AU2019204295A1

AU2019204295A1 - Plasma kallikrein binding proteins

Info

Publication number: AU2019204295A1
Application number: AU2019204295A
Authority: AU
Inventors: Daniel J. Sexton; Malini VISWANATHAN
Original assignee: Dyax Corp
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 2010-01-06
Filing date: 2019-06-19
Publication date: 2019-07-04
Anticipated expiration: 2031-01-06
Also published as: AU2016244213B2; AU2016244213A1; AU2018253476B2; AU2019204295B2; AU2018253476C1; AU2020204600B2; AU2018253476A1; AU2020204600A1

Abstract

PLASMA KALLIKREIN BINDING PROTEINS Plasma kallikrein binding proteins and methods of using such proteins are described.

Description

PLASMA KALLIKREIN BINDING PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of Australian Patent Application No. 2018253476, a divisional of Australian Patent Application No. 2016244213, which is a divisional of Australian Patent Application No. 2013205129, which in turn is a divisional application of Australian Patent Application No. 2011204349 (the national phase of PCT/US2011/020377), the entire contents of each of which is incorporated herein by reference in its entirety. The present application claims priority from USSN 61/292,614 the entire contents of which is hereby incorporated by reference.

BACKGROUND

Plasma kallikrein is a serine protease. Prekallikrein is the precursor of plasma kallikrein.

SUMMARY

Plasma kallikrein is a serine protease component of the contact system and a potential drug target for different inflammatory, cardiovascular, infectious (sepsis) and oncology diseases (Sainz l.M. et al., Thromb Haemost 98, 77-83, 2007). The contact system is activated by either factor Xlla upon exposure to foreign or negatively charged surfaces or on endothelial cell surfaces by prolylcarboxypeptidases (FIGURE 1) (Sainz l.M. et al., Thromb Haemost 98, 77-83, 2007).

Activation of the plasma kallikrein amplifies intrinsic coagulation via its feedback activation of factor Xll and enhances inflammation via the production of the proinflammatory nonapeptide bradykinin. As the primary kininogenase in the circulation, plasma kallikrein is largely responsible for the generation of bradykinin in the vasculature. A genetic deficiency in the Cl-inhibitor protein (C1-1NH), the major natural inhibitor of plasma kallikrein, leads to hereditary angioedema (HAE). Patients with HAE suffer from acute attacks of painful edema often precipitated by unknown triggers (Zuraw B.L. et al., N Engl J Med 359, 1027-1036, 2008). Through the use of pharmacological agents or genetic studies in animal models, the plasma kallikrein-kinin system (plasma KKS) has been implicated in various diseases.

Plasma kallikrein binding proteins (e.g., antibodies, e.g., inhibitory antibodies) are useful therapeutic agents for a variety of diseases and conditions, e.g., diseases and conditions that involve plasma kallikrein activity, due to their high potency, specificity, and prolonged serum residency. High potency can translate to efficacy and a low drug dosage, and high specificity can reduce side effects due to the inhibition of related off target serine proteases. In general, small molecule serine proteases are not as specific as antibody inhibitors. Prolonged serum residency can permit infrequent dosing.

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In some aspects, the disclosure features an isolated protein (e.g., antibody, e.g., human antibody) that binds to the active form of plasma kallikrein (e.g., human plasma kallikrein and/or mouse plasma kallikrein), and, e.g., does not bind preplasma kallikrein (e.g., human preplasma kallikrein and/or mouse preplasma kallikrein).

In some embodiments, the plasma kallikrein binding protein binds the same epitope or competes for binding with a kallikrein binding protein described herein. In some embodiments, the plasma kallikrein binding protein binds the same epitope or competes for binding with a protein (e.g., epi-Kal2) and/or a small molecule (e.g., AEBSF) described herein and does not bind pre-plasma kallikrein.

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, M06D09 and M35-G04.

In some embodiments, the plasma kallikrein binding protein competes with or binds the same epitope as X81-B01 and, e.g., does not bind pre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein competes with or binds the same epitope as X67-D03 and, e.g., does not bind pre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein competes with or binds to the same site as X101-A01 and, e.g., does not bind pre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein competes with or binds to the same site as M162-A04 and, e.g., does not bind pre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein competes with or binds to the same site as X63-G06 and, e.g., does not bind pre-plasma kallikrein.

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

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 an epitope that overlaps with the active site of plasma kallikrein and, e.g., does not bind pre-plasma kallikrein.

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

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

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

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

In some embodiments, the plasma kallikrein binding protein decreases Factor Xlla and/or bradykinin production by greater 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% as compared to a standard, e.g., the Factor Xlla and/or bradykinin production under the same conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has an apparent inhibition constant (Ki_iapp) of less than 1000, 500, 100, 10, 1, 0.5 or 0.2 nM.

In one embodiment, the HC and FC variable domain sequences are components of the same polypeptide chain.

In another embodiment, the HC and EC variable domain sequences are components of different polypeptide chains. For example, the plasma kallikrein binding protein is an IgG., e.g., IgGl, IgG2, IgG3, or IgG4. The plasma kallikrein binding protein can be a soluble Fab (sFab).

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In some embodiments, the plasma kallikrein binding protein has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, in vivo, e.g., in humans. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an

IgGl, IgG2, IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3 weeks, weeks, 5 weeks or more in vivo, e.g., in humans.

In some embodiments, the plasma kallikrein bindihng protein is physically associated with a moiety that improves serum residence time, e.g., a moiety described herein.

In other embodiments, the plasma kallikrein binding protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as 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 appropriate construction.

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

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

In one embodiment, the plasma kallikrein binding protein is a primate or primatized antibody or is non-immunogenic in a human. For example, the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

In one embodiment, the plasma kallikrein binding protein includes 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 (bonobos)), gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

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In one embodiment, the plasma kallikrein binding protein includes human framework regions, or framework regions that are at least 95, 96, 97, 98, or 99% identical to human framework regions.

In certain embodiments, the plasma kallikrein binding protein includes no sequences from mice or rabbits (e.g., is not a murine or rabbit antibody).

In certain embodiments, the plasma kallikrein binding protein is capable of binding to a cell or tissue, e.g., that expresses plasma kallikrein.

In one embodiment, the plasma kallikrein binding protein is physically associated with a nanoparticle, and can be used to guide a nanoparticle to a cell or tissue expressing plasma kallikrein.

In some aspects, the disclosure features an isolated protein (e.g., antibody, e.g., human antibody) that binds the same epitope or competes for binding with a kallikrein binding protein described herein.

In some embodiments, the protein binds the same epitope or competes for binding with a protein (e.g., epi-Kal2) and/or a small molecule (e.g., AEBSF) described herein.

In some embodiments, the isolated protein comprises a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein:

the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the 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 the light chain variable domain of a protein described herein, wherein the protein binds to plasma kallikrein.

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 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, XI15D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

2019204295 19 Jun 2019 the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of M162-A04,

M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04,

M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In some embodiments, the protein inhibits plasma kallikrein (e.g., human plasma kallikrein and/or mouse plasma kallikrein).

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

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

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

In some embodiments, the one, two, or three (e.g., three) CDR regions from the heavy chain variable domain are from M162-A04 and/or the one, two, or three (e.g., three) CDR regions from the light chain variable domain are from MJ162-A04.

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

In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of M162-A04, M160-G12, Ml 42H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-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, M142H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X1156

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E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

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

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

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

In some embodiments, the protein comprises the heavy chain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

and/or the light chain of M162-A04, M160-G12, M142-H08, X63-G06, X101A01, 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).

In some embodiments, the protein comprises the heavy chain ofX81-B01, and/or the light chain ofX81-B01.

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

In some embodiments, the plasma kallikrein binding protein decreases Factor Xlla and/or bradykinin production by greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about

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55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% as compared to a standard, e.g., the Factor Xlla and/or bradykinin production under the same conditions but in the absence of the protein.

In some embodiments, the protein includes one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain 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 to aLC variable domain described herein (e.g., overall or in framework regions or CDRs); (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 a HC variable domain described herein (e.g., overall or in framework regions or CDRs); (f) the protein binds 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 comprises a CDR1 that differs by at least one amino acid but by no more than 2 or 3 amino acids from the CDR1 of a HC variable domain described herein; (i) the HC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variable domain described herein; (j) the HC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one amino acid but by no more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variable domain described herein; (k) the LC immunoglobulin variable domain sequence comprises a CDR1 that differs by at least one amino acid but by no more than 2, 3, 4, or 5 amino acids from the CDR1 of a LC variable domain described herein; (1) the LC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid but by no more than 2, 3, or 4 amino acids from the CDR2 of a LC variable domain described herein; (m) the LC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one

2019204295 19 Jun 2019 amino acid but by no more than 2, 3, 4, or 5 amino acids from the CDR3 of a LC variable domain described herein ; (n) the LC immunoglobulin variable domain sequence differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a LC variable domain described herein (e.g., overall or in framework regions or CDRs); and (o) the HC immunoglobulin variable domain sequence differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a HC variable domain described herein (e.g., overall or in framework regions or CDRs).

In some embodiments, the protein has an apparent inhibition constant (K_papp) of less than 1000, 500, 100, 10, 1, 0.5 or 0.2 nM.

In some embodiments, the antibody does not bind prekallikrein (e.g., human prekallikrein and/or mouse prekallikrein), but binds to the active form of plasma kallikrein (e.g., human plasma kallikrein and/or mouse plasma kallikrein).

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67G04, 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.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the 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, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the 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, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X679

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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.

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, X67D03, 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.

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, X67D03, 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.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1,2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains 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, M06D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains 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, M06D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains 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, M06D09 and M35-G04.

2019204295 19 Jun 2019 and one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains 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, M145Dll, M06-D09 and M35-G04 (respectively).

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of X81 -BO 1.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chain ofX81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the heavy chain of X81-B01 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chain of X81 -BO 1.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the heavy chain of X67-D03.

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In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a heavy chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having a light chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the corresponding CDRs of the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs from the heavy chain of X67-D03 and one or more (e.g., 1, 2, or 3) light chain CDRs from the corresponding CDRs of the light chain of X67-D03.

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

In some embodiments, the plasma kallikrein binding protein has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, in vivo, e.g., in humans. In one embodiment, the plasma kallikrein binding protein is an IgG, e.g., an IgGl, IgG2, IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.

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., IgGl, IgG2, IgG3, or IgG4. The protein can be a soluble Fab (sFab).

2019204295 19 Jun 2019

In other embodiments, the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as 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 appropriate construction.

In one embodiment, the protein is a human or humanized antibody or is nonimmunogenic in a human. For example, the protein includes one or more human antibody framework regions, e.g., all human framework regions.

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

In one embodiment, the protein is a primate or primatized antibody or is nonimmunogenic in a human. For example, the protein includes one or more primate antibody framework regions, e.g., all primate framework regions.

In one embodiment, the protein includes 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 (bonobos)), gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

In one embodiment, the protein includes human framework regions, or framework regions that are at least 95, 96, 97, 98, or 99% identical to human framework regions.

In certain embodiments, the protein includes no sequences from mice or rabbits (e.g., is not a murine or rabbit antibody).

In certain embodiments, the protein is capable of binding to a cell or tissue, e.g., that expresses plasma kallikrein.

In one embodiment, protein is physically associated with a nanoparticle, and can be used to guide a nanoparticle to a cell or tissue expressing plasma kallikrein.

In some aspects, the disclosure features a pharmaceutical composition comprising a kallikrein binding protein described herein, e.g., including a pharmaceutically acceptable carrier. In some embodiments, the composition can be at least 10, 20, 30, 50,

2019204295 19 Jun 2019

75, 85, 90, 95, 98, 99, or 99.9% free of other protein species. In one embodiment, the pharmaceutical composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99, or

99.9% free of fragments of the binding protein that do not binding plasma kallikrein (e.g., human plasma kallikrein) or bind plasma kallikrein (e.g., human plasma kallikrein with a

Ki, app of 5000 nM or greater.

In some aspects, the disclosure features a method of treating or preventing a plasma kallikrein associated disorder in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody) that binds plasma kallikrein (e.g., human plasma kallikrein and/or mouse plasma kallikrein) and, e.g., does not bind prekallikrein (e.g., human prekallikrein and/or mouse prekallikrein) to the subject,

In some embodiments, the protein binds the same epitope or competes for binding with a kallikrein binding protein described herein.

In some embodiments, the plasma kallikrein associated disorder is selected from the group consisting of rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting on ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, and burn injury. In some embodiments, the plasma kallikrein binding protein reduces abherent clotting associated with the contact activation system (i.e., intrinsic activation system) by at least 10% as measured by e.g., an APTT clotting assay. In other embodiments, the plasma kallikrein binding protein reduces abherent 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 even 100% (i.e., no detectable abherent clotting).

2019204295 19 Jun 2019

In some embodiments, the plasma kallikrein binding protein is administered in combination with another treatment for the disorder.

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, X67D03, 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.

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

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

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 an epitope that overlaps with the active site of plasma kallikrein.

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

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

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

In some embodiments, the plasma kallikrein binding protein decreases Factor Xlla and/or bradykinin production by greater 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%,

2019204295 19 Jun 2019 or about 95% as compared to a standard, e.g., the Factor Xlla and/or bradykinin production under the same conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has an apparent inhibition constant (Κ_ραρρ) of less than 1000, 500, 100, 10, 5, 1, 0.5, or 0.2 nM.

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, e.g., IgGl, IgG2, IgG3, or IgG4. The plasma kallikrein binding protein can be a soluble Fab (sFab).

In other implementations the plasma kallikrein binding protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as 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 appropriate construction.

2019204295 19 Jun 2019

A method of treating or preventing a plasma kallikrein associated disorder in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein:

the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the heavy chain variable domain of a protein described herein, and/or

2019204295 19 Jun 2019 the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of a protein described herein, wherein the protein binds to plasma kallikrein (e.g., human plasma kallikrein and/or mouse plasma kallikrein).

In some embodiments, the plasma kallikrein associated disorder is selected from the group consisting of rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting of ventrical assistance devices or stents, head trauma or peritumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, and burn injury. In some embodiments, the plasma kallikrein binding protein reduces abherent clotting associated with the contact activation system (i.e., intrinsic activation system) by at least 10% as measured by e.g., an APTT clotting assay (e.g., 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 even 100% (i.e., no detectable abherent clotting)).

In some embodiments, the protein is administered in combination with another treatment for the disorder.

In some embodiments, the protein is administered in combination with a second agent selected from the group consisting of ecallantide, a Cl esterase inhibitor, aprotinin, a bradykinin B2 receptor inhibitor (e.g., icatibant).

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 domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67D03, 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 domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X11518

2019204295 19 Jun 2019

B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04,

M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In some embodiments, the protein inhibits plasma kallikrein.

In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of M162-A04, M160-G12, Ml 42H08 X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, XI15E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-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, M142H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

2019204295 19 Jun 2019

In some embodiments, the protein comprises the heavy chain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

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

In some embodiments, the protein includes one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the LC immunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variable domain described herein; (d) the LC immunoglobulin variable domain sequence is at

2019204295 19 Jun 2019 least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain described herein (e.g., overall or in framework regions or CDRs); (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 a HC variable domain described herein (e.g., overall or in framework regions or CDRs); (f) the protein binds 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.

In some embodiments, the protein has an apparent inhibition constant (K_papp) of less than 1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.

In some embodiments, the antibody does not bind prekallikrein (e.g., human prekallikrein), but binds to the active form of plasma kallikrein (e.g., human plasma kallikrein).

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X6721

2019204295 19 Jun 2019

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains 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, M06D09 and M35-G04 and one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of M162-A04, M160-G12,

2019204295 19 Jun 2019

M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,

2019204295 19 Jun 2019

In other implementations the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that

2019204295 19 Jun 2019 comprises the antigen combining site of one of the binding proteins herein. The VH and

VL regions of these Fabs can be provided as 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 appropriate construction.

In some aspects, the disclosure features a method of promoting wound healing in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody) that binds plasma kallikrein (e.g., human plasma kallikrein and/or mouse plasma kallikrein) and,

2019204295 19 Jun 2019

e.g., does not bind prekallikrein (e.g., human prekallikrein and/or mouse prekallikrein) to the subject.

In some embodiments, the protein binds the same epitope or competes for binding with a kallikrein binding protein described herein. In some embodiments, the protein binds the same epitope or competes for binding with a protein (e.g., epi-Kal2) and/or a small molecule (e.g., AEBSF) described herein.

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

In some embodiments, the plasma kallikrein binding protein does not bind prekallikrein (e.g., human prekallikrein), but binds to the active form of plasma kallikrein (e.g., human plasma kallikrein).

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

2019204295 19 Jun 2019

In some embodiments, the plasma kallikrein binding protein has an apparent inhibition constant (Κ_ραρρ) of less than 1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.

In other implementations the plasma kallikrein binding protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as 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 appropriate construction.

In one embodiment, the plasma kallikrein binding protein is a human or humanized antibody or is non-immunogenic in a human. For example, the protein

2019204295 19 Jun 2019 includes one or more human antibody framework regions, e.g., all human framework regions.

In some aspects, the disclosure features a method promoting wound healing in a subject, the method comprising:

2019204295 19 Jun 2019 the heavy chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the 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 the light chain variable domain of a protein described herein, wherein the protein binds to plasma kallikrein.

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

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 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, XI15D01, 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 domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In some embodiments, the protein inhibits plasma kallikrein.

2019204295 19 Jun 2019

In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises the heavy chain variable domain of M162-A04, M160-G12, Ml 42H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-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, M142H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, XI15-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

2019204295 19 Jun 2019

In some embodiments, the protein includes one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the FC immunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a FC variable domain described herein; (d) the FC 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 FC variable domain described herein (e.g., overall or in framework regions or CDRs); (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 a HC variable domain described herein (e.g., overall or in framework regions or CDRs); (f) the protein binds 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.

In some embodiments, the protein has an apparent inhibition constant (K_papp) of less than 1000, 500, 100, 5, 1, 0.5 or 0.2 nM.

In some embodiments, the antibody does not bind prekallikrein (e.g., human prekallikrein and/or murine prekallikrein), but binds to the active form of plasma kallikrein (e.g., human plasma kallikrein and/or murine plasma kallikrein).

2019204295 19 Jun 2019

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the light and heavy chains of antibodies selected from the group consisting of

M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67G04, 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.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06,, X101-A01, X81-B01, X67D03, 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.

2019204295 19 Jun 2019

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1,2, or 3) heavy chain CDRs selected from the corresponding CDRs of the group of heavy chains 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, M06D09 and M35-G04 and one or more (e.g., 1,2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains 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, M145Dll, M06-D09 and M35-G04 (respectively).

2019204295 19 Jun 2019

In other implementations the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as 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 appropriate construction.

2019204295 19 Jun 2019

In some aspects, the disclosure features a method of treating or preventing rheumatoid arthritis in a subject, the method comprising:

In some embodiments, the plasma kallikrein binding protein does not bind prekallikrein (e.g., human prekallikrein and/or murine prekallikrein), but binds to the

2019204295 19 Jun 2019 active form of plasma kallikrein (e.g., human plasma kallikrein and/or murine plasma kallikrein).

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

In some embodiments, the protein inhibits plasma kallikrein (e.g., human plasma kallikrein and/or murine plasma kallikrein).

In some aspects, the disclosure features a method of treating or preventing gout in a subject, the method comprising:

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

2019204295 19 Jun 2019

In some aspects, the disclosure features a method of treating or preventing intestinal bowel disease in a subject, the method comprising:

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

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

2019204295 19 Jun 2019 the light chain immunoglobulin variable domain sequence comprises the light chain variable domain of a protein described herein.

In some aspects, the disclosure features a method of treating or preventing oral mucositis in a subject, the method comprising:

In some embodiments, the protein is administered in combination with another treatment for oral mucositis.

2019204295 19 Jun 2019

In some aspects, the disclosure features a method of treating or preventing neuropathic pain in a subject, the method comprising:

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

In some aspects, the disclosure features a method of treating or preventing inflammatory pain in a subject, the method comprising:

2019204295 19 Jun 2019 administering an isolated protein (e.g., antibody, e.g., human antibody) comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence to the subject, wherein:

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

In some aspects, the disclosure features a method of treating or preventing spinal stenosis-degenerative spine disease in a subject, the method comprising:

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

In some aspects, the disclosure features a method of treating or preventing arterial or venous thrombosis in a subject, the method comprising:

2019204295 19 Jun 2019 the light chain immunoglobulin variable domain sequence comprises one, two, or three (e.g., three) CDR regions from the light chain variable domain of a protein described herein, wherein the protein binds to plasma kallikrein.

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

In some aspects, the disclosure features a method of treating or preventing post operative ileus in a subject, the method comprising:

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

In some aspects, the disclosure features a method of treating or preventing aortic aneurysm in a subject, the method comprising:

2019204295 19 Jun 2019

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

In some aspects, the disclosure features a method of treating or preventing osteoarthritis in a subject, the method comprising:

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

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In some aspects, the disclosure features a method of treating or preventing vasculitis in a subject, the method comprising:

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

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In some aspects, the disclosure features a method of treating or preventing head trauma or peri-tumor brain edema in a subject, the method comprising:

In some embodiments, the protein is administered in combination with another treatment for head trauma or peri-tumor brain edema.

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In some aspects, the disclosure features a method of treating or preventing sepsis in a subject, the method comprising:

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

In some aspects, the disclosure features a method of treating or preventing acute middle cerebral artery (MCA) ischemic event (stroke) in a subject, the method comprising:

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

In some aspects, the disclosure features a method of treating or preventing restenosis (e.g., after angioplasty) in a subject, the method comprising:

In some embodiments, the protein is administered in combination with another treatment for restenosis (e.g., after angioplasty).

In some aspects, the disclosure features a method of treating or preventing systemic lupus erythematosis nephritis in a subject, the method comprising:

In some embodiments, the protein is administered in combination with another treatment for systemic lupus erythematosis nephritis.

In some aspects, the disclosure features a method of treating or preventing burn injury in a subject, the method comprising:

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

In some aspects, the disclosure features a method of detecting plasma kallikrein in a sample, the method comprising: contacting the sample with a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein); and detecting an interaction between the protein and the plasma kallikrein, if present.

In some embodiments, the protein includes a detectable label.

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

In some aspects, the disclosure features a method of detecting plasma kallikrein in a subject, the method comprising: administering a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) to a subject; and detecting an

2019204295 19 Jun 2019 interaction between the protein and the plasma kallikrein in the subject, if present. For example, the detecting comprises imaging the subject.

In some embodiments, the protein further includes a detectable label.

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 disorder. The method includes: contacting plasma kallikrein with a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein) (e.g., in a human subject), thereby modulating plasma kallikrein activity.

In some embodiments, the plasma kallikrein associated disorder is selected from the group consisting of rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting induced by ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis/vasculitis, and burn injury.

In some embodiments, the plasma kallikrein binding protein reduces abherent clotting associated with the contact activation system (i.e., intrinsic activation system) by at least 10% as measured by e.g., an APTT clotting assay. In other embodiments, the

2019204295 19 Jun 2019 plasma kallikrein binding protein reduces abberent 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 even 100% (i.e., no detectable abberent clotting).

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

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

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

In some embodiments, the protein inhibits plasma kallikrein activity (e.g., human plasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein may include a detectable label (e.g., a radionuclide or an MRI-detectable label).

In some embodiments, the subject has or is suspected of having a plasma kallikrein associated disorder. The method is useful, e.g., for diagnosis of a plasma kallikrein associated disorder.

In some embodiments, the plasma kallikrein associated disorder is selected from the group consisting of rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting induced by ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, and burn injury.

In some embodiments, the plasma kallikrein binding protein reduces abherent clotting associated with the contact activation system (i.e., intrinsic activation system) by at least 10% as measured by e.g., an APTT clotting assay (e.g., 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 even 100% (i.e., no detectable abherent clotting)).

In some aspects, the disclosure features a method of imaging plasma kallikrein, e.g., in a subject or sample (e.g., biopsy sample). The method includes administering a plasma kallikrein binding protein (e.g., a plasma kallikrein binding protein described herein), e.g., to the subject or the sample, and detecting an interaction between the protein and the plasma kallikrein, if present.

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

2019204295 19 Jun 2019 kallikrein). In some embodiments, the plasma kallikrein binding protein binds prekallikrein (e.g., human prekallikrein and/or murine prekallikrein) and the active form of plasma kallikrein (e.g., human plasma kallikrein and/or murine plasma kallikrein).

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

In one aspect, the disclosure features the use of a plasma kallikrein binding protein described herein for the treatment of a disorder described herein, e.g., rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema,

2019204295 19 Jun 2019 cerebral edema, pulmonary embolism, stroke, clotting induced by ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, or burn injury; or to promote wound healing.

In one aspect, the disclosure features the use of a plasma kallikrein binding protein described herein for the manufacture of a medicament for the treatment of a disorder described herein, e.g., rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting induced by ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, or burn injury; or for the manufacture of a medicament for wound healing.

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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 be apparent from the description and drawings, and from the claims.

The contents of all cited references including literature references, issued patents, published or non-published patent applications cited throughout this application as well as those listed below are hereby expressly incorporated by reference in their entireties. In case of conflict, the present application, including any definitions herein, will control.

BRIEF DESCRIPTION OF DRAWINGS

FIGURE 1 is a schematic representation of the role of plasma kallikrein (pKal) in intrinsic coagulation pathway and inflammation.

FIGURE 2 depicts the effect of M162-A04 on carrageenan-induced rat paw edema. Paw swelling was measured by water displacement.

FIGURE 3 depicts the effect of M162-A04 on carrageenan-induced thermal hyperalgesia. Pain latency was measured by the Hargreaves method after carrageenan injection.

FIGURE 4 depicts the alignment of the light chain DNA sequence of nongermlined (X63-G06) and germlined, codon optimized (X81-B01) versions of the same antibody discovered using ROLIC affinity maturation. Positions indicated with an asterisk (*) are conserved, whereas blank spaces correspond to bases changed in X81-B01 due to either codon optimization or germlining.

FIGURE 5 depicts the alignment of the light chain amino acid sequence of nongermlined (X63-G06) and germlined, codon optimized (X81-B01) versions of the same antibody discovered using ROLIC affinity maturation. Positions indicated with an asterisk (*) are conserved, whereas blank spaces correspond to amino acids changed in X81-B01 due to germlining. A total of 11 amino acids differ between the nongermlined (X63-G06) and germlined, codon optimized antibody (X81-B01).

FIGURE 6 depicts the alignment of the heavy chain DNA sequence of nongermlined (X63-G06) and germlined, codon optimized (X81-B01) versions of the same antibody

2019204295 19 Jun 2019 discovered using ROLIC affinity maturation. Positions indicated with an asterisk (*) are conserved, whereas blank spaces correspond to DNA bases changed in X81-B01 due to codon optimization.

FIGURE 7 depicts the alignment of the heavy chain amino acid sequence of nongermlined (X63-G06) and germlined, codon optimized (X81-B01) versions of the same antibody discovered using ROLIC affinity maturation. Positions indicated with an asterisk (*) are conserved. The two antibodies have the same amino acid sequence in the heavy chain.

FIGURE 8A depicts the EPI-KAL2 competition for X81-B01 binding pKal. X81-B01 (IgG) was captured on an anti-human Fc fragment specific surface of a CM5 BIACORE® chip. pKal (100 nM) was flowed over the surface in the presence (lower sensorgram in the figure) or absence of 1 μΜ EPI-KAL2 (upper sensorgram in the figure).

FIGURE 8B depicts the EPI-KAL2 competition for X67-D03 binding pKal. X67-D03 (IgG) was captured on an anti-human Fc fragment specific surface of a CM5 Biacore chip. pKal (100 nM) was flowed over the surface in the presence (lower sensorgram in the figure) or absence of 1 μΜ EPI-KAL2 (upper sensorgram in the figure).

FIGURE 9 depicts the results of CLIPS epitope mapping for antibodies listed in

Table 12.

FIGURES 10A-10C depict ClustalW alignment of pKal sequences from different species. Positions indicated by a are conserved positions between, whereas positions indicated indicate conservative substitutions between species. Positions indicated by a have nonconservative substitutions in some species. Stretches of amino acids indicated by the symbol “@” were shown to be highly solvent exposed by solvent accessible surface area calculation. Stretches of amino acids indicated by a “+” were identified as potential epitopes of antibodies listed in Table 12. Amino acids highlighted in grey were found by solvent accessible surface area calculation to be buried when complexed with a Kunitz domain active site inhibitor. The underlined positions are the amino acids that form the catalytic triad (His434, Asp483, and Ser578, numbering based on the human sequence).

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FIGURES 11A and 11B depict a Biacore competition analysis with epi-kal2, as described herein in Example 12, for (i) DX-2922, and (ii) M6-D09 antibodies.

FIGURE 12 depicts a Biacore competition analysis with AEBSF, as described herein in

Example 12, for (i) DX-2911, and (ii) M6-D09 antibodies.

FIGURE 13 depicts a Biocore analysis showing that DX-2922 binds to plasma kallikrein that bound to hign molecular weight kininogen (HMWK).

FIGURE 14 depicts a graph showing dose dependent inhibition of edema by XI01-AO 1 in carrageenan-induced paw edema (CPE) in rats.

DETAILED DESCRIPTION

Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are defined here. Other terms are defined as they appear in the specification.

The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

The term “agonist,” as used herein, is meant to refer to an agent that mimics or up-regulates (e.g., potentiates or supplements) the bioactivity of a protein. An agonist can be a wild-type protein or derivative thereof having at least one bioactivity of the wild-type protein. An agonist can also be a compound which increases at least one bioactivity of a protein. An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, e.g., a target peptide or nucleic acid.

“Antagonist” as used herein is meant to refer to an agent that downregulates (e.g., suppresses or inhibits) at least one bioactivity of a protein. An antagonist can be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a target peptide or enzyme substrate. An antagonist can also be a compound which reduces the amount of expressed protein present.

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

2019204295 19 Jun 2019 (abbreviated herein as VH or HV), and a light (L) chain variable region (abbreviated herein as VL or LV). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDRs”), interspersed with regions that are more conserved, termed “framework regions” (“FRs”). The extent of the framework region and CDRs have been defined (see, Rabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Rabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain such that one or more CDR regions are positioned in a conformation suitable for an antigen binding site. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form an antigen binding site, e.g., a structure that preferentially interacts with plasma kallikrein.

The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy

2019204295 19 Jun 2019 immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CHI, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., 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. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and/or FR4 of the HC and/or LC. For example, the Fc region can be human. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. In one embodiment, the framework (FR) residues of a selected Fab can be converted to the amino-acid type of the corresponding residue in the most similar primate germline gene, especially the human germline gene. One or more of the constant regions can be human or effectively human. For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of an immunoglobulin variable domain, the constant region, the constant domains (CHI, CH2, CH3, and/or CL1), or the entire antibody can be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the many immunoglobulin variable region genes. Full-length

2019204295 19 Jun 2019 immunoglobulin “light chains” (about 25 KDa or about 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. 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 one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The length of human HC varies considerably because HC CDR3 varies from about 3 amino-acid residues to over 35 amino-acid residues.

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 binding fragments encompassed within the term “antigenbinding fragment” of a full length antibody and that retain functionality include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains;

(iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., U.S. Pat. Nos. 5,260,203,4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art. The term “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a “monoclonal antibody” or “monoclonal antibody composition,” which as used herein refers to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.

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Antibodies are “germlined” by reverting one or more non-germline amino acids in framework regions to corresponding germline amino acids of the antibody, so long as binding properties are substantially retained.

The inhibition constant (Ki) provides a measure of inhibitor potency; it is the concentration of inhibitor required to reduce enzyme activity by half and is not dependent on enzyme or substrate concentrations. The apparent Ki (Ki_iapp) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of inhibitor (e.g., inhibitory binding protein) on the extent of the reaction (e.g., enzyme activity); fitting the change in pseudo-first order rate constant as a function of inhibitor concentration to the Morrison equation (Equation 1) yields an estimate of the apparent Ki value. The Ki is obtained from the y-intercept extracted from a linear regression analysis of a plot of Ki,app versus substrate concentration.

+' + £>- #,,,+0 A-4·'A

V = v —v ---------------²----------------------Ο Ο Ί—Ι

E k J

Equation 1

Where v = measured velocity; vo = velocity in the absence of inhibitor; Ki_iapp = apparent inhibition constant; I = total inhibitor concentration; and E = total enzyme concentration.

As used herein, “binding affinity” refers to the apparent association constant or Ka. The Ka is the reciprocal of the dissociation constant (Kd). A binding protein may, for example, have a binding affinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, IO¹⁰ and 10¹¹ M’¹for a particular target molecule, e.g., plasma kallikrein. Higher affinity binding of a binding protein to a first target relative to a second target can be indicated by a higher Ka (or a smaller numerical value Kd) for binding the first target than the Ka (or numerical value K_D) for binding the second target. In such cases, the binding protein has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

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Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound and free binding protein 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 where (N) is the number of binding sites per target molecule by the following equation:

[Bound] = N · [Free]/((1/KA) + [Free]).

It is not always necessary to make an exact determination of Ka, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to Ka, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

The term “binding protein” refers to a protein that can interact with a target molecule. This term is used interchangeably with “ligand.” A “plasma kallikrein binding protein” refers to a protein that can interact with (e.g., bind) plasma kallikrein, and includes, in particular, proteins that preferentially or specifically interact with and/or inhibit plasma kallikrein. A protein inhibits plasma kallikrein if it causes a decrease in the activity of plasma kallikrein as compared to the activity of plasma kallikrein in the absence of the protein and under the same conditions. In some embodiments, the plasma kallikrein binding protein is an antibody.

A “conservative amino acid substitution” is one in which the 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. These families include amino acids with 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,

2019204295 19 Jun 2019 valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

It is possible for one or more framework and/or CDR amino acid residues of a binding protein to include one or more mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) relative to a binding protein described herein. A plasma kallikrein binding protein may have mutations (e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) (e.g., at least one, two, three, or four, and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations) relative to a binding protein described herein, e.g., mutations which do not have a substantial effect on protein function. The mutations can be present in framework regions, CDRs, and/or constant regions. In some embodiments, the mutations are present in a framework region. In some embodiments, the mutations are present in a CDR. In some embodiments, the mutations are present in a constant region. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as binding activity, can be predicted, e.g., by evaluating whether the mutation is conservative or by the method of Bowie, et al. (1990) Science 247:1306-1310.

Motif sequences for biopolymers can include positions which can be varied amino acids. For example, the symbol “X” in such a context generally refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified, e.g., to refer to any non-cysteine amino acid. Other allowed amino acids can also be indicated for example, using parentheses and slashes. For example, “(A/W/F/N/Q)” means that alanine, tryptophan, phenylalanine, asparagine, and glutamine are allowed at that particular position.

An “effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An “effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.

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An “epitope” refers to the site on a target compound that is bound by a binding protein (e.g., an antibody such as a Fab or full length antibody). In the case where the target compound is a protein, the site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature.

A first binding protein (e.g., antibody) “binds to the same epitope” as a second binding protein (e.g., antibody) if the first binding protein binds to the same site on a target compound that the second binding protein binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group)) with the site that the second binding protein binds.

A first binding protein (e.g., antibody) “competes for binding” with a second binding protein (e.g., antibody) if the binding of the first binding protein to its epitope decreases (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 its epitope. The competition can be direct (e.g., the first binding protein binds to an epitope that is the same as, or overlaps with, the epitope bound by the second binding protein), or indirect (e.g., the binding of the first binding protein to its epitope causes a steric change in the target compound that decreases the ability of the second binding protein to bind to its epitope).

Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and nonhomologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as

2019204295 19 Jun 2019 the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

In a preferred embodiment, 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%, and even more preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% of the length of the reference sequence. For example, the reference sequence may be the length of the immunoglobulin variable domain sequence.

A “humanized” immunoglobulin variable region is an immunoglobulin variable region that is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of “humanized” immunoglobulins include, for example, U.S. 6,407,213 and U.S. 5,693,762.

As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.Ιό.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); (2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified. The disclosure includes nucleic acids that hybridize with

2019204295 19 Jun 2019 low, medium, high, or very high stringency to a nucleic acid described herein or to a complement thereof, e.g., nucleic acids encoding a binding protein described herein. The nucleic acids can be the same length or within 30, 20, or 10% of the length of the reference nucleic acid. The nucleic acid can correspond to a region encoding an immunoglobulin variable domain sequence described herein.

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

An “isolated” protein refers to a protein that is removed from at least 90% of at least one component of a natural sample from which the isolated protein can be obtained. Proteins can be “of at least” a certain degree of purity if the species or population of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-weight basis.

The term “modulator” refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species or the like (naturally-occurring or non-naturally-occurring), or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, that may be capable of causing modulation. Modulators may be evaluated for potential activity as inhibitors or activators (directly or indirectly) of a functional property, biological activity or process, or combination of them, (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, antimicrobial agents, inhibitors of microbial infection or proliferation, and the like) by inclusion in assays. In such assays, many modulators may be screened at one time. The activity of a modulator may be known, unknown or partially known.

A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas changing an “essential” amino acid residue results in a substantial loss of activity.

