725) >gi|162138904|ref|NM_012725.2|Rattus norvegicus kallikrein B, plasma 1 (Klkb1), mRNA TGAAGACTAGCTTCATGTGAAGACTCCTTCTCCTCCAGCAGCACAAAGCAACCATCCTTCCAGGATGATT TTATTCAAACAAGTGGGTTATTTTGTTTCCTTGTTCGCTACAGTTTCCTGTGGGTGTCTGTCACAACTGT ATGCAAATACCTTCTTCAGAGGTGGGGATCTGGCTGCCATCTACACCCCGGATGCCCAGCACTGTCAGAA GATGTGCACGTTTCACCCCAGGTGCCTGCTCTTCAGCTTCCTTGCCGTGAGTCCAACCAAGGAGACAGAT AAAAGGTTTGGGTGCTTCATGAAAGAGAGCATTACAGGGACTTTGCCAAGAATACACCGGACAGGGGCCA TTTCTGGTCATTCTTTAAAACAGTGTGGCCATCAATTAAGTGCTTGCCACCAAGACATATACGAAGGACT GGATATGAGAGGGTCCAACTTTAATATATCTAAGACCGACAGTATTGAAGAATGCCAGAAACTGTGCACA AATAATATTCACTGCCAATTTTTCACATATGCTACAAAAGCATTTCACAGACCAGAGTACAGGAAGAGTT GCCTGCTGAAGCGCAGTTCAAGTGGAACGCCCACCAGTATAAAGCCAGTGGACAACCTGGTGTCTGGATT CTCACTGAAGTCCTGTGCTCTCTCAGAGATCGGTTGCCCCATGGATATTTTCCAGCACTTTGCCTTTGCA GACCTGAATGTAAGCCATGTCGTCACCCCCGATGCCTTCGTGTGTCGCACCGTTTGCACCTTCCATCCCA ACTGCCTCTTCTTCACATTCTACACGAATGAGTGGGAGACGGAATCACAGAGGAATGTTTGTTTTCTTAA GACATCTAAAAGTGGAAGACCAAGTCCCCCTATTATTCAAGAAAATGCTGTATCTGGATACAGTCTCTTC ACCTGCAGAAAAGCTCGCCCTGAACCCTGCCATTTCAAGATTTACTCTGGAGTTGCCTTCGAAGGGGAAG AACTGAACGCGACCTTCGTGCAGGGAGCAGATGCGTGCCAAGAGACTTGTACAAAGACCATCCGCTGTCA GTTTTTTACTTACTCATTGCTTCCCCAAGACTGCAAGGCAGAGGGGTGTAAATGTTCCTTAAGGTTATCC ACGGATGGCTCTCCAACTAGGATCACCTATGAGGCACAGGGGAGCTCTGGTTATTCTCTGAGACTGTGTA AAGTTGTGGAGAGCTCTGACTGTACGACAAAAATAAATGCACGTATTGTGGGAGGAACAAACTCTTCTTT AGGAGAGTGGCCATGGCAGGTCAGCCTGCAAGTAAAGTTGGTTTCTCAGAATCATATGTGTGGAGGGTCC ATCATTGGACGCCAATGGATACTGACGGCTGCCCATTGCTTTGATGGGATTCCCTATCCAGACGTGTGGC GTATATATGGCGGGATTCTTAATCTGTCAGAGATTACAAACAAAACGCCTTTCTCAAGTATAAAGGAGCT TATTATTCATCAGAAATACAAAATGTCAGAAGGCAGTTACGATATTGCCTTAATAAAGCTTCAGACACCG TTGAATTATACTGAATTCCAAAAACCAATATGCCTGCCTTCCAAAGCTGACACAAATACAATTTATACCA ACTGCTGGGTGACTGGATGGGGCTACACAAAGGAACGAGGTGAGACCCAAAATATTCTACAAAAGGCAAC TATTCCCTTGGTACCAAATGAAGAATGCCAGAAAAAATATAGAGATTATGTTATAACCAAGCAGATGATC TGTGCTGGCTACAAAGAAGGTGGAATAGATGCTTGTAAGGGAGATTCCGGTGGCCCCTTAGTTTGCAAAC ATAGTGGAAGGTGGCAGTTGGTGGGTATCACCAGCTGGGGCGAAGGCTGTGCCCGCAAGGAGCAACCAGG AGTCTACACCAAAGTTGCTGAGTACATTGACTGGATATTGGAGAAGATACAGAGCAGCAAGGAAAGAGCT CTGGAGACATCTCCAGCATGAGGAGGCTGGGTACTGATGGGGAAGAGCCCAGCTGGCACCAGCTTTACCA CCTGCCCTCAAGTCCTACTAGAGCTCCAGAGTTCTCTTCTGCAAAATGTCGATAGTGGTGTCTACCTCGC ATCCTTACCATAGGATTAAAAGTCCAAATGTAGACACAGTTGCTAAAGACAGCGCCATGCTCAAGCGTGC TTCCTGCCTTGAGCAACAGGAACGCCAATGAGAACTATCCAAAGATTACCAAGCCTGTTTGGAAATAAAA TGGTCAAAGGATTTTTATTAGGTAGTGAAATTAGGTAGTTGTCCTTGGAACCATTCTCATGTAACTGTTG ACTCTGGACCTCAGCAGATCACAGTTACCTTCTGTCCACTTCTGACATTTGTGTACTGGAACCTGATGCT GTTCTTCCACTTGGAGCAAAGAACTGAGAAACCTGGTTCTATCCATTGGGAAAAAGAGATCTTTGTAACA TTTCCTTTACAATAAAAAGATGTTCTACTTGGACTTGAAAAAAAAAAAAAAAAAAAAAAAAAA

[0506] Display Library A display library is a collection of entities, each of which contains an available polypeptide component and a recoverable component that encodes or identifies the polypeptide component. The polypeptide components are diverse, and therefore exhibit a variety of amino acid sequences. The polypeptide components can be of any length, for example, from 3 amino acids to over 300 amino acids. A display library entity can contain two or more polypeptide components, for example, two polypeptide chains of an sFab. In one embodiment, a display library can be used to identify proteins that bind to plasma kallikrein. During selection, the polypeptide component of each member of the library is probed with plasma kallikrein (or a fragment thereof), and if the polypeptide component binds to plasma kallikrein, the display library member is identified, typically by being retained on a support.

[0507] The retained display library members are recovered from the support and analyzed. Analysis can include amplification followed by selection under similar or different conditions. For example, positive and negative selection can be alternated. Analysis can also include determining the amino acid sequence of the polypeptide components and purifying the polypeptide components for detailed characterization.

[0508] Various formats of display libraries can be used, including, for example:

[0509] Phage display: The protein component is usually covalently linked to a bacteriophage coat protein as a result of translation of a nucleic acid encoding the protein component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display has been described, for example, in U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; WO 90 / 02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005) Nat. Biotechnol. 23(3)344-8. Bacteriophage displaying the protein component can be grown and recovered using standard phage preparation methods, such as PEG precipitation from the growth medium. After selection of individual display phages, nucleic acid encoding the selected protein component can be isolated after amplification from cells infected with the selected phage or from the phage itself. Individual colonies or plaques are picked, the nucleic acid isolated and sequenced.

[0510] Other Display Formats Other display formats include cellular display (see, e.g., WO 03 / 029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No. 6,207,446), ribosome display (see, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and E. coli periplasmic display (J Immunol Methods. 2005 Nov 22; PMID: 16337958).

[0511] scaffold Scaffolds useful for display include: antibodies (e.g., Fab fragments, single chain Fv molecules (scFv), single domain antibodies, camel antibodies and camelized antibodies); T cell receptors; MHC proteins; extracellular domains (e.g., fibronectin type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, etc.); TPR repeats; trefoil structures; zinc finger domains; DNA binding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, such as proteases (particularly inactivating proteases), RNases; chaperones, such as thioredoxin and heat shock proteins; intracellular signaling domains (such as SH2 and SH3 domains); linear and constrained peptides; and linear peptide substrates. Display libraries can include synthetic and / or natural diversity. See, for example, U.S. Patent Application Publication No. 2004-0005709.

[0512] Display techniques can also be used to obtain binding proteins (e.g., antibodies) that bind to specific epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the specific epitope or are mutated within the epitope, for example, by alanine. Such non-target molecules can be used as competitor molecules when binding the display library to the target, and in the negative selection methods described below, for example, as pre-eluting agents to capture dissociated display library members that are not specific for the target in a wash solution.

[0513] Iterative Selection In a preferred embodiment, display library technology is used in an iterative manner. A first display library is used to identify one or more binding proteins for a target. These identified proteins are then mutated using mutagenesis to form a second display library. High affinity binding proteins are then selected from the second library, for example, by using high stringency or high competitive binding and washing conditions.

[0514] In some embodiments, mutagenesis targets the region of the binding surface. For example, if the identified binding protein is an antibody, mutagenesis can be directed to the CDR region of the heavy or light chain described herein. Furthermore, mutagenesis can be directed to the framework region adjacent or adjacent to the CDR. In the case of an antibody, mutagenesis can be limited to one or a few CDRs, for example, to achieve precise stepwise improvement. Examples of mutagenesis techniques include error-prone PCR, recombination, DNA shuffling, site-directed mutagenesis and cassette mutagenesis.

[0515] In one example of iterative selection, the methods described herein are first used to identify proteins from a display library that bind plasma kallikrein with at least a minimal binding specificity for the target or with a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 0.5 nM, 1 nM, 10 nM, or 100 nM. The nucleic acid sequence encoding the initially identified protein is used as a template nucleic acid for the introduction of mutations to identify a second protein that has, for example, enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial protein.

[0516] Off Speed ​​Selection Because a slow off-rate can predict high affinity, particularly for interactions between a polypeptide and its target, the methods described herein can be used to isolate binding proteins that have desirable (e.g., slow) kinetic off-rates of their binding interactions to a target.

[0517] To select the slowly dissociating binding protein from the display library, the library is contacted with immobilized target.The immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules.The bound binding protein is then eluted with a second solution that contains a saturating amount of free target or a high affinity competitive monoclonal antibody specific to the target, i.e., a copy of the target that is not bound to the particle.The free target binds to the biomolecules that dissociate from the target.The saturating amount of free target effectively inhibits rebinding compared to a much lower concentration of immobilized target.

[0518] The second solution can have a solution condition that is substantially physiological or stringent.Usually, the solution condition of the second solution is the same as that of the first solution.The fractions of the second solution are collected in a time sequence to distinguish between early fractions and later fractions.Later fractions contain biomolecules that dissociate from the target at a slower rate than the biomolecules in early fractions.

[0519] Furthermore, it is possible to recover display library members that remain bound to the target after extended incubation. They can be dissociated using chaotropic conditions or amplified while still bound to the target. For example, target-bound phage can be contacted with bacterial cells.

[0520] Specificity Selection or Screening The display library screening methods described herein can include a selection or screening step to discard display library members that bind non-target molecules, such as streptavidin on magnetic beads, blocking agents (e.g., bovine serum albumin, non-fat bovine milk, soy protein, etc.), any capture or target immunized monoclonal antibody, or non-transfected cells that do not express the target.

[0521] In one embodiment, a so-called "negative selection" step is used to distinguish between the target, related non-target molecules, and related but different non-target molecules. The display library or a pool thereof is contacted with the non-target molecules. Members of the sample that do not bind to the non-target are collected and used in a subsequent selection for binding to the target molecule or in a further negative selection. The negative selection step may be performed before or after the selection of library members that bind to the target molecule.