A “patient,” “subject” or “host” (these terms are used interchangeably) to be treated by the subject method may mean either a human or non-human animal.

2019204295 19 Jun 2019

The terms “prekallikrein” and “preplasma kallikrein” are used interchangeably herein and refer to the zymogen form of active plasma kallikrein, which is also known as prekallikrein.

The term “preventing” or to “prevent” a disease in a subject refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition. “Preventing” a disease may also be referred to as “prophylaxis” or “prophylactic treatment.”

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 (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to 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) similar activities, e.g., a binding activity, a binding preference, or a biological activity. In the case of antibodies, the second antibody has the same specificity and has at least 50%, at least 25%, or at least 10% of the affinity relative to the same antigen.

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 higher. In some embodiments, a plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a binding protein described herein. In some embodiments, a plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the HC and/or LC framework regions (e.g., HC and/or LC FR 1, 2, 3, and/or 4) to a binding protein described herein. In some embodiments, a plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the HC and/or LC CDRs (e.g., HC and/or LC CDR1, 2, and/or 3) to a binding protein described herein. In some

2019204295 19 Jun 2019 embodiments, a plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the constant region (e.g., CHI, CH2, CH3, and/or CL1) to a binding protein described herein.

In addition, substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. Particular binding proteins may show a difference, e.g., in specificity or binding that are statistically significant (e.g., P value < 0.05 or 0.02). The terms “induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, e.g., which denote distinguishable qualitative or quantitative differences between two states, may refer to a difference, e.g., a statistically significant difference, between the two states.

A “therapeutically effective dosage” preferably modulates a measurable parameter, e.g., plasma kallikrein activity, by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to modulate a measurable parameter, e.g., a disease-associated parameter, can be evaluated in an animal model system predictive of efficacy in human disorders and conditions, e.g., rheumatoid arthritis or oral mucositis. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate a parameter in vitro.

“Treating” a disease (or condition) in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is cured, alleviated or decreased.

The term “preventing” a disease in a subject refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one

2019204295 19 Jun 2019 symptom of the disease is prevented, that is, administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) so that it protects the host against developing the unwanted condition. “Preventing” a disease may also be referred to as “prophylaxis” or “prophylactic treatment.”

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, because a prophylactic dose 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.

As used herein the term “DX-2922” as used inchangeably with the term “XI01A01”. Other variants of this antibody are described below.

Antibody Identification	Description
X63-G06	Non-germlined Fab discovered using ROLIC, same HC but different LC as M160-G12
X81-B01	Germlined IgG produced in HEK 293T cells
X101-A01	Germlined IgG produced in CHO cells, same HC and LC sequence as X81-B01
DX-2922	Alternate nomenclature for XI01-AO 1

Plasma Kallikrein Binding Proteins

Plasma kallikrein binding proteins can be full-length (e.g., an IgG (e.g., an IgGl, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgAl, IgA2), IgD, and IgE) or can include only an antigen-binding fragment (e.g., a Fab, F(ab j2 or scFv fragment. The binding protein can include two heavy chain immunoglobulins and two light chain immunoglobulins, or can be a single chain antibody. Plasma kallikrein binding proteins can be recombinant proteins such as humanized, CDR grafted, chimeric, deimmunized, or in vitro generated antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences. In one embodiment, the plasma kallikrein binding protein is a monoclonal antibody.

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 murine kallikrein) and includes

2019204295 19 Jun 2019 at least one immunoglobulin variable region. For example, the protein includes a heavy chain (HC) immunoglobulin variable domain sequence and/or a light chain (FC) immunoglobulin variable domain sequence. In one embodiment, the protein binds to and inhibits plasma kallikrein, e.g., human plasma kallikrein and/or murine kallikrein.

The protein can include one or more of the following characteristics: (a) a human CDR or human framework region; (b) the HC immunoglobulin variable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC variable domain described herein; (c) the FC immunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a FC variable domain described herein; (d) the FC 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 FC variable domain described herein (e.g., overall or in framework regions or CDRs); (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 a HC variable domain described herein (e.g., overall or in framework regions or CDRs); (f) the protein binds 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 comprises a CDR1 that differs by at least one amino acid but by no more than 2 or 3 amino acids from the CDR1 of a HC variable domain described herein; (i) the HC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variable domain described herein; (j) the HC immunoglobulin variable domain sequence comprises a CDR3 that differs by at least one amino acid but by no more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variable domain described herein; (k) the FC immunoglobulin variable domain sequence comprises a CDR1 that differs by at least one amino acid but by no more than 2, 3, 4, or 5 amino acids from the CDR1 of a FC variable domain described herein; (1) the FC immunoglobulin variable domain sequence comprises a CDR2 that differs by at least one amino acid but by no more than 2, 3, or 4 amino acids from the CDR2 of a FC variable domain described herein; (m) the FC immunoglobulin variable domain sequence

2019204295 19 Jun 2019 comprises a CDR3 that differs by at least one amino acid but by no more than 2, 3, 4, or 5 amino acids from the CDR3 of a LC variable domain described herein ; (n) the LC immunoglobulin variable domain sequence differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a LC variable domain described herein (e.g., overall or in framework regions or CDRs); and (o) the HC immunoglobulin variable domain sequence differs by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a HC variable domain described herein (e.g., overall or in framework regions or CDRs).

The plasma kallikrein binding protein may 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 isolated from antibody cleavage fragments (e.g., cleaved DX-2922) that are inactive or partially active (e.g., bind plasma kallikrein with a Ki, app of 5000 nM or greater) compared to the plasma kallikrein binding protein. 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 even 100% free from antibody cleavage fragments that are inactive or partially active.

The plasma kallikrein binding protein may additionally inhibit plasma kallikrein, e.g., human plasma kallikrein.

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

In some aspects, the protein binds the same epitope or competes for binding with a protein described herein.

In some embodiments, the protein competes with or binds the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67G04, XI15-B07, XI15-D05, XI15-E09, XI15-H06, XI15-A03, XI15-D01, XI15-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

2019204295 19 Jun 2019

In some embodiments, the protein binds to (e.g., positions on plasma kallikrein corresponding to) CLIPS peptide Cl, C2, C3, C4, C5, C6, or Cl, or more than one of these peptides, e.g., the protein binds to C5 and C6. CLIPS peptides C1-C7 are peptides in plasma kallikrein identified by CLIPS epitope mapping (see FIGURES 9 and 10A10C). Cl corresponds to positions 55-67 of the catalytic domain, C2 to positions 81-94, C3 to positions 101-108, C4 to positions 137-151, C5 to positions 162-178, C6 to positions 186-197, and C7 to positions 214-217 of plasma kallikrein.

In some embodiments, the protein binds to an epitope shown in FIGURE 9.

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

The active site cleft of plasma kallikrein contains three amino acids that form the catalytic triad (His434, Asp483, and Ser578) and result in enzymatic hydrolysis of bound substrate (catalytic triad residues are underlined in FIGURE 10). The peptides selected for the CLIPS epitope mapping analysis were determined to be surface accessible and either form or surround the vicinity of the active site. Peptide Cl contains the active site histidine 434. Peptide C3 contains the active site aspartate 483. Peptide C6 contains the active site serine 578. It is possible for an antibody to bind multiple surface exposed amino acids that are discontinuous in amino acid sequence. For example, by CLIPs analysis, X81-B01 appears to bind the C2, C3, C5 and the C6 peptides.

In some embodiments, the protein binds to an epitope that includes one or more amino acids from CLIPS peptide Cl, peptide C2, peptide C3, peptide C4, peptide C5, peptide C6, or peptide Cl.

2019204295 19 Jun 2019

In some embodiments, the protein binds to an epitope that includes amino acids from at least 2 different CLIPS peptides, e.g., from at least two of peptide Cl, peptide C2, peptide C3, peptide C4, peptide C5, peptide C6, or peptide Cl.

The protein can bind to plasma kallikrein, e.g., human plasma kallikrein, with a binding affinity of at least ΙΟ⁵, ΙΟ⁶, ΙΟ⁷, ΙΟ⁸, 10⁹, IO¹⁰ and 10¹¹ M’¹. In one embodiment, the protein binds to human plasma kallikrein with a Koff slower than 1 x 1 O’³, 5 x 1 O’⁴ s’¹, or 1 x 10’⁴ s’¹. In one embodiment, the protein binds to human plasma kallikrein with a Kon faster than 1 x 10², lx 10³, or 5 x 10³ M's’¹. In one embodiment, the protein binds to plasma kallikrein, but does not bind to tissue kallikrein and/or plasma prekallikrein (e.g., the protein binds to tissue kallikrein and/or plasma prekallikrein less effectively (e.g., 5-, 10-, 50-, 100-, or 1000-fold less or not at all, e.g., as compared to a negative control) than it binds to plasma kallikrein.

In one embodiment, the protein inhibits human plasma kallikrein activity, e.g., with a Ki of less than 10’⁵, 10’⁶, 10’⁷, 10’⁸, 10’⁹, and IO’¹⁰ M. The protein can have, for example, an IC50 of 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 Xlla (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 Xlla (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 can be characterized by a Kd of less than 100 nm, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM. In one embodiment, the protein inhibits plasma kallikrein, but does not inhibit tissue kallikrein (e.g., the protein inhibits tissue kallikrein less effectively (e.g., 5-, 10-, 50-, 100-, or 1000-fold less or not at all, e.g., as compared to a negative control) than it inhibits plasma kallikrein.

Plasma kallikrein binding proteins may be antibodies. Plasma kallikrein binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e.g., IgG or Fab).

2019204295 19 Jun 2019

M162-A04, M160-G12, M142-H08 X63-G06, X101-A01, X81-B01, X67-D03, X67G04, 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.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having the 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, M29D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having light and heavy antibody variable regions of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67D03, 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.

2019204295 19 Jun 2019

In a preferred embodiment, the protein is an antibody (e.g., a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs and one or more (e.g., 1, 2, or 3) light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67D03, 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.

In one embodiment, the HC and LC variable domain sequences are components of the same polypeptide chain. In another, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is an IgG, e.g., IgGl, IgG2, IgG3, or IgG4. The protein can be a soluble Fab. In other implementations the protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that comprises the antigen combining site of one of the binding proteins herein. The VH and VL regions of these Fabs can be provided as 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 appropriate construction.

In one embodiment, the protein is a human or humanized antibody or is nonimmunogenic in a human. For example, the protein includes one or more human antibody framework regions, e.g., all human framework regions, or framework regions at

2019204295 19 Jun 2019 least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to human framework regions. In one embodiment, the protein includes a human Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99% identical to a human Fc domain.

In one embodiment, the protein is a primate or primatized antibody or is nonimmunogenic in a human. For example, the protein includes one or more primate antibody framework regions, e.g., all primate framework regions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to primate framework regions. In one embodiment, the protein includes 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 (bonobos)), gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and tarsiers.

In some embodiments, the affinity of the primate antibody for human plasma kallikrein is characterized by a K_D 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.

In some aspects, the disclosure provides the use of proteins (e.g., binding proteins, e.g., antibodies) (e.g., the proteins described herein) that bind to plasma kallikrein (e.g., human plasma kallikrein) and include at least one immunoglobin variable region in methods for treating (or preventing) a plasma kallikrein associated disorder or condition. For example, the plasma kallikrein binding protein includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. A number of exemplary plasma kallikrein binding proteins are described herein.

The plasma kallikrein binding protein may be an isolated protein (e.g., at least 70, 80, 90, 95, or 99% free of other proteins).

The plasma kallikrein binding protein may additionally inhibit plasma kallikrein, e.g., human plasma kallikrein and/or murine plasma kallikrein. In some embodiments, it may be preferred to have an plasma kallikrein binding protein bind to both human and murine plasma kallikrein, as these antibodies can be tested for efficacy in a mouse model.

2019204295 19 Jun 2019

Plasma Kallikrein

Exemplary plasma kallikrein sequences against which plasma kallikrein binding proteins may be developed can include human, mouse, or rat plasma kallikrein amino acid sequences, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., of a sequence provided below.

The sequence of human plasma kallikrein that was used in selections and subsequent screening is shown below (accession number NP_000883.2). The human plasma kallikrein (86 kDa) that was used was purified from human plasma and activated with factor Xlla by a commercial vendor. Factor Xlla activates prekallikrein by cleaving the polypeptide sequence at a single site (between Arg371-Ile372, cleavage site marked by in the sequence below) to generate active plasma kallikrein, which then consists of two disulfide linked polypeptides; a heavy chain of approximately 52 kDa and a catalytic domain of approximately 34 kDa [Colman and Schmaier, (1997) “Contact System: A Vascular Biology Modulator With Anticoagulant, Pro fibrinolytic, Antiadhesive, and Proinflammatory Attributes” Blood, 90, 3819-3843]

GCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKV HRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAE YRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICT YHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFG GEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRL CNTGDNSVCTTKTSTR/IVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDV WRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYT NCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKH NGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA

The human, mouse, and rat prekallikrein amino acid sequences, and the mRNA sequences encoding the same, are illustrated below. The sequences of prekallikrein are the same as plasma kallikrein, except that active plasma kallikrein (pkal) has the single polypeptide chain cleaved at a single position (indicated by the “/”) to generate two chains. The sequences provided below are full sequences that include signal sequences. On secretion from the expressing cell, it is expected that the signal sequences are removed.

2019204295 19 Jun 2019

Human plasma kallikrein (ACCESSION: NP 000883.2) >giI 78191798|ref|NP_000 883.2 | plasma kallikrein Bl precursor [Homo sapiens]

MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSIND MEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKR CTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLA FSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYS LLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLR LSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCG GSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQ APLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQR MVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDG KAQMQSPA

Human plasma kallikrein mRNA (ACCESSION: NM 000892) >giI 78191797|ref|NM_000 8 92.3| Homo sapiens kallikrein B, plasma (Fletcher factor) 1 (KLKB1), mRNA

AGAACAGCTTGAAGACCGTTCATTTTTAAGTGACAAGAGACTCACCTCCAAGAAGCAATTGTGTTTTCAG AATGATTTTATTCAAGCAAGCAACTTATTTCATTTCCTTGTTTGCTACAGTTTCCTGTGGATGTCTGACT CAACTCTATGAAAACGCCTTCTTCAGAGGTGGGGATGTAGCTTCCATGTACACCCCAAATGCCCAATACT GCCAGATGAGGTGCACATTCCACCCAAGGTGTTTGCTATTCAGTTTTCTTCCAGCAAGTTCAATCAATGA CATGGAGAAAAGGTTTGGTTGCTTCTTGAAAGATAGTGTTACAGGAACCCTGCCAAAAGTACATCGAACA GGTGCAGTTTCTGGACATTCCTTGAAGCAATGTGGTCATCAAATAAGTGCTTGCCATCGAGACATTTATA AAGGAGTTGATATGAGAGGAGTCAATTTTAATGTGTCTAAGGTTAGCAGTGTTGAAGAATGCCAAAAAAG GTGCACCAGTAACATTCGCTGCCAGTTTTTTTCATATGCCACGCAAACATTTCACAAGGCAGAGTACCGG AACAATTGCCTATTAAAGTACAGTCCCGGAGGAACACCTACCGCTATAAAGGTGCTGAGTAACGTGGAAT CTGGATTCTCACTGAAGCCCTGTGCCCTTTCAGAAATTGGTTGCCACATGAACATCTTCCAGCATCTTGC GTTCTCAGATGTGGATGTTGCCAGGGTTCTCACTCCAGATGCTTTTGTGTGTCGGACCATCTGCACCTAT CACCCCAACTGCCTCTTCTTTACATTCTATACAAATGTATGGAAAATCGAGTCACAAAGAAATGTTTGTC TTCTTAAAACATCTGAAAGTGGCACACCAAGTTCCTCTACTCCTCAAGAAAACACCATATCTGGATATAG CCTTTTAACCTGCAAAAGAACTTTACCTGAACCCTGCCATTCTAAAATTTACCCGGGAGTTGACTTTGGA GGAGAAGAATTGAATGTGACTTTTGTTAAAGGAGTGAATGTTTGCCAAGAGACTTGCACAAAGATGATTC GCTGTCAGTTTTTCACTTATTCTTTACTCCCAGAAGACTGTAAGGAAGAGAAGTGTAAGTGTTTCTTAAG ATTATCTATGGATGGTTCTCCAACTAGGATTGCGTATGGGACACAAGGGAGCTCTGGTTACTCTTTGAGA TTGTGTAACACTGGGGACAACTCTGTCTGCACAACAAAAACAAGCACACGCATTGTTGGAGGAACAAACT CTTCTTGGGGAGAGTGGCCCTGGCAGGTGAGCCTGCAGGTGAAGCTGACAGCTCAGAGGCACCTGTGTGG AGGGTCACTCATAGGACACCAGTGGGTCCTCACTGCTGCCCACTGCTTTGATGGGCTTCCCCTGCAGGAT GTTTGGCGCATCTATAGTGGCATTTTAAATCTGTCAGACATTACAAAAGATACACCTTTCTCACAAATAA AAGAGATTATTATTCACCAAAACTATAAAGTCTCAGAAGGGAATCATGATATCGCCTTGATAAAACTCCA GGCTCCTTTGAATTACACTGAATTCCAAAAACCAATATGCCTACCTTCCAAAGGTGACACAAGCACAATT TATACCAACTGTTGGGTAACCGGATGGGGCTTCTCGAAGGAGAAAGGTGAAATCCAAAATATTCTACAAA AGGTAAATATTCCTTTGGTAACAAATGAAGAATGCCAGAAAAGATATCAAGATTATAAAATAACCCAACG GATGGTCTGTGCTGGCTATAAAGAAGGGGGAAAAGATGCTTGTAAGGGAGATTCAGGTGGTCCCTTAGTT TGCAAACACAATGGAATGTGGCGTTTGGTGGGCATCACCAGCTGGGGTGAAGGCTGTGCCCGCAGGGAGC AACCTGGTGTCTACACCAAAGTCGCTGAGTACATGGACTGGATTTTAGAGAAAACACAGAGCAGTGATGG AAAAGCTCAGATGCAGTCACCAGCATGAGAAGCAGTCCAGAGTCTAGGCAATTTTTACAACCTGAGTTCA AGTCAAATTCTGAGCCTGGGGGGTCCTCATCTGCAAAGCATGGAGAGTGGCATCTTCTTTGCATCCTAAG GACGAAAAACACAGTGCACTCAGAGCTGCTGAGGACAATGTCTGGCTGAAGCCCGCTTTCAGCACGCCGT AACCAGGGGCTGACAATGCGAGGTCGCAACTGAGATCTCCATGACTGTGTGTTGTGAAATAAAATGGTGA AAGATCAAAAAA

2019204295 19 Jun 2019

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

Mouse plasma kallikrein mRNA (ACCESSION: NM_008455.2) >giI 236465804|ref|NM_008455.2| Mus musculus kallikrein B, plasma 1 (Klkbl), mRNA

AGACCGCCCTCGGTGCCATATTCAGAGGGCTTGAAGACCATCTTCATGTGAAGACTCCCTCTCCTCCAGA ACCACAACGTGACCATCCTTCCAGGATGATTTTATTCAACCGAGTGGGTTATTTTGTTTCCTTGTTTGCT ACCGTCTCCTGTGGGTGTATGACTCAACTGTATAAAAATACCTTCTTCAGAGGTGGGGATCTAGCTGCCA TCTACACCCCAGATGCCCAGTACTGTCAGAAGATGTGCACTTTTCACCCCAGGTGCCTGCTGTTCAGCTT TCTCGCCGTGACTCCACCCAAAGAGACAAATAAACGGTTTGGTTGCTTCATGAAAGAGAGCATTACAGGG ACTTTGCCAAGAATACACCGGACAGGGGCCATTTCTGGTCATTCTTTAAAGCAGTGTGGCCATCAAATAA GTGCTTGCCACCGAGACATATACAAAGGACTTGATATGAGAGGGTCCAACTTTAATATCTCTAAGACCGA CAATATTGAAGAATGCCAGAAACTGTGCACAAATAATTTTCACTGCCAATTTTTCACATATGCTACAAGT GCATTTTACAGACCAGAGTACCGGAAGAAGTGCCTGCTGAAGCACAGTGCAAGCGGAACACCCACCAGCA TAAAGTCAGCGGACAACCTGGTGTCTGGATTCTCACTGAAGTCCTGTGCGCTTTCGGAGATAGGTTGCCC CATGGATATTTTCCAGCACTCTGCCTTTGCAGACCTGAATGTAAGCCAGGTCATCACCCCCGATGCCTTT GTGTGTCGCACCATCTGCACCTTCCATCCCAACTGCCTTTTCTTCACGTTCTACACGAATGAATGGGAGA CAGAATCACAGAGAAATGTTTGTTTTCTTAAGACGTCTAAAAGTGGAAGACCAAGTCCCCCTATTCCTCA AGAAAACGCTATATCTGGATATAGTCTCCTCACCTGCAGAAAAACTCGCCCTGAACCCTGCCATTCCAAA ATTTACTCTGGAGTTGACTTTGAAGGGGAAGAACTGAATGTGACCTTCGTGCAAGGAGCAGATGTCTGCC AAGAGACTTGTACAAAGACAATCCGCTGCCAGTTTTTTATTTACTCCTTACTCCCCCAAGACTGCAAGGA GGAGGGGTGTAAATGTTCCTTAAGGTTATCCACAGATGGCTCCCCAACTAGGATCACCTATGGCATGCAG GGGAGCTCCGGTTATTCTCTGAGATTGTGTAAACTTGTGGACAGCCCTGACTGTACAACAAAAATAAATG CACGTATTGTGGGAGGAACAAACGCTTCTTTAGGGGAGTGGCCATGGCAGGTCAGCCTGCAAGTGAAGCT GGTATCTCAGACCCATTTGTGTGGAGGGTCCATCATTGGTCGCCAATGGGTACTGACAGCTGCCCATTGC TTTGATGGAATTCCCTATCCAGATGTGTGGCGTATATATGGCGGAATTCTTAGTCTGTCCGAGATTACGA AAGAAACGCCTTCCTCGAGAATAAAGGAGCTTATTATTCATCAGGAATACAAAGTCTCAGAAGGCAATTA TGATATTGCCTTAATAAAGCTTCAGACGCCCCTGAATTATACTGAATTCCAAAAACCAATATGCCTGCCT TCCAAAGCTGACACAAATACAATTTATACCAACTGTTGGGTGACTGGATGGGGCTACACGAAGGAACAAG GTGAAACGCAAAATATTCTACAAAAGGCTACTATTCCTTTGGTACCAAATGAAGAATGCCAGAAAAAATA CAGAGATTATGTTATAAACAAGCAGATGATCTGTGCTGGCTACAAAGAAGGCGGAACAGACGCTTGTAAG GGAGATTCCGGTGGCCCCTTAGTCTGTAAACACAGTGGACGGTGGCAGTTGGTGGGTATCACCAGCTGGG GTGAAGGCTGCGCCCGCAAGGACCAACCAGGAGTCTACACCAAAGTTTCTGAGTACATGGACTGGATATT GGAGAAGACACAGAGCAGTGATGTAAGAGCTCTGGAGACATCTTCAGCCTGAGGAGGCTGGGTACCAAGG AGGAAGAACCCAGCTGGCTTTACCACCTGCCCTCAAGGCAAACTAGAGCTCCAGGATTCTCGGCTGTAAA ATGTTGATAATGGTGTCTACCTCACATCCGTATCATTGGATTGAAAATTCAAGTGTAGATATAGTTGCTG AAGACAGCGTTTTGCTCAAGTGTGTTTCCTGCCTTGAGTCACAGGAGCTCCAATGGGAGCATTACAAAGA TCACCAAGCTTGTTAGGAAAGAGAATGATCAAAGGGTTTTATTAGGTAATGAAATGTCTAGATGTGATGC AATTGAAAAAAAGACCCCAGATTCTAGCACAGTCCTTGGGACCATTCTCATGTAACTGTTGACTCTGGAC CTCAGCAGATCTCAGAGTTACCTGTCCACTTCTGACATTTGTTTATTAGAGCCTGATGCTATTCTTTCAA GTGGAGCAAAAAAAAAAAAAAA

2019204295 19 Jun 2019

Rat plasma kallikrein (ACCESSION: NP_036857.2) >giI 162138905|ref|NP_036857.2| kallikrein B, plasma 1 [Rattus norvegicus]

MILFKQVGYFVSLFATVSCGCLSQLYANTFFRGGDLAAIYTPDAQHCQKMCTFHPRCLLFSFLAVSPTKE TDKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQLSACHQDIYEGLDMRGSNFNISKTDSIEECQKL CTNNIHCQFFTYATKAFHRPEYRKSCLLKRSSSGTPTSIKPVDNLVSGFSLKSCALSEIGCPMDIFQHFA FADLNVSHWTPDAFVCRTVCTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIIQENAVSGYS LFTCRKARPEPCHFKIYSGVAFEGEELNATFVQGADACQETCTKTIRCQFFTYSLLPQDCKAEGCKCSLR LSTDGSPTRITYEAQGSSGYSLRLCKWESSDCTTKINARIVGGTNSSLGEWPWQVSLQVKLVSQNHMCG GSIIGRQWILTAAHCFDGIPYPDVWRIYGGILNLSEITNKTPFSSIKELIIHQKYKMSEGSYDIALIKLQ TPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKERGETQNILQKATIPLVPNEECQKKYRDYVITKQ MICAGYKEGGIDACKGDSGGPLVCKHSGRWQLVGITSWGEGCARKEQPGVYTKVAEYIDWILEKIQSSKE RALETSPA

Rat plasma kallikrein mRNA (ACCESSION: NM_012725) >giI 162138904|ref|NM_012725.2| Rattus norvegicus kallikrein B, plasma 1 (Klkbl), 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

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Display Libraries

A display library is a collection of entities; each entity includes an accessible polypeptide component and a recoverable component that encodes or identifies the polypeptide component. The polypeptide component is varied so that different amino acid sequences are represented. The polypeptide component can be of any length, e.g. from three amino acids to over 300 amino acids. A display library entity can include more than one polypeptide component, for example, the two polypeptide chains of a sFab. In one exemplary implementation, a display library can be used to identify proteins that bind to plasma kallikrein. In a selection, the polypeptide component of each member of the library is probed with plasma kallikrein (or fragment thereof) and if the polypeptide component binds to the plasma kallikrein, the display library member is identified, typically by retention on a support.

Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the polypeptide component and purification of the polypeptide component for detailed characterization.

A variety of formats can be used for display libraries. Examples include the following.

Phage Display: The protein component is typically covalently linked to a bacteriophage coat protein. The linkage results from 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 is 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 harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the

2019204295 19 Jun 2019 nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.

Other Display Formats. Other display formats include cell based 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):11935), and E. coli periplasmic display (J Immunol Methods. 2005 Nov 22;PMID: 16337958).

Scaffolds. Scaffolds useful for display include: antibodies (e.g., Fab fragments, single chain Fv molecules (scFv), single domain antibodies, camelid 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, and so forth); TPR repeats; trifoil structures; zinc finger domains; DNAbinding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., 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, e.g., U.S. 2004-0005709.

Display technology can also be used to obtain binding proteins (e.g., antibodies) that bind particular epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target.

Iterative Selection. In one preferred embodiment, display library technology is used in an iterative mode. A first display library is used to identify one or more binding proteins for a target. These identified binding proteins are then varied using a mutagenesis method to form a second display library. Higher affinity binding proteins

2019204295 19 Jun 2019 are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.

In some implementations, the mutagenesis is targeted to regions at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements. Exemplary mutagenesis techniques include: error-prone PCR, recombination, DNA shuffling, site-directed mutagenesis and cassette mutagenesis.

In one example of iterative selection, the methods described herein are used to first identify a protein from a display library that binds plasma kallikrein, with at least a minimal binding specificity for a target or 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 sequences encoding the initial identified proteins are used as a template nucleic acid for the introduction of variations, e.g., to identify a second protein that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial protein.

Off-Rate Selection. Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between polypeptides and their targets, the methods described herein can be used to isolate binding proteins with a desired (e.g., reduced) kinetic dissociation rate for a binding interaction to a target.

To select for slow dissociating binding proteins from a display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules. Then the bound binding proteins are eluted with a second solution that includes a saturating amount of free target or a target specific high-affinity competing monoclonal antibody, i.e., replicates of the target that are not attached to the particle. The free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.

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The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions.

Further, it is also possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.

Selecting or Screening for Specificity. The display library screening methods described herein can include a selection or screening process that discards display library members that bind to a non-target molecule. Examples of non-target molecules include streptavidin on magnetic beads, blocking agents such as bovine serum albumin, non-fat bovine milk, soy protein, any capturing or target immobilizing monoclonal antibody, or non-transfected cells which do not express the target.

In one implementation, a so-called “negative selection” step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecule. The display library or a pool thereof is contacted to the non-target molecule. Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.

In another implementation, a screening step is used. After display library members 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., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target as well as for cross species reactivity to related targets or subunits of the target (e.g., plasma kallikrein) and also under different condition such as pH 6 or pH 7.5. The

2019204295 19 Jun 2019 non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target.

Other Exemplary Expression Libraries

Other types of collections of proteins (e.g., expression libraries) can be used to identify proteins with a particular property (e.g., ability to bind plasma kallikrein), including, e.g., protein arrays of antibodies (see, e.g., De Wildt et al. (2000) Nat. Biotechnol. 18:989-994), lambda gtl 1 libraries, two-hybrid libraries and so forth.

Exemplary Libraries

It is possible to immunize a non-human primate and recover primate antibody genes that can be displayed on phage (see below). From such a library, one can select antibodies that bind the antigen used in immunization. See, for example, Vaccine. (2003) 22(2):257-67 or Immunogenetics. (2005) 57(10):730-8. Thus one could obtain primate antibodies that bind and inhibit plasma kallikrein by immunizing a chimpanzee or macaque and using a variety of means to select or screen for primate antibodies that bind and inhibit plasma kallikrein. One can also make chimeras of primatized Fabs with human constant regions, see Curr Opin Mol Ther. (2004) 6(6):675-83. “PRIMATIZED antibodies, genetically engineered from cynomolgus macaque monkey and human components, are structurally indistinguishable from human antibodies. They may, therefore, be less likely to cause adverse reactions in humans, making them potentially suited for long-term, chronic treatment” Curr Opin Investig Drugs. (2001) 2(5):635-8.

One exemplary type of library presents a diverse pool of polypeptides, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain. Of interest are display libraries where the members of the library include primate or “primatized” (e.g., such as human, non-human primate or “humanized”) immunoglobin domains (e.g., immunoglobin variable domains) or chimeric primatized Fabs with human constant regions. Human or humanized immunoglobin domain libraries may be used to identify human or “humanized” antibodies that, for example, recognize human antigens. Because the constant and framework regions of the antibody are human, these antibodies may avoid themselves being recognized and targeted as antigens when administered to humans. The constant regions may also be optimized to recruit effector functions of the

2019204295 19 Jun 2019 human immune system. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self-antigens.

A typical antibody display library displays a polypeptide that includes a VH domain and a VL domain. An “immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay, 1988, Ann. Rev. Immunol. 6:381-405). The display library can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.

As in the case of the Fab and other formats, the displayed antibody can include one or more constant regions as part of a light and/or heavy chain. In one embodiment, each chain includes one constant region, e.g., as in the case of a Fab. In other embodiments, additional constant regions are displayed.

Antibody libraries can be constructed by a number of processes (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. Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL). The variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and/or FR4, referring to such regions of either and both of heavy and light chain variable domains. For example, the variation(s) may be introduced into all three CDRs of a given variable domain, or into CDR1 and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible. In one process, antibody libraries are constructed by inserting diverse oligonucleotides that encode 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 describe a method for constructing CDR encoding oligonucleotides using

2019204295 19 Jun 2019 trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.

In another process, an animal (e.g., a rodent) is immunized with plasma kallikrein. The animal is optionally boosted with the antigen to further stimulate the response. Then spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library.

In yet another process, antibody libraries are constructed from nucleic acid amplified from naive germline immunoglobulin genes. The amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulinencoding nucleic acids are described below. Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.

Nucleic acid encoding immunoglobulin domains can be obtained from the immune cells of, e.g., a primate (e.g., a human), mouse, rabbit, camel, or rodent. In one example, the cells are selected for a particular property. B cells at various stages of maturity can be selected. In another example, the B cells are naive.

In one embodiment, fluorescent-activated cell sorting (FACS) is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated. In another preferred embodiment, the B or T cells are cultured in vitro. The cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin, or pokeweed mitogen.

In another embodiment, the cells are isolated from a subject that has a disease of condition described herein, e.g., a plasma kallikrein associated disease or condition.

In one preferred embodiment, the cells have activated a program of somatic hypermutation. Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, 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.

The nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method. First, RNA is isolated

2019204295 19 Jun 2019 from the immune cell. Full length (i.e., capped) mRNAs are separated (e.g. by degrading uncapped RNAs with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to produce the cDNAs.

The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et al., 1999, J. Biol. Chem. 274:18218-30. The primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific to a particular isotype of immunoglobulin. Typically, the primer is specific for a region that is 3 ’ to a sequence encoding at least one CDR. In another embodiment, poly-dT primers may be used (and may be preferred for the heavychain genes).

A synthetic sequence can be ligated to the 3’ end of the reverse transcribed strand. The synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription. The use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.

The variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity. The amplified nucleic acid is then cloned into a display library vector.

Secondary Screening Methods

After selecting candidate library members that bind to a target, each candidate library member can be further analyzed, e.g., to further characterize its binding properties for the target, e.g., plasma kallikrein. Each candidate library member can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, an inhibitory property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.

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As appropriate, the assays can use a display library member directly, a recombinant polypeptide produced from the nucleic acid encoding the selected polypeptide, or a synthetic peptide synthesized based on the sequence of the selected polypeptide. In the case of selected Fabs, the Fabs can be evaluated or can be modified and produced as intact IgG proteins. Exemplary assays for binding properties include the following.

ELISA. Binding proteins can be evaluated using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the binding protein bound to the target on the plate is determined by probing the plate with an antibody that can recognize the binding protein, e.g., a 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 horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided).

Homogeneous Binding Assays. The ability of a binding protein described herein to bind a target can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103). A fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means, e.g., using a fluorimeter. By titrating the amount of the first or second

2019204295 19 Jun 2019 binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.

Another example of a homogenous assay is ALPHASCREEN™ (Packard Bioscience, Meriden CT). ALPHASCREEN ™ uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity. One bead can be attached to the display library member, the other to the target. Signals are measured to determine the extent of binding.

Surface Plasmon Resonance (SPR). The interaction of binding protein and a target can be analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Patent 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 on-line resources provide by BIAcore International AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Kd), and kinetic parameters, including K_on and Koff, for the binding of a binding protein to a target. Such data can be used to compare different biomolecules. For example, selected proteins from an expression library can be compared to identify proteins that have high affinity for the target or that have a slow Κ_οΠ·. This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow K_Off. This information can be

2019204295 19 Jun 2019 combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by x-ray crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.

Cellular Assays. Binding proteins can be screened for ability to bind to cells which transiently or stably express and display the target of interest on the cell surface. For example, plasma kallikrein binding proteins can be fluorescently labeled and binding to plasma kallikrein in the presence of absence of antagonistic antibody can be detected by a change in fluorescence intensity using flow cytometry e.g., a FACS machine.

Other Exemplary Methods for Obtaining Plasma Kallikrein Binding Proteins

In addition to the use of display libraries, other methods can be used to obtain a plasma kallikrein binding protein (e.g., antibody). For example, plasma kallikrein protein or a fragment thereof can be used as an antigen in a non-human animal, e.g., a rodent.

In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies (Mabs) derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al., 1994, Nat. Gen. 7:13-21; U.S. 2003-0070185, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized. Winter describes a CDRgrafiting method that may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; U.S. Pat. No. 5,225,539. All of the CDRs of a particular human antibody may be replaced with at least a portion of a nonhuman CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

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Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Numerous sources of such nucleic acid are available. For example, nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Reducing Immunogenicity of Plasma Kallikrein Binding Proteins

Immunoglobin plasma kallikrein binding proteins (e.g., IgG or Fab plasma kallikrein binding proteins) may be modified to reduce immunogenicity. Reduced immunogenicity is desirable in plasma kallikrein binding proteins intended for use as therapeutics, as it reduces the chance that the subject will develop an immune response against the therapeutic molecule. Techniques useful for reducing immunogenicity of plasma kallikrein binding proteins include deletion/modification of potential human T cell epitopes and “germlining” of sequences outside of the CDRs (e.g., framework and Fc).

A plasma kallikrein-binding antibody may be modified by specific deletion of human T cell epitopes or “deimmunization,” e.g., by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody are analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed “peptide threading” can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected

2019204295 19 Jun 2019 can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible conservative substitutions are made, often but not exclusively, an amino acid common at this position in human germline antibody sequences may be used. Human germline sequences are disclosed in Tomlinson, I.A. et al., 1992, J. Mol. Biol. 227:776-798; Cook, G. P. et al., 1995, Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al., 1992, J. Mol. Bio. 227:799-817. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, I.A. et al. MRC Centre for Protein Engineering, Cambridge, UK). After the deimmunizing changes are identified, nucleic acids encoding V_H and Vl can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth). Mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGl or κ constant regions.

In some cases a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs were eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution should be tested. In other cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding. In these cases, variants with and without this substitution should be tested. Thus, in some cases several variant deimmunized heavy and light chain variable regions were designed and various heavy/light chain combinations tested in order to identify the optimal deimmunized antibody. The choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, i.e., the number of potential T cell epitopes remaining in the variable region. Deimmunization can be used to modify any antibody, e.g., an antibody that includes a non-human sequence, e.g., a synthetic

2019204295 19 Jun 2019 antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.

Plasma kallikrein binding antibodies are “germlined” by reverting one or more non-germline amino acids in framework regions to corresponding germline amino acids of the antibody, so long as binding properties are substantially retained. Similar methods can also be used in the constant region, e.g., in constant immunoglobulin domains.

Antibodies that bind to plasma kallikrein, e.g., an antibody described herein, may be modified in order to make the variable regions of the antibody more similar to one or more germline sequences. For example, an antibody can include one, two, three, or more amino acid substitutions, e.g., in a framework, CDR, or constant region, to make it more similar to a reference germline sequence. One exemplary germlining method can include identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Mutations (at the amino acid level) are then made in the isolated antibody, either incrementally or in combination with other mutations. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a framework and/or constant region. For example, a germline framework and/or constant region residue can be from a germline sequence that is similar (e.g., most similar) to the non-variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated (i.e., do not abrogate activity). Similar mutagenesis can be performed in the framework regions.

Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed using at

2019204295 19 Jun 2019 least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion of the sequence. In other implementations more than one or two germline sequences are used, e.g., to form a consensus sequence.

In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related 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 residues in the reference CDR sequences, residues that are identical to residues at corresponding positions in a human germline sequence (i.e., an amino acid sequence encoded by a human germline nucleic acid).

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

Accordingly, it is possible to isolate an antibody which has similar activity to a given antibody of interest, but is more similar to one or more germline sequences, particularly one or more human germline sequences. For example, an antibody can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to a germline sequence in a region outside the CDRs (e.g., framework regions). Further, an antibody can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified. Germline sequences of primary interest are human germline sequences. The activity of the antibody (e.g., the binding activity as measured by Ka) can be within a factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody.

Germline sequences of human immunoglobin genes have been determined and are available from a number of sources, including the INTERNATIONAF IMMUNOGENETICS INFORMATION SYSTEM® (IMGT), and the V BASE directory

2019204295 19 Jun 2019 (compiled by Tomlinson, I.A. et al. MRC Centre for Protein Engineering, Cambridge, UK).

Exemplary germline reference sequences for Vk_apPa include: 012/02, 018/08, A20, A30, L14, LI, L15, L4/18a, L5/L19, L8, L23, L9 ,L24, Lil, L12, 011/01, A17, Al, A18, A2, A19/A3, A23, A27, All, L2/L16, L6, L20, L25, B3, B2, A26/A10, and A14. See, e.g., Tomlinson et al., 1995, EMBO J. 14(18):4628-3.

A germline reference sequence for the HC variable domain can be based on a sequence that has particular canonical structures, e.g., 1-3 structures in the Hl and H2 hypervariable loops. The canonical structures of hypervariable loops of an immunoglobulin variable domain can be inferred from its 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. Exemplary sequences with 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, DP58, DP-49, DP-50, DP-51, DP-53, and DP-54.

Protein Production

Standard recombinant nucleic acid methods can be used to express a protein that binds to plasma kallikrein. Generally, a nucleic acid sequence encoding the protein is cloned into a nucleic acid expression vector. Of course, if the protein includes multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells.

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 ):21929). For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the periplasm and/or media.

Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (e.g., scFv’s) are expressed in a yeast cell such as Pichia (see, e.g., Powers et

2019204295 19 Jun 2019 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):15764; 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. One of skill in the art can optimize antibody production in yeast by optimizing, for example, oxygen conditions (see e.g., Baumann K., et al. 2010 BMC Syst. Biol. 4:141), osmolarity (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), strain of yeast (see e.g., Kozyr, AV et al. 2004 Mol Biol (Mosk) 38(6): 1067-75; Horwitz, AH. et al., 1988 Proc Natl Acad SciU S A 85(22): 8678-82; Bowdish, K. et al. 1991 J Biol Chem 266(18):11901-8), overexpression of proteins to enhance antibody production (see e.g., Gasser, B. et al., 2006 Biotechol. Bioeng. 94(2):353-61), level of acidity of the culture (see e.g., Kobayashi H., et al., 1997 FEMS Microbiol Lett 152(2):235-42), concentrations of substrates and/or ions (see e.g., Ko JH. et al., 2996 Appl Biochem Biotechnol 60(1):418). In addition, yeast systems can be used to produce antibodies with an extended half-life (see e.g., Smith, BJ. et al. 2001 Bioconjug Chem 12(5):750-756),

In one preferred embodiment, antibodies are produced in mammalian cells. Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(1-2):65-80), and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.

In some embodiments, plasma kallikrein binding proteins are produced in a plant or cell-free based system (see e.g., Galeffi, P., et al., 2006 J Transl Med 4:39).

In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication)

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2019204295 19 Jun 2019 and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

In an exemplary system for recombinant expression of an antibody, or antigenbinding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of 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 for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

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 IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcg receptors and complement Clq (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

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2019204295 19 Jun 2019 produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic posttranslational modifications.

Antibodies can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly.

Characterization of Plasma Kallikrein Binding Proteins

IC50 (Inhibitory Concentration 50%) and EC50 (Effective Concentration 50%). Within a series or group of binding proteins, those having lower IC50 or EC50 values are considered more potent inhibitors of plasma kallikrein than those binding proteins having higher IC50 or EC50 values. Exemplary binding proteins have an IC50 value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of plasma kallikrein activity when the plasma kallikrein is at 2 pM.

Plasma kallikrein binding proteins may also be characterized with reference to the activity of Factor XII and HMWK (high-molecular-weight kininogen) signaling events, e.g., the production of Factor Xlla and/or bradykinin.

The binding proteins can also be evaluated for selectivity toward plasma kallikrein. For example, a plasma kallikrein binding protein can be assayed for its potency toward plasma kallikrein and a panel of kallikreins and an IC50 value or EC50 value can be determined for each kallikrein. In one embodiment, a compound that demonstrates a low IC50 value or EC50 value for the plasma kallikrein, and a higher IC50 value or EC50 value, e.g., at least 2-, 5-, or 10- fold higher, for another kallikrein within the test panel is considered to be selective toward plasma kallikrein.

A pharmacokinetics study in rat, mice, or monkey can be performed with plasma kallikrein binding proteins for determining plasma kallikrein half-life in the serum.

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Likewise, the effect of the binding protein can be assessed in vivo, e.g., in an animal model for a disease (e.g., carrageenin-induced edema in rat hind paw (Winter et al. Proc

Soc Exp Biol Med. 1962; 111:544-7)), for use as a therapeutic, for example, to treat a disease or condition described herein, e.g., a plasma kallikrein associated disorder.

Pharmaceutical Compositions

Proteins (e.g., binding proteins) that bind to plasma kallikrein (e.g., human plasma kallikrein and/or murine plasma kallikrein) and, e.g., include at least one immunoglobin variable region can be used in methods for treating (or preventing) a plasma kallikrein associated disease or condition. The binding proteins can be present in a composition, e.g., a pharmaceutically acceptable composition or pharmaceutical composition, which includes a plasma kallikrein-binding protein, e.g., an antibody molecule or other polypeptide or peptide identified as binding to plasma kallikrein, as described herein. The plasma kallikrein binding protein can be formulated together with a pharmaceutically acceptable carrier. Pharmaceutical compositions include therapeutic compositions 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) a plasma kallikrein associated disease.

A pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is 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, the plasma kallikrein binding protein may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

A pharmaceutically acceptable salt is a salt that retains the desired biological activity of the compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include

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2019204295 19 Jun 2019 acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, 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, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form can depend on the intended mode of administration and therapeutic application. Many compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies. An exemplary 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.

The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared

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2019204295 19 Jun 2019 by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

A plasma kallikrein binding protein can be administered by a variety of methods, although for many applications, the preferred route/mode of administration is intravenous injection or infusion. For example, for therapeutic applications, the plasma kallikrein binding protein can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m² or 7 to 25 mg/m². The route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are available. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., 1978, Marcel Dekker, Inc., New York.

Pharmaceutical compositions can be administered with medical devices. For example, in one embodiment, a pharmaceutical composition disclosed herein can be administered with a device, e.g., a needleless hypodermic injection device, a pump, or implant.

In certain embodiments, a plasma kallikrein binding protein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed herein cross the BBB (if desired), they can be formulated, for example, in

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2019204295 19 Jun 2019 liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;

5,374,548; and 5,399,331. The liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, J. Clin. Pharmacol. 29:685).

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting 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. An anti- plasma kallikrein antibody can be administered, e.g., by intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m² or about 5 to 30 mg/m². For binding proteins smaller in molecular weight than an antibody, appropriate amounts can be proportionally less. Dosage values may vary with the type and severity of the condition to be alleviated. For a particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

The pharmaceutical compositions disclosed herein may include a “therapeutically effective amount” or a “prophylactically effective amount” of a plasma kallikrein binding protein disclosed herein. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic

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2019204295 19 Jun 2019 result. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein to elicit 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.

A “therapeutically effective dosage” preferably modulates a measurable parameter, e.g., levels of circulating IgG antibodies by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to modulate a measurable parameter, e.g., a diseaseassociated parameter, can be evaluated in an animal model system predictive of efficacy in human disorders and conditions, e.g., a plasma kallikrein associated disease. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate a parameter in vitro.

Stabilization and Retention

In one embodiment, a plasma kallikrein binding protein is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, a plasma kallikrein binding protein can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as polyalkylene oxides or polyethylene oxides. Suitable polymers will vary substantially by weight. Polymers having molecular number average 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, a plasma kallikrein binding protein can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g., polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of such polymers include

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2019204295 19 Jun 2019 polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.

A plasma kallikrein binding protein can also be associated with a carrier protein, e.g., a serum albumin, such as a human serum albumin (see e.g., Smith, BJ. et al., 2001 Bioconjug Chem 12(5): 750-756). For example, a translational fusion can be used to associate the carrier protein with the plasma kallikrein binding protein.

A plasma kallikrein binding protein can also be modified as a HESylation derivative. Processes for HESylation of a plasma kallikrein binding protein utilize hydroxyethyl starch to modify the protein. HESylation of a protein can extend the circulating half-life of the protein and also reduce renal clearance.

Kits

A plasma kallikrein binding protein described herein can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) a plasma kallikrein binding protein, e.g., a composition (e.g., a pharmaceutical composition) that includes a plasma kallikrein binding protein, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to a method described herein and/or the use of a plasma kallikrein binding protein, e.g., for a method described herein.

The informational material of the kit is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to using the binding protein to treat, prevent, or diagnosis of disorders and conditions, e.g., a plasma kallikrein associated disease or condition.

In one embodiment, the informational material can include instructions to administer a plasma kallikrein binding protein in a suitable manner to perform 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 can include instructions to administer a plasma

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2019204295 19 Jun 2019 kallikrein binding protein to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition described herein, e.g., a plasma kallikrein associated disease or condition. For example, the material can include instructions to administer a plasma kallikrein binding protein to a patient with a disorder or condition described herein, e.g., a plasma kallikrein associated disease. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but may also be in other formats, such as computer readable material.

A plasma kallikrein binding protein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a plasma kallikrein binding protein be substantially pure and/or sterile. When a plasma kallikrein binding protein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When a plasma kallikrein binding protein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition containing a plasma kallikrein binding protein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in association with the container. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a plasma kallikrein binding protein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a plasma kallikrein binding protein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In one embodiment, the device is an

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2019204295 19 Jun 2019 implantable device that dispenses metered doses of the binding protein. The disclosure also features a method of providing a kit, e.g., by combining components described herein.

Treatments

Proteins that bind to plasma kallikrein, e.g., as described herein, have therapeutic and prophylactic utilities, particularly in human subjects. These binding proteins are administered to a subject to treat, prevent, and/or diagnose a variety of disorders and conditions, including e.g., a plasma kallikrein associated disease, or even to cells in culture, e.g., in vitro or ex vivo. For example, these binding proteins can be used to modify the effects of plasma kallikrein released from cells in culture (Lilia et al., J Biol Chem. 284(20):13792-13803 (2009)). Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. The treatment may also delay onset, e.g., prevent onset, or prevent deterioration of a disease or condition.

As used herein, an amount of a target-binding agent effective to prevent a disorder, or a prophylactically effective amount of the binding agent refers to an amount of a target binding agent, e.g., an plasma kallikrein binding protein, e.g., an anti- plasma kallikrein antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, for preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., a disorder described herein, e.g., a plasma kallikrein associated disease.

Methods of administering plasma kallikrein binding proteins and other agents are also described in “Pharmaceutical Compositions.” Suitable dosages of the molecules used can depend on the age and weight of the subject and the particular drug used. The binding proteins can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between plasma kallikrein and its substrate (e.g., Factor XII or HMWK). The dose of the plasma kallikrein binding protein can be the amount sufficient to block 90%, 95%, 99%, or 99.9% of the activity of plasma kallikrein in the patient, especially at the site of disease. Depending on the disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0

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2019204295 19 Jun 2019 mg/Kg. For an IgG having a molecular mass of 150,000 g/mole (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μΜ, and 1.8 μΜ of binding sites for a 5 L blood volume.

In one embodiment, the plasma kallikrein binding proteins are used to inhibit an activity (e.g., inhibit at least one activity of plasma kallikrein, e.g., reduce Factor Xlla and/or bradykinin production) of plasma kallikrein, e.g., in vivo. The binding proteins can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope. This method includes: administering the binding protein alone or attached to an agent (e.g., a cytotoxic drug), to a subject requiring such treatment. For example, plasma kallikrein binding proteins that do not substantially inhibit plasma kallikrein may be used to deliver nanoparticles containing agents, such as toxins, to plasma kallikrein associated cells or tissues, e.g., to treat a plasma kallikrein-associate disorder.

Because the plasma kallikrein binding proteins recognize plasma kallikrein expressing cells and can bind to cells that are associated with (e.g., in proximity of or intermingled with) a plasma kallikrein associated disorder or condition, plasma kallikrein binding proteins can be used to inhibit an activity (e.g., inhibit at least one activity of plasma kallikrein, e.g., reduce Factor Xlla and/or bradykinin production) any such cells and inhibit the plasma kallikrein associated disease. Reducing plasma kallikrein activity can indirectly inhibit cells which may be dependent on the plasma kallikrein activity for the development and/or progression of a plasma kallikrein-associated disorder.

The binding proteins may be used to deliver an agent (e.g., any of a variety of cytotoxic and therapeutic drugs) to cells and tissues where plasma kallikrein is present. Exemplary agents include a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as toxins short range radiation emitters, e.g., short range, high energy a-emitters.

To target plasma kallikrein expressing cells, a prodrug system can be used. For example, a first binding protein is conjugated with a prodrug which is activated only when in close proximity with a prodrug activator. The prodrug activator is conjugated with a second binding protein, preferably one which binds to a non competing site on the

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2019204295 19 Jun 2019 target molecule. Whether two binding proteins bind to competing or non competing binding sites can be determined by conventional competitive binding assays. Exemplary drug prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287

3292.

The plasma kallikrein binding proteins can 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 can include complement binding effector domain, such as the Fc portions from IgGf, -2, or -3 or corresponding portions of IgM which bind complement. In one embodiment, a population of target cells is ex vivo treated with a binding agent described herein and appropriate effector cells. The treatment can be supplemented by the addition of complement or serum containing complement. Further, phagocytosis of target cells coated with a binding protein described herein can be improved by binding of complement proteins. In another embodiment target, cells coated with the binding protein which includes a complement binding effector domain are lysed by complement.

Methods of administering plasma kallikrein binding proteins are described in “Pharmaceutical Compositions.” Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The binding proteins can be used as competitive agents to inhibit or reduce an undesirable interaction, e.g., between a natural or pathological agent and the plasma kallikrein.

The plasma kallikrein binding protein can be used to deliver macro and micromolecules, e.g., a gene into the cell for gene therapy purposes into the endothelium or epithelium and target only those tissues expressing the plasma kallikrein. The binding proteins may be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short range radiation emitters, including, for example, short range, high energy a emitters, as described herein.

In the case of polypeptide toxins, recombinant nucleic acid techniques can be used to construct a nucleic acid that encodes the binding protein (e.g., antibody or antigen-binding fragment thereof) and the cytotoxin (or a polypeptide component thereof)

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2019204295 19 Jun 2019 as translational fusions. The recombinant nucleic acid is then expressed, e.g., in cells and the encoded fusion polypeptide isolated.

Alternatively, the plasma kallikrein binding protein can be coupled to high energy radiation emitters, for example, a radioisotope, such as ¹³¹I, a γ-emitter, which, when localized at a site, results in a killing of several cell diameters. See, e.g., S.E. Order, “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”, Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al. (eds.), pp 303 316 (Academic Press 1985). Other suitable radioisotopes include a emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, andb emitters, such as ¹⁸⁶Re and ⁹⁰Y. Moreover,¹⁷⁷ Lu may also be used as both an imaging and cytotoxic agent.

Radio immunotherapy (RIT) using antibodies labeled with ¹³¹1,⁹⁰Y, and ¹⁷⁷Lu is under intense clinical investigation. There are significant differences in the physical characteristics of these three nuclides and as a result, the choice of radionuclide is very critical in order to deliver maximum radiation dose to a tissue of interest. The higher beta energy particles of ⁹⁰Y may be good for bulky tumors. The relatively low energy beta particles of¹³'i are ideal, but in vivo dehalogenation of radioiodinated molecules is a major disadvantage for internalizing antibody. In contrast, ¹⁷⁷Lu has low energy beta particle with only 0.2-0.3 mm range and delivers much lower radiation dose to bone marrow compared to ⁹⁰Y. In addition, due to longer physical half-life (compared to ⁹⁰Y), the residence times are higher. As a result, higher activities (more mCi amounts) of ¹⁷⁷Lu labeled agents can be administered with comparatively less radiation dose to marrow. There have been several clinical studies investigating the use of ¹⁷⁷Lu labeled antibodies in the treatment of various cancers. (Mulligan T et al., 1995, Clin. Cane. Res. 1: 14471454; Meredith RF, et al., 1996, J. Nucl. Med. 37:1491-1496; Alvarez RD, et al., 1997, Gynecol. Oncol. 65: 94-101).

Exemplary Diseases and Conditions

A plasma kallikrein binding protein described herein is useful to treat (or prevent) a disease or condition in which plasma kallikrein activity is implicated, e.g., a disease or condition described herein, or to treat (or prevent) one or more symptoms associated

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2019204295 19 Jun 2019 therewith. In some embodiments, the plasma kallikrein binding protein (e.g., plasma kallikrein binding IgG or Fab) inhibits plasma kallikrein activity.

Examples of such diseases and conditions which can be treated (or prevented) by a plasma kallikrein binding protein described herein include: rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting induced by ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, and burn injury. A plasma kallikrein binding protein described herein can also be used to promote wound healing. A plasma kallikrein binding protein described herein can also be used as an oncology treatment by mechanisms that include, but are not limited to, blocking production of pro-angiogenic bradykinin.

A therapeutically effective amount of a plasma kallikrein binding protein can be administered to a subject having or suspected of having a disorder in which plasma kallikrein activity is implicated, thereby treating (e.g., ameliorating or improving a symptom or feature of a disorder, slowing, stabilizing and/or halting disease progression) the disorder.

The plasma kallikrein binding protein can be administered in a therapeutically effective amount. A therapeutically effective amount of a plasma kallikrein binding protein is the amount which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit 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. A therapeutically effective dosage preferably modulates a measurable parameter, favorably, relative to untreated subjects. The ability of a compound to affect (e.g., inhibit) a

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2019204295 19 Jun 2019 measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune, chronic inflammatory disease that causes joint swelling and pain and normally results in joint destruction. RA generally follows a relapsing/remitting course, with “flares” of disease activity interspersed with remissions of disease symptoms. RA is associated with a number of additional inflammatory disorders, including Sjogren’s syndrome (dry eyes and mouth caused by inflammation of tear and saliva glands), pleuritis (inflammation of the pleura that causes pain upon deep breath and coughing), rheumatoid nodules (nodular sites of inflammation that develop within the lungs), pericarditis (inflammation of the pericardium that causes pain when lying down or leaning forward), Felty syndrome (splenomegaly and leucopenia observed in conjunction with RA, making the subject prone to infection), and vasculitis (an inflammation of the blood vessels which can block blood flow). Plasma kallikrein has been implicated in rheumatoid arthritis.

Symptoms of active RA include fatigue, lack of appetite, low grade fever, muscle and joint aches, and stiffness. Muscle and joint stiffness are usually most notable in the morning and after periods of inactivity. During flares, joints frequently become red, swollen, painful, and tender, generally as a consequence of synovitis.

Treatment for rheumatoid arthritis involves a combination of medications, rest, joint strengthening exercises, and joint protection. Two classes of medications are used

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2019204295 19 Jun 2019 in treating rheumatoid arthritis: anti-inflammatory “first-line drugs,” and “DiseaseModifying Antirheumatic Drugs” (DMARDs). The first-line drugs 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 they are not anti-inflammatory agents.

The disclosure provides methods of treating (e.g., ameliorating, stabilizing, or eliminating one or more symptoms or ameliorating or stabilizing the subject’s score on a RA scale) rheumatoid arthritis 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 provided are methods of treating RAby 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., with at least one anti-inflammatory “first line drug” (e.g., anNSAID and/or cortisone) and/or a DMARD. The disclosure also provides methods of preventing rheumatoid arthritis 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 RA (e.g., a subject having a family member with RA or a genetic predisposition thereto).

Further provided are methods of treating (e.g., ameliorating, stabilizing, or eliminating one or more symptoms) rheumatoid arthritis associated disorders (Sjogren’s syndrome, pleuritis, pulmonary rheumatoid nodules, pericarditis, Felty 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.

Scales useful for assessing RA and symptoms of RA include, e.g., the Rheumatoid Arthritis Severity Scale (RASS; Bardwell et al., (2002) Rheumatology 41(1):38-45), SF-36 Arthritis Specific Health Index (ASHI; Ware et al., (1999) Med. Care. 37(5 Suppl):MS40-50), Arthritis Impact Measurement Scales or Arthritis Impact

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2019204295 19 Jun 2019

Measurement Scales 2 (AIMS or AIMS2; Meenan et al. (1992) Arthritis Rheum. 35(1): ΙΙΟ); the Stanford Health Assessment Questionnaire (HAQ), HAQII, or modified HAQ (see, e.g., Pincus et al. (1983) Arthritis Rheum. 26(11):1346-53).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of rheumatoid arthritis, such as collagen-induced arthritis (CIA), which is induced, typically in rodents, by immunization with autologous or heterologous type II collagen in adjuvant (Williams et al. Methods Mol Med. 98:207-16 (2004)).

Gout

Gout is a condition that results from crystals of uric acid depositing in tissues of the body. Gout is characterized by an overload of uric acid in the body and recurring attacks of joint inflammation (arthritis). Chronic gout can lead to deposits of hard lumps of uric acid in and around the joints, decreased kidney function, and kidney stones. Gout is often related to an inherited abnormality in the body's ability to process uric acid. Uric acid is a breakdown product of purines, which are part of many foods. An abnormality in handling uric acid can cause attacks of painful arthritis (gout attack), kidney stones, and blockage of the kidney filtering tubules with uric acid crystals, leading to kidney failure. Some patients may only develop elevated blood uric acid levels (hyperuricemia) without having arthritis or kidney problems.

Symptoms of gout include, e.g., excruciating and unexpected pain, swelling, redness, warmth and stiffness in the affected foot or other parts of the body, and lowgrade fever.

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

The disclosure provides methods of treating (e.g., ameliorating, stabilizing, or eliminating one or more symptoms or the worsening of) gout by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a plasma kallikrein

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2019204295 19 Jun 2019 binding protein) to a subject having or suspected of having gout. Additionally provided are methods of treating gout by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a kallikrein binding protein) in combination with a second therapy, e.g., an NSAID, a colchicine, an oral glucocorticoid, an intra-articular glucocorticoid administered via a joint injection, a xanthine oxidase inhibitor (e.g., allopurinol, febuxostat), a uricosuric (e.g., probenecid, EDTA), a urate oxidase (e.g., pegloticase), sodium bicarbonate, and/or low purine diet. The disclosure also provides methods of preventing gout 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 gout (e.g., a subject having a family member with gout or a genetic predisposition thereto).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a 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.

Intestinal Bowel Disease (IBD)

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the large intestine and, in some cases, the small intestine. The main forms of IBD are Crohn's disease and ulcerative colitis (UC). Accounting for far fewer cases are other forms of IBD: collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, infective colitis, and indeterminate colitis. The main difference between Crohn's disease and UC is the location and nature of the inflammatory changes. Crohn's can affect any part of the gastrointestinal tract, from mouth to anus (skip lesions), although a majority of the cases start in the terminal ileum. Ulcerative colitis, in contrast, is restricted to the colon and the rectum. Microscopically, ulcerative colitis is restricted to the mucosa (epithelial lining of the gut), while Crohn's disease affects the whole bowel wall. Finally, Crohn's disease and ulcerative colitis present with extra-intestinal manifestations (such as liver problems, arthritis, skin manifestations and eye problems) in different proportions.

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Symptoms of IBD include abdominal pain, vomiting, diarrhea, hematochezia, weight loss, weight gain and various associated complaints or diseases (arthritis, pyoderma gangrenosum, primary sclerosing cholangitis). Diagnosis is generally by colonoscopy with biopsy of pathological lesions. Rarely, a definitive diagnosis of neither Crohn's disease nor ulcerative colitis can be made because of idiosyncrases in the presentation. In this case, a diagnosis of indeterminate colitis may be made.

Treatment for IBD, depending on the level of severity, may require immunosuppression to control the symptoms. Immunosuppresives such as azathioprine, methotrexate, or 6-mercaptopurine can be used. More commonly, treatment of IBD requires a form of mesalamine. Often, steroids are used to control disease flares and were once acceptable as a maintenance drug. Biologicals, such as infliximab, have been used to treat patients with Crohn’s disease or Ulcerative Colitis. Severe cases may require surgery, such as bowel resection, strictureplasty or a temporary or permanent colostomy or ileostomy. Alternative medicine treatments for IBD exist in various forms however such methods concentrate on controlling underlying pathology in order to avoid prolonged steroidal exposure or surgical excision. Usually the treatment is started by administering drugs, such as prednisone, with high anti-inflammatory affects. Once the inflammation is successfully controlled, the patient is usually switched to a lighter drug, such as asacol- a mesalamine- to keep the disease in remission. If unsuccessful, a combination of the aforementioned immunosuppressant drugs with a mesalamine (which may also have an anti-inflammatory effect) may or may not be administered, depending on the patient.

The disclosure provides methods of treating (e.g., ameliorating, stabilizing, or eliminating one or more symptoms of) IBD 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 provided are methods of treating IBD by administering a plasma kallikrein binding protein (e.g., a therapeutically effective amount of a kallikrein binding protein) in combination with a second therapy, e.g., an immunosuppressive (e.g., azathioprine, methotrexate, 6-mercaptopurine), a mesalamine, a steroid, and/or infliximab. The disclosure also provides methods of preventing IBD or a symptom thereof by administering a plasma kallikrein binding

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2019204295 19 Jun 2019 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 having a family member with IBD or a genetic predisposition thereto).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a 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 references cited therein.

Oral Mucositis

Oral mucositis is the painful inflammation and ulceration of the mucous membranes in the mouth, usually as an adverse effect of chemotherapy and radiotherapy treatment for cancer.

Symptoms of oral mucositis include, e.g., ulcers, peripheral erythema, burning sensation accompanied by reddening, trouble speaking, eating, or even opening the mouth, and dyseusia (alteration in taste perception).

Treatment for oral mucositis includes oral hygiene (salt mouthwash, GELCLAIR®, CAPHOSOL®, MUGARD®), palifermin (a human keratinocyte growth factor), cytokines and other modifiers of inflammation (e.g., IL-1, IL-11, TGF-beta3), amino acid supplementation (e.g., glutamine), vitamins, colony-stimulating factors, cryotherapy, and laser therapy.

The disclosure provides methods of treating (e.g., ameliorating, reducing, or eliminating one or more symptoms, or stabilizing the subject’s score on a mucositis scale) oral mucositis 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 provided are 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, e.g., oral hygiene (salt mouthwash, GELCLAIR®, CAPHOSOL®, MUGARD®), palifermin (a human keratinocyte growth factor), a cytokine and/or a modifier of inflammation (e.g., IL-1, IL-11, TGF-beta3), an amino acid supplementation (e.g., glutamine), a vitamin, a colony-stimulating factor, cryotherapy, and/or laser

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2019204295 19 Jun 2019 therapy. The disclosure also provides methods of preventing oral mucositis 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 oral mucositis (e.g., a subject that has undergone or is undergoing chemotherapy or radiotherapy).

Scales useful 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), National Cancer Institute Common Toxicity Criteria (NCI-CTC) for Oral Mucositis (National Cancer Institute Common Toxicity Criteria. Version 2.0, June 1, 1999, Sonis et al., Cancer. 85:2103-2113 (1999)), and Oral Mucositis Assessment Scale (OMAS).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of oral mucositis, such as an animal model of oral mucositis induced by conditioning regimen of haematopoietic stem cell transplantation (Chen et al., Zhonghua Kou Qiang Yi Xue Za Zhi. 42(11):672-6 (2007)).

Neuropathic Pain

Neuropathic pain is a complex, chronic pain state that usually is accompanied by tissue injury. With neuropathic pain, the nerve fibers themselves may be damaged, dysfunctional or injured. These damaged nerve fibers send incorrect signals to other pain centers. The impact of nerve fiber injury includes a change in nerve function both at the site of injury and areas around the injury.

Symptoms of neuropathic pain include, e.g., shooting and burning pain and tingling and numbness.

Treatments for neuropathic pain include, e.g., medications (e.g., non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., ALEVE®, MOTRIN®, or morphine), anticonvulsant, and antidepressant drugs), and invasive or implantable devices (e.g., electrical stimulation).

The disclosure provides methods of treating (e.g., ameliorating, reducing, or eliminating one or more symptoms of or stabilizing the subject’s score on a pain scale)

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2019204295 19 Jun 2019 neuropathic pain 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 neuropathic pain. Additionally provided are methods of treating neuropathic 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, e.g., a nonsurgical treatment ((e.g., a non-steroidal antiinflammatory drug (NSAID) (e.g., ALEVE®, MOTRIN®, or morphine), an anticonvulsant, and/or an antidepressant drug), and/or an invasive or implantable device (e.g., electrical stimulation). The disclosure also provides methods of preventing neuropathic pain 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 neuropathic pain (e.g., a subject that has experienced tissue injury).

Scales useful for the assessment of neuropathic pain include, e.g., 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)), 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 .4««. Acad. Med. Singap. 23 (2): 129-38 (1994)).

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

Inflammatory Pain

Inflammatory pain is caused by an insult such as penetration wounds, burns, extreme cold, fractures, arthritis, autoimmune conditions, excessive stretching, infections

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2019204295 19 Jun 2019 and vasoconstriction to the integrity of tissues at a cellular level. During inflammation a complex neuro-immune interaction results in primary hyperalgesia, in which a large range of inflammatory molecules including prostaglandins and bradykinin induce and maintain the altered nociceptor sensitivity.

Treatments for inflammatory pain include, e.g., non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids.

The disclosure provides methods of treating (e.g., ameliorating, reducing, or eliminating one or more symptoms of) inflammatory pain 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 provided are 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, e.g., a non-steroidal anti-inflammatory drug (NSAID) and/or a corticosteroid. The disclosure also provides methods of preventing inflammatory pain 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 inflammatory pain (e.g., a subject that has experienced an insult, e.g., such as a penetration wound, a burn, extreme cold, a fracture, arthritis, an autoimmune condition, excessive stretching, or infection).

Scales useful for the assessment of inflammatory pain include, e.g., 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)), 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 .4««. Acad. Med. Singap. 23 (2): 129-38 (1994)).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal

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2019204295 19 Jun 2019 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)).

Spinal Stenosis

Spinal stenosis is a medical condition in which the spinal canal narrows and compresses the spinal cord and nerves. This is usually due to the common occurrence of spinal degeneration that occurs with aging. It can also sometimes be caused by spinal disc herniation, osteoporosis or a tumor. Spinal stenosis may affect the cervical, thoracic or lumbar spine. In some cases, it may be present in all three places in the same patient.

Symptoms of spinal stenosis include, e.g., pain or cramping in the legs, radiating back and hip pain, pain in the neck and shoulders, loss of balance, and loss of bowel or bladder function (cauda equina syndrome).

Treatments for spinal stenosis include, e.g., nonsurgical treatments (e.g., physical therapy, non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen and indomethacin), analgesics (e.g., acetaminophen), chondroitin sulfate, glucosamine, rest or restricted activity, back brace or corset, epidural steroid injections (e.g., corticosteroid)), and surgery (e.g., decompressive laminectomy, laminotomy and fusion).

The disclosure provides methods of treating (e.g., ameliorating, reducing, or eliminating one or more symptoms of) spinal stenosis 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 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., a nonsurgical treatment (e.g., physical therapy and/or a nonsteroidal anti-inflammatory drug (NSAID) (e.g., aspirin, ibuprofen or indomethacin), an analgesic (e.g., acetaminophen), chondroitin sulfate, glucosamine, rest or restricted activity, a back brace or corset, an epidural steroid injection (e.g., corticosteroid), and/or surgery (e.g., decompressive laminectomy, laminotomy and/or fusion). The disclosure also provides methods of preventing spinal stenosis or a symptom thereof by administering a plasma kallikrein binding protein (e.g.,

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2019204295 19 Jun 2019 a prophylactically effective amount of a plasma kallikrein binding protein) to a subject at risk of developing spinal stenosis (e.g., a subject that has spinal degeneration).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of spinal stenosis, such as a model of lumbar spinal stenosis (Sekiguchi et al., Spine 29, 1105-1111 (2004)).

Arterial and Venous Thrombosis

Arterial thrombosis is the formation of a thrombus within an artery. In most cases, arterial thrombosis follows rupture of atheroma, and is therefore referred to as atherothrombosis.

Arterial thrombosis is associated with a number of disorders, including stroke and myocardial infarction. In thrombotic stroke, a thrombus (blood clot) usually forms around atherosclerotic plaques. Since blockage of the artery is gradual, onset of symptomatic thrombotic strokes is slower. Thrombotic stroke can be divided into two categories - large vessel disease and small vessel disease. The former affects vessels such as the internal carotids, vertebral and the circle of Willis. The latter can affect smaller vessels such as the branches of the circle of Willis. Myocardial infarction (MI) is caused by an infarct (death of tissue due to ischemia), often due to the obstruction of the coronary artery by a thrombus. MI can quickly become fatal if emergency medical treatment is not received promptly.

Venous thrombosis is a blood clot that forms within a vein. If a piece of a blood clot formed in a vein breaks off, it can be transported to the right side of the heart, and from there into the lungs. A piece of thrombus that is transported in this way is an embolism and the process of forming a thrombus that becomes embolic is called a thromboembolism. An embolism that lodges in the lungs is a pulmonary embolism (PE). A pulmonary embolus is a very serious condition that can be fatal if not recognized and treated promptly.

Superficial venous thromboses can cause discomfort but generally do not cause serious consequences, unlike the deep venous thromboses (DVTs) that form in the deep veins of the legs or in the pelvic veins. Systemic embolisms of venous origin can occur

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2019204295 19 Jun 2019 in patients with an atrial or ventricular septal defect, through which an embolus may pass into the arterial system. Such an event is termed a paradoxical embolism.

Prevention of arterial and/or venous thrombosis includes medications (e.g., anticoagulants (e.g., heparin), aspirin, and vitamin E) and mechanical methods (e.g., mechanical leg pumps (pneumatic compression stockings)).

The disclosure provides methods of treating (e.g., ameliorating, reducing, or eliminating one or more symptoms of) arterial and/or venous thrombosis 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 provided are 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, e.g., an anticoagulant (e.g., heparin), aspirin, and/or vitamin E and/or a mechanical method (e.g., a mechanical leg pump (pneumatic compression stockings). The 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 that has experienced a stroke or myocardial infarction).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of arterial or venous thrombosis, such as a double-tuck model of arterial thrombosis (Gomez-Jorge et al., J. Vase. Inter. Rad. 9(4): 633-638 (1998), a model of venous thrombosis in rat with low flow conditions in the venous blood stream (Fredrich et al., Blood Coagul Fibrinolysis. 5(2):243-8 (1994)), and a canine model for venous thrombosis and spontaneous pulmonary embolism (Frisbiel, Spinal Cord 43, 635-639 (2005)).