[0522] In another embodiment, a screening step is used. After the members of the display library are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., the non-targets listed above). For example, high-throughput ELISA screening can be used to obtain this data. ELISA screening can also be used to obtain quantitative data on the binding of each library member to the target and the inter-species reactivity with related targets or subunits of the target (e.g., plasma kallikrein), and also under different conditions such as pH 6 or pH 7.5. Comparison of the non-target and target binding data (e.g., using a computer and software) is performed to identify library members that specifically bind to the target.

[0523] Other examples of expression libraries Other types of protein collections (e.g., expression libraries), including, for example, protein arrays of antibodies (see, e.g., De Wildt et al. (2000) Nat. Biotechnol. 18:989-994), lambda gt11 libraries, two-hybrid libraries, etc., can be used to identify proteins with particular properties (e.g., the ability to bind plasma kallikrein).

[0524] Library Example It is possible to immunize non-human primates to recover primate antibody genes that can be displayed on phage (see below). From such libraries, antibodies can be selected that bind to the antigen used for immunization. See, for example, Vaccine. (2003) 22(2): 257-67 or Immunogenetics. (2005) 57(10): 730-8. By immunizing chimpanzees or macaques in this manner and using various means to select or screen for primate antibodies that bind and inhibit plasma kallikrein, primate antibodies that bind and inhibit plasma kallikrein can be obtained. Also, chimeras of primatized Fabs with human constant regions can be made, see Curr Opin Mol Ther. (2004) 6(6): 675-83. "Primatized antibodies are genetically engineered from cynomolgus (macaque) and human components and are structurally indistinguishable from human antibodies. They are therefore less likely to cause adverse reactions in humans and may be suitable for long-term chronic treatment." (Curr Opin Investig Drugs. (2001) 2(5):635-8).

[0525] One exemplary type of library displays a diverse pool of polypeptides, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain. Of interest are display libraries in which library members include primate or "primatized" (e.g., human, non-human primate, or "humanized") immunoglobin domains (e.g., immunoglobin variable domains) or chimeric primatized Fabs having human constant regions. Human or humanized immunoglobin domain libraries can be used to identify human or "humanized" antibodies that recognize, for example, human antigens. These antibodies can avoid being recognized and targeted as antigens when administered to humans because the antibody constant and framework regions are human. The constant regions can also be optimized to recruit effector functions of the human immune system. The in vitro display selection process overcomes the inability of the normal human immune system to generate antibodies against self-antigens.

[0526] In a typical antibody display library, a polypeptide comprising a VH domain and a VL domain is displayed. An "immunoglobulin domain" refers to a domain derived from the variable or constant domain of an immunoglobulin molecule. An immunoglobulin domain usually comprises about seven beta strands and two beta sheets formed by conserved disulfide bonds (see, e.g., A.F. Williams and A.N. Barclay, 1988, Ann. Rev. Immunol. 6:381-405). In a display library, an antibody can be displayed as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using one polypeptide chain). Other formats can also be used.

[0527] As with Fab and other formats, the displayed antibody may include one or more constant regions as part of the light and / or heavy chain. In one embodiment, each chain includes one constant region, e.g., as with Fab. In other embodiments, additional constant regions are displayed.

[0528] Antibody libraries can be constructed in a number of steps (see, e.g., de Haard et al., 1999, J. Biol. Chem. 274:18218-30; Hoogenboom et al., 1998, Immunotechnology 4:1-20; Hoogenboom et al., 2000, Immunol. Today 21:371-378, and Hoet et al. (2005) Nat Biotechnol. 23(3):344-8). Moreover, elements of each step can be combined with elements of other steps. These steps can be used to introduce mutations within a single immunoglobulin domain (e.g., VH or VL) or within multiple immunoglobulin domains (e.g., VH and VL). Mutations can be introduced within one or more regions of an immunoglobulin variable domain that can be mutated, e.g., CDR1, CDR2, CDR3, FR1, FR2, FR3 and / or FR4, which are regions of either or both the heavy and light chain variable domains. For example, mutation(s) may be introduced in all three CDRs of a given variable domain, or, for example, in CDR1 and CDR2 of a heavy chain variable domain. Any combination is possible. In one step, an antibody library is constructed by inserting diverse oligonucleotides encoding the CDRs into the corresponding regions of the nucleic acid. The oligonucleotides can be synthesized using monomeric nucleotides or trinucleotides. For example, Knappik et al., 2000, J. Mol. Biol. 296:57-86, describes a method for constructing oligonucleotides encoding CDRs using trinucleotide synthesis and a template with designed restriction sites to accept the oligonucleotides.

[0529] In another process, an animal (e.g., a rodent) is immunized with plasma kallikrein. Optionally, the animal is boosted with an antigen to further stimulate the response. Spleen cells are then isolated from the animal and nucleic acids encoding the VH and / or VL domains are amplified and cloned for expression in a display library.

[0530] In yet another step, an antibody library is constructed from nucleic acids amplified from natural germline immunoglobulin genes. The amplified nucleic acids include nucleic acids encoding VH and / or VL domains. Sources of immunoglobulin-encoding nucleic acids are described below. Amplification can include, for example, PCR or another amplification method using primers that anneal to conserved constant regions.

[0531] Nucleic acids encoding immunoglobulin domains can be obtained from immune cells of, for example, primates (e.g., humans), mice, rabbits, camels or rodents. In one example, cells are selected for specific properties. B cells of various maturation stages can be selected. In another example, B cells are naive.

[0532] In one embodiment, B cells expressing surface-bound IgM, IgD or IgG molecules are sorted using fluorescence activated cell sorting (FACS). In addition, B cells expressing different isotypes of IgG can be separated. In another preferred embodiment, B or T cells are cultured in vitro. Cells can be stimulated in vitro, for example, by culturing with feeder cells or by adding mitogens or other regulatory reagents, such as antibodies against CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin or pokeweed mitogen.

[0533] In another embodiment, the cells are isolated from a subject having a condition described herein, eg, a plasma kallikrein-associated disease or condition.

[0534] In a preferred embodiment, the cells have an activated program of somatic hypermutation. For example, the cells can be stimulated to undergo multicellular mutation of immunoglobulin genes by treatment with anti-immunoglobulin, anti-CD40 and anti-CD38 antibodies (see, e.g., Bergthorsdottir et al., 2001, J. Immunol. 166:2228). In another embodiment, the cells are naive.

[0535] Nucleic acids encoding immunoglobulin variable domains can be isolated from natural repertoires by the following exemplary method: RNA is first isolated from immune cells. Full-length (e.g., capped) mRNA is isolated (e.g., by degrading uncapped RNA with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and cDNA is generated using reverse transcription.

[0536] Reverse transcription of the first (antisense) strand may be performed in any manner using any suitable primer. See, for example, de Haard et al., 1999, J. Biol. Chem. 274:18218-30. The primer binding region may be constant between different immunoglobulins, for example, to reverse transcribe different isotypes of immunoglobulins. The primer binding region may also be specific for a particular isotype of immunoglobulin. Usually, the primer is specific for a region 3' to the sequence encoding at least one CDR. In another embodiment, poly-dT primers may be used (and may be suitable for heavy chain genes).

[0537] A synthetic sequence can be linked to the 3' end of the reverse transcribed strand. This synthetic sequence can be used as the primer binding site for forward primer binding during PCR amplification after reverse transcription. The use of synthetic sequence eliminates the need to use a pool of different forward primers to fully capture available diversity.

[0538] The genes encoding the variable domains are then amplified, for example using one or more rounds. When multiple rounds are used, nested primers can be used to increase fidelity. The amplified nucleic acids are then cloned into a display library vector.

[0539] Secondary screening method After selecting the candidate library members that bind to the target, each candidate library member can be further analyzed to further characterize, for example, its binding properties to the target, for example, plasma kallikrein. Each candidate library member can be subjected to one or more secondary screening assays. The assays can be for binding properties, catalytic properties, inhibitory properties, physiological properties (e.g., cytotoxicity, renal clearance, immunogenicity) and structural properties (e.g., stability, conformation, oligomerization state) or other functional properties. The same assay can be used repeatedly and under different conditions to determine, for example, sensitivity to pH, ions or temperature.

[0540] If desired, the members of the display library can be used in the assay directly, or a recombinant polypeptide made from a nucleic acid encoding the selected polypeptide, or a synthetic peptide synthesized based on the sequence of the selected polypeptide. In the case of a selected Fab, the Fab can be evaluated or modified and made as an intact IgG protein. Examples of assays for binding properties are given below.

[0541] ELISA ELISA assays can be used to evaluate binding proteins. For example, each protein is contacted with a microtiter plate whose bottom surface is coated with the target, e.g., a limiting amount of the target. The plate is washed with a buffer to remove non-specifically bound polypeptides. The amount of binding protein bound to the target on the plate is then determined by probing the plate with an antibody that can recognize the binding protein, e.g., the tag or constant portion of the binding protein. The antibody is linked to a detection system (e.g., an enzyme such as alkaline phosphatase or horseradish peroxidase (HRP), which produces a colored product upon addition of an appropriate substrate).

[0542] Homogeneous binding assay The ability of the binding proteins described herein to bind to a target may be analyzed using a homogeneous assay (i.e., no additional liquid manipulations are required after all components of the assay have been added. For example, fluorescence resonance energy transfer (FRET) may be used as a homogeneous assay (see, e.g., Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,868,103). A fluorescent label on a first molecule (e.g., a target) is attached to the first molecule (e.g., a molecule identified in a fraction) such that if the second molecule (e.g., a target) is in close proximity to the first molecule (e.g., a molecule identified in a fraction), the emitted fluorescent energy can be absorbed by the fluorescent label on the second molecule. A fluorophore label is selected. The fluorescent label on the second molecule fluoresces upon absorbing the transferred energy. The efficiency of energy transfer between the labels is related to the distance of the separated molecules, so the spatial relationship between the molecules can be assessed. If binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. Binding events set up for monitoring by FRET can be conveniently measured by standard fluorometric detection means, for example with a fluorometer. Binding curves can be generated by titrating the amount of the first or second binding molecule to assess the equilibrium binding constant.

[0543] Another example of a homogeneous assay is ALPHASCREEN™ (Packard Bioscience, Meriden CT). ALPHASCREEN™ uses two labeled beads. One bead emits singlet oxygen when excited by a laser. The other bead emits a light signal when it is struck by singlet oxygen that diffuses from the first bead. This signal is only generated if the two beads are in close proximity. One bead can be bound to a member of the display library and the other bead to a target. The signal is measured to determine the degree of binding.

[0544] Surface Plasmon Resonance (SPR) SPR can be used to analyze the interaction of binding proteins with targets. SPR, or biomolecular interaction analysis (BIA), detects biospecific interactions in real time without labeling any of the interactants. A change in mass at the binding surface of the BIA chip (indicative of a binding event) changes the refractive index of light near the surface (an optical phenomenon called surface plasmon resonance (SPR)). This change in refractive index generates a detectable signal that is measured as an indication of real-time reactions between biomolecules. The use of SPR is described, for example, in U.S. Pat. No. 5,641,640; Raether, 1988, Surface Plasmons Springer Verlag; Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345; Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705, and online materials provided by BIAcore International AB (Uppsala, Sweden).

[0545] The information obtained from SPR is used to determine the equilibrium dissociation constant (K D ) and K on and K. offAccurate qualitative measurements of kinetic parameters, including K, are obtained. Such data can be used to compare different biomolecules. For example, proteins selected from an expression library can be compared to determine whether they have a high affinity for a target or a slow K off This information can also be used to determine structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of the mature form of a parent protein can be compared to those of the parent protein. Certain binding parameters, such as high affinity and slow K off Mutant amino acids at given positions associated with the target may be identified. This information can be combined with structural modeling (e.g., using homology modeling, energy minimization principles, or structure determination by X-ray crystallography or NMR). As a result, knowledge about the physical interactions between the protein and its target can be formulated and used to guide other design steps.

[0546] Cellular assays The binding protein can be screened for its ability to bind to cells that express the target of interest transiently or stably on the cell surface.For example, the plasma kallikrein binding protein can be fluorescently labeled, and the binding to plasma kallikrein in the absence of antagonistic antibody can be measured by flow cytometry, e.g., by the change in fluorescence intensity using a FACS instrument.

[0547] Examples of other methods for obtaining plasma kallikrein binding proteins In addition to the use of display libraries, other methods can be used to obtain plasma kallikrein binding proteins (e.g., antibodies). For example, plasma kallikrein protein or fragments thereof can be used as antigens in non-human animals, such as rodents.

[0548] In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, it is possible to engineer mouse strains that have large fragments of the human Ig locus and lack mouse antibody production. Hybridoma technology can be used to generate and select antigen-specific monoclonal antibodies (Mabs) derived from genes with the desired specificity. See, for example, XENOMOUSE™, Green et al., 1994, Nat. Gen. 7:13-21; U.S. Patent Application Publication No. 2003-0070185, International Publication No. WO 96 / 34096 published October 31, 1996, and International Application No. PCT / US96 / 05928 filed April 29, 1996.

[0549] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified, for example humanized or deimmunized. Winter describes a CDR grafting method that can be used to prepare humanized antibodies (GB2188638A filed March 26, 1987; U.S. Patent No. 5,225,539). All CDRs of a particular human antibody can be replaced with at least a portion of a non-human CDR, or only a portion of the CDRs can be replaced with a non-human CDR. It is only necessary to replace the number of CDRs required for the binding of the humanized antibody to a given antigen.

[0550] Humanized antibodies can be made by replacing sequences of Fv variable regions that are not directly involved in antigen binding with the corresponding sequences from human Fv variable regions. General methods for making humanized antibodies are described by Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762. The methods include isolating, manipulating, and expressing nucleic acid sequences that code for all or part of immunoglobulin Fv variable regions from at least one of the heavy or light chains. Numerous sources of such nucleic acids are available. For example, nucleic acids can be obtained from hybridomas that produce antibodies against a given target, as described above. The recombinant DNA encoding the humanized antibody or fragment thereof can then be cloned into an appropriate expression vector.

[0551] Reducing the immunogenicity of plasma kallikrein-binding proteins Immunoglobin plasma kallikrein binding proteins (e.g., IgG or Fab plasma kallikrein binding proteins) can be modified to reduce immunogenicity. Reduced immunogenicity is desirable in plasma kallikrein binding proteins intended for use as therapeutics, as it reduces the probability that a subject will develop an immune response to the therapeutic molecule. Techniques useful for reducing the immunogenicity of plasma kallikrein binding proteins include removing / modifying potential human T-cell epitopes and "germlining" sequences other than the CDRs (e.g., framework and Fc).

[0552] Plasma kallikrein-binding antibodies can be modified, for example, by specific removal of human T-cell epitopes, i.e., "deimmunization," by the methods disclosed in WO 98 / 52976 and WO 00 / 34317. Briefly, the heavy and light chain variable regions of the antibody are analyzed for peptides that bind to class II MHC; these peptides represent potential T-cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). Potential T-cell epitopes can be detected using a computer modeling method called "peptide threading," as described in WO 98 / 52976 and WO 00 / 34317, and in addition, motifs present in the VH and VL sequences can be searched against a database of class II human MHC-binding peptides. These motifs bind to any of the 18 major class II MHC DR allotypes, and thus constitute potential T-cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting a small number of amino acid residues in the variable regions, preferably by single amino acid substitutions. As long as possible conservative substitutions are made, often but not exclusively, amino acids common to this position in human germline antibody sequences can be used. Human germline sequences are disclosed in Tomlinson, IA et al., 1992, J. Mol. Biol. 227:776-798; Cook, GP et al., 1995, Immunol. Today Vol. 16(5):237-242; Chothia, D. et al., 1992, J. Mol. Bio. 227:799-817. The V Base Registry provides an extensive registry of human immunoglobulin variable region sequences (compiled by Tomlinson, IA et al., MRC Centre for Protein, Cambridge, UK). After identifying the changes that result in deimmunization, the V Base can be modified by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, etc.). H and V L A nucleic acid encoding the mutagenized variable sequence can be constructed. The mutagenized variable sequence can optionally be fused to a human constant region, such as a human IgG1 or kappa constant region.

[0553] In some cases, potential T cell epitopes include residues that are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased toward CDRs. In addition, potential T cell epitopes may occur within framework residues that are important for antibody structure and binding. Changes that remove these potential epitopes may need to be further investigated, for example, by making and testing altered and unaltered chains. If possible, potential T cell epitopes that overlap with CDRs are removed by substitutions outside the CDRs. In some cases, because modifications within the CDRs are the only option, variants with and without this substitution must be tested. In some cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that may be important for antibody binding. In such cases, variants with and without this substitution should be tested. Therefore, to identify the optimal deimmunized antibody, multiple variants of deimmunized heavy and light chain variable regions may be designed and various combinations of heavy / light chains may be tested. The final deimmunized antibody can then be selected by considering the binding affinity of the various variants together with the degree of deimmunization, i.e., the number of potential T cell epitopes remaining in the variable regions. Deimmunization can be used to modify any antibody, including antibodies that contain non-human sequences, such as synthetic antibodies, murine antibodies, other non-human monoclonal antibodies, or antibodies isolated from a display library.

[0554] A plasma kallikrein binding antibody is "germlined" by replacing one or more non-germline amino acids in the framework region with the corresponding germline amino acid of the antibody, so long as the binding properties are substantially retained. Similar methods can also be used in constant regions, e.g., constant immunoglobulin domains.

[0555] An antibody that binds plasma kallikrein, such as an antibody described herein, may be modified to make the variable region of the antibody more similar to one or more germline sequences. For example, the antibody may include one, two, three or more amino acid substitutions, for example in the framework, CDR or constant region, to make it more similar to a reference germline sequence. One example of a method for germlining may include identifying one or more germline sequences that are similar to the sequence of an isolated antibody (e.g., most similar in a particular database). The isolated antibody is then mutated (at the amino acid level) gradually or in combination with other mutations. For example, a nucleic acid library is generated that includes sequences that code for some or all of the possible germline mutations. The mutated antibodies are then evaluated to identify, for example, antibodies that have one or more additional germline residues compared to the isolated antibody and are still useful (e.g., have functional activity). In one embodiment, as many germline residues as possible are introduced into the isolated antibody.

[0556] In one embodiment, mutagenesis is used to replace or insert one or more germline residues into the framework and / or constant region. For example, the germline framework and / or constant region residues can be derived from a similar (e.g., most similar) germline sequence to the non-variable region to be modified. After mutagenesis, the activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine whether one or more germline residues are tolerated (i.e., do not abolish activity). Similar mutagenesis can be performed in the framework region.

[0557] Selection of germline sequences can be performed in various ways. For example, a germline sequence can be selected if it meets a certain criterion of selectivity or similarity, such as at least a certain percentage of identity, for example at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% identity. Selection can be performed using at least 2, 3, 5 or 10 germline sequences. In the case of CDR1 and CDR2, identification of a similar germline sequence can include selecting one such sequence. In the case of CDR3, identification of a similar germline sequence can include selecting one such sequence, but can include the use of two germline sequences that are separately involved in the amino-terminal and carboxy-terminal portions of the sequence. In other embodiments, two or more germline sequences are used, for example to form a consensus sequence.

[0558] In one embodiment, with respect to a particular reference variable domain sequence, such as a sequence described herein, the relevant variable domain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% of the CDR amino acid positions that are not identical to the residues in the reference CDR sequence, i.e., the residues that are identical to the residues at the corresponding positions in a human germline sequence (i.e., an amino acid sequence encoded by a human germline nucleic acid).

[0559] In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, the relevant variable domain sequence has at least 30, 50, 60, 70, 80, 90 or 100% of the FR regions that are identical to a FR sequence derived from a human germline sequence, e.g., the germline sequence related to the reference variable domain sequence.

[0560] Thus, it is possible to isolate antibodies that have similar activity to a given antibody of interest, but that are more similar to one or more germline sequences, particularly one or more human germline sequences. For example, the antibody can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to the germline sequence in regions other than the CDRs (e.g., framework regions). Furthermore, the antibody can contain at least 1, 2, 3, 4, or 5 germline residues in the CDR regions that are derived from the germline sequence that is similar (e.g., most similar) to the variable region to be modified. The germline sequences of primary interest are human germline sequences. The activity (e.g., K) of the antibody can be determined by the following steps: A The binding activity (as measured by ) can be within 1 or 100, 10, 5, 2, 0.5, 0.1 and 0.001 fold of the original antibody.

[0561] The germline sequences of human immunoglobin genes have been determined and are available from a number of sources, including the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT) and the V Base registry (compiled by Tomlinson, IA et al., MRC Centre for Protein Engineering, Cambridge, UK).

[0562] V kappa Examples of germline reference sequences include O12 / O2, O18 / O8, A20, A30, L14, L1, L15, L4 / 18a, L5 / L19, L8, L23, L9,L24, L11, L12, O11 / O1, A17, A1, A18, A2, A19 / A3, A23, A27, A11, L2 / L16, L6, L20, L25, B3, B2, A26 / A10, and A14. See, e.g., Tomlinson et al., 1995, EMBO J. 14(18):4628-3.

[0563] The germline reference sequence of the HC variable domain can be based on a sequence with a particular canonical structure, such as the 1-3 structure in the H1 and H2 hypervariable loops. The canonical structures of the hypervariable loops of immunoglobulin variable domains can be deduced from the sequence as described in Chothia et al., 1992, J. Mol. Biol. 227:799-817; Tomlinson et al., 1992, J. Mol. Biol. 227:776-798; and Tomlinson et al., 1995, EMBO J. 14(18):4628-38. Examples of sequences having a 1-3 structure include DP-1, DP-8, DP-12, DP-2, DP-25, DP-15, DP-7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3005f3, DP-46, DP-47, DP-58, DP-49, DP-50, DP-51, DP-53 and DP-54.

[0564] Protein production Standard recombinant nucleic acid methods can be used to express the protein that binds to plasma kallikrein.Generally, the nucleic acid sequence that codes for the protein is cloned into a nucleic acid expression vector.Of course, if the protein comprises multiple polypeptide chains, each chain is cloned into an expression vector, for example, into the same or different vectors, and expressed in the same or different cells.

[0565] Antibody production Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells (see, e.g., Nadkarni, A. et al., 2007 Protein Expr Purif 52(1):219-29). For example, if the Fab is encoded by a sequence in a phage display vector, which contains a suppressible stop codon between the display and the bacteriophage protein (or a fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress the stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the periplasm and / or medium.