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Postoperative Ileus

Postoperative ileus is a temporary paralysis of a portion of the intestines typically after an abdominal surgery. Postoperative ileus commonly occurs for 24 to 72 hours after abdominal surgery.

Symptoms of postoperative ileus include, e.g., moderate and diffuse abdominal discomfort, constipation, abdominal distension, nausea or vomiting, lack of bowel movement and/or flatulence, and excessive belching.

Treatments for postoperative ileus include, e.g., nil per os (NPO or Nothing by Mouth) until peristaltic sound is heard from auscultation of the area where this portion lies, nasogastric suction, parenteral feeds, and medications (e.g., lactulose and erythromycin).

The disclosure provides methods of treating (e.g., ameliorating, reducing, or eliminating one or more symptoms of) postoperative ileus 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 provided are 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 therapy, e.g., nil per os, nasogastric suction, parenteral feeds, and/or a medication (e.g., lactulose and/or erythromycin). The disclosure also provides methods of preventing postoperative ileus 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 postoperative ileus (e.g., a subject that has had abdominal surgery).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of postoperative ileus, such as a model to investigate postoperative ileus with strain gauge transducers in awake rats (Huge et al. J Surg Res. 74(2):112-8 (1998)).

Aortic Aneurysm

An aortic aneurysm is a general term for any swelling (dilatation or aneurysm) of the aorta, usually representing an underlying weakness in the wall of the aorta at that

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2019204295 19 Jun 2019 location. Types of aortic aneurysms include aortic root aneurysm, thoracic aortic aneurysm, abdominal aortic aneurysm, and thoracoabdominal aortic aneurysm.

Most intact aortic aneurysms do not produce symptoms. As they enlarge, symptoms of aortic aneurysm include, e.g., anxiety or feeling of stress, nausea or vomiting, clammy skin, rapid heart rate, abdominal pain, back pain may develop, leg pain or numbness, erythema nodosum (leg lesions typically found near the ankle region), and a hoarse voice as the left recurrent laryngeal nerve winding around the arch of the aorta is stretched. Once an aneurysm is ruptured, it can cause severe pain and massive internal hemorrhage, and is fatal in the absence of prompt treatment.

Treatments for aortic aneurysm include, e.g., medications, surgical treatment and endovascular treatment. Smaller aneurysms that are not at high risk for rupturing can be treated with drugs to treat high blood pressure, such as beta-blockers; or doxycycline for matrix metalloproteinase-9 inhibition. Surgical treatment typically involves opening up of the dilated portion of the aorta and insertion of a synthetic (Dacron or Gore-tex) patch tube. Endovascular treatment, as a minimally invasive alternative to open surgery repair, involves the placement of an endovascular stent via a percutaneous technique (usually through the femoral arteries) into the diseased portion of the aorta.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms of) aortic aneurysm 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 aortic aneurysm. Additionally provided are methods of treating aortic aneurysm 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., a medication (e.g., a drug to treat high blood pressure (e.g., a beta-blocker) or doxycycline), surgery, and/or an endovascular treatment. The disclosure also provides methods of preventing aortic aneurysm 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 aortic aneurysm (e.g., a subject that has high blood pressure).

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Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from an animal model of aortic aneurysm, e.g., a rat model of abdominal aortic aneurysm using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure (Tanaka et al. J Vase Surg. 50(6):1423-32 (2009)).

Osteoarthritis

Osteoarthritis, also known as degenerative arthritis, is characterized by the breakdown and eventual loss of the cartilage of one or more joints. Osteoarthritis occurs when the cartilage that cushions the ends of bones in the joints deteriorates over time. The smooth surface of the cartilage becomes rough, causing irritation. If the cartilage wears down completely, the ends of the bones will be damaged. Osteoarthritis commonly affects the hands, feet, spine, and large weight-bearing joints, such as the hips and knees.

Symptoms of osteoarthritis include, e.g., pain, tenderness, stiffness, loss of flexibility, grating sensation, and bone spurs.

Treatments for osteoarthritis include, e.g., conservative measures (e.g., rest, weight reduction, physical and occupational therapy) and medications (e.g., acetaminophen, pain-relieving creams applied to the skin over the joints (e.g., capsaicin, salycin, methyl salicylate, and menthol), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, nabumetone and naproxen), and Cox-2 inhibitors.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on an osteoarthritis scale) osteoarthritis 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 osteoarthritis. Additionally provided are methods of treating osteoarthritis 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., a conservative measure (e.g., rest, weight reduction, physical and/or occupational therapy) and/or a medication (e.g., acetaminophen, a topical painrelieving cream, an NSAID (e.g., aspirin, ibuprofen, nabumetone, or naproxen), and/or a Cox-2 inhibitor. The disclosure also provides methods of preventing osteoarthritis or a

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2019204295 19 Jun 2019 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 osteoarthritis (e.g., a subject that has had a joint injury).

Scales useful for the assessment of osteoarthritis include, e.g., the Knee Injury and Osteoarthritis Outcome Score (KOOS; Roos et al. (1998) J. Orthop. Sports Phys. Ther. 28(2):88-96), Western Ontario and McMaster Universities Osteoarthrtis Index (WOMAC; Roos et al. (2003) Health Qual. Life Outcomes 1(1):17), and the 36-item Short Form General Health Scale (SF-36 GHS), as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from an animal model of osteoarthritis, e.g., injection of mono-iodoacetate (MIA) into the femorotibial joint of rodents which promotes loss of articular cartilage similar to that noted in human osteoarthritis (Guzman et al. Toxicol Pathol. 31(6):619-24 (2003)), and transection of the anterior cruciate ligament (ACL) in canines to induce osteoarthritis (Fife and Brandt J Clin Invest. 84(5): 1432-1439 (1989)).

Vasculitis

Vasculitis refers to a heterogeneous group of disorders that are characterized by inflammatory destruction of blood vessels. Both arteries and veins can be affected. Lymphangitis is sometimes considered a type of vasculitis. Vasculitis is primarily due to leukocyte migration and resultant damage. Vasculitis can be classified by the underlying cause, the location of the affected vessels, or the type or size of the blood vessels. Vasculitis is associated with a number of additional disorders and conditions, e.g., Kawasaki disease, Behcet's disease, Polyarteritis nodosa, Wegener's granulomatosis, Cryoglobulinemia, Takayasu's arteritis, Churg-Strauss syndrome, Giant cell arteritis (temporal arteritis), Henoch-Schonlein purpura, Rheumatic diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), cancer (e.g., lymphomas), infections (e.g., hepatitis C), exposure to chemicals and drugs (e.g., amphetamines, cocaine, and anthrax vaccines which contain the Anthrax Protective Antigen as the primary ingredient).

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Symptoms of vasculitis include, e.g., fever, weight loss, palpable purpura, livedo reticularis, myalgia or myositis, arthralgia or arthritis, mononeuritis multiplex, headache, stroke, tinnitus, reduced visual acuity, acute visual loss, myocardial infarction, hypertension, gangrene, nose bleeds, bloody cough, lung infiltrates, abdominal pain, bloody stool, perforations, and glomerulonephritis.

Treatments for vasculitis include, e.g., cortisone-related medications (e.g., prednisone) and immune suppression drugs (e.g., cyclophosphamide).

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a vasculitis scale) 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 vasculitis. Additionally provided are methods of treating vasculitis 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., a cortisone-related medication (e.g., prednisone) and/or an immune suppression drug (e.g., cyclophosphamide)). The disclosure also provides methods of preventing vasculitis 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 vasculitis (e.g., a subject that has had Kawasaki disease, Behcet's disease, Polyarteritis nodosa, Wegener's granulomatosis, Cryoglobulinemia, or Takayasu's arteritis, and so forth).

The disclosure also provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a vasculitis scale) vasculitis associated with systemic lupus erythematosis 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 vasculitis associated with systemic lupus erythematosis. Additionally provided are methods of treating vasculitis associated with systemic lupus erythematosis 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., a cortisone-related medication (e.g., prednisone) and/or an immune suppression drug (e.g., cyclophosphamide).

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Further provided are methods of treating (e.g., ameliorating, stabilizing, or eliminating one or more symptoms) a vasculitis associated disorder (Kawasaki disease, Behcet's disease, Polyarteritis nodosa, Wegener's granulomatosis, Cryoglobulinemia, Takayasu's arteritis, Churg-Strauss syndrome, Giant cell arteritis (temporal arteritis), Henoch-Schonlein purpura, Rheumatic diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), cancer (e.g., lymphomas), infections (e.g., hepatitis C), exposure to chemicals and drugs (e.g., amphetamines, cocaine, and anthrax vaccines which contain the Anthrax Protective Antigen as the primary ingredient)) 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 a vasculitis associated disorder. The disclosure also provides methods of preventing a vasculitis associated disorder 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 a vasculitis associated disorder.

Scales useful for the assessment of osteoarthritis include, e.g., Birmingham Vasculitis Activity score (BVAS) version 3 (Mukhtyar et al. Ann Rheum Dis. 68(12):1827-32 (2009)), as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from an animal model of vasculitis, see e.g., those described in Katz et al., Clin Rev Allergy Immunol. 35(1-2):11-8 (2008) and references cited therein.

Head Trauma

Head trauma refers to trauma to the head, which may or may not include injury to the brain. Types of head trauma include concussion, epidural hematoma, subdural hematoma, cerebral contusion, and diffuse axonal injury.

Symptoms of head trauma include, e.g., coma, confusion, drowsiness, personality change, seizures, nausea and vomiting, headache and a lucid interval, during which a patient appears conscious only to deteriorate later, leaking cerebrospinal fluid, visible deformity or depression in the head or face, an eye that cannot move or is deviated to one side can indicate that a broken facial bone is pinching a nerve that innervates eye

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2019204295 19 Jun 2019 muscles, wounds or bruises on the scalp or face, basilar skull fractures, a subcutaneous bleed over the mastoid, hemotympanum, cerebrospinal fluid rhinorrhea, and otorrhea.

Treatments for head trauma include, e.g., controlling elevated intracranial pressure (e.g., sedation, paralytics, cerebrospinal fluid diversion), decompressive craniectomy, barbiturate coma, hypertonic saline, and hypothermia.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a head trauma scale) head trauma 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 head trauma. Additionally provided are methods of treating head trauma 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., controlling elevated intracranial pressure (e.g., sedation, a paralytic, and/or cerebrospinal fluid diversion), decompressive craniectomy, barbiturate coma, hypertonic saline, and/or hypothermia. The disclosure also provides methods of preventing head trauma 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 head trauma (e.g., a subject that will be participating in a dangerous activity or contact sport).

Scales useful for assessing head trauma and symptoms of head trauma include, e.g., the Glasgow Coma Scale (Teasdale and Jennett, Lancet 13;2(7872):81 -4 (1974)), as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of head trauma, see, e.g., those described in Cernak, NeuroRx. 2(3): 410-422 (2005) and references cited therein.

Brain Edema

Brain edema (cerebral edema) is an excess accumulation of water in the intracellular and/or extracellular spaces of the brain. Types of brain edema include, e.g.,

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2019204295 19 Jun 2019 vasogenic cerebral edema, cytotoxic cerebral edema, osmotic cerebral edema, and interstitial cerebral edema.

Vasogenic cerebral edema is due to a breakdown of tight endothelial junctions which make up the blood-brain barrier (BBB). This allows normally excluded intravascular proteins and fluid to penetrate into cerebral parenchymal extracellular space. Once plasma constituents cross the BBB, the edema spreads; this may be quite fast and widespread. As water enters white matter it moves extracellularly along fiber tracts and can also affect the gray matter. This type of edema is seen in response to trauma, tumors, focal inflammation, late stages of cerebral ischemia and hypertensive encephalopathy. Some of the mechanisms contributing to BBB dysfunction are: physical disruption by arterial hypertension or trauma, tumor-facilitated release of vasoactive and endothelial destructive compounds (e.g., arachidonic acid, excitatory neurotransmitters, eicosanoids, bradykinin, histamine and free radicals). Some of the special subcategories of vasogenic edema include: hydrostatic cerebral edema, cerebral edema from brain cancer, high altitude cerebral edema.

Cytotoxic cerebral edema is due to the derangement in cellular metabolism resulting in inadequate functioning of the sodium and potassium pump in the glial cell membrane. As a result there is cellular retention of sodium and water. Cytoxotic edema is seen with various intoxications (dinitrophenol, triethyltin, hexachlorophene, isoniazid), in Reye's syndrome, severe hypothermia, early ischemia, encephalopathy, early stroke or hypoxia, cardiac arrest, pseudotumor cerebri, and cerebral toxins.

Osmotic cerebral edema occurs when plasma is diluted by excessive water intake (or hyponatremia), syndrome of inappropriate antidiuretic hormone secretion (SIADH), hemodialysis, or rapid reduction of blood glucose in hyperosmolar hyperglycemic state (HHS), formerly hyperosmolar non-ketotic acidosis (HONK) and brain osmolality exceeds the serum osmolality creating an abnormal pressure.

Interstitial cerebral edema occurs in obstructive hydrocephalus. This form of edema is due to rupture of cerebral-spinal fluid (CSF)-brain barrier resulting in transependymal flow of CSF, which permits CSF to penetrate brain and spread in the extracellular space of white matter.

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Symptoms of brain edema (e.g., peritumoral brain edema) include, e.g., headache, loss of coordination (ataxia), weakness, and decreasing levels of consciousness including disorientation, loss of memory, hallucinations, psychotic behavior, and coma.

Treatments for brain edema (e.g., peritumoral brain edema) include, e.g., medications (e.g. dexamethasone, mannitol, diuretics) and surgical decompression.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms of) brain edema (e.g., peritumoral brain edema) 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 brain edema (e.g., peritumoral brain edema). Additionally provided are methods of treating brain edema (e.g., peritumoral brain edema) 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., a medication (e.g. dexamethasone, mannitol, and/or diuretics) and/or surgical decompression. The disclosure also provides methods of preventing brain edema 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 brain edema (e.g., a subject that has been diagnosed with a brain tumor).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of brain edema, e.g., a rat model of cerebral embolism in which recirculation can be introduced in the ischemic area (Koizumi et al., Jpn J Stroke 8: 1-8 (1986)).

Sepsis

Sepsis is a serious medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. This immunological response may be caused by microbes in the blood, urine, lungs, skin, or other tissues and can lead to widespread activation of acute-phase proteins, affecting the complement system and the coagulation pathways, which then cause damage to the vasculature as well as to the organs. Different levels of sepsis include systemic inflammatory response syndrome (SIRS), sepsis (SIRS in response to a confirmed

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2019204295 19 Jun 2019 infectious process), severe sepsis (sepsis with organ dysfunction, hypoperfusion, or hypotension), and septic shock (sepsis with refractory arterial hypotension or hypoperfusion abnormalities in spite of adequate fluid resuscitation).

Symptoms of sepsis include, e.g., general symptoms related to the infection, acute inflammation present throughout the entire body, hypothermia or fever, tachycardia, tachypnea or hypocapnia due to hyperventilation, leukopenia, leukocytosis, bandemia, and organ (e.g., lung, brain, liver, kidney, and/or heart) dysfunction.

Treatments for sepsis include, e.g., antibiotics, vasopressor drugs, insulin, corticosteroids, drotrecogin alfa, surgical drainage of infected fluid collections, fluid replacement, and appropriate support for organ dysfunction (e.g., hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure). Early Goal Directed Therapy (EGDT), a systematic approach to resuscitation, can be used to treat severe sepsis and septic shock.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a sepsis scale) sepsis 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 sepsis. Additionally provided are methods of treating sepsis 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 antibiotic, a vasopressor drug, insulin, a corticosteroid, drotrecogin alfa, surgical drainage of infected fluid collections, fluid replacement, an appropriate support for organ dysfunction (e.g., hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and/or drug and fluid therapy for circulatory failure), and/or an Early Goal Directed Therapy (EGDT). The disclosure also provides methods of preventing sepsis 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 sepsis (e.g., a subject that has been diagnosed as having an infection).

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Scales useful for assessing sepsis and symptoms of sepsis include, e.g., the

Baltimore Sepsis Scale (Meek et al. J Burn Care Rehabil. 12(6):564-8 (1991)) as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of sepsis, see, e.g., those described in U.S. Pat. No. 6,964,856, and Buras et al. Nat Rev Drug Discov. 4(10):854-65 (2005) and references cited therein.

Acute Middle Cerebral Artery (MCA) Ischemic Event (Stroke)

An acute middle cerebral artery (MCA) ischemic event (stroke) is the rapidly developing loss of brain function(s) due to disturbance in the blood supply to the brain due to ischemia (lack of glucose and oxygen supply) caused by thrombosis (e.g., venous thrombosis), embolism, or systemic hypoperfusion. As a result, the affected area of the brain is unable to function, leading to inability to move one or more limbs on one side of the body, inability to understand or formulate speech, or inability to see one side of the visual field. A stroke is a medical emergency and can cause permanent neurological damage, complications, and/or death.

Symptoms of acute middle cerebral artery (MCA) ischemic event (stroke) include, e.g., hemiplegia, decreased sensation and muscle weakness of the face, numbness, reduction in sensory or vibratory sensation, altered smell, taste, hearing or vision (total or partial), drooping of eyelid (ptosis) and weakness of ocular muscles, decreased reflexes, balance problems and nystagmus, altered breathing and heart rate, weakness in sternocleidomastoid muscle with inability to turn head to one side, weakness in tongue (inability to protrude and/or move from side to side), aphasia, apraxia, visual field defect, memory deficits, hemineglect, disorganized thinking, confusion, hypersexual gestures , anosognosia, trouble walking, altered movement coordination, and vertigo and/or disequilibrium.

Treatment for acute middle cerebral artery (MCA) ischemic event (stroke) includes, e.g., thrombolysis (e.g., tissue plasminogen activator (tPA)), thrombectomy, angioplasty and stenting, therapeutic hypothermia, and medications (e.g., aspirin, clopidogrel and dipyridamole).

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The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a stroke scale) acute middle cerebral artery (MCA) ischemic event (stroke) 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 acute middle cerebral artery (MCA) ischemic event (stroke). Additionally provided are methods of treating acute middle cerebral artery (MCA) ischemic event (stroke) 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., thrombolysis (e.g., tissue plasminogen activator (tPA)), thrombectomy, angioplasty and stenting, therapeutic hypothermia, and/or a medication (e.g., aspirin, clopidogrel and dipyridamole). The disclosure also provides methods of preventing acute middle cerebral artery (MCA) ischemic event (stroke) 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 acute middle cerebral artery (MCA) ischemic event (stroke) (e.g., a subject that has experienced systemic hypoperfusion).

Scales useful for assessing acute middle cerebral artery (MCA) ischemic event (stroke) and symptoms of acute middle cerebral artery (MCA) ischemic event (stroke) include, e.g., Oxford Community Stroke Project classification (OCSP, also known as the Bamford or Oxford classification) (Bamford et al., Lancet 337 (8756): 1521-6 (1991)), and TOAST (Trial of Org 10172 in Acute Stroke Treatment) (Adams et al., Stroke 24 (1): 35-41 (1993)).

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of acute middle cerebral artery (MCA) ischemic event (stroke), see, e.g., those described in Beech et al., Brain Res 895: 18-24 (2001), Buchan et al., Stroke 23 (2): 273-9 (1992), Carmichael, NeuroRx 2: 396X09 (2005), Chen et al., Stroke 17 (4): 73843 (1986), Dittmar et al., Stroke 34: 2252-7 (2003), Dittmar et al., JNeurosci Methods 156: 50 (2006), Gerriets et al., JNeurosci Methods 122: 201-11 (2003), Gerriets et al., Stroke 35: 2372-2377 (2004), Graham et al., Comp Med 54: 486X96 (2004), Koizumi et al., Jpn J Stroke 8: 1-8 (2004), Longa et al., Stroke 20 (1): 84-91 (1989), Mayzel-Oreg,

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Magn Reson Med 51: 1232-8 (2004), Schmid-Elsaesser et al., Stroke 29 (10): 2162-70 (1989), Tamura et al., J Cereb Blood Flow Metab 1: 53-60 (1981), Watson et al., Ann

Neurol 17: 497-504 (1985), and Zhang et al., J Cereb Blood Flow Metab 17: 123-35 (1997).

Restenosis

Restenosis is the reoccurrence of stenosis, a narrowing of a blood vessel, leading to restricted blood flow. Restenosis usually pertains to an artery or other large blood vessel that has become narrowed, received treatment to clear the blockage such as angioplasty, and subsequently become renarrowed. It can be defined as a reduction in the circumference of the lumen of 50% or more, and had a high incidence rate (25-50%) in patients who had undergone balloon angioplasty, with the majority of patients needing further angioplasty within 6 months.

Treatments for restenosis include, e.g., additional angioplasty if restenosis occurs without a stent or at either end of a stent, repeated angioplasty and insertion of another stent inside the original if restenosis occurs within a stent, drug-eluted stents, brachytherapy, and intracoronary radiation.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms of) restenosis (e.g., after angioplasty) 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 restenosis (e.g., after angioplasty). Additionally provided are methods of treating restenosis (e.g., after angioplasty) 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., angioplasty if restenosis occurs without a stent or at either end of a stent, repeated angioplasty and insertion of another stent inside the original if restenosis occurs within a stent, a drug-eluted stent, brachytherapy, and/or intracoronary radiation. The disclosure also provides methods of preventing restenosis 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 restenosis (e.g., a subject that has had stenosis).

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Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of restenosis, see, e.g., those described in U.S. Pat. Nos. 5,304,122 and 6,034,053, and Kantor et al., Cardiovasc Radiat Med. 1(1):48-54 (1999), and references cited therein.

Systemic Lupus Erythematosus Nephritis

Systemic lupus erythematosus nephritis is an inflammation of the kidney caused by systemic lupus erythematosus (SLE), a chronic autoimmune connective tissue disease. SLE can be associated with vasculitis which are disorders characterized by inflammatory destruction of blood vessels.

Symptoms of systemic lupus erythematosus nephritis include, e.g., general symptoms of kidney disease, weight gain, high blood pressure, darker foamy urine, and swelling around the eyes, legs, ankles or fingers.

Treatments for systemic lupus erythematosus nephritis include, e.g., steroid therapy (e.g., corticosteroids), chemotherapy (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, or cyclosporine), and immunosuppressant agents (e.g., mycophenolate mofetil and intravenous cyclophosphamide).

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a lupus scale) systemic lupus erythematosus nephritis 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 systemic lupus erythematosus nephritis. Additionally provided are methods of treating systemic lupus erythematosus nephritis 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., steroid therapy (e.g., a corticosteroid), chemotherapy (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, and/or cyclosporine), and/or an immunosuppressant agent (e.g., mycophenolate mofetil and/or intravenous cyclophosphamide). The disclosure also provides methods of preventing systemic lupus erythematosus nephritis or a symptom thereof by administering a plasma kallikrein binding protein (e.g., a prophylactically

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2019204295 19 Jun 2019 effective amount of a plasma kallikrein binding protein) to a subject at risk of developing systemic lupus erythematosus nephritis (e.g., a subject that has been diagnosed with lupus or a subject having a family member with lupus or a genetic predisposition thereto).

Scales useful for assessing systemic lupus erythematosus nephritis and symptoms of systemic lupus erythematosus nephritis include, e.g., World Health Organization (WHO) classification based on the biopsy (Weening et al., J. Am. Soc. Nephrol. 15 (2): 241-50 (2004)) as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of systemic lupus erythematosus nephritis, see, e.g., those described in U.S. Pat. No. 7,26,5261, Peng, Methods Mol Med. 102:227-72 (2004), and references cited therein.

Burn Injury and Wound Healing

A burn injury is a type of injury that may be caused by heat, electricity, chemicals, light, radiation, or friction. Muscle, bone, blood vessel, dermal and epidermal tissue can all be damaged with subsequent pain due to profound injury to nerves. Depending on the location affected and the degree of severity, a burn victim may experience a wide number of potentially fatal complications including shock, infection, electrolyte imbalance and respiratory distress. In burn injuries, the damage to epidermis and dermal elements is the result of several key insults which can be divided into initial (e.g., heat injury, inflammatory mediator injury, ischemia induced injury) and delayed insults. Excess heat causes rapid protein denaturation and cell damage. Much of the tissue damage, e.g., in the perfused subsurface burn, can be caused by toxic mediators of inflammation (e.g., oxidants and/or proteases) which are activated with the burn. Consumption of wound oxygen by neutrophils can lead to tissue hypoxia. Instant surface vascular thrombosis occurs along with cell death from the heat insult and causes ischemia and further tissue damage. Delayed injury after the initial heat and mediator damage includes, e.g., inflammation caused by neurotic tissue, bacteria on surface, caustic topical agents, and surface exudate; and continued damage to viable cells and new tissue growth by excess wound proteolytic activity and oxidant release.

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Treatments of burn injury include, e.g., intravenous fluids, dressings, pain management (e.g., analgesics (e.g., ibuprofen and acetaminophen), narcotics, and local anesthetics), inflammatory mediator inhibitors, and antibiotics.

The disclosure provides methods of treating (e.g., stabilizing, reducing, or eliminating one or more symptoms or stabilizing the subject’s score on a burn scale) a burn injury and/or promoting wound healing 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 a burn injury. Additionally provided are methods of treating a burn injury 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., intravenous fluid, a dressing, pain management (e.g., an analgesic (e.g., ibuprofen and acetaminophen), a narcotic, and a local anesthetic), an inflammatory mediator inhibitor, and an antibiotic. The disclosure also provides methods of preventing burn injuries 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 burn injuries (e.g., a subject whose occupation creates a risk of a burn injury, e.g., firefighter or cook).

Scales useful for assessing burns and symptoms of burns include, e.g., burn scales by degrees, by thickness, and by total body surface area (TBS A) (Meek et al. J Burn Care Rehabil. 12(6):564-8 (1991)) as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers a therapeutically effective amount of a plasma kallikrein binding protein may be obtained from animal models of burn, such as a porcine burn model (Singer and McClain, Methods Mol Med. 78:107-19 (2003), a sheep model of thermal injury (Jonkam et al., Shock, 28:704-709 (2007)), a rabbit model of thermal injury (Nwariaku et al., Burns, 22:324-327 (1996)), and a mouse model of burn wounding (Stevenson et al., Methods Mol Med. 78:95-105 (2003)).

Combination Therapies

A plasma kallikrein binding protein described herein, e.g., an anti-plasma kallikrein antibody, e.g., an anti- plasma kallikrein Fab or IgG, can be administered in

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2019204295 19 Jun 2019 combination with one or more of the other therapies for treating a disease or condition associated with plasma kallikrein activity, e.g., a disease or condition described herein. For example, a plasma kallikrein binding protein can be used therapeutically or prophylactically with surgery, another anti- plasma kallikrein Fab or IgG (e.g., another Fab or IgG described herein), another plasma kallikrein inhibitor, a peptide inhibitor, or small molecule inhibitor. Examples of plasma kallikrein inhibitors that can be used in combination therapy with a plasma kallikrein binding protein described herein include plasma kallikrein inhibitors described in, e.g., WO 95/21601 or WO 2003/103475.

One or more plasma kallikrein inhibitors can be used in combination with one or more plasma kallikrein binding proteins described herein. For example, the combination can result in a lower dose of the inhibitor being needed, such that side effects are reduced.

A plasma kallikrein binding protein described herein can be administered in combination with one or more current therapies for treating a plasma kallikrein associated disease or condition, including, but not limited to the current therapies for treating the disorder, e.g., a current therapy for rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, burn injury, or wound healing. For example, pKal inhibition is a novel mechanism of treating disease and therefore could provide effects that are synergistic or additive with other therapeutics. For example, a protein described herein that inhibits plasma kallikrein or that inhibits a downstream event of plasma kallikrein activity can also be used in combination with another treatment for a plasma kallikrein associated disease, such as surgery or administration of a second agent, e.g., as described herein. For example, the second agent can include ecallantide, a Cl esterase inhibitor (e.g., CINRYZE™), aprotinin (TRASYLOL®), a bradykinin B2 receptor inhibitor (e.g., icatibant (FIRAZYR®)).

The term “combination” refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or therapies overlap in time. The agents or therapies can be administered at the same time (e.g., as a single formulation

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2019204295 19 Jun 2019 that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times. The time between administration of the one agent and another agent can be minutes, hours, days, or weeks. The use of a plasma kallikrein binding protein described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side effects associated with another agent that is being administered. Accordingly, a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the plasma kallikrein binding protein.

The second agent or therapy can also be another agent for a plasma kallikrein associated therapy. Non-limiting examples of another treatment for a plasma kallikrein associated disease or condition include, e.g., ecallantide, a Cl esterase inhibitor (e.g., CINRYZE™), aprotinin (TRASYLOL®), a bradykinin B2 receptor inhibitor (e.g., icatibant (FIRAZYR®)) or a second binding protein described herein.

A combination therapy can include administering an agent that reduces the side effects of other therapies. The agent can be an agent that reduces the side effects of a plasma kallikrein associated disease treatment. For example, for inflammatory diseases, a pKal inhibitor could be steroid sparring. Also, there could be synergism with a TNFalpha inhibitor for treating inflammation or a VEGF blocker for treating cancer and/or angiogenesis.

Diagnostic Uses

A protein that binds to plasma kallikrein described herein can have in vitro and in vivo diagnostic utilities. A plasma kallikrein binding protein described herein (e.g., a protein that binds or binds and inhibits plasma kallikrein) can be used, e.g., for in vivo imaging, e.g., during a course of treatment for a disease or condition in which plasma kallikrein is active, e.g., a disease or condition described herein, or in diagnosing a disease or condition described herein.

In one aspect, the disclosure provides a diagnostic method for detecting the presence of plasma kallikrein, in vitro or in vivo (e.g., in vivo imaging in a subject). The

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2019204295 19 Jun 2019 method can include localizing plasma kallikrein within a subject or within a sample from a subject. With respect to sample evaluation, the method can include, for example: (i) contacting a sample with plasma kallikrein binding protein; and (ii) detecting the location of the plasma kallikrein binding protein in the sample.

A plasma kallikrein binding protein can also be used to determine the qualitative or quantitative level of expression of plasma kallikrein in a sample. The method can also include contacting a reference sample (e.g., a control sample, e.g., a negative control) with the binding protein, and determining a corresponding assessment of the reference sample. A difference (e.g., increase), e.g., a statistically significant difference, in the formation of the complex in the sample or subject relative to the control sample or subject can be indicative of the presence of plasma kallikrein in the sample. In one embodiment, the plasma kallikrein binding protein does not cross react with another kallikrein protein, such as tissue kallikrein and/or with plasma prekallikrein. E.g., the binding protein binds to another kallikrein protein or to prekallikrein 5- to 10-fold less well (or even less well) than it binds to plasma kallikrein. For example, the binding protein can bind to plasma kallikrein with a KD of-10-50 pM, whereas it binds to tissue kallikrein and/or prekallikrein at -10 nM.

The plasma kallikrein binding protein can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.

Complex formation between the plasma kallikrein binding protein and plasma kallikrein can be detected by evaluating the binding protein bound to the plasma kallikrein or unbound binding protein. Conventional detection assays can be used, e.g., an enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. Further to labeling the plasma kallikrein binding protein, the presence of plasma kallikrein can be assayed in a sample by a competition immunoassay utilizing standards labeled with a detectable substance and an unlabeled plasma kallikrein binding protein. In one example of this assay, the biological sample, the labeled standards, and the plasma kallikrein binding protein are combined and the amount of labeled standard bound to the unlabeled binding protein is determined. The amount of

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2019204295 19 Jun 2019 plasma kallikrein in the sample is inversely proportional to the amount of labeled standard bound to the plasma kallikrein binding protein.

Fluorophore and chromophore labeled proteins can be prepared. Because antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer, 1968, Science 162:526 and Brand, L. et al., 1972, Annu. Rev. Biochem. 41:843-868. The proteins can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. One group of fluorescers having a number of the desirable properties described above is the xanthene dyes, which include the fluoresceins and rhodamines. Another group of fluorescent compounds are the naphthylamines. Once labeled with a fluorophore or chromophore, the protein can be used to detect the presence or localization of the plasma kallikrein in a sample, e.g., using fluorescent microscopy (such as confocal or deconvolution microscopy).

Histological Analysis. Immunohistochemistry can be performed using the proteins described herein. For example, in the case of an antibody, the antibody can be synthesized with a label (such as a purification or epitope tag), or can be detectably labeled, e.g., by conjugating a label or label-binding group. For example, a chelator can be attached to the antibody. The antibody is then contacted to a histological preparation, e.g., a fixed section of tissue that is on a microscope slide. After an incubation for binding, the preparation is washed to remove unbound antibody. The preparation is then analyzed, e.g., using microscopy, to identify if the antibody bound to the preparation.

Of course, the antibody (or other polypeptide or peptide) can be unlabeled at the time of binding. After binding and washing, the antibody is labeled in order to render it detectable.

Protein Arrays. The plasma kallikrein binding protein can also be immobilized on a protein array. The protein array can be used as a diagnostic tool, e.g., to screen medical samples (such as isolated cells, blood, sera, biopsies, and the like). Of course, the protein array can also include other binding proteins, e.g., that bind to plasma kallikrein or to other target molecules.

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Methods of producing polypeptide arrays are described, e.g., in De Wildt et al., 2000, Nat. Biotechnol. 18:989-994; Lueking et al., 1999, Anal. Biochem. 270:103-111; Ge, 2000, Nucleic Acids Res. 28, e3,1-VII; MacBeath and Schreiber, 2000, Science 289:1760-1763; WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be spotted at high speed, e.g., using commercially available robotic apparati, e.g., from Genetic MicroSystems or BioRobotics. The array substrate can be, for example, nitrocellulose, plastic, glass, e.g., surface-modified glass. The array can also include a porous matrix, e.g., acrylamide, agarose, or another polymer.

For example, the array can be an array of antibodies, e.g., as described in De Wildt, supra. Cells that produce the proteins can be grown on a filter in an arrayed format. Polypeptide production is induced, and the expressed polypeptides are immobilized to the filter at the location of the cell. A protein array can be contacted with a labeled target to determine the extent of binding of the target to each immobilized polypeptide. Information about the extent of binding at each address of the array can be stored as a profile, e.g., in a computer database. The protein array can be produced in replicates and used to compare binding profiles, e.g., of a target and a non-target.

FACS (Fluorescence Activated Cell Sorting). The plasma kallikrein binding protein can be used to label cells, e.g., cells in a sample (e.g., a patient sample). The binding protein is also attached (or attachable) to a fluorescent compound. The cells can then be sorted using fluorescence activated cell sorter (e.g., using a sorter available from Becton Dickinson Immunocytometry Systems, San Jose CA; see also U.S. Pat. Nos. 5,627,037; 5,030,002; and 5,137,809). As cells pass through the sorter, a laser beam excites the fluorescent compound while a detector counts cells that pass through and determines whether a fluorescent compound is attached to the cell by detecting fluorescence. The amount of label bound to each cell can be quantified and analyzed to characterize the sample.

The sorter can also deflect the cell and separate cells bound by the binding protein from those cells not bound by the binding protein. The separated cells can be cultured and/or characterized.

In vivo Imaging. Also featured is a method for detecting the presence of plasma kallikrein expressing tissues in vivo. The method includes (i) administering to a subject

147

2019204295 19 Jun 2019 (e.g., a patient having, e.g., a plasma kallikrein associated disease or condition) an antiplasma kallikrein antibody, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to the plasma kallikrein expressing tissues or cells. For example, the subject is imaged, e.g., by NMR or other tomographic means.

Examples of labels useful for diagnostic imaging include radiolabels such as ¹³¹I, ⁱⁿIn, ¹²³I, ^99mTc, ³²P, ¹²⁵1,³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short range radiation emitters, such as isotopes detectable by short range detector probes can also be employed. The protein can be labeled with such reagents; for example, see Wensel and Meares, 1983, Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York for techniques relating to the radiolabeling of antibodies and D. Colcher et al., 1986, Meth. Enzymol. 121: 802-816.

The binding protein can be labeled with a radioactive isotope (such as ¹⁴C, ³H,

S, I, P, I). A radiolabeled binding protein can be used for diagnostic tests, e.g., an in vitro assay. The specific activity of a isotopically-labeled binding protein depends upon the half life, the isotopic purity of the radioactive label, and how the label is incorporated into the antibody.

In the case of a radiolabeled binding protein, the binding protein is administered to the patient, is localized to cells bearing the antigen with which the binding protein reacts, and is detected or “imaged” in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A.R. Bradwell et al., “Developments in Antibody Imaging”, Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al., (eds.), pp 65 85 (Academic Press 1985). Alternatively, a positron emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., ⁿC, ¹⁸F, ¹⁵O, and ¹³N).

MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses NMR to visualize internal features of living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for

148

2019204295 19 Jun 2019 obvious benefit. Some MRI techniques are summarized in EP-A-0 502 814. Generally, the differences related to relaxation time constants Tl and T2 of water protons in different environments are used to generate an image. However, these differences can be insufficient to provide sharp high resolution images.