[0566] Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibody (e.g., scFv) is expressed in a yeast cell such as Pichia (see, e.g., Powers et al., 2001, J. Immunol. Methods. 251:123-35; Schoonooghe S. et al., 2009 BMC Biotechnol. 9:70; Abdel-Salam, HA. et al., 2001 Appl Microbiol Biotechnol 56(1-2):157-64; Takahashi K. et al., 2000 Biosci Biotechnol Biochem 64(10):2138-44; Edqvist, J. et al., 1991 J Biotechnol 20(3):291-300), Hanseula, or Saccharomyces.Those skilled in the art will be familiar with the various aspects of the present invention, including, for example, oxygen conditions (see, e.g., Baumann K. et al., 2010 BMC Syst. Biol. 4:141), osmolality (see, e.g., Dragosits, M. et al., 2010 BMC Genomics 11:207), temperature (see, e.g., Dragosits, M. et al., 2009 J Proteome Res. 8(3):1380-92), fermentation conditions (see, e.g., Ning, D. et al., 2005 J. Biochem. and Mol. Biol. 38(3):294-299), yeast strains (see, e.g., Kozyr, AV et al., 2004 Mol Biol (Mosk) 38(6):1067-75; Horwitz, AH. et al., 1988 Proc Natl Acad Sci USA 85(22):8678-82; Bowdish, K. et al. 1991 J Biol Chem 266(18):11901-8), protein overexpression that enhances antibody production (see, e.g., Gasser, B. et al., 2006 Biotechol. Bioeng. 94(2):353-61), culture acidity levels (see, e.g., Kobayashi H. et al., 1997 FEMS Microbiol Lett 152(2):235-42), and substrate and / or ion concentrations (see, e.g., Ko JH. et al., 2996 Appl Biochem Biotechnol 60(1):41-8) can be optimized to maximize antibody production in yeast. Moreover, yeast systems can be used to produce antibodies with long half-lives (see, eg, Smith, BJ. et al., 2001 Bioconjug Chem 12(5):750-756).

[0567] In a preferred embodiment, the antibody is produced in a mammalian cell. Suitable mammalian host cells for the expression of a cloned antibody or an antigen-binding fragment thereof include Chinese Hamster Ovary (CHO) cells (including dhfr-CHO cells (e.g., with DHFR selection marker, as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621)), lymphoid cell lines (e.g., NS0 myeloma cells and SP2 cells), COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(1-2):65-80) and transgenic animals, such as cells derived from a transgenic mammal. For example, the cell is a mammary epithelial cell.

[0568] In some embodiments, plasma kallikrein binding proteins are produced in plant cells or in cell-free systems (see, eg, Galeffi, P. et al., 2006 J Transl Med 4:39).

[0569] In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vector may carry additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selection marker gene sequences. The selection marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, the selection marker gene usually confers resistance to drugs, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. Suitable selection marker genes include the dihydrofolate reductase (DHFR) gene (dhfr for methotrexate selection / amplification), and the like. - host cell) and the neo gene (for G418 selection).

[0570] In one example of a system for recombinant expression of an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is transfected with dhfr - The antibody heavy and light chain genes are introduced into CHO cells. In the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to an enhancer / promoter regulatory element (e.g., CMV enhancer / AdMLP promoter regulatory element or SV40 enhancer / AdMLP promoter regulatory element from SV40, CMV, adenovirus, etc.) to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformed cells, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies may be isolated by affinity chromatography using a matrix bound to protein A or protein G.

[0571] For antibodies that include an Fc domain, the antibody production system may produce antibodies in which the Fc region is glycosylated. For example, the Fc domain of an IgG molecule is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary oligosaccharides. This glycosylation has been shown to be necessary for effector functions mediated by Fcg receptors and complement C1q (Burton and Woof, 1992, Adv. Immunol. 51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76). In one embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain may also include other eukaryotic post-translational modifications.

[0572] Antibodies can also be produced in transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary gland of transgenic mammals. A transgene is constructed that includes a milk-specific promoter, a nucleic acid encoding the antibody of interest, and a signal sequence for secretion. The milk produced by the female transgenic mammal contains the antibody of interest secreted therein. The antibody can be purified from the milk or used directly depending on the application.

[0573] Characterization of plasma kallikrein-binding proteins I C 50 (50% inhibitory concentration) and EC 50 (50% effective concentration) Among a series or group of binding proteins, IC 50 or EC 50 Binding proteins with low IC 50 or EC 50 An exemplary binding protein may have an IC of 2 pM plasma kallikrein as measured in an in vitro assay for inhibition of plasma kallikrein activity. 50 The value is less than 800nM, 400nM, 100nM, 25nM, 5nM or 1nM.

[0574] Plasma kallikrein binding proteins can also be characterized with respect to activity of factor XII and HMWK (high molecular weight kininogen) signaling events, such as production of factor XIIa and / or bradykinin.

[0575] The binding proteins can also be evaluated for selectivity for plasma kallikrein. For example, a plasma kallikrein binding protein can be assayed for its potency against plasma kallikrein and a panel of kallikreins, and the IC for each kallikrein can be determined. 50 Value or EC 50 In one embodiment, a low IC value for plasma kallikrein can be determined. 50 Value or EC50 value and high IC against other kallikreins in the test panel 50 Value or EC 50 Compounds that exhibit a value, for example at least 2-fold, 5-fold or 10-fold higher, are considered to be selective for plasma kallikrein.

[0576] Pharmacokinetic studies with plasma kallikrein binding proteins can be performed in rats, mice or monkeys to determine the half-life of plasma kallikrein in blood. Similarly, the effect of the binding proteins can be evaluated in vivo, for example, in animal models of disease (e.g., carrageenan-induced edema of the rat hind paw (Winter et al., Proc Soc Exp Biol Med. 1962;111:544-7)) for use as therapeutic agents for treating, for example, diseases or conditions described herein (e.g., plasma kallikrein-related diseases).

[0577] Pharmaceutical Compositions Proteins (e.g., binding proteins) that bind to plasma kallikrein (e.g., human plasma kallikrein and / or mouse plasma kallikrein) and, for example, include at least one immunoglobin variable region may be used in methods of treating (or preventing) plasma kallikrein-related diseases or conditions. The binding proteins may be present as compositions, e.g., pharma- ceutically acceptable compositions or pharmaceutical compositions, that include plasma kallikrein-binding proteins, antibody molecules or other polypeptides or peptides that have been identified as binding to plasma kallikrein as described herein. The plasma kallikrein-binding proteins may be formulated with a pharma- ceutically acceptable carrier. Pharmaceutical compositions include therapeutic and diagnostic compositions, e.g., compositions that include labeled plasma kallikrein-binding proteins for in vivo imaging and compositions that include labeled plasma kallikrein-binding proteins for treating (or preventing) plasma kallikrein-related diseases.

[0578] Pharmaceutically acceptable carriers include any physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Carriers are preferably suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion), although carriers suitable for inhalation and intranasal administration are also contemplated. Depending on the route of administration, plasma kallikrein binding protein may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0579] A pharma- ceutically acceptable salt is a salt that retains the desired biological activity of a compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanes, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, and non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0580] The composition may be in various forms. Such forms include, for example, liquid, semi-solid and solid dosage forms, such as liquids (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form may depend on the intended mode of administration and therapeutic use. Many compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administering antibodies to humans. An example of a mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the plasma kallikrein binding protein is administered by intravenous infusion or injection. In another preferred embodiment, the plasma kallikrein binding protein is administered by intramuscular or subcutaneous injection.

[0581] The terms "parenteral administration" and "administering parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0582] The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions may be prepared by incorporating the required amount of binding protein into a suitable solvent with one or a combination of the above-listed ingredients as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile solvent containing the basic dispersion medium and the other required ingredients listed above. For sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, in which the powder of the active ingredient and any additional desired ingredients is obtained from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0583] Although plasma kallikrein binding protein can be administered in a variety of ways, for many applications a preferred route / mode of administration is intravenous injection or infusion. For example, in therapeutic applications, plasma kallikrein binding protein can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg / min, at a rate of about 1 to 100 mg / m 2 or 7 to 25 mg / m 2 The dosage may be administered until a dose of 100 mg / kg is reached. The route and / or mode of administration may vary depending on the desired result. In certain embodiments, the active compound may be prepared with a carrier, such as a sustained release formulation, including implants and microencapsulated delivery systems, which protect the compound from rapid release. Biodegradable biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Numerous methods are available for preparing such formulations. For example, see Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, 1978, Marcel Dekker, Inc., New York.

[0584] The pharmaceutical composition may be administered using a medical device. For example, in one embodiment, the pharmaceutical compositions disclosed herein may be administered using a device, such as a needleless hypodermic injection device, a pump, or an implant.

[0585] In certain embodiments, plasma kallikrein binding protein is formulated to obtain suitable in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed herein cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported into specific cells or organs, enhancing targeted drug delivery (see, for example, VV Ranade, 1989, J. Clin. Pharmacol. 29:685).

[0586] The dosage regimen is adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or multiple divided doses can be administered gradually, or the dose can be proportionally reduced or increased if indicated by the exigent therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form to facilitate administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable for unitary administration to the subject to be treated, each unit containing a predetermined amount of active compound calculated to obtain a desired therapeutic effect together with the necessary pharmaceutical carrier. The specifications of dosage unit form can be determined or directly determined by (a) the inherent properties of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technology of compounding such active compound to address the sensitivities in individuals.

[0587] A non-limiting example range for a therapeutically or prophylactically effective amount of a binding protein (e.g., an antibody) disclosed herein is 0.1-20 mg / kg, more preferably 1-10 mg / kg. Anti-plasma kallikrein antibodies can be administered, for example, by intravenous infusion at a rate of about 1-100 mg / m, for example, at a rate of less than 30, 20, 10, 5, or 1 mg / min. 2 or about 5-30 mg / m 2 The amount of the composition may be increased until a dose of 100 mg / kg is reached. For binding proteins with molecular weights smaller than antibodies, appropriate amounts may be proportionally smaller. The numerical dosage may vary depending on the type and severity of the condition to be alleviated. For a particular subject, a particular dosage regimen may be gradually adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the composition.

[0588] The pharmaceutical compositions disclosed herein may contain a "therapeutically effective amount" or a "prophylactically effective amount" of the plasma kallikrein binding protein disclosed herein. A "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve the desired prophylactic result. A therapeutically effective amount of a composition may vary depending on factors such as the individual's pathological condition, age, sex, and weight, and the ability of the protein to induce a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0589] A "therapeutically effective amount" preferably modulates a measurable parameter, such as circulating IgG antibody levels, to a statistically significant extent, or at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated subjects. The ability of a compound to modulate a measurable parameter, such as a disease-related parameter, can be evaluated in an animal model system that is predictive of efficacy in human diseases and conditions, such as plasma kallikrein-related diseases. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate the parameter in vitro.

[0590] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic amount is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0591] Stabilization and retention In one embodiment, the plasma kallikrein binding protein is physically conjugated to a moiety that enhances its stabilization and / or retention in the circulation, e.g., in blood, serum, lymph or other tissues, e.g., at least 1.5, 2, 5, 10 or 50 fold. For example, the plasma kallikrein binding protein can be conjugated to a polymer, e.g., a substantially non-immunogenic polymer, such as a polyalkylene oxide or polyethylene oxide. Suitable polymers vary widely in their weight. Polymers having number average molecular weights ranging from about 200 to about 35,000 (or about 1,000 to about 15,000 and 2,000 to about 12,500) can be used. For example, the plasma kallikrein binding protein can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol and polyvinylpyrrolidone. A non-limiting list of such polymers includes polyalkylene oxide homopolymers (such as polyethylene glycol (PEG) or polypropylene glycol), polyoxyethylenated polyols, copolymers thereof and block copolymers thereof (provided that the water solubility of the block copolymers is maintained).

[0592] The plasma kallikrein binding protein can also be conjugated to a carrier protein, e.g., a serum albumin, such as human serum albumin (see, e.g., Smith, BJ. et al., 2001 Bioconjug Chem 12(5):750-756). For example, a carrier protein is conjugated to the plasma kallikrein binding protein using a translational fusion.

[0593] Plasma kallikrein binding proteins can also be modified as HES derivatives. In the process of HES conversion of plasma kallikrein binding proteins, the proteins are modified with hydroxyethyl starch. HES conversion of proteins can extend the circulating half-life of the proteins and also reduce renal clearance.