The differences in these relaxation time constants can be enhanced by contrast agents. Examples of such contrast agents include a number of magnetic agents paramagnetic agents (which primarily alter Tl) and ferromagnetic or superparamagnetic (which primarily alter T2 response). Chelates (e.g., EDTA, DTPA andNTA chelates) can be used to attach (and reduce toxicity) of some paramagnetic substances (e.g., Fe⁺³, +2 +3

Mn , Gd ). Other agents can be in the form of particles, e.g., less than 10 mm to about 10 nM in diameter). Particles can have ferromagnetic, antiferromagnetic, or superparamagnetic properties. Particles can include, e.g., magnetite (FejOq), y-Fe2O3, ferrites, and other magnetic mineral compounds of transition elements. Magnetic particles may include: one or more magnetic crystals with and without nonmagnetic material. The nonmagnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch and the like.

The plasma kallikrein binding protein can also be labeled with an indicating group containing of the NMR active ¹⁹F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the ¹⁹F isotope and, thus, substantially all fluorine containing compounds are NMR active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost; and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body MRI is carried out using an apparatus such as one of those described by Pykett, 1982, Sci. Am. 246:78 88 to locate and image tissues expressing plasma kallikrein.

The following examples provide further illustration and are not limiting.

149

EXAMPLES

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EXAMPLE 1:

We have discovered several antibody inhibitors and binders of plasma kallikrein (pKal). The most potent of these have been further characterized and shown to have apparent inhibition constants (Ki,_app) <10 nM, to be specific pKal inhibitors with respect to other tested serine proteases, and to not bind prekallikrein. Amino acid sequences of the CDRs for the inhibitors and the binders are shown in Tables 1 and 2, respectively.

150

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Q fc ix § % l-l fc 0

l-l Q fc rt fc ω 0 fc fc fc fc 0 ω

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EGLLWFGENAFDI

K Q co ίχ fc 0 fc l-l ix Q ix co 0

NPQYSGYDRSLSDGAFDI

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DLFLYDFWSKGAFDI

DKFEWRLLFRGIGNDAFDI

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0 fc > co 9 ix ix Eh fc 0 0 CO ex H

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ix ix fc

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l-l fc fc ix fc

fc s fc ix Η

fc ix fc

ix 2 fc ix ex

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co fc fc ix fc

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Eh fc fc co fc Eh O O'

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0 rt Eh CO Eh Q fc rt ex

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0 rt Eh CO Eh Q fc rt O'

l-l > l-l co co Q fc rt O'

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l-l rt Eh CO CO Q fc rt O'

δ g ex co Q fc rt O'

C4 fc Q υ > j

co o J Eh co 3

co ex g fc

co ex g co Q o

co ex fc fc fc Q O

fc o fc fc co rt 0

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s fc fc co rt Q

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i—l fc Q 0 1 > J

fc J ix l-l CO O co rt fc

co > fc fc ix fc 0 > Q co fc Eh 0 Eh

u rt ix fc Q 0 J fc Q 0 CO

ix rt >-< fc s 0 J fc Q 0 CO

fc fc ix > co l-l co O co rt fc

rt fc ix co co co > co O' co rt fc

0 > ix fc Q 0 J O' Q 0 CO

0 rt ix fc Q 0 J fc Q 0 CO

0 > ix fc Q 0 J O' Q 0 CO

rt > ix fc Q 0 J fc § co

CO fc ix fc fc 0 J fc Q 0 CO

rt j ix co co > co o co rt fc

co > ix fc Q 0 J fc ω 0 co

co Eh ix fc Q 0 J fc Q 0 CO

Human. pKal· (Ki,app nM)

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152

2019204295 19 Jun 2019

GTPKWELLLRSIYIENAFDI

DSSGSPNPLFDY

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GGLLLWFRELKSNYFDY

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DKFEWRLLFRGIGNDAFDI

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EGPPYSSGWYRGLRQYHFDY

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GGLLLWFMELKSNYFDY

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153

2019204295 19 Jun 2019

Q	>Η Q	>Η Q	>Η Q	>Η Q	>Η
Cl	Cl	Cl	X	X	Q
>h	>η	>η	>Η	>Η	X
5	5	5	5	5	>Η
ω	ω	ω	ω	ω	ω
td	td	X	X	X	Η
j	χ	X	X	X	X
ω	ω	Μ	ω	ω	ω
cd	cd	X	X	X	X
Q	ο	Η		ω	3

j	χ	X	X	X	Η
j	χ	X	X	X	0
j	χ	X	X	X	X
0	0	0	0	0
0	0	0	0	0	>
0	0	0	0	0	0
td	td	X	X	X	X
>	>	>	>	>	>
ω Q	ω Q	ω Q	ω Q	ω Q	ω Q
S’	S’ >Η	S’ >Η	S’ >Η	S’ >Η	S’ >Η
H	Η	Η	Η	Η	Η
H	Η	Η	Η	Η	Η
cd	cd	X	X	X	X
0	0	0	0	0	0
0	0	0	0	0	0
ω	ω	ω	ω	ω	ω
X	X	X	X	X	X
cd	cd	X	X	X	ω
H	Η	Η	Η	Η	Η
	>Η	>Η	>Η	>Η	>Η
H	Η	Η	Η	Η	Η
s	S	S	S	S	S
ω	ω	ω	ω	ω	X
	>Η	>Η	>Η	>Η	>Η
					X
H	Η	Η	Η	Η	Η
	>Η	>Η	>Η	>Η
CL	X	X	X	X	X
>	>	>	>	>	Η
H	Η	Η	Η	Η	X
	>Η	>Η	>Η	>Η	ω
ω	ω	ω	ω	ω	ω
o	ο	ο	ο	ο	ο
ο	ο	ο	ο	ο	ο
ω	ω	ω	ω	ω	Η
ο	ο	ο	ο	ο
χ	χ	X	X	X	X
cd	cd	X	X	X	ω
ω	ω	ω	ω	ω	ω

					0
					X
X	X	X	X	X	>Η
>Η	>Η	>Η	>Η	>Η	5
5	5	5	5	5	ω
Q	Q	Q	Q	Q	5
Η	Η	Η	Η	Η	>
X	X	X	X	X	X
ο	ο	ο	ο	ο	ο
ω	ω	ω	ω	ω	ω
					Η
cd	cd	X	X	X	X
co	σι	CO	LD CO	<0	CM
Η	ο	Η	Ο	CO	ο
tJ	τ5	Τ5	Τ5	Τ5	τ5
S	S	S			S
	Η	ο			Η
ο	ο		Ο	ο	ο
ω	Cl	X	0	X	X
	γ-	Γ-	Γ-		Η
<0	<0	<0	<0	<0	co
χ	X	X	X	X	X

154

2019204295 19 Jun 2019

Amino acid sequences of light chain (LC) and heavy chain (HC) variable domain of pKal antibody inhibitors are shown below.

M6-D09 QDIQMTQSPS ARFSGSGSGT	SLSASVGDRV DFTLTISSLQ	LC TITCRASQSI PEDFATYYCQ	RNYLNWYQQK QLSGYPHTFG	PGKAPNLLIY QGTKLEIK	AASTLQSGVP	60 108
M6-D09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	FYYMVWVRQA	PGKGLEWVSV	IYPSGGITVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARDK	WAVMPPYYYY	AMDVWGQGTT	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M7-B04		LC
QSALTQPASV	SGSPGQSITI	SCTGTNSDVG	NYNLVSWYQQ	HPGEAPKLLI	YEVNKRPSGV	60
SNRFSGSKSG	NTASLTISGL	QAEDEADYLC	CSYAGNRNFY	VFGAGTKVTV	L	111
M7-B04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYSMVWVRQA	PGKGLEWVSS	ISPSGGLTNY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARHT	AARPFYYYYM	DVWGKGTTVT	120
VSSASTKGPS	VFPLAPSSKS					140
M7-E07		LC
QSELTQPPSV	SVSPGQTASI	TCSGDKLGDK	YACWYQQKPG	QSPVLVIYQD	SKRPSGIPER	60
FSGSNSGNTA	TLTISGTQAM	DEADYYCQAW	DSSTGVFGGG	TKLTVL		106
M7-E07		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYLMIWVRQA	PGKGLEWVSY	IYPSGGFTYY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	MAVYYCARTE	GPLSWGYGMD	VWGQGTTVTV	120
SSASTKGPSV	FPLAPSSKS					139
M8-A09		LC
QCELTQPPSE	SVSPGQTANI	TCSGDKLGNK	YAYWYQQKPG	QSPVLVIYQD	NNRPSGIPER	60
FSGSNSGNTA	TLTISGTQAI	DEANYYCQAW	DSRTWFGGG	TKLTVL		106
M8-A09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	TYFMLWVRQA	PGKGLEWVSS	IYPSGGNTVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARAA	SPVRNYYYYG	MDVWGQGTTV	120
TVSSASTKGP	SVFPLAPSSK	S				141
M10-F10		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI	SVYLNWYQHK	PGKAPKLLIY	GASNLQFGVP	60
SRFSGSGYGT	DFTLTISSLQ	PEDFATYHCQ	QTFSLFTFGG	GTKVEIK		107
M10-F10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	FYNMNWVRQA	PGKGLEWVSS	ISPSGGETNY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	GAYRNNWWGG	FDIWGLGTMV	120
TVSSASTKGP	SVFPLAPSSK	S				141
M10-H05		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRASQSV	SSSYLAWYQQ	KPGQAPRLLI	YGASSRATGI	60
PDRFSGSGSG	TDFTLTISRL	EPEDFAVYYC	QQYGSSPFTF	GPGTKVDIK		109
M10-H05		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	PYNMYWVRQA	PGKGLEWVSS	IRPSGGGTVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAGGF	IAARWYYFDY	WGQGTLVTVS	120

155

SASTKGPSVF PLAPSSKS

138

2019204295 19 Jun 2019

M12-D05		LC
QSVLTQPPSV	SVSPGQTATI	TCSGDQLGDK	YVGWYQQKPG	QSPILVIYQD	TKRPSGIPER	60
FSGSNSGNTA	TLTISGTHTV	DEAHYYCQAW	DTSTAGFGGG	TKLTVL		106
M12-D05		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYTMVWVRQA	PGKGLEWVSR	IYPSGGWTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TATYYCAREG	LLWFGENAFD	IWGQGTMVTV	120
SSASTKGPSV	FPLAPSSKS					139
M27-E05		LC
QSELTQPPSV	SVSPGQTASI	TCSGDKLGDK	YACWYQQKPG	QSPVLVIYQD	SKRPSGIPER	60
FSGSNSGNTA	TLTISGTQAM	DEADYYCQAW	DSSTGVFGGG	TKLTVL		106
M27-E05		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYLMIWVRQA	PGKGLEWVSY	IYPSGGFTYY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	MAVYYCARTE	GPLSWGYGMD	VWGQGTTVTV	120
SSASTKGPSV	FPLAPSSKS					139
M28-B11		LC
QSVLTQPPSV	SVSPGQTATI	TCSGDQLGDK	YVGWYQQKPG	QSPILVIYQD	TKRPSGIPER	60
FSGSNSGNTA	TLTISGTHTV	DEAHYYCQAW	DTSTAGFGGG	TKLTVL		106
M28-B11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYTMVWVRQA	PGKGLEWVSR	IYPSGGWTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TATYYCAREG	LLWFGENAFD	IWGQGTMVTV	120
SSASTKGPSV	FPLAPSSKS					139
M29-D09		LC
QSALTQPPTV	SVSPGQTARI	TCSGNKLGDK	YVAWYQQKPG	QSPMLVIYQD	TKRPSRVSER	60
FSGSNSANTA	TLSISGTQAL	DEADYYCQAW	DSSIVIFGGG	TRLTVL		106
M29-D09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYTMVWVRQA	PGKGLEWVSY	IYPSGGATFY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAMGS	YDYIWGFYSD	HWGQGTLVTV	120
SSASTKGPSV	FPLAPSSKS					139
M29-E09		LC
QYELTQPPSV	SVSPGQTATI	TCSGDNLGNK	YNSWYQQKPG	QSPLLVIYQD	TKRPSAIPER	60
FSGSNSGNTA	TLTISGTQAM	DEADYYCQAW	DGNWFGGGT	KLTVL		105
M29-E09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYEMGWVRQA	PGKGLEWVSS	IYSSGGGTMY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARNP	QYSGYDRSLS	DGAFDIWGQG	120
TMVTVSSAST	KGPSVFPLAP	SSKS				144
M35-G04		LC
QDIQMTQSPA	TLSLSPGERA	TLSCRASQSV	SSYLAWYQQK	PGQAPRLLIY	DASNRATGIP	60
ARFSGSGSGT	DFTLTISSLE	PEDFAVYYCQ	QRSNWPRGFT	FGPGTKVDIK		110
M35-G04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	YYHMSWVRQA	PGKGLEWVSV	ISPSGGSTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	SSDYAWGSYR	RPYYFDYWGQ	120
GTLVTVSSAS	TKGPSVFPLA	PSSKS				145

156

2019204295 19 Jun 2019

M38-F02		LC
QSVLTQPPSV	SVSPGQTASI	TCSGEKLGDK	YVSWYQQKPG	QSPSLVICED	SRRPSGIPER	60
FSGSNSGNTA	TLTISGAQPM	DEADYYCQAW	DSSTAIFGPG	TKVTVL		106
M38-F02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	YYMMVWVRQA	PGKGLEWVSY	IYSSGGHTVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARDL	FLYDFWSKGA	FDIWGQGTMV	120
TVSSASTKGP	SVFPLAPSSK	S				141
M41-A11		LC
QSVLTQPPSV	SVSPGQTASI	TCSGDKLGDK	YTSWYQQRPG	QSPVLVIYQD	IKRPSGIPER	60
FSGSNSGNTA	TLTISGTQAM	DEADYYCQAW	DSPNARVFGS	GTKVTVL		107
M41-A11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYRMSWVRQA	PGKGLEWVSS	IYPSGGRTVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAKDK	FEWRLLFRGI	GNDAFDIWGQ	120
GTMVTVSSAS	TKGPSVFPLA	PSSKS				145
M73-D06		LC
QSELTQPPSA	SETPGQRVTI	SCSGSSSNIG	SNTVSWFQQL	PGSAPRLLIY	NDHRRPSGVP	60
DRFSGSKSGT	SASLVISGLQ	SQDEADYYCS	AWDDSLNGVV	FGGGTKLTVL		110
M73-D06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	RYEMYWVRQA	PGKGLEWVSS	ISSSGGPTAY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAMYYCAKGT	PKWELLLRSI	YIENAFDIWG	120
QGTMVTVSSA	STKGPSVFPL	APSSKS				146
M76-D01		LC
QDIVMTQTPP	SLPVNPGEPA	SISCRSSQSL	SDDGNTYLDW	YLQRPGQSPQ	LLIHTLSYRA	60
SGVPDRFSGS	GSGTDFTLKI	SRVEAEDVGV	YYCMQGTHWP	PTFGQGTKVE	IK	112
M76-D01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	FYAMHWVRQA	PGKGLEWVSG	IVPSGGRTHY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCATDS	SGSPNPLFDY	WGQGTLVTVS	120
SASTKGPSVF	PLAPSPKS					138
M110-C12		LC
QDIQMTQSPL	SLSVTPGEPA	SISCRSSLSL	LHSNGYNYLD	WYVQRPGQSP	QLLMYLSSTR	60
ASGVPDRFSG	SGSGTDFTLE	ISRVEAEDVG	VYYCMQPLET	PPTFGGGTKV	EIK	113
M110-C12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	YYEMDWVRQA	PGKGLEWVSG	ISSSGGHTAY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TATYYCARER	RSSSRARYYY	GMDVWGQGTT	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M137-E12		LC
QSVLIQPPSV	SGIPGQRVTI	SCSGNNSNFG	SNTVTWYQQL	PGTAPKLLIY	SDSRRPSGVP	60
DRFSGSRSDT	SASLAISGLQ	SEDEAEYHCA	AWDDSLNGVF	GGGTKLTVL		109
M137-E12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	DYRMQWVRQA	PGKGLEWVSV	IVPSGGNTMY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	PGSSIAARRA	PTGYYGMDVW	120
GQGTTVTVSS	ASTKGPSVFP	LAPSSKS				147
M142-H08		LC
QDIQMTQSPS	SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
SRLSGSGFGT	DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108

157

2019204295 19 Jun 2019

M142-H08 EVQLLESGGG ADSVKGRFTI LVTVSSASTK	LVQPGGSLRL SRDNSKNTLY GPSVFPLAPS	HC SCAASGFTFS LQMNSLRAED SKS	AYSMIWVRQA TAVYYCARGG	PGKGLEWVSY LLLWFRELKS	IRPSGGRTTY NYFDYWGQGT	60 120 143
M145-D01		LC
QDIQMTQSPA	TLSLSPGERA	TLSCRASQSV	SSYLAWYQQK	PGQAPRLLIY	DASNRATGIP	60
ARFSGSGSGT	DFTLTISSLE	PEDFAVYYCQ	QRSNWPRGFT	FGPGTKVDIK		110
M145-D01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	YYHMSWVRQA	PGKGLEWVSV	ISPSGGSTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	SSDYAWGSYR	RPYYFDYWGQ	120
GTLVTVSSAS	TKGPSVFPLA	PSSKS				145
M145-D11		LC
QSVLTQPPSV	SVSPGQTASI	TCSGDKLGDK	YTSWYQQRPG	QSPVLVIYQD	IKRPSGIPER	60
FSGSNSGNTA	TLTISGTQAM	DEADYYCQAW	DSPNARVFGS	GTKVTVL		107
M145-D11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYRMSWVRQA	PGKGLEWVSS	IYPSGGRTVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAKDK	FEWRLLFRGI	GNDAFDIWGQ	120
GTMVTVSSAS	TKGPSVFPLA	PSSKS				145
M146-E12		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASGDI	GNALGWYQQK	PGKAPRLLIS	DASTLQSGVP	60
LRFSGSGSGT	EFTLTISSLQ	PEDFATYYCL	QGYNYPRTFG	QGTKLEIR		108
M146-E12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	RYIMHWVRQA	PGKGLEWVSS	ISPSGGLTSY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAREF	ENAYHYYYYG	MDVWGQGTTV	120
TVSSASTKGP	SVFPLAPSSK	S				141
M152-A12		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI	SSYLSWYQQR	PGKAPNLLIY	AASSLQSGVP	60
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ	QSISIPRTFG	QGTKVEVK		108
M152-A12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	PYFMGWVRQA	PGKGLEWVSG	IGPSGGSTTY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAREG	PPYSSGWYRG	LRQYHFDYWG	120
QGTLVTVSSA	STKGPSVFPL	APSSKS				146
M160-G12		LC
QDIQMTQSPS	FLSASVGDRV	TITCRASQGI	SSYLAWYQQK	PGKAPKLLIY	AASTLQSGVP	60
SRFSGSGSGT	EFTLTISSLQ	PEDFATYYCQ	QLNSYPLTFG	GGTKVEIK		108
M160-G12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M161-C11		LC
QSALTQPPSV	SVSPGQTASI	TCSGDKLGDK	YVSWYQQRPG	QSPVLVIYQD	TKRPSGIPER	60
FSGSNSGNTA	TLTISGTQAV	DEADYYCQAW	DSSTYVFGGG	TKVTVL		106
M161-C11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	DYAMKWVRQA	PGKGLEWVSS	ISSSGGVTQY	60

158

2019204295 19 Jun 2019

ADSVKGRFTI

GQGTTVTVSS

M162-A04

QDIQMTQSPS

SRFSGSGSGT

M162-A04

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

SRDNSKNTLY ASTKGPSVFP	LQMNSLRAED LAPSSKS	TAVYYCAREE	DYSSSWYSRR	FDYYYGMDVW	120 147
TLSASVGDRV	LC TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
LVQPGGSLRL	HC SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139

X67-B03 LC QDIQMTQSPS SRLSGSGFGT

X67-B03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-C03 LC QDIQMTQSPS SRLSGSGFGT

X67-C03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-C09 LC QDIQMTQSPS SRLSGSGFGT

X67-C09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-D03 LC QDIQMTQSPS SRLSGSGFGT

X67-D03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-E04 LC QDIQMTQSPS SRLSGSGFGT

X67-E04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWSRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWMRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWGRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWNRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWDRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143

159

X67-F01 LC

QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108

2019204295 19 Jun 2019

X67-F01 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWQRELKS NYFDYWGQGT 120

LVTVSSASTK GPSVFPLAPS SKS143

X67-F10 LC

QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR108

X67-F10 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWTRELKS NYFDYWGQGT120

LVTVSSASTK GPSVFPLAPS SKS143

X67-G04 LC

QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR108

X67-G04 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWARELKS NYFDYWGQGT120

LVTVSSASTK GPSVFPLAPS SKS143

X67-H04 LC

QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR108

X67-H04 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWERELKS NYFDYWGQGT 120

LVTVSSASTK GPSVFPLAPS SKS143

Note: X81-B01 is a germilined IgG derived from X63-G06 which is shown in

Table 7.

160

19 Jun 2019

HV-CDR3

VSTAVADNDY

DDYGGKGGAFDI

VSTAVADNDY

Οι Q pH ο Η Η ω

DAVGIGDAFDI

LFYGSGSVGFEY

DLDLGILDY

DSYSSSWFDI

SFSGFGEIDY

Η Q Ρη Ο % Η ω Q Q

Η Q 01 >Η £ ω

GNSSSFDAFDI

VDYTGDGLGY

Η Q 01 Ph Ο 3 ο > ω

GSPWGDDAFDI

>Η Q Η > σ Q

Η Q 01 Q J >Η ω Ο ω

DDWNVGMDV

QGLLTAFDI

VDTAMAMIDY

GTVGASDAFDI

> Q S Ο >Η >Η >Η >Η ω > Ο Οι Q

>Η Q > ω <2 ω ω Q >Η Η

DDYGGKGGAFDI

DTDYGMDV

TSITTVGMDV

Η Q 01 Q ω ω <2 >

GYLGY

EYPSTYYFDY

19204295 1

(N Q U g

GIWPSGGFTDYADSVKG |

Ο > ω § >Η ω Η S ο ο ω ω ω Η ω

G > ω § ω H G G ω ω Ο Η >

ο > ω § >Η Η Ο Ο ω & ω Η ω

Ο > ω § s Η S Ο Ο ω Οι >Η Η ω

Ο > ω § >Η ω Η j Ο Ο ω Οι >Η Η >Η

Ο > ω § Q H Ph G G ω Oi ω Η ω

Ο > ω § >Η ο Η S ο ο ω Οι > Η ω

G > ω § Q H >H G G ω Oi H >

ο > ω § >Η σ Η ο ο ω ω ω Η ω

ο > ω § >Η Q Η >Η Ο Ο ω ω >Η Η ω

Ο > ω § ω Η ω Ο Ο ω ω Ο Η >Η

Ο > ω § >Η >Η Η J Ο Ο ω Οι > Η >Η

ο > ω § >Η Ρη Η S Ο Ο ω ω Ο Η ω

Ο > ω § >Η Η Ο Ο ω Οι >Η Η ω

ο > ω § >Η >Η Η S Ο Ο ω ω Ο Η ω

Ο > ω § >Η Q Η Ρη Ο Ο ω Οι >Η Η >

Ο > ω § >Η Η Η S Ο Ο ω ω ω Η

Ο > ω § >Η Η Η ω Ο Ο ω Οι ω Η ω

Ο > ω § >Η S Η Ρη Ο Ο ω Οι >Η Η ω

VIYPSGGVTMYADSVKG |

Ο > ω § >Η S Η >Η Ο Ο ω Οι > Η >Η

ο > ω § >Η Ρη Η ω Ο Ο ω Οι Η ω

Ο > ω § >Η ω Η S ο ο ω ω ω Η ω

Ο > ω § Η Η Ph Ο Ο ω ω >Η Η ω

RIWPSGGVTMYADSVKG |

Ο > ω § >Η Q Η S Ο Ο ω Οι ω Η >Η

Ο > ω § >Η ω Η 5 Ο Ο ω ω ω Η

Ο > ω § >Η S Η Οι Ο Ο ω ω Η ω

20 s of PKal

rd Q U 1 > K

S o J

ο 5 6 >Η &

Ο S pH >Η

Ε >Η

>Η

ο S Οι >Η >Η

>Η j

Η S Ρη >Η Ε

J s ω >H j

Ο S >Η Ο

S S Ρη >Η ο

S ω >Η

Ο >Η Ε

σ S Οι >Η J

S S >Η

S S Q >Η ω

Η Ξ >Η 01

Ο S Οι >Η ω

ω S Q >Η Q

j S Η >Η >Η

Η S Ξ >Η

Ο S ω >Η

S Η >Η

σ S Οι >Η Οι

Ε S J >Η J

ω S ω >Η

S S Οι >Η S

5 S Οι >Η

Ρη S >Η J

^? Antibody Binder

Q U 1 > J

H Oi ω >H ω o ο

Η 5 Λ Η ω Ρη Η ο ο

Η Η ω >Η ω ο ο

Η Η >Η ω ο ο

Η Η Οι Η Η >Η ω ο ο

> Η ω ω Q § ο

Η j Οι Ρη ο Q ο ο

Η >Η S J Η ω >Η ω ο ο

H Oi Oi >H H σ σ

Η Ρη Οι Η Η Ρη ω σ σ

> Οι Η Ο >Η ω σ σ

η Οι Η 5 >Η ω σ σ

Η J Οι Η ω Ρη ω σ σ

Η Ρη J Η ω >Η ω ω σ

Η Οι ω ω Ρη ω σ σ

Η Οι Οι Η Ε ω σ σ

Η >Η 01 Οι Η ω >Η ω σ σ

Η Οι Η Η >Η Η σ σ

Η >Η Οι Η ω >Η ω σ σ

Η Η Οι Η ω >Η ω σ σ

Η >Η 01 Q ω Ρη ω σ σ

> >Η Η 5 ω Q j σ

Η Οι Οι Η ω >Η ω σ σ

Η 5 Οι Η ω Ρη Η σ σ

Η Ρη Οι Η ω >Η ω σ σ

Η Οι Η ω Ρη ω σ σ

Η Οι Οι Η ω Ε ω σ σ

Η J Οι Η σ J σ S

Η >Η 01 Q ω >Η ω σ σ

.ISA Signal ol

(N Q U 1 > J

ω ο j ω ω Η Ο

ω ο j ω ω Ο

ω ο j ω ω 3

ω ο j ω ω Ο

ω S ω Ρη g

ω Οι ω Q ο

ω ο j 5 ω Q

ω ο j ω ω 3

ω σ j ω ω Ο

ω σ j ω ω Η Ο

ω σ j 5 >Η ο

ω σ j ω ω Ο

σ j ω ω 3

ω σ j ω ω Ο

ω Ε j ω ω Ο Ο

Η σ j 5 ω 3

ω σ j ω ω 3

ω ω j Η ω 3

ω σ j ω ω 3

ω σ j Η ω ο

ω Οι 5 Q σ

ω σ j ω ω 3

ω σ j ω ω ο

ω σ j ω ω Ο

ω σ j Η ω 3

ω g 5 ω Ο j

ω σ j ω ω Ο

Amino Acid Sequences and EL

rd Q U 1 > J

5 >Η S ω Η ω ο ω g

5 >Η Ρη ω Η ο ω g

5 >Η ω ω Η ω ο ω g

5 j >Η ω ω Η ω ο ω g

5 J >Η Η ω Η ω ο ω g

ω Η 8 J Q Ο ω

> j ω ω Η Q ο ω g

5 J >Η ω ω Η ω ο ω g

j >Η >Η ω 8 σ ω g

5 j >Η Η ω Η >Η σ ω g

5 J >Η Η Q Η 5 σ ω g

5 j >Η Η ω Η σ ω g

>Η Η ω Η ω σ ω g

5 j >Η ω ω Η Ο σ ω g

5 j >Η Ρη ω Η Η ω g

5 J >Η Ο ω Η ω σ ω g

5 J >Η ω ω Η ω σ ω g

5 J >Η Η 5 Η ω σ ω g

5 J >Η ω ω Η ω σ ω g

5 j >Η Η Η ω ω g

ω Η >Η § J Q Ο ω

5 J >Η ω ω Η ω σ ω g

5 j >Η Ρη ω Η σ ω g

5 j >Η > ω Η Q ω g

5 j >Η Η ω Η ω σ ω g

5 J >Η Η ω Η ω σ ω g

Q J >Η >Η Ο 5 ω Ε j j ω σ ω ω

5 j >Η Η ω Η ω σ ω g

Human pKal ELISA (T/B)

> Η γΗ

rt¹ co (Ί

LD LD Η

to Η

LD co Η

Γ-

σι Η γΗ

co co

ΓΊ co ΓΊ

ΠΊ ΠΊ

rt¹ rt¹ Η

LD LD

σι ο Η

co

σι LD co

co co Η

σι ο Η

rt¹ co

co rt¹

LD CO

Η to Η

> co co

co ο ΓΊ

Η Η

co LT)

to

co ΓΊ CO

Table 2. CDR

Φ S al S 1—1 al -rt JJ -rt 0 H

<0 ο <0 S

co ο <c to S

CO Ο Q to S

co ο Q to S

LD ο ο to S

ο υ co S

LD Ο Q co S

to ο ω co S

σι ο Ο co S

Ο Ε co S

co Ο <c σι S

co ο <c σι S

co ο υ σι S

ο Η υ σι S

co ο Q σι S

rt¹ο ω σι S

co ο Ρη σι S

σι ο Ρη σι S

ο Η Ρη σι S

co ο Ο σι S

ΓΊ Ο Ε σι S

co ο Ε σι S

ο Ε σι S

co ο ο Η s

ΓΊ Η Ο Η S

σι ο ffl ο Η S

Η γΗ υ ο Η S

Η γΗ Q ο Η S

to ο ω ο Η S

1094814.1 - 160161

2019204295 19 Jun 2019

HV-CDR3

ω Q &4 E ω Λ 0 5 H Q

DGYYDSSGYPDY

GSYSDYGVFES

Ρη ο j 3 Η 0 Οι ο ω

ITTVTTGGAFDI

LGGPLDAFDI

5 J J ω 0 0 0 0

DTGFWSADAFDI

EEQQGGFDY

TTVTTGDAFDI

DPLYSSGDV

Η Q 01 Q 0 0 0 J 5 S

DLKWGGRGSPDWYFDL

DTSGSYLGFDY

APWFGELGMDV

GWAPGGDAFDI

Η Q Ρη 0 0 Ξ rtj Η § ω

> Q S 0 >Η >Η >Η 0 ω ω Ρί Q

LVAGLDAFDI

EYGDYGGGFDP

DGYHYGDYTYFQH

EYPSTYYFDY

>Η Q 01 ω ω ω ω Η ω

>Η Η

HNWNDGAFDI

DLSNGDDVFDI

GSPWGDDAFDI

SSGWLGDAFDI

DGSGSYLGFDY

DDWNVGMDV

HV-CDR2

0 > § Ρί H &4 0 0 0 Λ 0 H >H

SIRSSGGATRYADSVKG |

0 > § >Η 0 Η Ρη 0 0 0 0 0 Η 0

0 l: > § >Η S Η S 0 0 0 0 0 Η 0

0 > § >Η Ρη Η > 0 0 0 0 > Η 0

WIYFGGLTTYADSVKG |

0 l: > § >Η J Η Ρη 0 0 0 0ι >Η Η 0

0 tii > § >Η Ε Η 0 0 0 0 0ι >Η Η >Η

0 tii > ω § >Η Η Η 01 0 0 ω ω ω Η ω

WIVSSGGLTLYADSVKG |

RIRPSGGGTVYADSVKG |

SIGSSGGFTLYADSVKG |

0 > ω § >Η σ 0 0 ω 0ι ω Η 0

0 > ω § >Η Η 0 0 ω ω ω Η ω

0 > ω § Η J 0 0 ω 0ι 0 Η 0

SIWPSGGLTSYADSVKG |

0 l: > ω § >Η 01 Η Η 0 0 ω ω ω Η ω

0 l: > ω § >Η ω Η Η 0 0 ω 0ι ω Η ω

GIYPSGGWTDYADSVKG |

0 > ω § >Η Η Η σ 0 0 ω 0ι 0 Η ω

SIYPSGGATMYADSVKG |

0 l: > ω § >Η S Η 0ι 0 0 ω ω Η ω

0 l: > ω § >Η σ Η σ 0 0 ω 0ι ω Η >Η

0 l: > ω § >Η ω Η 01 0 0 ω ω > Η >Η

0 > ω § >Η Q Η >Η 0 0 ω ω >Η Η >

0 > ω § δ Η >Η 0 0 ω ω >Η Η 0

0 l: > ω § >Η Q Η S 0 0 ω 0ι >Η Η ω

0 > ω § >Η Q Η 0 0 0 ω 0ι >Η Η >Η

VIYPSGGPTSYADSVKG |

0 l: > ω § >Η Η Η S 0 0 ω ω ω Η Ρί

HV-CDR1

s s Q 5

o S 0 o

S S >Η

>Η S ω >Η Η

Ε >Η S

0 S Η >Η

Ε S >Η ο

Η S 5 >Η J

0ι >Η ο

0 S >Η >Η

>Η S ω >Η j

>Η ω

Ε S ω >Η Q

Q S 0ι >Η 5

J S l: >Η σ

Q S Ρί >Η

S Η >Η Q

S S σ >Η

Ε S S >Η ω

Η S 0ι >Η

S S Ε >Η ω

01 S Ρί >Η J

>Η >Η

Q >Η ω

σ >Η

Ε Ξ >Η

0 S σ >Η >Η

σ S 0ι >Η

Ε S Η >Η

0 S 0ι >Η ω

LV-CDR3

H Pi Oi H 5 >H 0 o o

H 01 Oi >H > o o

0 >Η Οι >Η >Η ο

Η Οι Οι Η 0 >Η 0 ο ο

Η Pi Οι J 0 Ρη Η ο ο

Η >Η 0ι 01 Ph 0 ο ο

>Η Η 01 0 ω ο

Η >Η 01 0 Η >Η Η ο ο

Η 01 0ι Η ω >Η ω ο ο

Η >Η 01 Η 0 >Η 0 ο ο

Η j 0ι >Η ω >Η Q σ j

Η 01 0ι Η ω >Η ω Ε σ

> > Η >Η ω Q § σ

Η 0ι 0ι Η ω >Η ω σ σ

Η 0ι 0ι Η ω Ε ω σ σ

Η > 0ι Η Η >Η 0 σ σ

Η >Η 0ι Η ω >Η ω σ σ

> δ 0 σ

Η Ρί 0ι Η ω >Η ω σ σ

Η 0ι 0ι Η ω >Η ω σ σ

Η Ρί J Η 5 >Η ω σ σ

Η >Η 01 Q ω >Η ω σ σ

Η Ρη 0ι Η ω 01 σ σ

Η 01 0ι Η Q >Η ω σ σ

Η σ 0ι Η ω >Η ω σ σ

Η J Η ω >Η ω σ σ

Η >Η 0ι ω >Η ω σ j

Η 0ι ω ω 01 ω σ σ

Η ω Ρί Η ω >Η ω σ σ

Η 0ι Η Η >Η Η σ σ

LV-CDR2

0 ο j 0 0 3

5 0 Q

0 ο j 0 0 3

Η ο j 1

0 ω j Η Ε

Η ω j 0 0 3

Η ο j ω 0 3

ω ο j ω ω 0

Η ω j Η 0 3

ω σ j Η ω 3

ω σ j ω ω 0

ω 0ι Ρί l: 5 Q σ

ω σ j ω ω 3

ω σ j ω 01 0

ω σ j ω 0 > 0 >Η

ω σ j ω ω 3

ω 0ι Ρί l: Ρί Q σ

ω σ j ω ω 3

ω σ j ω ω 0

ω ω j ω ω 3

ω σ j ω ω 3

ω ω j ω ω £

ω σ j ω ω 0

ω σ j ω ω 3

LV-CDR1

Η J Q Q Q H H o 0 g

5 Η >H 0 0 Η 0 ο 0 g

5 >Η Η Q Η 0 ο 0 g

5 >Η 0 0 Η 0 ο 0 g

Η >Η Η 0 Η 0 ο 0 g

j >Η 5 5 Η Η ο 0 Η Pi

5 J >Η Η 0 Η 0 Pi 0 g

5 J >Η Ε 5 Η 0 ο 0 Η Pi

>Η > ω Η Η ο ω g

5 J >Η Pi 0 Η 0 ο 0

0 J Q 5 Ρί Η 0 σ ω g

5 j >Η Η 0 Η ω σ ω g

ω > ω 0 j l: Q 0 ω

5 J >Η ω ω Η ω σ ω g

5 j >Η Η ω Η ω σ ω g

J ω Η ω σ ω g

5 J >Η ω ω Η ω σ ω g

% >Η ω Q 0 j l: Q 0 ω

5 J >Η ω ω Η ω σ ω g

J >Η ω ω Η 0 σ ω g

5 j >Η Η ω Η ω σ ω g

Η J >Η >Η ω Η Ρί σ ω g

5 J >Η ω Η ω σ Ρί Η Ρί

5 J >Η ω Η ω σ ω g

5 J >Η ω ω Η ω σ ω g

J >Η Ρη 5 Η ω σ ω g

5 j >Η Η 0 Η Ρί Ρί ω Η Ρί

5 J >Η Η Η ω σ ω g

5 J >Η ω ω Η ω σ ω g

Human pKal ELISA (T/B)

co

Η Γ-

LD η

Γ- Ι—Ι γΗ

η C0

Η

<0 η Η

<0 C0 η

σι

ο

co Η

ΠΊ co

σι σι

co

ΓΊ ΠΊ

Η ΠΊ ΠΊ

ΠΊ η

σι LD ΠΊ

LD Ο Η

ο Η

Η ΠΊ

Η

η η

η Η

σι Η

η η

Γ- η

<0 Η

Η

Initial Name

σι o 01 o r-1 s

σι ο 0 ο Η S

Ο Η Η γΗ S

γΗ ο ω Η γΗ S

Ο ω Η γΗ S

LD ο ω Η γΗ S

<0 ο ω Η γΗ S

σι ο 0 Η γΗ S

η Η 0 Η γΗ S

co ο η Η S

ο ffl ΓΊ Η S

σι ο 0 (Μ Η S

Ο Η 0 ΓΊ Η S

ο Η Q (Μ Η S

<0 ο ω η Η S

Ο Η rtj ΓΓΊ S

Η Ο ffl Γη S

η Η ffl Γη S

ΠΊ ο ω Γη S

ο Ρη Γη S

γΗ γΗ Ρη Γη S

Η ο 0 Γη S

ο 0 Γη S

σι ο 0 Γη S

ο Η Ε Γη S

γΗ ο co η S

ΠΊ ο υ co η S

η ο Q co η S

η Η Q co η S

Η ο ω co η S

1094814.1 - 161

162

2019204295 19 Jun 2019

HV-CDR3

H Q Q 0 ω ω Q H

PGGLDAFDI

ELEGLGGFDY

DHLSSWYGGFFDY

ESSGLYYFDY

WADYGGSLDY

ω j Q Q 0

0 J Oi to >H 0 to to Q Q

DRVDLGYLDY

VSTAVADNDY

H Q 01 to H s Oi σ

YRVGVAATDY

Q H g 5 Oi Q

H Q 01 Q 0 >H to 0 to Oi

ESSGVAAPDY

Q S 0 H to 0 >H to >H Pi to 0 Q

HV-CDR2

0 > § H > 0 0 0 0 0 H 0

0 > § σ H >H 0 0 0 Qi 0 H 0

0 > to § σ H >H 0 0 to to >H H 0

SIVPSGGHTLYADSVKG |

0 > to § s H 5 0 0 to to to H >

0 > to § Ol H Oi 0 0 to to 0 H to

0 > to § 01 H Q 0 0 to Oi >H H >

GISPSGGFTFYADSVKG |

0 > to § σ H to 0 0 to to to H to

0 > to § to H >H 0 0 to Oi H to

0 > to § s H σ 0 0 to Oi to H 0

0 > to § to H 01 0 0 to to to H

WIYPSGGWTEYADSVKG |

0 > to § s H Oi 0 0 to Oi >H H

0 > to § s H 01 0 0 to Oi >H H 0

0 > to § Pi H 0 0 0 to to Pi H 0

HV-CDR1

H S Q >H J

E S J >

S H >H Pi

S H >H Q

S S Q >H 0

>H S Oi >H

s s Oi Pi

5 S E

H S 01

S S J σ

Q J

S J

E S H >H 0

σ s s

0 S H >H J

H s H

LV-CDR3

H Λ J 0 >H 0 σ σ

H Oi Oi H 0 >H 0 σ σ

H J Oi H to >H to σ σ

H J Oi Ph to σ σ

H >H Pi H to >H to σ σ

H J Oi H Q >H to σ σ

> ft! 0 5 J to Q Q

H E Oi Q Ph σ σ

H Ol > >H to σ σ

H Oi to to >H to σ σ

H σ Oi H Q >H to σ σ

H Oi Oi H to >H to σ σ

H E Oi H to >H to σ σ

H 01 Oi J to >H to σ σ

H Pi Oi H to >H to σ σ

H Pi Pi σ j σ S

LV-CDR2

Η σ j 0 0 3

0 ω j Η 0 3

to σ j to to 3

to σ j 0 to

to σ j to to 3

to E j 5 to 0

to Oi j j Q Q >H

g H to

to ω j σ to 0

Pi E J to to 5

to σ j to to q

to σ j to to 3

to σ j to 0 0

to σ j to to 3

to g 5 to 0 J

LV-CDR1

J 5 0 H Q σ 0 g

5 J >H H 5 H 0 σ 0 g

5 J >H to to H to σ to g

j to to H 0 σ to g

5 J >H to to H to σ to g

5 J H H H to σ to g

to i § H 5 to to to 0 to

£ Q 5 0 H 5 σ to g

5 j H to H to σ to g

5 J >H H 5 H 5 σ to H Pi

E j 5 Q H H σ to g

5 J >H to to H to σ to g

5 J >H H to H to σ to g

5 J > to H Q σ to g

5 J >H to to H 5 σ to g

Q J >H >H 0 Pi to E J J to σ to to Pi

Human pKal ELISA (T/B)

co σι (N

LT) r-1

r-1 CO

co co

(N to

to co (N

(N

o

<0 r-1

<0 co

<0

co o r-1

co r-1 r-1

co r-1

r-1 <0 r-1

<0

Initial Name

r-1 r-1 ω co (N s

r-1 O Ph co (N S

to o 01 co (N S

> o Ph co (N S

> o 0 σι (N S

O r-1 Q σι (N S

(N o ω σι (N S

co o 0 σι (N S

o r-1 0 σι (N S

ro > S

(N O 01 <0 > s

ΓΊ o 0 <0 > s

<0 O 0 <0 > S

ΓΊ o E <0 > S

> O 0 > > S

o E > > S

1094814.1 - 162163

2019204295 19 Jun 2019

Amino acid sequences of light chain (LC) and heavy chain (HC) variable domain of pKal antibody binders are shown below,

M6-A06

QDIQMTQSPS

SRFSGSGPGG

SLSASVGDSV

TDFTLTISSL

LC

TISCPASQSI

QPEDFATYYC

SMYLNWYQHK QQSYSAPWTF

PGKAPKLLIY

GQGTKVEIK

GTSSLQSGVP

109

M6-A06

EVQLLESGGG ADSVKGRFTI STKGPSVFPL

LVQPGGSLRL SRDNSKNTLY APSSKS

HC

SCAASGFTFS

LQMNSLRAED

LYQMTWVRQA

TAVYYCARVS

PGKGLEWVSG

TAVADNDYWG

IWPSGGFTDY

QGTLVTVSSA

120

136

M6-A08

QDIQMTQSPS

SRFSGSGSGT

SLSASVGDRV

DFTLTISSLQ

LC

TITCPASQRI

PKDFGTYYCQ

SFYLNWFQQK QTFSTPNTFG

PGKAPNLLIY

QGTKLEIK

GASSLQSGVP

M6-A08

EVQLLESGGG

ADSVKGRFTI

SASTKGPSVF

LVQPGGSLRL SRDNSKNTLY PLAPSSKS

HC

SCAASGFTFS

LQMNSLRAED

PYPMQWVRQA

TAVYYCARDD

PGKGLEWVSS

YGGKGGAFDI

ISSSGGMTEY

WGQGTMVTVS

M6-D03

QDIQMTQSPS

SRFSGSGSGT

SLSASVGDRV

DFTLTISSLQ

LC

TITCPASQSI

PEDFATYYCQ

SSYLNWYQQK QSYSTLWTFG

PGKAPKLLIY

QGTKVEIK

AASSLQSGVP

M6-D03

EVQLLESGGG ADSVKGRFTI STKGPSVFPL

LVQPGGSLRL SRDNSKNTLY APSSKS

HC

SCAASGFTFS

LQMNSLRAED

KYFMGWVRQA TAVYYCARVS

PGKGLEWVSV

TAVADNDYWG

IGSSGGWTSY

QGTLVTVSSA

120

136

M6-D08

QDIQMTQSPS

SRFSGSGSGT

SLSASVGDRV

DFTLTISSLQ

LC

TITCPASQSI

PEDSATYYCQ

SSYLNWYqQK QSYTRWTFGQ

PGKAPKLLIY

GTKVEIK

GASSLQSGVP

107

M6-D08

EVQLLESGGG ADSVKGRFTI ASTKGPSVFP

LVQPGGSLRL SRDNSKNTLY LAPSSKS

HC

SCAASGFTFS

LQMNSLRAED

P.YHMVWVP.QA

TAMYYCAP.EM

PGKGLEWVSS

ATIAGQFDPW

ISPSGGWTNY

GQGTLVTVSS

120

137

M6-G05

QDIQMTQSPS

STISGSGSGT

SLSASVGDRV

DFTLTISSLQ

LC

TITCPASQSI

PEDFATYYCQ

STYLNWYQLK QSYTTPTTFG

PGKAPKLLIY

QGTKVEIK

NAFSMEP.GVP

M6-G05

EVQLLESGGG

ADSVKGRFTI

ASTKGPSVFP

LVQPGGSLRL SRDNSKNTLY LAPSSKS

HC

SCAASGFTFS

LQMNSLRAED

P.YP.MVWVP.QA

TAVYYCARDA

PGKGLEWVSS

VGIGDAFDIW

IYPSGGMTAY

GQGTMVTVSS

120

137

M8-C04

QSALTQPPSV

FSGSNSGNTA

SVSPGQTASI

TLTISGTQAM

LC

TCSGDKLGDK

DEADYYCQAW

YTSWHQQKPG DSSTVFGGGT

QSPVLVIYQD

RLTVL

SKP.PSGIPEP.

105

M8-C04

EVQLLESGGG

LVQPGGSLRL

HC

SCAASGFTFS

YYPMQWVRQA

PGKGLEWVSY

IYPSGGLTSY

164

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLF YGSGSVGFEY WGQGTLVTVS 120

SASTKGPSVF PLAPSSKS 138

2019204295 19 Jun 2019

M8-D05 LC

QDIQMTQSPS FVSASVGDRV TITCRASQDI SSWLVWYQQK PGKGPKLLIY DASNLQSGVP 60

SRFSGGGSGT HFTLTISSLQ PEDFATYYCQ QADGFPLTFG GGTKVEMK 108

M8-D05

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYNMNWVRQA PGKGLEWVSS ISPSGGFTDY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL DLGILDYWGQ GTLVTVSSAS 120

TKGPSVFPLA PSSKS

135

M8-E06 LC

QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60

SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLMYTF GQGTKLEIK 109

M8-E06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
STKGPSVFPL	APSSKS
M8-G09		LC
QDIQMTQSPS	SLSASVGDTV	TITCRASQGV
SKFSGSGSGT	VFTLTISSLQ	PDDFATYYCQ
M8-G09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
STKGPSVFPL	APSSKS
M8-H04		LC
QDIQMTQSPS	SLSASIGDRV	TITCRASQYI
SRFSGSGSGT	EFSLTISSLQ	PEDFATYYCQ
M8-H04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
SASTKGPSVF	PLAPSSKS
M9-A03		LC
QDIQMTQSPS	SLSASLGDRV	TITCRASQNI
SRFSGSGSGT	DFTLTISSLQ	PEDFGTYYCQ
M9-A03		HC

HYFMTWVRQA PGKGLEWVSS IVPSGGMTQY 60

TAVYYCARDS YSSSWFDIWG QGTMVTVSSA 120

136

SYYLAWFQQK PGKAPKSLIY GASSLQSGVP 60

QYNTYPPTFG QGTRLDIK 108

LYEMLWVRQA PGKGLEWVSV IYPSGGYTDY 60

MAVYYCARSF SGFGEIDYWG QGTLVTVSSA 120

136

STYLNWYEQK PGKAPKLLIY GTSSLQSGVP 60

QSFTTPFTFG QGTKLEIK 108

GYWMGWVRQA PGKGLEWVSS ISSSGGWTQY 60

TATYYCARDD EIAAGGAFDI WGQGAMVTVS 120

138

DIYLNWYQQT PGKAPKLLIY GAYNLQSGVP 60

QSYGTPVFGQ GTKLEIK 107

EVQLLESGGG LVQPGGSLRL SCAASGFTFS ADSVKGRFTI SRDNSKNTLY LQMNSLRAED STKGPSVFPL APSSKS

GYFMMWVRQA PGKGLEWVSS IYSSGGYTDY 60

TAVYYCAREV AGTYAFDIWG QGTMVTVSSA 120

136

M9-A08

QDIQMTQSPS

SRFSGSGSGT

SLSASVGDRV

DFTLTISSLQ

LC

TVTCRASQRI PDDFATYYCQ

STYLNWYQQK QSYNTPRTFG

PGKAPKLLIS

QGTKVEIR

GASSLQSGVP

M9-A08

EVQLLESGGG

ADSVKGRFTI

ASTKGPSVFP

LVQPGGSLRL SRDNSKNTLY LAPSSKS

HC

SCAASGFTFS

LQMNSLRAED

AYEMWWVRQA

TAVYYCTGGN

PGKGLEWVSY

SSSFDAFDIW

IGSSGGSTSY

GQGTMVTVSS

120

137

165

M9-C08 LC

QDIQMTQSPS SLSASVGDRV TITCRASQSI SIYVNWYQQK PGKAPNLLIF AASSLQRGVP 60

SRFSGSGSGA DFTLTISSLQ PEDFATYYCQ QSFSTPLTFG GGTKVEIK 108

2019204295 19 Jun 2019

M9-C08

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYGMVWVRQA PGKGLEWVSY IVPSGGLTYY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVD YTGDGLGYWG QGTLVTVSSA 120

STKGPSVFPL APSSKS

136

M9-C10		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQGI
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ
M9-C10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
ASTKGPSVFP	LAPSSKS
M9-D08		LC
QDIQMTQSPS	SLSASVGDRV	TLTCRASRTI
SRFSGSGSGT	DFSLTISNLQ	PEDIAVYYCQ
M9-D08		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
ASTKGPSVFP	LAPSSKS
M9-E04		LC
QDIQMIQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DFTLTINNLQ	PEDFATYYCQ
M9-E04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
STKGPSVFPL	APSSKS
M9-F08		LC
QDIQMTqSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ
M9-F08		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
ASTKGPSVFP	LAPSSKS
M9-F09		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DYTLTISSLQ	PEDFATYYCQ
M9-F09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
TKGPSVFPLA	PSSKS
M9-F10		LC

SSYLNWYQQK PGNAPNLLIY GASSLQSGVP 60

ESYSTLFTFG PGTTVEIK 108

LYPMQWVRQA TAVYYCTREV	PGKGLEWVSS GAAGFAFDIW	IGSSGGMTFY GQGTMVTVSS	60 120 137
SFYLNWYQQK	AGKAPELLIY	GGSSLHSGVP	60
QSFSSPWTFG	QGTKVEIK		108
WYKMMWVRQA	PGKGLEWVSS	IYPSGGWTNY	60
TAVYYCTRGS	PWGDDAFDIW	GQGTMVTVSS	120 137
SGYLNWYQQR	SGKAPKLLIF	AASNLQTGVP	60
QSHTPPKTFG	PGTKVDIK		108
EYDMMWVRQA	PGKGLEWVSS	IGSSGGMTYY	60
TAVYYCARDQ	VAAAAIDYWG	QGTLVTVSSA	120 136
SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
QSYSTPPYTF	GQGTKLEIK		109
PYAMTWVRQA	PGKGLEWVSV	IYPSGGFTDY	60
TAMYYCARAS	GSYLDAFDIW	GQGTMVTVSS	120 137
SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
QTYTTPWTFG	QGTKVEIK		108
SYPMGWVRQA	PGKGLEWVSR	ISSSGGMTIY	60
TAVYYCARDD	WNVGMDVWGQ	GTTVTVSSAS	120 135

QDIQMTQSPS SLSASVGDRV TITCRASQSI

SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ

NTYLNWYQQK PGKAPKVLIH AASTLESGVP 60

QSYSTPYTFG QGTKLEVR 108

166

2019204295 19 Jun 2019

M9-F10

EVQLLESGGG ADSVKGRFTI TKGPSVFPLA

M9-G08

QDIQMTQSPS

SRFSGSGSGT

M9-G08

EVQLLESGGG

ADSVKGRFTI STKGPSVFPL

M9-H02

QDIQMTQSPS

SRFSGSGSGT

M9-H02

EVQLLESGGG

ADSVKGRFTI ASTKGPSVFP

M9-H03

QYELTQAPSV FSGSNSGNTA

M9-H03

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M9-H04

QDIQMTQSPS

SRFSGSGSGT

M9-H04

EVQLLESGGG

ADSVKGRFTI ASTKGPSVFP

MIO-AO3

QDIQMTQSPS SRFSGSGSGT

MIO-AO3

EVQLLESGGG

ADSVKGRFTI

SASTKGPSVF

M10-A12

QDIQMTQSPL

SRFSGSGSGT

M10-A12

EVQLLESGGG

LVQPGGSLRL SRDNSKNTLY PSSKS	HC SCAASGFTFS LQMNSLRAED	DYDMEWVRQA TAVYYCARQG	PGKGLEWVSS LLTAFDIWGQ	ISPSGGSTIY GTMVTVSSAS	60 120 135
	LC
SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
DFTLTISSLQ	PEDFATYYCQ	QSYSTPITFG	GGTKVEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	YYTMLWVRQA	PGKGLEWVSS	IYPSGGFTMY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARVD	TAMAMIDYWG	QGTLVTVSSA	120
APSSKS					136
	LC
SLSASVGDRV	IITCRASRSI	ATYLNWYQQK	PGKAPNLLIF	GASTLQSGVP	60
DFTLTISDLQ	PEDFATYYCQ	QSFSDPYTFG	QGTNLEMK		108
	HC
LVQPGGSLRL	SCAASGFTFS	AYMMIWVRQA	PGKGLEWVSV	IYPSGGVTMY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGT	VGASDAFDIW	GQGTMVTVSS	120
LAPSSKS					137
	LC
SVAPGQTASI	TCSGDKLGNR	YTSWYQQKPG	QSPVLVIFQD	NKRPSGIPER	60
TLTISGTQAM	DEADYYCQAL	DSNTYVFGTG	TKVTVL		106
	HC
LVQPGGSLRL	SCAASGFTFS	WYSMGWVRQA	PGKGLEWVSY	IVPSGGYTMY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARDP	GVSYYYYGMD	VWGQGTTVTV	120
FPLAPSSKS					139
	LC
SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
DFTLTISSLQ	PEDFATYYCQ	QSYSTPPTFG	QGTRLEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	AYTMWWVRQA	PGKGLEWVSS	IWPSGGSTFY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARTY	DSSAGEVDYW	GQGTLVTVSS	120
LAPSSKS					137
	LC
SLSASVGDRV	TITCRASQRI	SFYLNWFQQK	PGKAPNLLIY	GASSLQSGVP	60
DFTLTISSLQ	PKDFGTYYCQ	QTFSTPNTFG	QGTKLEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	PYPMQWVRQA	PGKGLEWVSS	ISSSGGMTEY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARDD	YGGKGGAFDI	WGQGTMVTVS	120
PLAPSSKS					138
	LC
SLSAFVGDRV	TITCRASRDI	SVYLNWYQLK	SGKAPKLLIY	GASSLQSGVP	60
DFTLTITSLQ	PEDFATYYCQ	QSYSIPFTFG	GGTKVETK		108
	HC
LVQPGGSLRL	SCAASGFTFS	LYLMHWVRQA	PGKGLEWVSS	IYSSGGFTTY	60

167

2019204295 19 Jun 2019

ADSVKGRFTI

KGPSVFPLAP

M10-B09

QDIQMTQSPS SRFSGSGSGT

M10-B09

EVQLLESGGG ADSVKGRFTI STKGPSVFPL

M10-C11

QDIQMTQSPS SRFSGSGSGT

M10-C11

EVQLLESGGG ADSVKGRFTI STKGPSVFPL

M10-D11

QDIQMTQSPL ASGVPDRFSG

M10-D11

EVQLLESGGG ADSVKGRFTI SVFPLAPSSK

M10-E06

QDIQMTQSPS

SRFSGSGSGT

M10-E06

EVQLLESGGG ADSVKGRFTI STKGPSVFPL

M10-F09

QDIQMTQSPS SRFSGSGSGT

M10-F09

EVQLLESGGG ADSVKGRFTI TVSSASTKGP

M10-G09

QDIQMTQSPS

SRFSGSGSGT

M10-G09

EVQLLESGGG ADSVKGRFTI SASTKGPSVF

SRDNSKNTLY

SSKS

LQMNSLRAED

TAVYYCARDT

DYGMDVWGQG

TTVTVSSAST

120

134

SLSASVGDGV

DFTLTISSLQ

LVQPGGSLRL SRDNSKNTLY APSSKS

LC

TITCRASQSI

REDFATYYCQ

HC

SCAASGFTFS

LqMNSLRAED

STYLNWYQQR

QSFSTPWTFG

WYEMSWVRQA TAVYYCTRTS

SLSASVGDRV

DFTLTISNLQ

LVQPGGSLRL SRDNSKNTLY APSSKS

SLPVTPGEPA

SGSGTDFTLK

LVQPGGSLRL SRDNSKNTLY S

SLSASVGDRV

DFTLTISSLQ

LVQPGGSLRL SRDNSKNTLY APSSKS

SLSASVGDRV

DFTLTITSLQ

LVQPGGSLRL SRDNSKNTLY SVFPLAPSSK

SLSASVGDSV

HFTLTISSLQ

LVQPGGSLRL SRDNSKNTLY PLAPSSKS

PGKAPKLLIY

QGTRVEIK

PGKGLEWVSR

ITTVGMDVWG

GASSLQSGVP

IWPSGGVTMY

QGTTVTVSSA

108

120

136

LC

TITCRASQSI

PEDFATYYCQ

HC

SCAASGFTFS

LQMNSLRAED

LC

SISCRSSQSL

ISRVEAEDVG

HC

SCAASGFTFS

LQMNSLRAED

LC

TITCRASQSI

PEDFTIYYCQ

HC

SCAASGFTFS

LQMNSLRAED

LC

TITCRASQTI

PEDFATYYCQ

HC

SCAASGFTFS LQMNSLRAED S

LC

TITCRASQSI

PEDFATYYCQ

HC

SCAASGFTFS LQMNSLRAED

SIYLNWYQQK QSHSIPPTFG

PEKAPKLLIF

LGTKVEVK

AASTLQSGVP

108

MYPMMWVRQA

MAVYYCARVA

LHSNGYNYLD

VYYCMQALQT

AYPMNWVRQA

TAVYYCALGY

STYLNWYQQK QSYSDPYTFG

LYRMFWVRQA TAVYYCAREY

DDDLIWYQQK

QSYNIPRTFG

NYDMMWVRQA TATYYCAKDI

SGYINWYQQK QYVSYPFTFG

QYGMQWVRQA TAVYYCARDG

PGKGLEWVSY

GSSDAFDIWG

ISPSGGMTDY

QGTMVTVSSA

120

136

WYLQKPGQSP

PLTFGPGTKV

QLLIYLGSNR

HIK

113

PGKGLEWVSR

LGYWGQGTLV

PGKAPKLLIY

QGTKLDIK

PGKGLEWVSS

PSTYYFDYWG

PGRAPKLLIY

QGTKLESK

PGKGLEWVSY

YYYNWGPSHY

AGKAPKLLIY

PGTKVDIK

PGKGLEWVSS

YYDSSGYPDY

ISSSGGNTSY

TVSSASTKGP

GASSLQSGVP

IWSSGGPTMY

QGTLVTVSSA

AASSLQSGVP

ISPSGGFTRY FDSWGQGTLV

AASSLQSGVP

IRSSGGATRY

WGQGTLVTVS

120

131

108

120

136

108

120

141

108

120

138

168

2019204295 19 Jun 2019

M11-A10

QDIQMTQSPS

SRFSGSGSGT

M11-A10

EVQLLESGGG

ADSVKGRFTI ASTKGPSVFP

M11-E01

QDIQMTQSPS

SRFSGSGSGT

M11-E01

EVQLLESGGG

ADSVKGRFTI STKGPSVFPL

M11-E04

QDIQMTQSPS

SRFSGSGSGT

M11-E04

EVQLLESGGG

ADSVKGRFTI

SASTKGPSVF

M11-E05

QDIQMTQSPS

SRFSGSGSGT

M11-E05

EVQLLESGGG DSVKGRFTIS TKGPSVFPLA

M11-E06

QDIQMTQSPS

PRFSGSGSGT

M11-E06

EVQLLESGGG

ADSVKGRFTI ASTKGPSVFP

M11-G09

QDIQMTQSPS

SRFSGTGSGT

M11-G09

EVQLLESGGG

ADSVKGRFTI

SASTKGPSVF

M11-G12

QDIQMTQSPS SRFSGSGSGT

SLSASVGDRV DFTFIINSLQ	LC
AITCRASQSI PEDVATYYCQ	DTYLNWYQQK HYLYAPYSFG	PGKAPKLLIY QGTKLEIK	DASNLEIGVP	60 108
	HC
LVQPGGSLRL	SCAASGFTFS	NYWMMWVRQA	PGKGLEWVSG	IGSSGGFTSY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAKGS	YSDYGVFESW	GQGTLVTVSS	120
LAPSSKS					137
	LC
SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
DFTLTISSLQ	PEDFATYYCQ	QSYSTPPTFG	QGTKVEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	TYEMYWVRQA	PGKGLEWVSG	IGSSGGMTMY	60
SRDNSKNTLY	LQMNSLRAED	TAMYYCAREQ	PGIAALQFWG	QGTLVTVSSA	120
APSSKS					136
	LC
SLSASVGDRV	TITCRASQSI	SIYLTWYQHR	PGKAPNLLIY	GAATLQTGVP	60
DFTLTIRGLQ	PEDFATYYCQ	QTFSLPRTFG	QGTKLEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	MYHMNWVRQA	PGKGLEWVSG	IVSSGGVTFY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARIT	TVTTGGAFDI	WGQGTMVTVS	120
PLAPSSKS					138
	LC
SLSASVGDTV	TITCRTSQTI	NNYLNWYQQR	PGEAPKVLIY	ATHTLESGVP	60
DFTLTIGSLQ	PEDFATYYCQ	QSFAFPYTFG	QGTKVEIT		108
	HC
LVQPGGSLRL	SCAASGFTFS	WYTMGWVRQA	PGKGLEWVSW	IYFGGLTTYA	60
RDNSKNTLYL	QMNSLRAEDT	AVYYCARLGG	PLDAFDIWGQ	GTMVTVSSAS	120
PSSKS					135
	LC
SLSASIGDRV	TISCRASRGI	GTYLNWYQQH	AGKAPKLLIR	AASSLETGVP	60
DFTLTISSLQ	SDDFATYYCQ	ESFTNVYNFG	QGTKLEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	QYAMHWVRQA	PGKGLEWVSS	IYPSGGFTLY	60
SRDNSKNTLY	LQMNSLRAED	TAMYYCARGG	WLAGGELLNW	GQGTLVTVSS	120
LAPSSKS					137
	LC
SLSASVGDRV	TITCRTSQGI	NHYLNWYQQK	PGKAPKILVF	AASELQTGVP	60
SYTLTITSLQ	PEDVATYYCQ	QTYTSPYTFG	QGTKLEVK		108
	HC
LVQPGGSLRL	SCAASGFTFS	LYNMTWVRQA	PGKGLEWVSY	IYPSGGGTHY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARDT	GFWSADAFDI	WGQGTMVTVS	120
PLAPSSKS					138

LC

SLSAFVGDRV SITCRASQTI SVYVNWYQHK SGQAPKLLIY GASSLQSGVP 60

DFTLTISSLQ PEDFATYFCQ QSYSIPFTFG GGTDVQIR 108

169

2019204295 19 Jun 2019

M11-G12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	QYPMNWVRQA	PGKGLEWVSS	ISSSGGFTTY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAREE	QQGGFDYWGQ	GTLVTVSSAS	120
TKGPSVFPLA	PSSKS					135
M12-A08		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI	SRYLNWYQQK	PGKAPKLLIY	AASTLETGVP	60
SRFSGSGSGT	DFTLTITTLQ	PEDFVIYYCQ	QSYSTPYTFG	QGTKLEIK		108
M12-A08		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	WYYMGWVRQA	PGKGLEWVSW	IVSSGGLTLY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAMYYCARTT	VTTGDAFDIW	GQGTMVTVSS	120
ASTKGPSVFP	LAPSSKS					137
M12-B04		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQGI	RNDLGWYQHK	PGKAPKLLIY	AASILQSGVP	60
SRFSGTASGT	DFTLTISSLQ	PEDFATYFCL	QDYEYPLTFG	GGTKLDIK		108
M12-B04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	LYSMYWVRQA	PGKGLEWVSR	IRPSGGGTVY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARDP	LYSSGDVWGQ	GTTVTVSSAS	120
TKGPSVFPLA	PSSKS					135
M12-C09		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI	GIYLNWYHQK	PGKAPNLLIY	GASSLQSGVP	60
SRFSGSGSGT	DFTLTISSLQ	PGDFATYYCQ	HSYSTPFTFG	GGTKVEIK		108
M12-C09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	SYAMVWVRQA	PGKGLEWVSS	IGSSGGFTLY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCASMN	LGGGDAFDIW	GQGTMVTVSS	120
ASTKGPSVFP	LAPSSKS					137
M12-C10		LC
QSALTQPPSV	SVSPGQTASI	TCSGDKLGEK	YVSWYQQKPG	QSPVWIYQD	NKRPSGIPER	60
FSGSNSGNTA	TLTISGTQAV	DEADYYCQAW	DSYTWFGGG	SKLTVLGQPK		110
M12-C10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	DYEMHWVRQA	PGKGLEWVSG	ISPSGGKTQY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARDL	KWGGRGSPDW	YFDLWGRGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M12-D10		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ	QSYSTPPTFG	GGTKVEIK		108
M12-D10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	NYPMDWVRQA	PGKGLEWVSS	ISSSGGWTNY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCATDT	SGSYLGFDYW	GQGTLVTVSS	120
ASTKGPSVFP	LAPSSKS					137
M12-E06		LC
QDIQMTQSPS	SLSASVGDRV	SITCRASQSI	STYLNWYQHK	PGKAPTLLIY	GAFSLQSGVP	60
SRFSGSGSGT	DFALTISSLQ	PEDFATYYCQ	QSHSTPPTFG	QGTRVEIK		108
M12-E06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	QYKMLWVRQA	PGKGLEWVSG	IGPSGGLTAY	60

170

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARAP WFGELGMDVW GQGTTVTVSS 120

ASTKGPSVFP LAPSSKS 137

2019204295 19 Jun 2019

M27-Al0 LC

QDIQMTQSPS SLSASVGDRV TITCRASQSI SAYLNWYQQK PGKAPQLLMY GVGSLQSGVP 60

SRFSGSGSGT DFTLTISSLQ PEDFATYFCQ QGYTTPVTFG GGTKVEIK 108

M27-Al0

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYRMDWVRQA PGKGLEWVSS IWPSGGLTSY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGW APGGDAFDIW GQGTMVTVSS 120

ASTKGPSVFP LAPSSKS

137

M27-B01 LC

QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60

SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPYTFG QGTKLEIK 108

M27-B01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
SASTKGPSVF	PLAPSSKS
M27-B12		LC
QYELTQPPAV	SVSPGQTATI	TCSGDKLGDE
FSGSNFGNTA	TLTITGTQVM	DEADYYCQAW
M27-B12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
SSASTKGPSV	FPLAPSSKS
M27-E03		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ
M27-E03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
STKGPSVFPL	APSSKS
M27-F04		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ
M27-F04		HC

DYTMWWVRQA PGKGLEWVSS ISSSGGITFY 60

TAVYYCARSA DTAMGGAFDI WGQGTMVTVS 120

138

YAAWYQQKPG QSPVLVIYQD RKRPSGIPER 60

GKRNWFGGG TKLTVL 10 6

WYQMMWVRQA PGKGLEWVSS ISPSGGITEY 60

TAVYYCARDR SSGWYYYGMD VWGQGTTVTV 120

139

SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60

QSYSTPRTFG QGTKVEIK 108

SYMMHWVRQA PGKGLEWVSG IYPSGGWTDY 60

TATYYCARLV AGLDAFDIWG QGTMVTVSSA 120

136

SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60

QSYSTPPTFG QGTKVEIK 108

EVQLLESGGG LVQPGGSLRL SCAASGFTFS ADSVKGRFTI SRDNSKNTLY LQMNSLRAED ASTKGPSVFP LAPSSKS

WYPMTWVRQA PGKGLEWVSS IGPSGGQTIY 60

TAVYYCTTEY GDYGGGFDPW GQGTLVTVSS 120

137

M27-F11 LC

QDIQMTQSPS FLSASVGDRV TITCRASQGI SSYLAWYQQK PGKAPKLLIY AASSLQSGVP 60

SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYNTLRTFG PGTKVDLK 108

M27-F11 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYHMMWVRQA PGKGLEWVSS IYPSGGATMY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARDG YHYGDYTYFQ HWGQGTLVTV 120

SSASTKGPSV FPLAPSSKS 139

171

M27-G01 LC

QDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQQK PGKAPKLLIY GASSLQSGVP 60

SRFSGSGSGT DFTLTISSLQ PEDFTIYYCQ QSYSDPYTFG QGTKLDIK 108

2019204295 19 Jun 2019

M27-G01

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYRMFWVRQA PGKGLEWVSS IWSSGGPTMY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREY PSTYYFDYWG QGTLVTVSSA 120

STKGPSVFPL APSSKS

136

M27-G04 LC

QDIQMTQSPS SLSASVGDRV TITCRASQRI SYYLTWYQQK PGKVPKLLIY AASSLESGVP 60

SRFSGSGSGT DFTLTISNLQ PEDFATYYCQ QAFSTPFTFG GGTKVEIK 108

M27-G04 EVQLLESGGG ADSVKGRFTI STKGPSVFPL	HC
LVQPGGSLRL SRDNSKNTLY APSSKS	SCAASGFTFS LQMNSLRAED
M27-G09		LC
QDIQMTQSPS	SVSASVGDRI	TITCRTRQSI
SRFSGSGTGT	EFTLTISSLQ	PEDLAIYYCQ
M27-G09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
STKGPSVFPL	APSSKS
M27-H10		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DFTLSISSLQ	PEDFATYYCQ
M27-H10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
STKGPSVFPL	APSSKS
M28-AO 1		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ
M28-AO 1		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
ASTKGPSVFP	LAPSSKS
M28-C03		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI
SRFSGSASGT	EFTLTISSLQ	PEDFATYYCL
M28-C03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED
ASTKGPSVFP	LAPSSKS
M28-D02		LC

AYYMVWVRQA TAVYYCAREA	PGKGLEWVSY ISSSSFDYWG	ISPSGGQTQY QGTLVTVSSA	60 120 136
SNYLNWYQQK	PGEPPKLLIF	AASSLQSGVP	60
QSYDIPFTFG	QGTKLEIK		108
EYDMAWVRQA	PGKGLEWVSY	IVSSGGFTSY	60
TAVYYCTTWA	GWIAAADYWG	QGTLVTVSSA	120 136
SNYLNWYQQK	PGKAPKFLIY	AASSLQSGVP	60
QSYSTPQTFG	QGTKVEMK		108
AYQMAWVRQA	PGKGLEWVSV	IYSSGGYTDY	60
TAVYYCARHN	WNDGAFDIWG	QGTMVTVSSA	120 136
SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
QSYSTLTFGG	GTKVEIK		107
WYAMHWVRQA	PGKGLEWVSG	IYSSGGYTKY	60
TAVYYCARDL	SNGDDVFDIW	GQGTMVTVSS	120 137
NFYLNWYQQK	PGKAPKLLIY	VASSLESGVP	60
QSYSAPYTFG	QGTKVEIT		108
YYQMGWVRQA	PGKGLEWVSS	IYPSGGMTDY	60
TAVYYCTRGS	PWGDDAFDIW	GQGTMVTVSS	120 137