[0594] kit The plasma kallikrein binding proteins described herein may be provided in a kit, e.g., as a component of a kit. For example, the kit may include (a) the plasma kallikrein binding protein, e.g., a composition (e.g., a pharmaceutical composition) that includes the plasma kallikrein binding protein, and optionally (b) informational material. The informational material may be descriptive, explanatory, marketing, or other material related to the methods described herein and / or uses of the plasma kallikrein binding protein (e.g., in the methods described herein).

[0595] The informational material of the kit is not limited in its form. In one embodiment, the informational material can include information regarding the manufacture of the compound, the molecular weight of the compound, concentration, expiration date, batch or place of manufacture information, etc. In one embodiment, the informational material relates to the treatment, prevention or diagnosis of diseases and conditions using the binding protein, e.g., plasma kallikrein-associated diseases and conditions.

[0596] In one embodiment, the informational material may include instructions for administering the plasma kallikrein binding protein in a suitable manner for carrying out the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material may include instructions for administering the plasma kallikrein binding protein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disease or condition described herein (e.g., a plasma kallikrein-associated disease or condition). For example, the material may include instructions for administering the plasma kallikrein binding protein to a patient having a disease or condition described herein, e.g., a plasma kallikrein-associated disease. The informational material of the kit is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed form, but may be in other formats, such as computer readable material.

[0597] The plasma kallikrein binding protein may be provided in any form, for example in liquid, dry or lyophilized form. The plasma kallikrein binding protein is preferably substantially pure and / or sterile. When the plasma kallikrein binding protein is provided in a liquid solution, the liquid solution is preferably an aqueous solution, and the aqueous solution is preferably a sterile aqueous solution. When the plasma kallikrein binding protein is provided in a dry form, reconstitution is generally achieved by the addition of a suitable solvent. Optionally, a solvent, for example sterile water or a buffer, may be provided in the kit.

[0598] The kit may include one or more containers for the composition containing the plasma kallikrein binding protein. In some embodiments, the kit includes separate containers, dividers, or compartments for the composition and the informational material. For example, the composition may be contained in a bottle, vial, or syringe, and the informational material may be contained associated with the container. In other embodiments, the separate elements of the kit are contained in a single undivided container. For example, the composition is contained in a bottle, vial, or syringe that has informational material in the form of a label attached thereto. In some embodiments, the kit includes a plurality of separate containers (e.g., a pack), each of which contains one or more unit dosage forms (e.g., dosage forms described herein) of the plasma kallikrein binding protein. For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each of which contains a single dose of the plasma kallikrein binding protein. The containers of the kit may be airtight, water-resistant (e.g., impermeable to moisture change or evaporation), and / or light-tight.

[0599] The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, an inhaler, a dropper (e.g., an eye dropper), a swab (e.g., a cotton swab or a wooden swab), or any such delivery device. In one embodiment, the device is an implantable device that administers a metered amount of the binding protein. The disclosure also features methods of combining components, e.g., as described herein, to provide a kit.

[0600] treatment Proteins that bind to plasma kallikrein, such as those described herein, are particularly useful for treatment and prevention in human subjects. These binding proteins are administered to subjects to be treated, prevented and / or diagnosed for various diseases and conditions, including, for example, plasma kallikrein-related diseases, or to cultured cells, for example, in vitro or ex vivo. For example, these binding proteins are used to change the action of plasma kallikrein released from cultured cells (Lilla et al., J Biol Chem. 284(20):13792-13803 (2009)). Treatment includes administering an amount effective to relieve, alleviate, alter, correct, improve, improve or affect a disease, a symptom of a disease, or a predisposition to a disease. Treatment can also delay the onset of a disease or disorder, for example, prevent the onset or prevent the worsening.

[0601] As used herein, a disease-preventive effective amount of a targeted binding agent or a prophylactically effective amount of a binding agent refers to an amount of a targeted binding agent, e.g., a plasma kallikrein binding protein, e.g., an anti-plasma kallikrein antibody as described herein, that is effective in preventing or delaying the onset or recurrence of a disease, e.g., a disease described herein, e.g., a plasma kallikrein-associated disease, upon administration to a subject in single or multiple doses.

[0602] Methods for administering plasma kallikrein binding proteins and other drugs are also described in the "Pharmaceutical Compositions" section. The appropriate dose of the molecules used may depend on the age and weight of the subject, as well as the specific drug used. The binding proteins may be used as competitive drugs, for example, to inhibit and reduce undesirable interactions between plasma kallikrein and its substrates (e.g., factor XII or HMWK). The dose of plasma kallikrein binding protein may be sufficient to block 90%, 95%, 99% or 99.9% of the activity of plasma kallikrein in the patient, particularly at the disease site. Depending on the disease, 0.1, 1.0, 3.0, 6.0 or 10.0 mg / kg may be required. For an IgG with a molecular mass of 150,000 g / mol (two binding sites), the above doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM and 1.8 μM binding sites for a blood volume of 5 L.

[0603] In one embodiment, the plasma kallikrein binding protein is used to inhibit the activity of plasma kallikrein, for example in vivo (e.g., inhibit at least one activity of plasma kallikrein, for example, reduce factor XIIa and / or bradykinin production). The binding protein may be used alone or conjugated with an agent, for example, a cytotoxic drug, a cytotoxic enzyme, or a radioisotope. The method includes administering the binding protein alone or in combination with an agent (e.g., a cytotoxic drug) to a subject in need of such treatment. For example, the plasma kallikrein binding protein that does not substantially inhibit plasma kallikrein is used to deliver nanoparticles containing an agent, such as a toxin, to cells or tissues associated with plasma kallikrein, for example, to treat a plasma kallikrein-related disease.

[0604] Because plasma kallikrein binding proteins can recognize plasma kallikrein-expressing cells and bind to cells associated with (e.g., adjacent or intermingled with) plasma kallikrein-associated diseases or conditions, plasma kallikrein binding proteins can be used to inhibit the activity of any such cells (e.g., inhibit at least one activity of plasma kallikrein, e.g., reduce factor XIIa and / or bradykinin production) to suppress plasma kallikrein-associated diseases. Reducing plasma kallikrein activity can indirectly inhibit cells whose development and / or progression of plasma kallikrein-associated diseases may depend on plasma kallikrein activity.

[0605] The binding protein can be used to deliver drugs (e.g., any of a variety of cytotoxic and therapeutic drugs) to cells and tissues where plasma kallikrein is present. Examples of drugs include compounds that emit radiation, molecules of plant, fungal or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drug can be a cytotoxic drug that acts intracellularly, such as a short-range radiation-emitting toxin, e.g., a short-range high-energy alpha emitter.

[0606] Prodrug systems can be used to target plasma kallikrein-expressing cells. For example, a first binding protein is conjugated with a prodrug that is activated only when it is in close proximity to a prodrug activator. The prodrug activator is conjugated with a second binding protein, preferably a binding protein that binds to a non-competing site on the target molecule. Whether the two binding proteins bind to competing or non-competing binding sites can be determined by conventional competitive binding assays. Examples of drug-prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287 3292.

[0607] Plasma kallikrein binding proteins may be used directly in vivo to eliminate antigen-expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). The binding proteins described herein may contain a complement-binding effector domain, such as the Fc portion from IgG1, IgG2, or IgG3, or the corresponding portion of IgM that binds complement. In one embodiment, a target cell population is treated ex vivo with a binding agent described herein and appropriate effector cells. This treatment may be complemented by the addition of complement or serum containing complement. Furthermore, phagocytosis of target cells coated with the binding proteins described herein may be improved by binding of complement proteins. In another embodiment, target cells coated with binding proteins that contain a complement-binding effector domain are lysed by complement.

[0608] The administration method of plasma kallikrein binding protein is described in the "pharmaceutical composition" section. The appropriate dose of the molecule used can depend on the age and weight of the subject and the specific drug used. The binding protein can be used as a competitive drug, for example, to inhibit or reduce the undesirable interaction between natural or pathological agents and plasma kallikrein.

[0609] Plasma kallikrein binding proteins can be used to deliver macromolecular and micromolecular molecules, such as genes, into endothelial or epithelial cells for gene therapy purposes, targeting only those tissues that express plasma kallikrein. Binding proteins can be used to deliver a variety of cytotoxic drugs, including compounds that emit radiation, molecules of plant, fungal or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drug can be a cytotoxic drug that acts intracellularly, such as a short-range radiation-emitting toxin, including the short-range high-energy alpha-emitters described herein.

[0610] In the case of polypeptide toxins, recombinant nucleic acid techniques can be used to construct a nucleic acid that encodes a binding protein (e.g., an antibody or antigen-binding fragment thereof) and a cytotoxin (or a polypeptide component thereof) as a translational fusion. The recombinant nucleic acid is then expressed, e.g., intracellularly, and the encoded fusion polypeptide is isolated.

[0611] Alternatively, plasma kallikrein binding protein can be treated with a high-energy radiation emitter, e.g., a gamma emitter, which, when localized to a site, causes some loss of cell diameter. 131 The antibody may be coupled to a radioisotope such as I. See, for example, Order, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, RW Baldwin et al. (eds.), pp 303-316 (Academic Press 1985). Other suitable radioisotopes include: 212 Bi, 213 Bi and 211 α-emitters such as At and 186 Re and 90 β-emitters such as Y. 177 Lu may also be used as both an imaging agent and a cytotoxic agent.

[0612] 131 I, 90 Y and 177 Radioimmunotherapy (RIT) using Lu-labeled antibodies is currently undergoing active clinical research. There are significant differences in the physical properties of these three nuclides, and as a result, the choice of radionuclide is very important to deliver the maximum radiation dose to the target tissue. For larger tumors, the higher beta energy particles are preferred. 90 Y would be preferred. It is a relatively low energy beta particle. 131I is preferred, but in vivo dehalogenation of radioiodinated molecules is highly detrimental to antibody internalization. 177 Lu has low-energy beta particles with diameters in the range of only 0.2-0.3 mm, 90 It delivers a significantly lower dose of radiation to the bone marrow than Y. In addition, it has a long physical half-life ( 90 Y) for a longer residence time. As a result, it has a higher activity (higher mCi content). 177 Lu-labeled drugs can be administered to the bone marrow with a relatively small radiation dose. 177 There are several clinical studies that have examined the use of Lu-labeled antibodies (Mulligan T et al., 1995, Clin. Canc. Res. 1:1447-1454; Meredith RF et al., 1996, J. Nucl. Med. 37:1491-1496; Alvarez RD et al., 1997, Gynecol. Oncol. 65:94-101).

[0613] Examples of Diseases and Conditions The plasma kallikrein binding proteins described herein are useful for treating (or preventing) diseases or conditions in which plasma kallikrein activity is implicated, such as the diseases or conditions described herein, or for treating (or preventing) one or more symptoms associated with these diseases or conditions. In some embodiments, the plasma kallikrein binding protein (e.g., plasma kallikrein binding IgG or Fab) inhibits plasma kallikrein activity.

[0614] Examples of the above diseases and conditions that can be treated (or prevented) by the plasma kallikrein binding proteins described herein include rheumatoid arthritis, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal canal 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 blood clots, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic events (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, and burns.The plasma kallikrein binding proteins described herein can also be used to promote wound healing.The plasma kallikrein binding proteins described herein can also be used as oncological treatments, including, but not limited to, mechanisms that include blocking proangiogenic bradykinin production.

[0615] By administering a therapeutically effective amount of plasma kallikrein binding protein to a subject having or suspected of having a disease in which plasma kallikrein activity is implicated, the disease can be treated (e.g., to ameliorate symptoms or characteristics of the disease, slow, stabilize and / or halt disease progression).

[0616] The plasma kallikrein binding protein may be administered in a therapeutically effective amount. A therapeutically effective amount of plasma kallikrein binding protein is an amount that is effective in treating a subject, for example, in administering one or more doses to a subject to cure, alleviate, reduce or improve at least one symptom of a disease in the subject more than would be expected without such treatment. The therapeutically effective amount of a composition may vary depending on factors such as the individual's pathological condition, age, sex and weight, and the ability of the compound to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects. A therapeutically effective amount preferably favorably regulates a measurable parameter compared to untreated subjects. The ability of a compound to affect (e.g., inhibit) a measurable parameter can be evaluated in an animal model system that is predictive of efficacy in human disease.

[0617] The dosage regimen can be adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or multiple divided doses can be administered gradually, or the dose can be proportionally reduced or increased if indicated by the exigent therapeutic situation. It is particularly advantageous for parenteral compositions to be formulated in dosage unit form to facilitate administration and uniformity of dosage. As used herein, dosage unit form refers to a physically separate unit suitable for unitary administration to the subject to be treated, each unit containing a predetermined amount of active compound calculated to obtain a desired therapeutic effect together with the necessary pharmaceutical carrier.

[0618] Rheumatoid arthritis Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that causes joint swelling and pain, usually resulting in joint destruction. RA generally follows a relapsing / remitting course, with remissions of disease symptoms between "flares" of disease activity. RA is associated with a number of additional inflammatory diseases, including Sjogren's syndrome (dry eyes and dry mouth caused by inflammation of the tear and salivary glands), pleurisy (inflammation of the pleura that causes pain when breathing deeply and coughing), rheumatoid nodules (inflammation of nodules that develop in the lungs), pericarditis (inflammation of the pericardium that causes pain when lying down or leaning forward), Felty's syndrome (splenomegaly and leukopenia seen with RA, making the subject more susceptible to infections), and vasculitis (inflammation of blood vessels that can block blood flow). Plasma kallikrein is thought to be involved in rheumatoid arthritis.

[0619] Symptoms of active RA include fatigue, loss of appetite, low-grade fever, and muscle and joint pain and stiffness. Muscle and joint stiffness is usually most noticeable in the morning and after periods of inactivity. During flare-ups, joints often become red, swollen, painful, and tender, typically as a result of synovitis.

[0620] Rheumatoid arthritis treatment includes a combination of medications, rest, strengthening exercises, and joint protection. Two classes of medications are used in the treatment of rheumatoid arthritis: anti-inflammatory "first-line" and "disease-modifying antirheumatic drugs" (DMARDs). First-line medications include NSAIDS (e.g., aspirin, naproxen, ibuprofen, and etodolac) and cortisone (corticosteroids). DMARDs, such as gold (e.g., gold salts, gold thioglucose, gold thiomalate, oral gold), methotrexate, sulfasalazine, D-penicillamine, azathioprine, cyclophosphamide, chlorambucil, and cyclosporine, leflunomide, etanercept, infliximab, anakinra, and adalimumab, and hydroxychloroquine, promote disease remission and prevent progressive joint destruction, but DMARDs are not anti-inflammatory drugs.

[0621] The present disclosure provides methods of treating rheumatoid arthritis (e.g., improving, stabilizing or eliminating one or more symptoms, or improving or stabilizing a subject's score on a RA scale) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having RA. Additionally, methods of treating RA by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second therapy, such as at least one anti-inflammatory "first-line treatment" (e.g., an NSAID and / or cortisone) and / or a DMARD. The present disclosure also provides methods of preventing rheumatoid arthritis or symptoms thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing RA (e.g., a subject with a family member with RA or a genetic predisposition thereto).

[0622] Additionally, methods are provided for treating (e.g., improving, stabilizing or eliminating one or more symptoms of) rheumatoid arthritis-related diseases (Sjogren's syndrome, pleurisy, pulmonary rheumatoid nodules, pericarditis, Felty's syndrome and vasculitis) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having RA.

[0623] Useful scales for assessing RA and RA symptoms include, for example, the Rheumatoid Arthritis Severity Scale (RASS; Bardwell et al., (2002) Rheumatology 41(1):38-45), the SF-36 Arthritis Specific Health Index (ASHI; Ware et al., (1999) Med. Care. 37(5 Suppl):MS40-50), the Arthritis Impact Measurement Scales or Arthritis Impact Measurement Scales2 (AIMS or AIMS2; Meenan et al. (1992) Arthritis Rheum. 35(1):1-10); the Stanford Health Assessment Questionnaire (HAQ), HAQII or revised HAQ (e.g., Pincus et al. (1983) Arthritis Rheum.26(11):1346-53).

[0624] Guidance for determining doses that deliver a therapeutically effective amount of plasma kallikrein binding protein can be obtained from animal models of rheumatoid arthritis, such as collagen-induced arthritis (CIA), which is induced by immunization with autologous or heterologous type II collagen in adjuvant, usually in rodents (Williams et al., Methods Mol Med. 98:207-16 (2004)).

[0625] gout Gout is a condition caused by the deposition of uric acid crystals in body tissues. Gout is characterized by an overload of uric acid in the body and recurrent attacks of joint inflammation (arthritis). Chronic gout can cause hard clumps of uric acid to be deposited in and around the joints, as well as reduced kidney function and kidney stones. Gout is often associated with a genetic abnormality in the body's ability to process uric acid. Uric acid is a breakdown product of purines, which are part of many foods. Abnormal uric acid processing can lead to painful attacks of arthritis (gout attacks), kidney stones, and blockage of the kidney's filtering tubules by uric acid crystals, which can lead to kidney failure. Some patients develop elevated levels of uric acid in the blood (hyperuricemia) without arthritis or kidney damage.

[0626] Symptoms of gout include, for example, sudden, severe pain, swelling, redness, warmth and stiffness in the affected foot or other body part, as well as a low-grade fever.

[0627] Treatments for gout include, for example, nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine and oral glucocorticoids, intra-articular glucocorticoids administered by joint injection, xanthine oxidase inhibitors (e.g., allopurinol, febuxostat), uricosurics (e.g., probenecid, EDTA), urate oxidase (e.g., pegloticase), sodium bicarbonate, and a low-purine diet.

[0628] The present disclosure provides methods of treating gout (e.g., ameliorating, stabilizing, or eliminating one or more symptoms or exacerbations) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having gout. Additionally, methods of treating gout by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second therapy, e.g., an NSAID, colchicine, oral glucocorticoids, intra-articular glucocorticoids administered by joint injection, xanthine oxidase inhibitors (e.g., allopurinol, febuxostat), uricosurics (e.g., probenecid, EDTA), urate oxidase (e.g., pegloticase), sodium bicarbonate, and / or a low purine diet are provided. The present disclosure also provides methods of preventing gout or symptoms thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing gout (e.g., a subject who has a family member with gout or a genetic predisposition thereto).

[0629] Guidance for determining a dose that delivers a therapeutically effective amount of plasma kallikrein binding protein may be obtained from animal models of gout, see, e.g., Reginato and Olsen, Curr Opin Rheumatol. 19(2):134-45 (2007) and references cited therein.

[0630] Intestinal Disease (IBD) Inflammatory bowel disease (IBD) refers to a group of inflammatory conditions of the large and sometimes small intestine. The main forms of IBD are Crohn's disease and ulcerative colitis (UC). Much less common are other forms of IBD: collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's syndrome, infectious colitis, and indeterminate colitis. The main difference between Crohn's disease and UC is the location and nature of the inflammatory changes. Crohn's disease can affect any part of the digestive tract from the mouth to the anus (stepping stone lesions), but the majority of cases begin in the terminal ileum. In contrast, ulcerative colitis is restricted to the colon and rectum. Microscopically, ulcerative colitis is restricted to the mucosa (the epithelial lining of the digestive tract), whereas Crohn's disease affects the entire intestinal wall. Finally, Crohn's disease and ulcerative colitis present with different rates of extraintestinal symptoms (e.g. liver damage, arthritis, skin symptoms, and eye problems).

[0631] Symptoms of IBD include abdominal pain, vomiting, diarrhea, bloody stools, weight loss, weight gain, and various complaints or illnesses (arthritis, pyoderma gangrenosum, primary sclerosing cholangitis). Diagnosis is usually made by colonoscopy with biopsy of lesions. Rarely, specific symptoms may make the diagnosis of either Crohn's disease or ulcerative colitis unclear. In such cases, a diagnosis of indeterminate colitis may be made.

[0632] Treatment of IBD may require immunosuppression to control symptoms, depending on the level of severity. Immunosuppressants such as azathioprine, methotrexate, or 6-mercaptopurine may be used. More commonly, a form of mesalamine is required to treat IBD. Steroids are often used to control disease flares, but steroids were once tolerated as maintenance medications. Biologics such as infliximab have been used to treat patients with Crohn's disease or ulcerative colitis. In severe cases, surgery may be required, such as bowel resection, strictureplasty, or temporary or permanent colostomy or ileostomy. Alternative medical treatments for IBD exist in various forms, but these methods focus on controlling the underlying condition to avoid long-term steroid exposure or surgical resection. Treatment usually begins with the administration of anti-inflammatory drugs such as prednisone. Once inflammation is well controlled, patients are usually switched to milder drugs such as Asacol (a mesalamine) to keep the disease in remission. If not well controlled, combinations of the above immunosuppressants and mesalamine (which may also have anti-inflammatory effects) may or may not be given depending on the patient.

[0633] The present disclosure provides methods of treating IBD (e.g., improving, stabilizing, or eliminating one or more symptoms) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having IBD. Additionally, methods of treating IBD by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second therapy, such as immunosuppression (e.g., azathioprine, methotrexate, 6-mercaptopurine), mesalamine, steroids, and / or infliximab. The present disclosure also provides methods of preventing IBD or a symptom thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing IBD (e.g., a subject with a family member with IBD or a genetic predisposition thereto).

[0634] Guidance for determining a dose that delivers a therapeutically effective amount of plasma kallikrein binding protein may be obtained from animal models of IBD, see, e.g., those described in U.S. Pat. No. 6,114,382, WO 2004 / 071186 and the references cited therein.

[0635] Oral mucositis Oral mucositis is a painful inflammation and ulceration of the mucous membranes inside the mouth that usually occurs as a side effect of cancer chemotherapy and radiation therapy.

[0636] Symptoms of oral mucositis include, for example, ulcers, erythema around the edges, a burning sensation with redness, difficulty speaking, eating or opening one's mouth, and dysgeusia (altered sense of taste).

[0637] Treatments for oral mucositis include oral hygiene (saline mouthwash, GELCLAIR®, CAPHOSOL®, MUGARD®), palifermin (a human keratinocyte growth factor), cytokines and other inflammatory regulators (e.g., IL-1, IL-11, TGF-beta 3), amino acid supplementation (e.g., glutamine), vitamins, colony-stimulating factors, cryotherapy, and laser therapy.

[0638] The present disclosure provides methods of treating oral mucositis (e.g., improving, reducing or eliminating one or more symptoms, or stabilizing the subject's score on a mucositis scale) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having oral mucositis. Additionally, methods of treating oral mucositis by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second therapy, such as oral hygiene (saline mouthwash, GELCLAIR®, CAPHOSOL®, MUGARD®), palifermin (a type of human keratinocyte growth factor), cytokines and / or inflammation regulators (e.g., IL-1, IL-11, TGF-beta3), amino acid supplementation (e.g., glutamine), vitamins, colony stimulating factors, cryotherapy, and / or laser therapy. The disclosure also provides methods for preventing oral mucositis or symptoms thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing oral mucositis (e.g., a subject that has undergone or is currently undergoing chemotherapy or radiation therapy).