QDIqMTQSPS SLSASEGDMV TITCRTSRRI

SRFSGSGSGT DFTLTVSSLQ PEDVGTYYCQ

GTYLNWYQQK PGKAPKLLIY GASSLQSGVP 60

QSFSSPWTFG PGTKVEIK 108

172

2019204295 19 Jun 2019

M28-D02

EVQLLESGGG

ADSVKGRFTI ASTKGPSVFP

M28-D12

QDIQMTQSPS

SRFVGGGSGS

M28-D12

EVQLLESGGG

ADSVKGRFTI ASTKGPSVFP

M28-E01

QDIQMTQSPS

SKFSGSGSGT

M28-E01

EVQLLESGGG

ADSVKGRFTI TKGPSVFPLA

M28-E11

QDIQMTQSPS

SRFSGSGSGT

M28-E11

EVQLLESGGG

ADSVKGRFTI

TVSSASTKGP

M28-F01

QDIQMTQSPS

SRFSGSGSGT

M28-F01

EVQLLESGGG

ADSVKGRFTI TKGPSVFPLA

M28-F05

QDIQMTQSPS

SRFSGSGSGT

M28-F05

EVQLLESGGG

ADSVKGRFTI STKGPSVFPL

M28-F07

QDIQMTQSPS

SRFSGSGSGT

M28-F07

EVQLLESGGG

	HC
LVQPGGSLRL	SCAASGFTFS	WYPMQWVRQA	PGKGLEWVSY	IYPSGGGTDY	60
SRDNSKNTLY	LQMNSLRAED	TATYYCATSS	GWLGDAFDIW	GQGTMVTVSS	120
LAPSSKS					137
	LC
SLSASVGDRV	TITCRASQSI	ATYLNWYQQK	PGRAPKLLIY	AASSLQSGVP	60
GTHFTLTISS	LQPEDFATYY	CQQSYSTRET	FGQGTKVEIK		110
	HC
LVQPGGSLRL	SCAASGFTFS	WYTMHWVRQA	PGKGLEWVSV	IYPSGGPTSY	60
SRDNSKNTLY	LQMNSLRAED	TATYYCARDG	SGSYLGFDYW	GQGTLVTVSS	120
LAPSSKS					137
	LC
SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
DYTLTISSLQ	PEDFATYYCQ	QTYTTPWTFG	QGTKVEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	SYPMGWVRQA	PGKGLEWVSR	ISSSGGMTIY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARDD	WNVGMDVWGQ	GTTVTVSSAS	120
PSSKS					135
	LC
SVSASVGDRV	TINCRASQDI	SNWLAWYQQK	PGKAPNLLIY	AASSLQTGAP	60
DFTLTISSLQ	PEDFGTYVCQ	QSYSLPWTFG	LGTKVEVR		108
	HC
LVQPGGSLRL	SCAASGFTFS	LYDMTWVRQA	PGKGLEWVSG	ISSSGGVTIY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARTY	YYDSSGYADA	FDIWGQGTMV	120
SVFPLAPSSK	S				141
	LC
SLSASVGDRV	TITCRASQSI	NTYLNWYQQK	PGKAPKVLIH	AASTLESGVP	60
DFTLTISSLQ	PEDFATYYCQ	QSYSTPPTFG	QGTKVEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	VYLMHWVRQA	PGKGLEWVSG	ISPSGGYTQY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARPG	GLDAFDIWGQ	GTMVTVSSAS	120
PSSKS					135
	LC
SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
DFTLTISSLQ	PEDFATYYCQ	QSYSTPLTFG	GGTKVEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	RYIMWWVRQA	PGKGLEWVSG	IYSSGGYTQY	60
SRDNSKNTLY	LQMNSLRAED	TAMYYCAREL	EGLGGFDYWG	QGTLVTVSSA	120
APSSKS					136
	LC
SVSASVGDRV	TITCRASQGI	SSWLAWYQQK	PGKAPKLLIY	ATSGLQSGVP	60
DFTLTISSLQ	PEDFATYYCQ	QAKSFPLTFG	GGTRVEIK		108
	HC
LVQPGGSLRL	SCAASGFTFS	DYTMYWVRQA	PGKGLEWVSS	IVPSGGHTLY	60

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ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDH LSSWYGGFFD YWGQGTLVTV 120

SSASTKGPSV FPLAPSSKS 139

M29-C07LC

QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP60

SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTRYTFG QGTKLEIK108

M29-C07HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYDMMWVRQA PGKGLEWVSV ISSSGGNTAY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARES SGLYYFDYWG QGTLVTVSSA 120

STKGPSVFPL APSSKS136

M29-D10LC

QDIQMTQSPS SLSASVGDTV SITCRASQSI TIYLNWYQHK PGKAPNLLIY GASNLHSGVP60

SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYDTPLTFG GGTKVEIK108

M29-D10HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMYWVRQA PGKGLEWVSS IGSSGGPTPY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARWA DYGGSLDYWG QGTLVTVSSA 120

STKGPSVFPL APSSKS136

M29-E02LC

QSVLTQPPSV SEAPRQRVTI SCSGSSSNIG NNAVSWYQQL PGKAPKLLIY YDDLLPSGVS60

DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSLNGFV FGTGTKVTVL110

M29-E02HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYPMMWVRQA PGKGLEWVSV IYPSGGDTFY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASGD DYLWEAAVYW GQGTLVTVSS 120

ASTKGPSVFP LAPSSKS137

M29-G08LC

QDIQMTQSPA TLSASPGETV TLSCRASQNI GNDVAWYRQR PGQAPRLLIH HASTRAYGIP60

ARLRGSGSAT EFTLTITSLE PEDFAIYYCQ QFYDWPAHTF ALGTRLEIKR110

M29-G08HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYHMWWVRQA PGKGLEWVSG ISPSGGFTFY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY YYDSSGYSPL GYWGQGTLVT 120

VSSASTKGPS VFPLAPSSKS140

M29-G10LC

QDIQMTQSPS SLSSSVGDSA TITCRASQSI SIYLNWYQQK PGKAPKILIY GASQLESGVP60

SRFSGSGSGT DFTLTVSGLQ PEDFATYWCQ QSYNVPYTFG QGTKLEIK108

M29-G10HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS FYKMIWVRQA PGKGLEWVSS ISSSGGSTQY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR VDLGYLDYWG QGTLVTVSSA 120

STKGPSVFPL APSSKS136

M74-A07LC

QDIQMTQSPS SLSASVRDRV TITCRTSQNI NTYLNWYYQA PGRAPKLLIF GVSSLHRGVS60

SRFSGSGDGT EFTLTISSLQ PEDIGTYFCQ QSYSSPWTFG QGTKVEIK108

M74-A07 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYLMMWVRQA PGKGLEWVSS IYPSGGYTSY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVS TAVADNDYWG QGTLVTVSSA 120

STKGPSVFPL APSSKS 136

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M76-F02		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQTI	DNYLHWYQQK	PGKAPKVLIH	DASSLQSGVP	60
PRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ	QSYDTPQYTF	GQGTKLEIK		109
M76-F02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	LYDMNWVRQA	PGKGLEWVSG	ISPSGGQTMY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARQP	MISAFDIWGQ	GTMVTVSSAS	120
TKGPSVFPLA	PSSKS					135
M76-G02		LC
QDIQMTQSPS	SLSASVGDRV	TITCRASQSI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
SRFSGSGSGT	DFTLTISSLQ	PEDFATYYCQ	QSYSTPPWTF	GQGTKVEIK		109
M76-G02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	LYAMWWVRQA	PGKGLEWVSY	ISSSGGFTSY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARYR	VGVAATDYWG	QGTLVTVSSA	120
STKGPSVFPL	APSSKS					136
M7 6-GO 6		LC
QDIQMTQSPS	SLSASVRDRV	TITCRASQSI	STYLNWYQQK	PGEAPKLLVF	AASSLQSGVP	60
SRFSGSGSGT	DFTLSISSLQ	PEDFATYYCQ	QSYSTPHTFG	QGAKVEIK		108
M7 6-GO 6		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	GYIMHWVRQA	PGKGLEWVSW	IYPSGGWTEY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARDA	PGVGAIDYWG	QGTLVTVSSA	120
STKGPSVFPL	APSSKS					136
M76-H02		LC
QDIQMTQSPS	SLSASEGDRV	TITCRASQDI	SVYLNWYQMK	SGKAPKLLIY	GGASLQSGVP	60
ARFSGSGYGT	DFTLTITDLR	PEDFATYYCQ	QSYSLPFTFG	GGTKVEIK		108
M76-H02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	MYWMQWVRQA	PGKGLEWVSY	IYPSGGPTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARPS	GSYGDAFDIW	GQGTMVTVSS	120
ASTKGPSVFP	LAPSSKS					137
M77-C07		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQNI	SSYLNWYQQK	PGKAPKLLIY	AASSLQSGVP	60
SRFSGSGSGT	DFTLTISSLQ	PEDFATYSCQ	QSYSTPRTFG	QGTKVEIK		108
M77-C07		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	LYIMGWVRQA	PGKGLEWVSG	IYPSGGFTMY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARES	SGVAAPDYWG	QGTLVTVSSA	120
STKGPSVFPL	APSSKS					136
M77-H04		LC
QDIQMTQSPL	SLPVTPGEPA	SISCRSSQSL	LHSRGYNYLD	WYLQKPGQSP	QLLIYLGSNR	60
ASGVPDRFSG	SGSGTDFTLK	ISRVEAEDVG	VYYCMQALQR	RTFGQGTKLE	IK	112
M77-H04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	YYTMIWVRQA	PGKGLEWVSG	IRSSGGGTRY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAKDG	SRYSYGSIYY	YYGMDAWGQG	120
TTVTVSSAST	KGPSVFPLAP	SSKS				144

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EXAMPLE 2: Lead Antibody Inhibitors

Antibodies were selected as lead plasma kallikrein inhibitors on the basis of apparent inhibition constant (Κ_ρ£φρ), specificity with respect to lack of inhibition of other serine proteases, inhibition of bradykinin generation, and lack of binding to plasma prekallikrein (Table 3). Plasma kallikrein circulates in the plasma as an inactive zymogen (prekallikrein) at a concentration of approximately 500 nM. Antibodies that bound prekallikrein may be rendered inaccessible towards active plasma kallikrein inhibition and could substantially increase the in vivo dose required for efficacy. Therefore, a surface plasmon resonance (SPR) assay was used to identify antibodies that do not bind prekallikrein (data not shown). Specifically, human IgGs (X81-B01, M162A04 (R84-H05); M160-G12 (R84-D02); and M142-H08) were captured on a CM5 chip using an anti-human Fc surface and 100 nM of plasma kallikrein or 100 nM or 500 nM prekallikrein. The prekallikrein was treated with aprotinin-sepharose to remove active plasma kallikrein. The prekallikrein used for X81-B01 was buffer exchanged into the exact preparation of SPR running buffer (HEPES buffered saline) to avoid the refractive index shift that was observed with three other antibodies that were tested: M162-A04 (R84-H05); M160-G12 (R84-D02); andM142-H08.

Of the antibodies listed in Table 3, only M142-H08 inhibits human plasma kallikrein with a subnanomolar Κ;_ι£ΐρρ. However, when M142-H08 was produced as an IgG it was found to be cleaved in the CDR3 of the heavy chain. Consequently, we decided to undertake two approaches to improve the affinity: 1) affinity maturation of M162-A04 and M160-G12 using a novel form of light chain shuffling called ROLIC (Rapid Optimization of Light Chains) (see, e.g., WO 2009/102927 and U.S. 20090215119); and 2) sequence optimization of M142-H08 in order to prevent the cleavage of the IgG that occurs while retaining the binding and inhibitor properties of M142-H08.

Table 3. Top Ranking Antibody Inhibitors of PKal Before Affinity Maturation or Sequence Optimization

Criteria	M162-A04	M160-G12	M142-H08^a
Ki,app human pKal	2 nM (as an IgG)	5.6 nM (as an IgG)	0.6 nM (as a Fab)

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Ki,app rodent pKal	2 nM (mouse and rat)	<1 nM (mouse)	~ 1 nM (mouse and rat)
Binds prekallikrein?	No	No	No
Specific inhibitor with respect to fXIa, plasmin, and trypsin	Yes	Yes	Yes
Inhibits bradykinin generation	Yes	Yes	Yes

^aWhen M142-H08 was produced as an IgG it was determined to be cleaved in the CDR3 of its heavy chain (GGLLLWFR-ELKSNYFDY).

EXAMPLE 3: Sequence Optimization of M142-H08

Of the antibodies listed in Table 3, only M142-H08 inhibits human pKal with a subnanomolar Ki_iapp. However, when M142-H08 was produced as an IgG it was found to be cleaved in the CDR3 of the heavy chain. M142-H08 was found by mass spectrometry to be cleaved after the arginine in the “WFR” sequence of the HC-CDR3 sequence (GGLLLWFRELKSNYFDY). This cleavage suggests that a protease from the cells used to express the antibody (both CHO and 293T human kidney cells) is enzymatically cleaving the antibody at a single specific site. We mutated the HC-CDR3 sequence of M142-H08 in order to identify amino acid substitutions that prevent the cleavage of the IgG that occurs while retaining the binding and inhibitor properties of M142-H08. Previous experience with similarly “clipped” antibodies suggested that focusing simply on the putative Pl position (protease subsite 1, see Table 4) may not be sufficient to identify antibodies that retain potent inhibition of the target enzyme while not being clipped by a host cell protease. Therefore, we created a small library of single point mutations in the region around the cleavage site in order to identify variants of Ml42H08 that are not clipped but are still potent pKal inhibitors. We refer to this library as the “CDR3 by Design” library. The small library was constructed using a PCR primer that contains the randomized codon NNK at either the P3, the P2, the Pt, or the Pt ’ site. This results in a small library where each of the 4 positions may contain any of the 20 amino acids (20 +20 + 20 + 20 = 80 members). Using PCR, this library was cloned into the M142-H08 Fab sequence in the pMid21 vector, which is a standard phagemid vector.

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Table 4. Primer sequences

Primer Name	Sequence	N
	P3 P2 Pl Pl' P2' GGLLLWFRELK S N Y
559A. Pl. top	GGC GGT CTA TTA CTA TGG TTC NNK GAG CTG AAG TCT AAC TAC	20
559A.P2. top	GGC GGT CTA TTA CTA TGG NNK AGG GAG CTG AAG TCT AAC TAC	20
559A.P3. top	GGC GGT CTA TTA CTA NNK TTC AGG GAG CTG AAG TCT AAC TAC	20
55 9A. Pip . top	GGC GGT CTA TTA CTA TGG TTC AGG NNK CTG AAG TCT AAC TAC	20

By DNA sequencing, we recovered 61 of the possible 80 antibodies (Table 5). These antibodies were produced as Fab fragments in small scale (~20 pg) and tested for inhibition against human pKal in an in vitro protease cleavage assay using Pro-Phe-Argaminomethylcoumarin as the synthetic peptide substrate. The Fabs that were found to be inhibitors of human pKal were subcloned into our pBRH If vector (a vector for transient expression of IgGs in 293T cells) for conversion to full length human IgGl antibodies. Five antibodies were then expressed in 293T cells and purified by protein A sepharose chromatography. The antibodies were analyzed by SDS-PAGE to determine which of the inhibitory mutants are not cleaved by the host cell protease(s) (data not shown). The cleaved antibodies (559A-X67-G05, 559A-X67-H01, 559A-X67-G09) had an extra band that migrated between the 38 and the 49 kDa molecular weight marker. This band is absent in the 559A-X67-H04 and 559A-X67-D03 antibodies, which indicates that these antibodies are intact.

Ki,ap_P values were determined by steady state enzyme kinetics for those that were shown by SDS-PAGE to be not cleaved (Table 5). Interestingly, the P2 position was the only position where amino acid substitutions yielded intact antibody inhibitors of pKal.

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Of the 14 different mutations that were recovered at the P3 position (Table 5), only one mutant (W to L) was found to be a pKal inhibitor as a Fab but it was subsequently shown to be clipped as an IgG. None of the 16 different mutations at the Pl position (Table 5) were found to be pKal inhibitors. Eight of the 15 different mutations at the Pl ’ position were found to be inhibitors of pKal as a Fab but all were clipped as an IgG. Consequently, only mutations at the P2 position led to antibody inhibitors that were not clipped during expression. Of the 16 different mutations that were recovered at the P2 position (Table 5), eight mutants were found to be a pKal inhibitor as a Fab but it was subsequently shown to be clipped as an IgG. Four mutants at the P2 position were found to have subnanomolar Ki_iapp values: X67-G04 (F to A), X67-C03 (F to M), X67-F01 (F to Q) and X67-D03 (F to N). The antibody with the highest potency is X67-D03 (Ki,_app = 0.1 nM). The two antibodies shown in Table 6 were not cleaved when expressed as IgGs and were found to inhibit pKal with a subnanomolar Κ_ρ£φρ.

DNA and amino acid sequence alignments of the light chains of nongermlined (X63-G06) and germlined, codon optimized (X81-B01) versions of the same antibody discovered using ROEIC affinity maturation are shown in FIGURES 4 and 5, respectively. DNA and amino acid sequence alignments of the heavy chains of nongermlined (X63-G06) and germlined, codon optimized (X81-B01) versions of the same antibody discovered using ROEIC affinity maturation are shown in FIGURES 6 and 7, respectively.

Table 5. HV-CDR3 Sequences Obtained from “CDR3 by Design” Library*

Mutation Site	Antibody I.D.	HV-CDR3	Inhibit as a Fab?	Intact as an IgG?	Ki,app as an IgG (nM)
Parental	X69-C09	GGLLLWFRELKSNYFDY	Yes	No	0.2
P3	X68-E07	GGLLLAFRELKSNYFDY	No	n/a	n/a
P3	X68-E12	GGLLLCFRELKSNYFDY	No	n/ a	n/a

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P3	X68-A03	GGLLLDFRELKSNYFDY	No	n/ a	n/a
P3	X68-E03	GGLLLEFRELKSNYFDY	No	n/ a	n/a
P3	X68-A12	GGLLLGFRELKSNYFDY	No	n/ a	n/a
P3	X68-D11	GGLLLKFRELKSNYFDY	No	n/ a	n/a
P3	X68-E01	GGLLLLFRELKSNYFDY	Yes	No	n/a
P3	X68-F05	GGLLLMFRELKSNYFDY	No	n/a	n/a
P3	X68-D10	GGLLLPFRELKSNYFDY	No	n/a	n/a
P3	X68-F10	GGLLLQFRELKSNYFDY	No	n/a	n/a
P3	X68-G01	GGLLLRFRELKSNYFDY	No	n/a	n/a
P3	X68-G05	GGLLLSFRELKSNYFDY	No	n/a	n/a
P3	X68-F12	GGLLLTFRELKSNYFDY	No	n/a	n/a
P3	X68-H04	GGLLLVFRELKSNYFDY	No	n/a	n/a
P2	X67-G04	GGLLLWARELKSNYFDY	Yes	Yes	0.35
P2	X67-G01	GGLLLWCRELKSNYFDY	No	n/a	n/a
P2	X67-E04	GGLLLWDRELKSNYFDY	Yes	Yes	1.3
P2	X67-H04	GGLLLWERELKSNYFDY	Yes	Yes	3.6
P2	X67-C09	GGLLLWGRELKSNYFDY	Yes	Yes	8.6
P2	X67-B04	GGLLLWKRELKSNYFDY	Yes	No	n/a
P2	X67-G09	GGLLLWLRELKSNYFDY	Yes	No	n/a
P2	X67-C03	GGLLLWMRELKSNYFDY	Yes	Yes	0.7
P2	X67-D03	GGLLLWNRELKSNYFDY	Yes	Yes	0.1
P2	X67-B05	GGLLLWPRELKSNYFDY	No	n/a	n/a
P2	X67-F01	GGLLLWQRELKSNYFDY	Yes	Yes	0.9
P2	X67-G05	GGLLLWRRELKSNYFDY	Yes	No	n/a
P2	X67-B03	GGLLLWSRELKSNYFDY	Yes	Yes	2.1
P2	X67-F10	GGLLLWTRELKSNYFDY	Yes	Yes	1.3

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P2	X67-H01	GGLLLWWRELKSNYFDY	Yes	No	n/a
P2	X67-F08	GGLLLWYRELKSNYFDY	Yes	No	n/a
Pl	X66-E09	GGLLLWFAELKSNYFDY	No	n/a	n/a
Pl	X66-B05	GGLLLWFCELKSNYFDY	No	n/a	n/a
Pl	X66-D03	GGLLLWFEELKSNYFDY	No	n/a	n/a
Pl	X66-H04	GGLLLWFFELKSNYFDY	No	n/a	n/a
Pl	X66-H02	GGLLLWFGELKSNYFDY	No	n/a	n/a
Pl	X66-C11	GGLLLWFHELKSNYFDY	No	n/a	n/a
Pl	X66-A07	GGLLLWFKELKSNYFDY	No	n/a	n/a
Pl	X66-C03	GGLLLWFLELKSNYFDY	No	n/a	n/a
Pl	X66-G05	GGLLLWFMELKSNYFDY	No	n/a	n/a
Pl	X66-F10	GGLLLWFPELKSNYFDY	No	n/a	n/a
Pl	X66-E04	GGLLLWFQELKSNYFDY	No	n/a	n/a
Pl	X66-F01	GGLLLWFSELKSNYFDY	No	n/a	n/a
Pl	X66-H11	GGLLLWFTELKSNYFDY	No	n/a	n/a
Pl	X66-C02	GGLLLWFVELKSNYFDY	No	n/a	n/a
Pl	X66-F09	GGLLLWFWELKSNYFDY	No	n/a	n/a
Pl	X66-G08	GGLLLWFYELKSNYFDY	No	n/a	n/a
Pl'	X69-D08	GGLLLWFRALKSNYFDY	No	n/a	n/a
Pl'	X69-B02	GGLLLWFRCLKSNYFDY	No	n/a	n/a
Pl'	X69-D09	GGLLLWFRGLKSNYFDY	Yes	No	n/a
Pl'	X69-D02	GGLLLWFRHLKSNYFDY	No	n/a	n/a
Pl'	X69-A12	GGLLLWFRKLKSNYFDY	No	n/a	n/a
Pl'	X69-F05	GGLLLWFRLLKSNYFDY	Yes	No	n/a
Pl'	X69-B08	GGLLLWFRNLKSNYFDY	Yes	No	n/a
Pl'	X69-A10	GGLLLWFRPLKSNYFDY	No	n/a	n/a

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Pl'	X69-A09	GGLLLWFRQLKSNYFDY	Yes	No	n/a
Pl'	X69-E05	GGLLLWFRRLKSNYFDY	No	n/a	n/a
Pl'	X69-F09	GGLLLWFRSLKSNYFDY	Yes	No	n/a
Pl'	X69-F01	GGLLLWFRTLKSNYFDY	Yes	No	n/a
Pl'	X69-C12	GGLLLWFRVLKSNYFDY	Yes	No	n/a
Pl'	X69-E01	GGLLLWFRWLKSNYFDY	Yes	No	n/a
Pl'	X69-H10	GGLLLWFRYLKSNYFDY	No	n/a	n/a

*A11 of these antibodies are single point mutations of the M142-H08 sequence.

Amino acid sequences of light chain (LC) and heavy chain (HC) variable domain of pKal antibodies with designed HC CDR3s are shown below.

X68-E07 LC

QDIQMTQSPS SLSAFVGDRV TITCPASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR108

X68-E07 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLAFRELKS NYFDYWGQGT 120

LVTVSSASTK GPSVFPLAPS SKS143

X68-E12 LC

QDIQMTQSPS SLSAFVGDRV TITCPASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR108

X68-E12 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLCFRELKS NYFDYWGQGT 120

LVTVSSASTK GPSVFPLAPS SKS143

X68-A03 LC

QDIQMTQSPS SLSAFVGDRV TITCPASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR108

X68-A03 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLDFRELKS NYFDYWGQGT 120

LVTVSSASTK GPSVFPLAPS SKS143

X68-E03 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY 60

X68-E03 LC

QDIQMTQSPS SLSAFVGDRV TITCPASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60

SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108

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ADSVKGRFTI

LVTVSSASTK

X68-A12 LC QDIQMTQSPS SRLSGSGFGT

X68-A12 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-D11 LC QDIQMTQSPS SRLSGSGFGT

X68-D11 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-E01 LC QDIQMTQSPS SRLSGSGFGT

X68-E01 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-F05 LC QDIQMTQSPS SRLSGSGFGT

X68-F05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-D10 LC QDIQMTQSPS SRLSGSGFGT

X68-D10 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-F10 LC QDIQMTQSPS SRLSGSGFGT

X68-F10 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

SRDNSKNTLY GPSVFPLAPS	LQMNSLRAED SKS	TAVYYCARGG	LLLEFRELKS	NYFDYWGQGT	120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLGFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLKFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLLFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLMFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLPFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLQFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143

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X68-G01 LC QDIQMTQSPS SRLSGSGFGT

X68-G01 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-G05 LC QDIQMTQSPS SRLSGSGFGT

X68-G05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-F12 LC QDIQMTQSPS SRLSGSGFGT

X68-F12 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X68-H04 LC QDIQMTQSPS SRLSGSGFGT

X68-H04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-G04 LC QDIQMTQSPS SRLSGSGFGT

X67-G04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-G01 LC QDIQMTQSPS SRLSGSGFGT

X67-G01 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-E04 LC

QDIQMTQSPS SRLSGSGFGT

SLSAFVGDRV DFTLTISSLQ	TITCRASQPI PEDFGNYYCQ	DNYLNWYHQK QSYTVPYTFG	PGKAPKLLIY GGTKVEIR	AASRLQSGVP	60 108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLRFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNILY	LQMNSLRAED	TAVYYCARGG	LLLSFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLTFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLVFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWARELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWCRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108

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X67-E04 HC

EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-H04 LC QDIQMTQSPS SRLSGSGFGT

X67-H04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-E09 LC QDIQMTQSPS SRLSGSGFGT

X66-E09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-B05 LC QDIQMTQSPS SRLSGSGFGT

X66-B05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-D03 LC QDIQMTQSPS SRLSGSGFGT

X66-D03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-H04 LC QDIQMTQSPS SRLSGSGFGT

X66-H04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-H02 LC QDIQMTQSPS SRLSGSGFGT

X66-H02 HC EVQLLESGGG

LVQPGGSLRL SRDNSKNTLY GPSVFPLAPS	SCAASGFTFS LQMNSLRAED SKS	AYSMIWVRQA TAVYYCARGG	PGKGLEWVSY LLLWDRELKS	IRPSGGRTTY NYFDYWGQGT	60 120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWERELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFAELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFCELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFEELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFFELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60

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ADSVKGRFTI

LVTVSSASTK

X66-C11 LC QDIQMTQSPS SRLSGSGFGT

X66-C11 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-A07 LC QDIQMTQSPS SRLSGSGFGT

X66-A07 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-C03 LC QDIQMTQSPS SRLSGSGFGT

X66-C03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-G05 LC QDIQMTQSPS SRLSGSGFGT

X66-G05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-F10 LC QDIQMTQSPS SRLSGSGFGT

X66-F10 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-E04 LC QDIQMTQSPS SRLSGSGFGT

X66-E04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

SRDNSKNTLY GPSVFPLAPS	LQMHSLRAED SKS	TAVYYCARGG	LLLWFGELKS	NYFDYWGQGT	120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFHELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFKELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFLELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFMELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFPELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFQELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143

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X69-D08 LC QDIQMTQSPS SRLSGSGFGT

X69-D08 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-B02 LC QDIQMTQSPS SRLSGSGFGT

X69-B02 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-C09 LC QDIQMTQSPS SRLSGSGFGT

X69-C09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-D09 LC QDIQMTQSPS SRLSGSGFGT

X69-D09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-D02 LC QDIQMTQSPS SRLSGSGFGT

X69-D02 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-A12 LC QDIQMTQSPS SRLSGSGFGT

X69-A12 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-F05 LC

QDIQMTQSPS SRLSGSGFGT

SLSAFVGDRV DFTLTISSLQ	TITCRASQPI PEDFGNYYCQ	DNYLNWYHQK QSYTVPYTFG	PGKAPKLLIY GGTKVEIR	AASRLQSGVP	60 108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRALKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRCLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRGLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRHLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRKLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108

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X69-F05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-B08 LC QDIQMTQSPS SRLSGSGFGT

X69-B08 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-A10 LC QDIQMTQSPS SRLSGSGFGT

X69-A10 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-A09 LC QDIQMTQSPS SRLSGSGFGT

X69-A09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-E05 LC QDIQMTQSPS SRLSGSGFGT

X69-E05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-F09 LC QDIQMTQSPS SRLSGSGFGT

X69-F09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-F01 LC QDIQMTQSPS SRLSGSGFGT

X69-F01 HC EVQLLESGGG

LVQPGGSLRL SRDNSKNTLY GPSVFPLAPS	SCAASGFTFS LQMNSLRAED SKS	AYSMIWVRQA TAVYYCARGG	PGKGLEWVSY LLLWFRLLKS	IRPSGGRTTY NYFDYWGQGT	60 120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRNLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRPLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRQLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRRLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRSLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60

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ADSVKGRFTI

LVTVSSASTK

X69-C12 LC QDIQMTQSPS SRLSGSGFGT

X69-C12 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-E01 LC QDIQMTQSPS SRLSGSGFGT

X69-E01 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X69-H10 LC QDIQMTQSPS SRLSGSGFGT

X69-H10 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-F01 LC QDIQMTQSPS SRLSGSGFGT

X66-F01 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-H11 LC QDIQMTQSPS SRLSGSGFGT

X66-H11 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-C02 LC QDIQMTQSPS SRLSGSGFGT

X66-C02 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

SRDNSKNTLY GPSVFPLAPS	LQMNSLRAED SKS	TAVYYCARGG	LLLWFRTLKS	NYFDYWGQGT	120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRVLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRWLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFRYLKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFSELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFTELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFVELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143

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2019204295 19 Jun 2019

X66-F09 LC QDIQMTQSPS SRLSGSGFGT

X66-F09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X66-G08 LC QDIQMTQSPS SRLSGSGFGT

X66-G08 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-C09 LC QDIQMTQSPS SRLSGSGFGT

X67-C09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-B04 LC QDIQMTQSPS SRLSGSGFGT

X67-B04 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-G09 LC QDIQMTQSPS SRLSGSGFGT

X67-G09 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-C03 LC QDIQMTQSPS SRLSGSGFGT

X67-C03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-D03 LC

QDIQMTQSPS SRLSGSGFGT

SLSAFVGDRV DFTLTISSLQ	TITCRASQPI PEDFGNYYCQ	DNYLNWYHQK QSYTVPYTFG	PGKAPKLLIY GGTKVEIR	AASRLQSGVP	60 108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFWELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWFYELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWGRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWKRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWLRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWMRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108

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2019204295 19 Jun 2019

X67-D03 HC

EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-B05 LC QDIQMTQSPS SRLSGSGFGT

X67-B05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-F01 LC QDIQMTQSPS SRLSGSGFGT

X67-F01 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-G05 LC QDIQMTQSPS SRLSGSGFGT

X67-G05 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-B03 LC QDIQMTQSPS SRLSGSGFGT

X67-B03 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-F10 LC QDIQMTQSPS SRLSGSGFGT

X67-F10 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

X67-H01 LC QDIQMTQSPS SRLSGSGFGT

X67-H01 HC EVQLLESGGG

LVQPGGSLRL SRDNSKNTLY GPSVFPLAPS	SCAASGFTFS LQMNSLRAED SKS	AYSMIWVRQA TAVYYCARGG	PGKGLEWVSY LLLWNRELKS	IRPSGGRTTY NYFDYWGQGT	60 120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWPRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWQRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWRRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWSRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWTRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60

190

2019204295 19 Jun 2019

ADSVKGRFTI

LVTVSSASTK

X67-F08 LC QDIQMTQSPS SRLSGSGFGT

X67-F08 HC EVQLLESGGG ADSVKGRFTI LVTVSSASTK

SRDNSKNTLY GPSVFPLAPS	LQMNSLRAED SKS	TAVYYCARGG	LLLWWRELKS	NYFDYWGQGT	120 143
SLSAFVGDRV	TITCRASQPI	DNYLNWYHQK	PGKAPKLLIY	AASRLQSGVP	60
DFTLTISSLQ	PEDFGNYYCQ	QSYTVPYTFG	GGTKVEIR		108
LVQPGGSLRL	SCAASGFTFS	AYSMIWVRQA	PGKGLEWVSY	IRPSGGRTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCARGG	LLLWYRELKS	NYFDYWGQGT	120
GPSVFPLAPS	SKS				143

191

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HV-CDR2	YISPSGGHTIYADSVKG	YISPSGGHTIYADSVKG	YISPSGGHTIYADSVKG	YISPSGGHTIYADSVKG	YISPSGGHTIYADSVKG	YISPSGGHTIYADSVKG
HV- CDR1	HYLMT	HYLMT	HYLMT	HYLMT	HYLMT	HYLMT
LV-CDR3	QQSQSTPYT	QQTYSSPFT	QQSYTSPYT	QQSFNIPYT	QQSSRTPWT	QQSSRTPWT
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Initial Name	\| X59-C07	\| X60-D01	\| X63-G10	\| X64-F04	\| X63-G06	\| Χ81-Β0Γ

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193

2019204295 19 Jun 2019

Amino acid sequences of light chain (LC) and heavy chain (HC) variable domain of affinity matured antibody inhibitors of pKal discovered using ROLIC are shown below.

X59-C07 LC

QDIQMTQSPS SLSASVGDRV TVTCRAGRSI STYVNWYQQK PGKAPKLLIY AASSLQSGVP 60

SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ QSQSTPYTFG QGTKLEVK108

X59-C07 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL120

VTVSSASTKG PSVFPLAPSS KS142

X60-D01 LC

QDIQMTQSPG TLSLSPGERA TLSCRASQIV SSRYLAWYQQ RPGQAPRLLI YGAASRATGI60

PDRFSGSGSG TDFTLTISSL QAEDFATYYC QQTYSSPFTF GQGTKMEIK109

X60-D01 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL120

VTVSSASTKG PSVFPLAPSS KS142

X63-G06 LC

QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI60

PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK109

X63-G06 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL120

VTVSSASTKG PSVFPLAPSS KS142

X63-G10 LC

QDIQMTQSPD SLSASVGDRV TITCRASQSI SNYLNWYQQK PGKAPKLLIY AASSLQSGVP60

SRFSGSGSGT DFTLTISGLQ PEDFASYYCQ QSYTSPYTFV QGTKLEIKRT110

X63-G10 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL120

VTVSSASTKG PSVFPLAPSS KS142

X64-F04 LC

QDIQMTQSPA TLSLSPGERA TLSCRASQIV SSNYLAWYQQ KPGQAPRLLI YGASNRATGI60

PDRFSGSGSG TEFTLTISSL QSEDFAIYYC QQSFNIPYTF GQGTRVDIK109

X64-F04 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120

VTVSSASTKG PSVFPLAPSS KS142

X81-B01 is the germlined IgG produced in HEK 293T cells version of the X63-G06 Fab, as indicated above.

X101-A01 (aka DX-2922) is the germlined IgG produced in CHO cells version of the X63-G06 Fab

194

2019204295 19 Jun 2019

EXAMPLE 4: Affinity Maturation

In addition to optimizing the sequence of the clipped antibody (M142-H08), we also performed affinity maturation on two of the antibodies identified by phage display (M162-A04 and M160-G12). Both of these antibodies inhibit human pKal with single digit nanomolar potency, appear specific to pKal, and do not bind prekallikrein (Table 3). We first performed a novel form of light chain shuffling called ROLIC (Rapid Optimization of Light Chains) on M162-A04 and M160-G12 (see, e.g., WO 2009/102927 and U.S. 2009-0215119). From the screening of the antibodies discovered by ROLIC we identified one antibody with subnamolar potency (X63-G06) that shared the same heavy chain as M160-G12. We then constructed HV-CDR3 spiking affinity maturation libraries based on CDR3 sequences in M162-A04 and X63-G06 (described below).

Affinity Maturation by ROLIC. We used ROLIC to affinity mature the two leads from Table 3 that were not cleaved (M162-A04 and M160-G12). This process identified one antibody that inhibits pKal with a subnanomolar Ki_iapp (Table 7). X63G06 inhibits pKal with a Κ_ραρρ of approximately 0.4 nM as a Fab fragment. When this antibody was converted to an IgG that is germlined and sequenced optimized for CHO cell expression (X81-B01) it was found to inhibit pKal with a Κ_ραρρ of approximately 0.2 nM.

EXAMPLE 5: Affinity Maturation of Heavy Chain CDR1/2 and CDR3

We used two additional affinity maturation strategies to identify highly potent antibodies based on two different parental antibody inhibitor leads: M162-A04 and X63G06. One approach was to generate libraries that shuffled the CDR1/2 of the HC of two different parental antibody inhibitor leads (M162-A04 and X63-G06) against additional CDR1/2 diversity. Another approach was to create heavy chain CDR3 spiking libraries based on these leads.

The 82 antibodies that were discovered based on improvements in M162-A04 due to modifications in either the CDR1/2 and CDR3 region are shown in Table 8. Inhibition screening with 10 nM antibody (as Fab fragments) revealed that there were 33 antibodies that inhibited pKal activity by over 90%. Several antibodies were shown to be

195

2019204295 19 Jun 2019 subnanomolar inhibitors of human pKal.

The 62 antibodies that were discovered based on improvements in X63-G06 due to modifications in either the CDR1/2 and CDR3 region are shown in Table 9. Inhibition screening with 10 nM antibody (as Fab fragments) revealed that there were 24 antibodies that inhibited pKal activity by over 90%. Several antibodies were shown to be subnanomolar inhibitors of human pKal.