[0639] Useful scales for assessing oral mucositis include the World Health Organization (WHO) Oral Toxicity Score (Handbook for reporting results of cancer treatment. Geneva, Switzerland: World Health Organization; 1979:15-22), the National Cancer Institute Common Toxicity Criteria for Oral Mucositis (NCI-CTC) (National Cancer Institute Common Toxicity Criteria. Version 2.0, June 1, 1999, Sonis et al., Cancer. 85:2103-2113 (1999)) and the Oral Mucositis Assessment Scale (OMAS).

[0640] Guidance for determining a dose that delivers a therapeutically effective amount of plasma kallikrein binding protein can be obtained from animal models of oral mucositis, such as that induced by hematopoietic stem cell transplant conditioning regimens (Chen et al., Zhonghua Kou Qiang Yi Xue Za Zhi. 42(11):672-6 (2007)).

[0641] Neuropathic pain Neuropathic pain is a complex chronic pain condition that usually involves tissue damage. In neuropathic pain, the nerve fibers themselves may be damaged, dysfunctional or injured. These damaged nerve fibers send incorrect signals to other pain centers. The effects of nerve fiber injury include changes in nerve function both at the site of injury and in the area surrounding the injury.

[0642] Symptoms of neuropathic pain include, for example, shooting, burning, tingling, and numbness.

[0643] Treatments for neuropathic pain include, for example, medications (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ALEVE®, MOTRIN®, or morphine), anticonvulsants, and antidepressants) and invasive or implanted devices (e.g., electrical stimulation).

[0644] The present disclosure provides methods of treating neuropathic pain (e.g., improving, reducing or eliminating one or more symptoms, or stabilizing the subject's pain score) by administering plasma kallikrein binding protein (e.g., a therapeutically effective amount of plasma kallikrein binding protein) to a subject having or suspected of having neuropathic pain. Additionally, methods of treating neuropathic pain by administering plasma kallikrein binding protein (e.g., a therapeutically effective amount of plasma kallikrein binding protein) in combination with a second therapy, such as, for example, a non-surgical therapy (e.g., a non-steroidal anti-inflammatory drug (NSAID) (e.g., ALEVE®, MOTRIN®, or morphine), an anticonvulsant drug, and / or an antidepressant) and / or an invasive or implanted device (e.g., electrical stimulation). The present disclosure also provides methods of preventing neuropathic pain or symptoms thereof by administering plasma kallikrein binding protein (e.g., a prophylactically effective amount of plasma kallikrein binding protein) to a subject at risk of developing neuropathic pain (e.g., a subject who has experienced tissue injury).

[0645] Useful scales for assessing neuropathic pain include, for example, the Wong-Baker FACES Pain Rating Scale (Wong-Baker FACES Pain Rating Scale Foundation), visual analog scale (VAS) (Huskisson, J. Rheumatol. 9(5):768-9(1982)), McGill Pain Questionnaire (MPQ) (Melzack, Pain 1(3):277-99(1975)), Descriptor differential scale (DDS) (Gracely and Kwilosz, Pain 35(3):279-88(1988)), and the Faces Pain Scale-Revised (FPS-R) (Hicks et al., Pain 93(2):173-83 (2001)), Numerical 11 point box (BS-11) (Jensen et al., Clin J Pain 5(2):153-9 (1989)), Numeric Rating Scale (NRS-11) (Hartrick et al., Pain Pract 3(4):310-6 (2003)), Dolorimeter Pain Index (DPI) (Hardy et al., (1952). Pain Sensations and Reactions. Baltimore: The Williams & Wilkins Co.), and Brief Pain Inventory (BPI) (Cleeland and Ryan Ann. Acad. Med. Singapore. 23(2):129-38 (1994)).

[0646] Guidance for determining a dose that delivers a therapeutically effective amount of plasma kallikrein binding protein may be obtained from animal models of neuropathic pain, see, e.g., Martin et al., Methods Mol Med. 84:233-42 (2003) and references cited therein.

[0647] inflammatory pain Inflammatory pain is caused by injuries to tissue integrity at the cellular level, such as penetrating wounds, burns, extreme cold, fractures, arthritis, autoimmune conditions, overstretching, infections and vasoconstriction. In inflammation, primary hyperalgesia occurs as a result of complex neuroimmune interactions in which a wide range of inflammatory molecules, including prostaglandins and bradykinin, induce and maintain altered sensitivity of nociceptors.

[0648] Treatments for inflammatory pain include, for example, nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids.

[0649] The present disclosure provides methods of treating inflammatory pain (e.g., improving, reducing or eliminating one or more symptoms) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having inflammatory pain. Additionally, methods of treating inflammatory pain by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second therapy, such as, for example, a nonsteroidal anti-inflammatory drug (NSAID) and / or a corticosteroid. The present disclosure also provides methods of preventing inflammatory pain or symptoms thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing inflammatory pain (e.g., a subject who has experienced an injury, such as a penetrating wound, a burn, extreme cold, a fracture, arthritis, an autoimmune condition, overstretching or an infection, etc.).

[0650] Useful scales for assessing inflammatory pain include, for example, the Wong-Baker FACES Pain Rating Scale (Wong-Baker FACES Pain Rating Scale Foundation), visual analog scale (VAS) (Huskisson, J. Rheumatol. 9(5):768-9(1982)), McGill Pain Questionnaire (MPQ) (Melzack, Pain 1(3):277-99(1975)), Descriptor differential scale (DDS) (Gracely and Kwilosz, Pain 35(3):279-88(1988)), and the Faces Pain Scale-Revised (FPS-R) (Hicks et al., Pain 93(2):173-83 (2001)), Numerical 11 point box (Jensen et al., Clin J Pain 5(2):153-9 (1989)), Numeric Rating Scale (Hartrick et al., Pain Pract 3(4):310-6 (2003)), Dolorimeter Pain Index (Hardy et al., (1952). Pain Sensations and Reactions. Baltimore: The Williams & Wilkins Co.) and Brief Pain Inventory (BPI) (Cleeland and Ryan Ann. Acad. Med. Singapore. 23(2):129-38 (1994)).

[0651] Guidance for determining dosages that deliver a therapeutically effective amount of plasma kallikrein binding protein can be obtained from animal models of inflammatory pain, such as an animal model of chronic inflammatory pain (Wilson et al., Eur J Pain. 10(6):537-49 (2006)) and an inflammatory model of pain and hyperalgesia (Ren and Dubner, ILAR J. 40(3):111-118 (1999)).

[0652] spinal canal stenosis Spinal stenosis is a medical condition in which the spinal canal narrows, compressing the spinal cord and nerves. It is usually due to general spinal degeneration that occurs with aging. Spinal stenosis can also be caused by a herniated spinal disc, osteoporosis, or a tumor. Spinal stenosis can affect the cervical, thoracic, or lumbar spine. The same patient may have spinal stenosis in all three places.

[0653] Symptoms of spinal stenosis include, for example, pain or cramps in the legs, radiating pain in the back and buttocks, pain in the neck and shoulders, loss of balance, and loss of bowel or bladder function (cauda equina syndrome).

[0654] Treatments for spinal stenosis include, for example, non-surgical treatments (e.g., physical therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, and indomethacin), painkillers (e.g., acetaminophen), chondroitin sulfate, glucosamine, rest or activity restriction, back braces or corsets, epidural steroid injections (e.g., corticosteroids)) and surgery (e.g., decompression laminectomy, laminectomy, and fusion).

[0655] The present disclosure provides methods of treating spinal stenosis (e.g., ameliorating, reducing or eliminating one or more symptoms) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having spinal stenosis. Additionally provided are methods of treating spinal stenosis by administering plasma kallikrein binding protein (e.g., a therapeutically effective amount of plasma kallikrein binding protein) in combination with a second therapy, such as, for example, a non-surgical treatment (e.g., physical therapy and / or a nonsteroidal anti-inflammatory drug (NSAID) (e.g., aspirin, ibuprofen, or indomethacin), analgesics (e.g., acetaminophen), chondroitin sulfate, glucosamine, bed rest or activity restriction, a back brace or corset, epidural steroid injections (e.g., corticosteroids), and / or surgery (e.g., decompression laminectomy, laminectomy, and / or fusion). The present disclosure also provides methods of preventing spinal stenosis or symptoms thereof by administering plasma kallikrein binding protein (e.g., a prophylactically effective amount of plasma kallikrein binding protein) to a subject at risk of developing spinal stenosis (e.g., a subject having spinal cord degeneration).

[0656] Guidance for determining dosages that deliver a therapeutically effective amount of plasma kallikrein binding protein can be obtained from animal models of spinal stenosis, such as the lumbar spinal stenosis model (Sekiguchi et al., Spine 29, 1105-1111 (2004)).

[0657] Arterial and venous thrombosis Arterial thrombosis is the formation of a blood clot in an artery. In most cases, arterial thrombosis occurs after the rupture of an atheroma, hence the name atherothrombosis.

[0658] Arterial thrombosis is associated with numerous diseases, including stroke and myocardial infarction. In thrombotic stroke, a thrombus (blood clot) usually forms around an atherosclerotic plaque. Symptomatic thrombotic stroke develops late because of gradual arterial obstruction. Thrombotic stroke can be divided into two categories: large vessel disease and small vessel disease. The former affects blood vessels such as the internal carotid artery, vertebral artery, and the Circle of Willis. The latter can affect small vessels such as the branches of the Circle of Willis. Myocardial infarction (MI) is caused by infarction (death of tissue due to ischemia), often due to occlusion of a coronary artery by a thrombus. If emergency medical care is not immediately sought, MI can quickly become fatal.

[0659] A venous thrombosis is a blood clot that forms in a vein. When a clot forms in a vein, it can break off and travel to the right heart and then into the lungs. This travel of clot fragments is called an embolism, and the process of forming an embolic clot is called thromboembolism. An embolism that lodges in the lungs is called a pulmonary embolism (PE). A pulmonary embolism is a very serious condition that can be fatal if not recognized and treated promptly.

[0660] Although superficial venous thrombosis may cause discomfort, it generally does not have serious consequences and is distinct from deep venous thrombosis (DVT), which forms in the deep veins of the legs or in the pelvic veins. In patients with atrial or ventricular septal defects, emboli can enter the arterial system, causing systemic embolism of venous origin. Such an event is called paradoxical embolism.

[0661] Prophylaxis of arterial and / or venous thrombosis includes pharmacological therapy (eg, anticoagulants (eg, heparin), aspirin and vitamin E) and mechanical methods (eg, mechanical leg pumps (pneumatic stockings)).

[0662] The present disclosure provides methods of treating arterial and / or venous thrombosis (e.g., improving, reducing or eliminating one or more symptoms) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having arterial and / or venous thrombosis. Additionally, methods of treating arterial and / or venous thrombosis by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second therapy, such as an anticoagulant (e.g., heparin), aspirin and / or vitamin E, and / or a mechanical method (e.g., a mechanical leg pump (pneumatic stockings)). The present disclosure also provides methods of preventing arterial and / or venous thrombosis or a symptom thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing arterial and / or venous thrombosis (e.g., a subject who has experienced a stroke or myocardial infarction).

[0663] Guidance for determining dosages that deliver a therapeutically effective amount of plasma kallikrein binding protein can be obtained from animal models of arterial or venous thrombosis, such as the double-tuck arterial thrombosis model (Gomez-Jorge et al., J. Vasc. Inter. Rad. 9(4):633-638 (1998), the rat venous thrombosis model in which venous blood flow is in a low-flow state (Fredrich et al., Blood Coagul Fibrinolysis. 5(2):243-8 (1994)), and the canine model of venous thrombosis and spontaneous pulmonary embolism (Frisbiel, Spinal Cord 43,635-639 (2005)).

[0664] Postoperative ileus Postoperative ileus is the temporary paralysis of a portion of the intestine, usually after abdominal surgery. Postoperative ileus generally occurs 24 to 72 hours after abdominal surgery.

[0665] Symptoms of post-operative ileus include, for example, moderate generalized abdominal discomfort, constipation, abdominal distension, nausea or vomiting, lack of bowel motility and / or flatulence, and excessive eructation.

[0666] Treatments for postoperative ileus include, for example, nil per os (NPO, or "nothing by mouth") until peristalsis is heard by auscultation over the surgical site, nasogastric suction, parenteral feeding, and drug therapy (e.g., lactulose and erythromycin).

[0667] The present disclosure provides methods of treating postoperative ileus (e.g., improving, reducing or eliminating one or more symptoms) by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) to a subject having or suspected of having postoperative ileus. Additionally, methods of treating postoperative ileus by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein binding protein) in combination with a second treatment, such as fasting, nasogastric suction, parenteral feeding and / or drug therapy (e.g., lactulose and / or erythromycin). The present disclosure also provides methods of preventing postoperative ileus or symptoms thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing postoperative ileus (e.g., a subject who has undergone abdominal surgery).

[0668] Guidance for determining a dose that will deliver a therapeutically effective amount of plasma kallikrein binding protein can be obtained from animal models of postoperative ileus, such as a model for studying postoperative ileus in conscious rats using strain gauge transducers (Huge et al., J Surg Res. 74(2):112-8 (1998)).

[0669] aortic aneurysm Aortic aneurysm is a general term used to describe any aortic swelling (dilation or aneurysm) that usually indicates an underlying weakness in the aortic wall at that location. Types of aortic aneurysms include aortic root aneurysms, thoracic aortic aneurysms, abdominal aortic aneurysms, and thoracic-abdominal aortic aneurysms.

[0670] Most intact aortic aneurysms do not cause symptoms. As they expand, symptoms of an aortic aneurysm may occur, including, for example, feelings of anxiety or stress, nausea or vomiting, cold sweats, rapid heart rate, abdominal pain, back pain, leg pain or numbness, eryth...

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to plasma kallikrein (pKal) but not to prekallikrein.

2. 1. An isolated antibody or antigen-binding fragment thereof that binds to plasma kallikrein (pKal) and does not bind to prekallikrein, said isolated antibody comprising: (i) a heavy chain immunoglobulin variable domain (V) comprising the heavy chain complementarity determining region (HC CDR) 1 sequence of PYNMY, the HC CDR2 sequence of SIRPSGGGTVYADSVKG, and the HC CDR3 sequence of GFIAARWYYFDY; Ｈ A light chain immunoglobulin variable domain (V CDR 1 ) having a sequence of RASQSVSSSYLA, a LC CDR2 sequence of GASSRAT, and a LC CDR3 sequence of QQYGSSPFT. Ｌ )array; (ii) a V comprising the HC CDR1 sequence of WYTMV, the HC CDR2 sequence of RIYPSGGWTKYADSVKG, and the HC CDR3 sequence of EGLLWFGENAFDI; Ｈ and a V sequence including an LC CDR1 sequence of SGDQLGDKYVG, an LC CDR2 sequence of QDTKRPS, and an LC CDR3 sequence of QAWDTSTAG. Ｌ array; (iii) a V comprising the HC CDR1 sequence of WYLMI, the HC CDR2 sequence of YIYPSGGFTYYADSVKG, and the HC CDR3 sequence of TEGPLSWGYGMDV; Ｈ and a V comprising the LC CDR1 sequence of SGDKLGDKYAC, the LC CDR2 sequence of QDSKRPS, and the LC CDR3 sequence of QAWDSSTGV. Ｌ array; (iv) a V comprising the HC CDR1 sequence of WYTMV, the HC CDR2 sequence of RIYPSGGWTKYADSVKG, and the HC CDR3 sequence of EGLLWFGENAFDI; Ｈ and a V sequence including an LC CDR1 sequence of SGDQLGDKYVG, an LC CDR2 sequence of QDTKRPS, and an LC CDR3 sequence of QAWDTSTAG. Ｌ array; (v) V comprising a HC CDR1 sequence of WYEMG, a HC CDR2 sequence of SIYSSGGGTMYADSVKG, and a HC CDR3 sequence of NPQYSGYDRSLSDGAFDI. Ｈ and a V comprising the LC CDR1 sequence of SGDNLGNKYNS, the LC CDR2 sequence of QDTKRPS, and the LC CDR3 sequence of QAWDGNVV. Ｌ array; (vi) a V comprising a HC CDR1 sequence of YYMMV, a HC CDR2 sequence of YIYSSGGHTVYADSVKG, and a HC CDR3 sequence of DLFLYDFFWSKGAFDI; Ｈ and a V sequence including the LC CDR1 sequence of SGEKLGDKYVS, the LC CDR2 sequence of EDSRRPS, and the LC CDR3 sequence of QAWDSSTAI. Ｌ array; (vii) a V sequence comprising a HC CDR1 sequence of HYRMS, a HC CDR2 sequence of SIYPSGGRTVYADSVKG, and a HC CDR3 sequence of DKFEWRLLFRGIGNDAFDI; Ｈ and a V comprising an LC CDR1 sequence of SGDKLGDKYTS, an LC CDR2 sequence of QDIKRPS, and an LC CDR3 sequence of QAWDSPNARV. Ｌ array; (viii) a V comprising a HC CDR1 sequence of RYIMH, a HC CDR2 sequence of SISPSGGLTSYADSVKG, and a HC CDR3 sequence of EFENAYHYYYYGMDV; Ｈ and a V sequence including the LC CDR1 sequence of RASGDIGNALG, the LC CDR2 sequence of DASTLQS, and the LC CDR3 sequence of LQGYNYPRT. Ｌ array; (ix) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including an LC CDR1 sequence of RAGRSISTYVN, an LC CDR2 sequence of AASSLQS, and an LC CDR3 sequence of QQSQSTPYT. Ｌ array; (x) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including an LC CDR1 sequence of RASQIVSSRYLA, an LC CDR2 sequence of GAASRAT, and an LC CDR3 sequence of QQTYSSPFT. Ｌ array; (xi) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including an LC CDR1 sequence of RASQSISNYLN, an LC CDR2 sequence of AASSLQS, and an LC CDR3 sequence of QQSYTSPYT. Ｌ array; (xii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including the LC CDR1 sequence of RASQIVSSNYLA, the LC CDR2 sequence of GASNRAT, and the LC CDR3 sequence of QQSFNIPYT. Ｌ array; (xiii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xiv) a V comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of QRTGVPRRDSFNI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xv) a V comprising a HC CDR1 sequence of IYSMH, a HC CDR2 sequence of SIYPSRGMTWYADSVKG, and a HC CDR3 sequence of RRTGIPRRDAFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xvi) a V comprising a HC CDR1 sequence of VYNMH, a HC CDR2 sequence of SIYPSGGMTYYADSVKG, and a HC CDR3 sequence of RRTGIPRRDAFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xvii) a V sequence comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRIGVPRRDEFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xviii) a V comprising an HC CDR1 sequence of HYIMM, an HC CDR2 sequence of GIYSSGGITVYADSVKG, and an HC CDR3 sequence of RRTGVPRWDDFDDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xix) a V comprising an HC CDR1 sequence of IYSMH, an HC CDR2 sequence of SIYSSGGPTKYADSVKG, and an HC CDR3 sequence of RRTGIPRRDAFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xx) a V comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRIGVPRRDSFDM; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxi) a V sequence comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRTGVPRRDEFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxii) a V comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRIGVPRRDSFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxiii) V comprising the HC CDR1 sequence of VYNMH, the HC CDR2 sequence of SIYPSGGMTTYADSVKG, and the HC CDR3 sequence of RRTGIPRRDAFDI. Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxiv) a V comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRTGVPRRDSFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxv) V comprising the HC CDR1 sequence of RYSMR, the HC CDR2 sequence of VIYPSGGQTYYADSVKG, and the HC CDR3 sequence of RRTGIPRRDAFDI. Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxvi) a V comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRTGVPRRDDFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxvii) a V comprising a HC CDR1 sequence of HYIMM, a HC CDR2 sequence of GIYSSGGITVYADSVKG, and a HC CDR3 sequence of RRIGVPRRDDFDI; Ｈ and a V sequence including the LC CDR1 sequence of RASQSISSWLA, the LC CDR2 sequence of KASTLES, and the LC CDR3 sequence of QQYNTYWT. Ｌ array; (xxviii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYLDQ; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxix) a V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VGQGIRGRSRTSYFAQ; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxx) V comprising the HC CDR1 sequence of DYMMA, the HC CDR2 sequence of SIVPSGGHTHYADSVKG, and the HC CDR3 sequence of VARGIAARSRTSYFDY. Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxi) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VAQGIAARSRTSSVDQ; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSFFDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxiii) a V comprising a HC CDR1 sequence of QYLMA, a HC CDR2 sequence of SIYPSGGWTKYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxiv) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIASRSRTRYCDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxv) V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VATGIVARSRTRYFDQ. Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxvi) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTRYFDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxvii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VAQGISARSRTSYFDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxviii) V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VAQGIVARSRTSYLHQ. Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxix) a V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VARGIAARSRTSYLDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxx) A V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VARGIAARSRTRYFDQ. Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxxi) a V comprising the HC CDR1 sequence of GYMMV, the HC CDR2 sequence of RISPSGGPTIYADSVKG, and the HC CDR3 sequence of VARGIAARSRTSYFDY; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxxii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDQ; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxxiii) a V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VARGIAARSRTILLDQ; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxxiv) V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VARGIAARSRTSFIDY. Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxxv) V comprising a HC CDR1 sequence of TYKMQ, a HC CDR2 sequence of SISPSGGPTNYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFDY. Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; (xxxxvi) a V comprising the HC CDR1 sequence of HYLMT, the HC CDR2 sequence of YISPSGGHTIYADSVKG, and the HC CDR3 sequence of VARGIAARSRTSYLDF; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ An array; or (xxxxvii) a V comprising a HC CDR1 sequence of HYLMT, a HC CDR2 sequence of YISPSGGHTIYADSVKG, and a HC CDR3 sequence of VARGIAARSRTSYFNQ; Ｈ and a V sequence including the LC CDR1 sequence of RTSQFVNSNYLA, the LC CDR2 sequence of GASSRAT, and the LC CDR3 sequence of QQSSRTPWT. Ｌ array; 2. An isolated antibody or antigen-binding fragment thereof comprising:

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a full-length antibody.

4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is an IgG antibody.

5. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody is a soluble Fab (sFab).

6. A nucleic acid encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

7. A vector comprising the nucleic acid of claim 6.

8. The vector of claim 7 , wherein the vector is an expression vector.

9. A host cell comprising the nucleic acid of claim 6.

10. The host cell of claim 9 , wherein the cell is a mammalian cell.

11. The host cell of claim 10, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

12. 1. A method for producing an antibody or antigen-binding fragment thereof, comprising: Cultivating the host cell of claim 11 in a medium, thereby producing the antibody or antigen-binding fragment thereof; The method includes:

13. The method of claim 12, further comprising purifying the antibody or antigen-binding fragment thereof from the medium.

14. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

15. A composition for use in a method for detecting plasma kallikrein in a subject, comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, The method includes administering the composition and detecting an interaction between the antibody or antigen-binding fragment thereof and plasma kallikrein in the subject. composition.

16. The composition of claim 15 , wherein the antibody or antigen-binding fragment thereof comprises a detectable label.

17. 1. A method for detecting plasma kallikrein in a sample, comprising: Contacting the sample with the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5; detecting an interaction, if any, between said antibody or antigen-binding fragment thereof and plasma kallikrein in said sample; A method comprising:

18. 20. The method of claim 17, wherein the sample is a sample obtained from a subject.

19. 20. The method of claim 18, wherein the sample is a biopsy sample.

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