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RRIGVPRRDEFDI

RRTGVPRWDDFDI

RRTGIPRRDAFDI

RRIGVPRRDSFDM

RRTGVPRWDDFDI

RRTGVPRRDEFDI

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RRIGVPRRDSFDI

RRTGIPRRDAFDI

RRTGVPRRDSFDI

RRTGIPRRDAFDI

HV-CDR2

GIYSSGGITVYAD SVKG

SIY P S RGMTWYADSVKG

SIYPSGGMTYYADSVKG

GIYSSGGITVYADSVKG

SIYSSGGPTKYADSVKG

GIYSSGGITVYADSVKG

SIYPSGGNTSYADSVKG

GIYSSGGITVYADSVKG

SIY P S GGMT TYADSVKG

SIYPSGGQTIYADSVKG

GIYSSGGITVYADSVKG

SIVPSGGKTNYADSVKG

VIYPSGGQTYYADSVKG

SIGSSGGKTLYADSVKG

HV- CDR1

HYIMM

IYSMH

VYNMH

HYIMM

IYSMH

HYIMM

IYDMI

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VYNMH

IYDMT

HYIMM

SYNMH

RYSMR

IYSMQ

LV-CDR3

QQYNTYWT

LV-CDR2

KASTLES

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RASQSISSWLA

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Ml 9 6-CO 6

M198-F09

M199-A08

M202-C01

M198-A06

M200-D03

M202-H03

M201-A07

M197-A01

M202-D09

M197-A09

M198-G07

M200-A10

M197-H10

M196-D12

M197-A08

197

2019204295 19 Jun 2019

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2019204295 19 Jun 2019

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199

2019204295 19 Jun 2019

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200

2019204295 19 Jun 2019

Amino acid sequences of light chain (LC) and heavy chain (HC) variable domain of pKal antibodies obtained from CDR1/2 and CDR3 spiking affinity maturation libraries based on M162-A04.

M195-A02 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M195-A02

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYMMMWVRQA PGKGLEWVSG ISSSGGHTDY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M195-A12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M195-A12

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYMMMWVRQA PGKGLEWVSG IYPSGGYTVY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M195-B02 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M195-B02

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYLMWWVRQA PGKGLEWVSY IGPSGGPTHY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M195-B12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M195-B12

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS KYWMYWVRQA PGKGLEWVSY IRPSGGQTYY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M195-C12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M195-C12

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS MYQMFWVRQA PGKGLEWVSS ISPGGGTQYA 60

DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120

SASTKGPSVF PLAPSSKS

138

M195-D12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

201

2019204295 19 Jun 2019

M195-D12 EVQLLESGGG ADSVKGRFTI SSASTKGPSV	LVQPGGSLRL SRDNSKNTLY FPLAPSSKS	HC SCAASGFTFS LQMNSLRAED	IYGMFWVRQA TAVYYCAYRR	PGKGLEWVSG TGIPRRDAFD	IGPSGGPTKY IWGQGTMVTV	60 120 139
M195-E12		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
SRFSGSGSGT	EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
M195-E12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	YYIMFWVRQA	PGKGLEWVSY	ISPSGGYTHY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
SSASTKGPSV	FPLAPSSKS					139
M195-F12		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
SRFSGSGSGT	EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107

M195-F12 EVQLLESGGG ADSVKGRFTI SSASTKGPSV	LVQPGGSLRL SRDNSKNTLY FPLAPSSKS	HC SCAASGFTFS LQMNSLRAED	HYVMMWVRQA TAVYYCAYRR	PGKGLEWVSY TGIPRRDAFD	IVPSGGVTAY IWGQGTMVTV	60 120 139
M0195-G02		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
SRFSGSGSGT	EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
M195-G02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	PYNMMWVRQA	PGKGLEWVSS	IWPSGGTTDY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
SSASTKGPSV	FPLAPSSKS					139
Ml 9 6-CO6		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
SRFSGSGSGT	EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
Ml 9 6-CO 6		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	IYSMHWVRQA	PGKGLEWVSS	IYPSRGMTWY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
SSASTKGPSV	FPLAPSSKS					139
M196-D02		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
SRFSGSGSGT	EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
M196-D02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	VYSMHWVRQA	PGKGLEWVSV	IGPSGGITLY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
SSASTKGPSV	FPLAPSSKS					139
M196-D12		LC
QDIQMTQSPS	TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
SRFSGSGSGT	EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107

202

2019204295 19 Jun 2019

M196-D12

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMRWVRQA PGKGLEWVSV IYPSGGQTYY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M196-G12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M196-G12

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYSMDWVRQA PGKGLEWVSR IYSSGGGTIY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M196-H03 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M196-H03

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYVMWWVRQA PGKGLEWVSS ISPSGDTHYA 60

DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120

SASTKGPSVF PLAPSSKS

138

M197-A01 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-A01

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMIWVRQA PGKGLEWVSS IYPSGGNTSY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-A08 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-A08

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMQWVRQA PGKGLEWVSS IGSSGGKTLY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-A09 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-A09

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYNMHWVRQA PGKGLEWVSS IYPSGGMTTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-C12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-C12 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYDMLWVRQA PGKGLEWVSY IVSSGGLTKY 60

203

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS 139

2019204295 19 Jun 2019

M197-E12 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-E12

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYTMRWVRQA PGKGLEWVSS IYPSGGKTQY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-F01 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-F01

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYHMYWVRQA PGKGLEWVSS IGPSGGPTGY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-F03 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-F03

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMFWVRQA PGKGLEWVSS IGPSGGVTHY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-G10 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-G10

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYAMRWVRQA PGKGLEWVSS IYPSGGKTWY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-G11 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

M197-G11

HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYAMVWVRQA PGKGLEWVSS IYPSGGITTY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

SSASTKGPSV FPLAPSSKS

139

M197-H10 HC

EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYNMHWVRQA PGKGLEWVSS IVPSGGKTNY 60

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

M197-H10 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

204

FPLAPSSKS

139

2019204295 19 Jun 2019

SSASTKGPSV

M197-H11

QDIQMTQSPS SRFSGSGSGT

M197-H11

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-A01

QDIQMTQSPS

SRFSGSGSGT

M198-A01

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-A02

QDIQMTQSPS

SRFSGSGSGT

M198-A02

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-A06

QDIQMTQSPS

SRFSGSGSGT

M198-A06

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-A07

QDIQMTQSPS

SRFSGSGSGT

M198-A07

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-A08

QDIQMTQSPS

SRFSGSGSGT

M198-A08

EVQLLESGGG

DSVKGRFTIS SASTKGPSVF

M198-A09

TLSASVGDRV EFTLTISSLQ	LC TITCRASQSI PDDFATYYCQ	SSWLAWYQQK QYNTYWTFGQ	PGKAPNLLIY GTKVEIK	KASTLESGVP	60 107
	HC
LVQPGGSLRL	SCAASGFTFS	IYNMIWVRQA	PGKGLEWVSS	IYPSGGWTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	PYTMIWVRQA	PGKGLEWVSS	ISSSGGMTPY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	RYSMIWVRQA	PGKGLEWVSS	IWSSGGATEY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	IYSMHWVRQA	PGKGLEWVSS	IYSSGGPTKY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	RYQMHWVRQA	PGKGLEWVSW	ISPSGGITGY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS WYMMQWVRQA PGKGLEWVSR ISPSGGTTYA 60

RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120

PLAPSSKS 138

LC

205

2019204295 19 Jun 2019

QDIQMTQSPS

SRFSGSGSGT

M198-A09

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-B09

QDIQMTQSPS

SRFSGSGSGT

M198-B09

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-B12

QDIQMTQSPS

SRFSGSGSGT

M198-B12

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-C03

QDIQMTQSPS

SRFSGSGSGT

M198-C03

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-C05

QDIQMTQSPS

SRFSGSGSGT

M198-C05

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-C06

QDIQMTQSPS

SRFSGSGSGT

M198-C06

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-C09

QDIQMTQSPS

SRFSGSGSGT

TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	IYFMSWVRQA	PGKGLEWVSS	IRSSGGPTWY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	VYSMTWVRQA	PGKGLEWVSS	IGSSGGSTTY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	KYSMAWVRQA	PGKGLEWVSG	IYPSGGRTLY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	RYSMSWVRQA	PGKGLEWVSG	ISPSGGETSY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	RYKMYWVRQA	PGKGLEWVSV	IGPSGGATFY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	QYFMHWVRQA	PGKGLEWVSY	IYPSGGMTEY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107

206

2019204295 19 Jun 2019

M198-C09

EVQLLESGGG ADSVKGRFTI SSASTKGPSV

M198-C10

QDIQMTQSPS

SRFSGSGSGT

M198-C10

EVQLLESGGG YADSVKGRFT VSSASTKGPS

M198-D12

QDIQMTQSPS

SRFSGSGSGT

M198-D12

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-E02

QDIQMTQSPS

SRFSGSGSGT

M198-E02

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-E09

QDIQMTQSPS

SRFSGSGSGT

M198-E09

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-E11

QDIQMTQSPS

SRFSGSGSGT

M198-E11

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-F04

QDIQMTQSPS

SRFSGSGSGT

M198-F04

EVQLLESGGG ADSVKGRFTI

LVQPGGSLRL SRDNSKNTLY FPLAPSSKS	HC SCAASGFTFS LQMNSLRAED	IYTMYWVRQA TAVYYCAYRR	PGKGLEWVSS TGIPRRDAFD	ISPSGGWTYY IWGQGTMVTV	60 120 139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYMGMNWVRQ	APGKGLEWVS	SIVPSGGWTQ	60
ISRDNSKNTL	YLQMNSLRAE	DTAVYYCAYR	RTGIPRRDAF	DIWGQGTMVT	120
VFPLAPSSKS	LC				140
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	LYVMYWVRQA	PGKGLEWVSY	IVPSGGPTAY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	IYSMYWVRQA	PGKGLEWVSY	IRPSGGNTKY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	IYDMNWVRQA	PGKGLEWVSS	IYPSGGRTRY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	TYSMYWVRQA	PGKGLEWVSS	IYPSGGLTWY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	IYDMWWVRQA	PGKGLEWVSS	IRPSGGITKY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120

207

FPLAPSSKS

139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

2019204295 19 Jun 2019

SSASTKGPSV

M198-F08

QDIQMTQSPS

SRFSGSGSGT

M198-F08

EVQLLESGGG ADSVKGRFTI SSASTKGPSV

M198-F09

QDIQMTQSPS SRFSGSGSGT

M198-F09

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-F12

QDIQMTQSPS SRFSGSGSGT

M198-F12

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-G03

QDIQMTQSPS SRFSGSGSGT

M198-G03

EVQLLESGGG SVKGRFTISR ASTKGPSVFP

M198-G07

QDIQMTQSPS SRFSGSGSGT

M198-G07

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M198-H02

QDIQMTQSPS SRFSGSGSGT

M198-H02

EVQLLESGGG DSVKGRFTIS SASTKGPSVF

HC

LVQPGGSLRL SCAASGFTFS VYSMWWVRQA PGKGLEWVSS ISSSGGMTEY 60

SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

FPLAPSSKS

139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS VYNMHWVRQA PGKGLEWVSS IYPSGGMTYY 60

SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

FPLAPSSKS

139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS IYTMMWVRQA PGKGLEWVSS IWSSGGQTKY 60

SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

FPLAPSSKS

139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS QYTMVWVRQA PGKGLEWVSW IYSSRANYAD 60

DNSKNTLYLQ MNSLRAEDTA VYYCAYRRTG IPRRDAFDIW GQGTMVTVSS 120

LAPSSKS

137

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS IYDMTWVRQA PGKGLEWVSS IYPSGGQTIY 60

SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120

FPLAPSSKS

139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS PYNMYWVRQA PGKGLEWVSW IVPGGVTKYA 60

RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120

PLAPSSKS 138

208

2019204295 19 Jun 2019

M198-H08

QDIQMTQSPS

SRFSGSGSGT

M198-H08

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M198-H09

QDIQMTQSPS

SRFSGSGSGT

M198-H09

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M199-A08

QDIQMTQSPS

SRFSGSGSGT

M199-A08

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M199-A11

QDIQMTQSPS

SRFSGSGSGT

M199-A11

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M199-B01

QDIQMTQSPS

SRFSGSGSGT

M199-B01

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M200-A10

QDIQMTQSPS

SRFSGSGSGT

M200-A10

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M200-B01

QDIQMTQSPS

SRFSGSGSGT

TLSASVGDRV EFTLTISSLQ	LC TITCRASQSI PDDFATYYCQ	SSWLAWYQQK QYNTYWTFGQ	PGKAPNLLIY GTKVEIK	KASTLESGVP	60 107
	HC
LVQPGGSLRL	SCAASGFTFS	IYDMYWVRQA	PGKGLEWVSS	IGPSGGPTAY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	IYLMIWVRQA	PGKGLEWVSY	IGPSGGPTEY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	IGVPRRDEFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	RGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAFRR	TGIPRRDAFD	NWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRRDSFD	IWGQGTMVTV	120
FPLAPSSKS					139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

209

2019204295 19 Jun 2019

M200-B01

EVQLLESGGG ADSVKGRFTI SSASTKGPSV

M200-D03

QDIQMTQSPS

SRFSGSGSGT

M200-D03

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M200-E11

QDIQMTQSPS

SRFSGSGSGT

M200-E11

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M200-F01

QDIQMTQSPS

SRFSGSGSGT

M200-F01

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M200-H07

QDIQMTQSPS

SRFSGSGSGT

M200-H07

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M201-A06

QDIQMTQSPS

SRFSGSGSGT

M201-A06

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M201-A07

QDIQMTQSPS SRFSGSGSGT

M201-A07

LVQPGGSLRL SRDNSKNTLY FPLAPSSKS	HC SCAASGFTFS LQMNSLRAED	HYIMMWVRQA TAVYYCAYRR	PGKGLEWVSG TGIPRRDAFD	IYSSGGITVY SWGQGTMVTV	60 120 139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAWRR	IGVPRRDSFD	MWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRRDAFD	NWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	MGIPRRNAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRRNAFD	NWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDVFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107

HC

210

2019204295 19 Jun 2019

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M201-F11

QDIQMTQSPS SRFSGSGSGT

M201-F11

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M201-H06

QDIQMTQSPS SRFSGSGSGT

M201-H06

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M201-H08

QDIQMTQSPS SRFSGSGSGT

M201-H08

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-A01

QDIQMTQSPS SRFSGSGSGT

M202-A01

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-A04

QDIQMTQSPS SRFSGSGSGT

M202-A04

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-A08

QDIQMTQSPS SRFSGSGSGT

M202-A08

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

LVQPGGSLRL SRDNSKNTLY FPLAPSSKS	SCAASGFTFS LQMNSLRAED	HYIMMWVRQA TAVYYCAYRR	PGKGLEWVSG TGVPRRDEFD	IYSSGGITVY IWGQGTMVTV	60 120 139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	SGIPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRRDALD	NWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	IGVPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	KGIPRRDDFD	IWGQGTMVTV	120
FPLAPSSKS	LC				139
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRWDAFD	IWGQGTMVTV	120
FPLAPSSKS					139

211

2019204295 19 Jun 2019

M202-A10

QDIQMTQSPS SRFSGSGSGT

M202-A10

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-A12

QDIQMTQSPS

SRFSGSGSGT

M202-A12

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-B03

QDIQMTQSPS

SRFSGSGSGT

M202-B03

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-B04

QDIQMTQSPS

SRFSGSGSGT

M202-B04

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-C01

QDIQMTQSPS

SRFSGSGSGT

M202-C01

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-C02

QDIQMTQSPS

SRFSGSGSGT

M202-C02

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

TLSASVGDRV EFTLTISSLQ	LC TITCRASQSI PDDFATYYCQ	SSWLAWYQQK QYNTYWTFGQ	PGKAPNLLIY GTKVEIK	KASTLESGVP	60 107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAFRR	TGIPRRDSFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYQR	TGVPRRDSFN	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRRDDFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	SGVPRRDDFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRWDDFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	PGVPRRDAFD	IWGQGTMVTV	120
FPLAPSSKS					139

M202-C09 LC

QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

212

2019204295 19 Jun 2019

SRFSGSGSGT

M202-C09

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-D09

QDIQMTQSPS

SRFSGSGSGT

M202-D09

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-E03

QDIQMTQSPS

SRFSGSGSGT

M202-E03

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-E06

QDIQMTQSPS

SRFSGSGSGT

M202-E06

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-F06

QDIQMTQSPS

SRFSGSGSGT

M202-F06

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-G03

QDIQMTQSPS

SRFSGSGSGT

M202-G03

EVQLLESGGG

ADSVKGRFTI SSASTKGPSV

M202-H03

QDIQMTQSPS

SRFSGSGSGT

EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK	107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	IGVPRRDDFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	IGVPRRDSFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGIPRRDAFE	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	RGVPRRDDFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAYRR	TGVPRWDAFD	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107
	HC
LVQPGGSLRL	SCAASGFTFS	HYIMMWVRQA	PGKGLEWVSG	IYSSGGITVY	60
SRDNSKNTLY	LQMNSLRAED	TAVYYCAFRR	TGVPRRDSFE	IWGQGTMVTV	120
FPLAPSSKS					139
	LC
TLSASVGDRV	TITCRASQSI	SSWLAWYQQK	PGKAPNLLIY	KASTLESGVP	60
EFTLTISSLQ	PDDFATYYCQ	QYNTYWTFGQ	GTKVEIK		107

213

2019204295 19 Jun 2019

M202-H03

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

M202-H05

QDIQMTQSPS

SRFSGSGSGT

M202-H05

EVQLLESGGG

ADSVKGRFTI

SSASTKGPSV

HC

LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY 60

SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGVPRWDDFD IWGQGTMVTV 120

FPLAPSSKS

139

LC

TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60

EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107

HC

LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY 60

SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGVPRRDVFD IWGQGTMVTV 120

FPLAPSSKS 139

214

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216

2019204295 19 Jun 2019

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X	X	X	X	X	X	X	X	X	X
ω	ω	ω	ω	σ	ω	ω	ω	ω	ω
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X	X	3	X	X	3	X	X	X	Χ
0	0	0	0	0	0	0	0	0	0
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ω	ω	ω	ω	ω	ω	ω	ω	ω	ω
X	X	ω	X	ω	X	X	X	X	X
ω	ω	X	ω	ω	ω	>	ω	ω	ω
1—I	1—I	1—I	1—I	1—I	1—I	1—1	1—I	1—I	1—I
X	X	ω	X	X	X	3	X	X	ω
Εη	Εη	>	Εη	1—I	>	<	Εη	Εη	>
3	3	3	3	3	3	3	3	3	3
X	X	3	X	Μ	Ο<	X	X	X	3
>1	>1	X	>1	X	ίΤ	>1	>1	>1	X
X	X	σ	X	ο	ω	3	X	X	ω
Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη
3	3	3	3	3	3	3	3	3	3
X	X	X	X	X	X	X	X	X	X
Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη
X	X	X	X	X	X	X	X	X	X
ω	ω	ω	ω	ω	ω	ω	ω	ω	ω
ω	ω	ω	ω	ω	ω	ω	ω	ω	ω
ΟΙ	ΟΙ	ΟΙ	ΟΙ	ΟΙ	ΟΙ	ΟΙ	ΟΙ	ΟΙ	ΟΙ
ο	ο	ο	ο	ο	ο	ο	ο	ο	ο
Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη
ω	ω	ω	ω	ω	ω	ω	ω	ω	ω
ω	ω	ω	ω	ω	ω	ω	ω	ω	ω

0	0	0	0	0	0	0	0	0	0
<	<	<	<	<	<	<	<	<	<
X	X	X	X	X	X	X	X	X	X
>1	>1	>1	>1	>1	>1	>1	>1	>1	>1
3	3	3	3	3	3	3	3	3	3
ω	ω	ω	ω	ω	ω	ω	ω	ω	ω
3	3	3	3	3	3	3	3	3	3
>	>	>	>	>	>	>	>	>	>
X	X	X	X	X	X	X	X	X	X
ο	ο	ο	ο	ο	ο	ο	ο	ο	ο
ω	ω	ω	ω	ω	ω	ω	ω	ω	ω
Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη	Εη
X	X	X	X	X	X	X	X	X	X

X	X	LO		00			Ο	X	Ο
X	X	LO	τ—I	03	03	τ—I	X	X	σδ
X	X			LO	LO	LO			τ—I
X	τ—1	X	LO	X	X	τ—1	τ—1	X
ο	ο	Ο	Ο	τ—1	Ο	ο	ο	τ—1	ο
ω	<	Ο	Ο	<	X	<	X	Q	X
σδ	X		X	LO	LO	X	σδ	03
ο	ο	ο	ο	Ο	ο	ο	ο	ο	ο
X	X	X	X	X	X	X	X	X	X
3	3	3	3	3	3	3	3	3	3

217

2019204295 19 Jun 2019

Amino acid sequences of light chain (LC) and heavy chain (HC) variable domain of pKal antibodies obtained from CDR1/2 and CDR3 spiking affinity maturation libraries based on X63-G06.

M203-A01 QDIQMTQSPG PDRFSGTGYG	TLSLSPGERA TDFTLTISRL	LC TLSCRTSQFV EPEDYGTYYC	NSNYLAWYQQ QQSSRTPWTF	TPGQAPRLLI GQGTRVEIK	YGASSRATGI	60 109
M203-A01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	NYLMAWVRQA	PGKGLEWVSW	IVPSGGYTEY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M203-A03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M203-A03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	VYMMIWVRQA	PGKGLEWVSS	ISPSGGQTTY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M204-A02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M204-A02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	DYMMTWVRQA	PGKGLqWVSY	ISPSGGLTSY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M204-E12		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M204-E12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	TYMMqWVRQA	PGKGLEWVSY	IGPSGGKTDY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-A02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-A02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	TYLMAWVRQA	PGKGLEWVSG	IVSSGGRTLY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-A12		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109

2019204295 19 Jun 2019

10280/177W01/D2033-7130WO

M205-A12 EVQLLESGGG ADSVKGRFTI VTVSSASTKG	LVQPGGSLRL SRDNSKNTLY PSVFPLAPSS	HC SCAASGFTFS LQMNSLRAED KS	QYDMIWVRQA TAVYYCARVA	PGKGLEWVSY RGIAARSRTS	ISSSGGFTRY YFDYWGQGTL	60 120 142
M205-B04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-B04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	NYDMIWVRQA	PGKGLEWVSS	ISSSGGTTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-C11		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-C11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	QYMMMWVRQA	PGKGLEWVSR	ISPSGGSTLY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-D04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-D04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	TYKMqWVRQA	PGKGLEWVSS	ISPSGGPTNY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-E11		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-E11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	NYTMGWVRQA	PGKGLEWVSS	ISPSGGKTDY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-F03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-F03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	SqQMVWVRQA	PGKGLEWVSY	ISPSGGNTYY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M205-H01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-H01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	NYTMQWVRQA	PGKGLqWVSY	ISPSGGYTGY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120

1094814.1

-218219

2019204295 19 Jun 2019

VTVSSASTKG	PSVFPLAPSS	KS	142
M205-H08		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M205-H08		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	DYMMMWVRQA	PGKGLEWVSS	IVPSGGHTqY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-A06		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-A06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	NYMMGWVRQA	PGKGLqWVSS	ISPSGGLTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-B10		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-B10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	qYLMAWVRQA	PGKGLEWVSS	IYPSGGWTKY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-C03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-C03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	qYMMVWVRQA	PGKGLEWVSS	IYSSGGNTPY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-E02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-E02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	EYMMMWVRQA	PGKGLEWVSV	ISPSGGQTHY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-F01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-F01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	GYMMVWVRQA	PGKGLEWVSR	ISPSGGPTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-F09		LC

220

2019204295 19 Jun 2019

QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-F09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	VYMMSWVRQA	PGKGLEWVSS	IVPSGGSTTY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-H01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-H01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	EYMMVWVRQA	PGKGLEWVSR	ISPSGGTTEY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-H04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-H04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	SYMMVWVRQA	PGKGLEWVSS	ISPSGGYTIq	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-H05		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-H05		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	GYKMqWVRQA	PGKGLEWVSS	ISPSGGITMY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M206-H08		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M206-H08		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	DYMMAWVRQA	PGKGLEWVSS	IVPSGGHTHY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M207-A01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M207-A01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGISARSRTS	CFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M207-A02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109

221

2019204295 19 Jun 2019

M207-A02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TALYYCARVA	RGIAARSRTI	NLDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M207-A04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109

M207-A04 EVQLLESGGG ADSVKGRFTI VTVSSASTKG	LVQPGGSLRL SRDNSKNTLY PSVFPLAPSS	HC SCAASGFTFS LQMNSLRAED KS	HYLMTWVRQA TAVYYCARVA	PGKGLEWVSY RGIAARSRTR	ISPSGGHTIY SFDYWGQGTL	60 120 142
M207-C05		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
R0121-D02 =	= M0207-C05		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVG	RGIAARSRTS	YFDKWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-A10		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-A10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	qFDHWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-B01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-B01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	FFDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-C06		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-C06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	FIDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-D12		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-D12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60

222

2019204295 19 Jun 2019

ADSVKGRFTI VTVSSASTKG	SRDNSKNTLY PSVFPLAPSS	LQMNSLRAED KS	TAVYYCARLA	RGIAARSRTS	YqDIWGQGTL	120 142
M208-E10		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-E10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	QGISARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-F04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-F04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	FFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-F10		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109

M208-F10 EVQLLESGGG ADSVKGRFTI VTVSSASTKG	LVQPGGSLRL SRDNSKNTLY PSVFPLAPSS	HC SCAASGFTFS LQMNSLRAED KS	HYLMTWVRQA TAVYYCARVA	PGKGLEWVSY RGIAARSRTS	ISPSGGHTIY YFDqWGQGTL	60 120 142
M208-G02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-G02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIADRSRTS	YSDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-G03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-G03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YLDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M208-H02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M208-H02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIASRSRTR	YCDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142

223

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M209-B01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK	109

M209-B01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIVARSRTS	NFDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-B09		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-B09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	QGIVARSRTS	YLHqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-B11		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-B11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTR	YIDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-C02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-C02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAYRRRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-C11		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-C11		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCAMVG	QGIRGRSRTS	YFAqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-C12		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-C12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVG	RGIAARSRTS	qLDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142

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M0209-D02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-D02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGITARSRTS	YFDDWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-D03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-D03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVG	RGIAARSRTS	FFDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-D12		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-D12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCATVA	RGIAARSRTS	YFDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-E02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-E02		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTI	LLDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-E03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-E03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARqA	RGIAARSRTS	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-F04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-F04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YLDqWSQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-G01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109

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M209-G01 EVQLLESGGG ADSVKGRFTI VTVSSASTKG	LVQPGGSLRL SRDNSKNTLY PSVFPLAPSS	HC SCAASGFTFS LQMNSLRAED KS	HYLMTWVRQA TAVYYCARVA	PGKGLEWVSY qGISGRSRLS	ISPSGGHTIY YVDYWGQGTL	60 120 142
M209-G07		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-G07		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YFDTWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-H03		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-H03		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	QGIAARSRTT	qFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-H07		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-H07		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARqRTS	YYDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M209-H09		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M209-H09		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTV	YFDHWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-A06		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-A06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	qFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-B02		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109

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M210-B02 EVQLLESGGG ADSVKGRFTI VTVSSASTKG	LVQPGGSLRL SRDNSKNTLY PSVFPLAPSS	HC SCAASGFTFS LQMNSLRAED KS	HYLMTWVRQA TAVYYCASVA	PGKGLEWVSY RGIAARSRTS	ISPSGGHTIY YFNqWGQGTL	60 120 142
M210-C12		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-C12		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	QGIAARSRTS	SVDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-G04		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-G04		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	TGIVARSRTR	YFDqWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-G10		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-G10		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTS	YLDFWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-H01		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-H01		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRNS	qQDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-H06		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-H06		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTR	YFDYWGQGTL	120
VTVSSASTKG	PSVFPLAPSS	KS				142
M210-H07		LC
QDIQMTQSPG	TLSLSPGERA	TLSCRTSQFV	NSNYLAWYQQ	TPGQAPRLLI	YGASSRATGI	60
PDRFSGTGYG	TDFTLTISRL	EPEDYGTYYC	QQSSRTPWTF	GQGTRVEIK		109
M210-H07		HC
EVQLLESGGG	LVQPGGSLRL	SCAASGFTFS	HYLMTWVRQA	PGKGLEWVSY	ISPSGGHTIY	60
ADSVKGRFTI	SRDNSKNTLY	LQMNSLRAED	TAVYYCARVA	RGIAARSRTR	YFDqWGQGTL	120

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VTVSSASTKG PSVFPLAPSS KS 142

EXAMPLE 6: In Vivo Testing of M162-A04 (IgG) and X101-A01

Bradykinin and other bioactive kinins have been previously implicated in carrageenan-induced edema and inflammatory pain (Sharma J.N. et al. (1998) Inflammopharmacology 6, 9-17; Asano M. et al. (1997) Br J Pharmacol 122, 1436-1440; De Campos R.O. et al. (1996) Eur J Pharmacol 316, 277-286). Plasma kallikrein and tissue kallikrein 1 are the two primary kininogenases in mammals (Schmaier A.H. (2008) Int Immunopharmacol 8, 161-165). M162-A04 (M162-A4) (IgG), a specific plasma kallikrein inhibitor, was tested to determine whether it would be effective in carrageenan induced edema. The study design is outlined in Table 10. The route of administration (ROA) for the vehicle (PBS), the antibody, and the positive control (indomethacin) was intra-peritoneal (IP) and was given 30 minutes prior to carrageenan injection (0.1 mL of a 2% carrageenan solution). It is evident from FIGURE 2 that antibody doses at 10 mg/kg and above were equally effective in reducing carrageenan-induced edema as the positive control (indomethacin). However, the antibody was not effective in reducing carrageenan-induced thermal hyperalgesia (FIGURE 3). The reason for the dissociation between effectiveness in edema and hyperalgesia are not obvious but may be due to differences in the bioactivity of different kinin metabolites. Lys-desArg9-bradykinin is the most potent agonist of the Bl receptor, which is believed to be primarily involved in pain hypersensitivity (Leeb-Lundberg L.M et al. (2005) Pharmacol Rev 57, 27-77). This kinin metabolite is generated by tissue kallikrein 1, not plasma kallikrein (Schmaier A.H. (2008) Int Immunopharmacol 8,161-165). This difference in kinin generation and resulting bradykinin receptor activation may account for the unexpected decoupling of edema and hyperalgesia in this model.

Another pKal antibody inhibitor X101-A01 was also tested in the CPE model using the study design shown in Table 10B. The data obtained in Figure 14 shows that

XI01-A01 inhibited edema in a dose-dependent manner to an extent that is comparable to that of the positive control (indomethacin).

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Table 10A. Carrageenan-Induced Paw Edema Study Design to test M162-A04

Group #	Number of Rats	Treatment	Dose (mg/kg)	ROA	Timing relative to carrageenan	Dose Volume (mL/kg)
1	6	Vehicle	N/A	IP	T -30 minutes	20
2	6	559A-M162-A4	3	IP	T -30 minutes	20
3	6	559A-M162-A4	10	IP	T -30 minutes	20
4	6	559A-M162-A4	30	IP	T -30 minutes	20
5	6	Indomethacin	5	IP	T -30 minutes	20

Table 10B. Carrageenan-Induced Paw Edema Study Design to Test X101-

Group	Treatment	n	Dose (mg/Kg)	ROA	Timing *	Vol (mL/Kg)
1	Vehicle	10	N/A	IP	-30 min	20
2	X101-A01	10	1	IP	-30 min	20
3	X101-A01	10	3	IP	-30 min	20
4	X101-A01	10	10	IP	-30 min	20
5	X101-A01	10	30	IP	-30 min	20
6	Indomethacin	10	5	IP	-30 min	20

229

EXAMPLE 7: Evaluation of Selected Antibody Inhibitors of Plasma Kallikrein

Evaluation of selected optimized antibodies (X81-B01 andX67-D03) is shown in

Table 11. Neither antibody has any putative deamidation, isomerization, or oxidation sites.

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Table 11

Criteria	X81-B01 (IgG)	X67-D03 (IgG)
< nM Ki,app against human pKal	0.2 nM	0.1 nM
< nM Ki,app against rodent pKal	mouse - 11 pM rat - 0.14 nM	mouse - 0.7 nM rat - 0.34 nM
prekallikrein binding	no	no
Specific inhibitor with respect to fXIa, plasmin, and trypsin	yes	yes
Inhibits bradykinin generation	yes	yes
Inhibits pKal in presence of prekallikrein	yes	yes
Competition for binding with aprotinin	yes	yes
Stability in human serum	yes	nd*

* not done; a parental form of this antibody was shown to be stable in serum

EXAMPLE 8: Epitope Mapping

The region of pKal bound by selected anti-pKal antibodies was investigated using several methods. First, competition assays were used to determine whether the antibodies competed for binding to pKal with known active site-directed inhibitors. Second, antibodies were grouped according to whether they were inhibitors or just binders to pKal. Third, epitopes were investigated using synthetic peptides and peptidic structures based on the sequence and 3-dimensional structure of pKal. These peptidic structures are called “CLIPS” (Chemically Linked Peptides on Scaffolds) and the testing was performed by a fee for service company called Pepscan .

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Fourth, antibodies were tested for their ability to inhibit pKal from other species, besides human, where the amino acid sequence of pKal has been determined in order to identify amino acids that may account for the differences in inhibition.

Competition Assays

Using a BIACORE® SPR assay antibodies of interest were tested for competition with a known active site inhibitor of pKal. EPI-KAL2 is potent (K_papp = 0.1 nM) active site inhibitor of pKal and a Kunitz domain inhibitor based on the first domain of tissue factor pathway inhibitor (Markland (1996) Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin. Biochemistry. 35(24):8058-67). Kunitz domains are known active site inhibitors of serine proteases, such as pKal.

The sequence of EPI-KAL2 is:

EAMHSFCAFKADDGPCRArtT/PRfrEFNIFTRQCEEFriYGGCGGNONRFESL

EECKKMCTRD (amino acids in italics are those that differ from TFPI)

As shown in FIGURES 8A-8B, the antibodies X8f-B0f andX67-D03 were competed for binding to pKal in the presence or EPI-KAE2. This result indicates that these antibodies either bind in vicinity of the active site or allosteric changes in the conformation of the pKal-EPI-KAE2 complex prevent antibody binding.

Antibody Binders vs Inhibitors

As shown in Tables 1 and 2, all the unique antibodies discovered by phage display were characterized as being either pKal inhibitors or binders but not inhibitors. Antibodies that inhibit the activity of pKal either bind near the active site and preclude substrate interactions (competitive inhibitors) or that bind away from the active site and induce allosteric changes in the structure of the active site (noncompetitive inhibitors). Antibodies that bind but do not inhibit pKal are unlikely to bind near the active site and may bind the non-catalytic domain (i.e. the apple domain). Table 12 categorizes selected antibodies as being either inhibitors or binders of pKal. Also shown in Table 12 for the

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2019204295 19 Jun 2019 listed antibodies, is a demonstration of whether they cross-react with mouse pKal as inhibitors and whether they bind prekallikrein.

Table 12. Binding Properties of Selected Anti-pKal Antibodies

Number	Antibody	Binding Category	human Ki,app (nM)	mouse Ki,app (nM)	CLIPS Peptide(s) Identified
1	M6-A06	1) Binder only	no	no	C4
2	M6-D09	2) inhibitor, prekallikrein binder, inhibits mouse and human pKal	5.9	3.9	Cl, C5
3	M8-C04	1) Binder only	no	no
4	M8-G09	1) Binder only	no	no	Cl, C4, C6, C7
5	M29-D09	3) inhibitor, does not bind prekallikrein, does not inhibit mouse pKal	0.7	no	Cl, C4, C7
6	M35-G04	2) inhibitor, prekallikrein binder, inhibits mouse and human pKal	2.9	8	Cl, C4
7	M145- Dll	3) inhibitor, does not bind prekallikrein, weak inhibitor of mouse pKal	0.79	800	Cl, C4
8	M160- G12	4) inhibitor of both mouse and human pKal, does not bind prekallikrein	5	0.2	C2
9	X55-F01	4) inhibitor of both mouse and human pKal, does not bind prekallikrein	0.4	2	C2, C3
10	X73-H09	4) inhibitor, does not bind prekallikrein, weak inhibitor of human and mouse pKal	20	70	C6
11	X81-B01	4) inhibitor of both mouse and human pKal, does not bind prekallikrein	0.1	0.011	C2, C3, C5, C6
12	A2	5) Negative control, does not bind pKal, binds streptavidin	No binding	No binding	No binding

C1-C7: peptides in pKal identified by CLIPS epitope mapping (see FIGURES 9 and

10A-10C). Cl corresponds to positions 55-67 of the catalytic domain, C2 to positions

81-94, C3 to positions 101-108, C4 to positions 137-151, C5 to positions 162-178, C6 to

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2019204295 19 Jun 2019 positions 186-197, and C7 to positions 214-217.

Epitope Mapping Using CLIPS

The 11 anti-pKal antibodies listed in Table 12, plus one negative control (A2) were tested for binding to 5000 different synthetic CLIPS (Chemically Linked Peptides on Scaffolds) by Pepscan as described below in the CLIP METHODS sections. This analysis led to the identification of peptide regions in pKal that are likely to be a part of the antibody epitope for each of the tested antibodies (FIGURE 9).

CLIPS METHODS

The linear and CLIPS peptides were synthesized based on the amino acid sequence of the target protein using standard Fmoc-chemistry and deprotected using trifluoric acid with scavengers. The constrained peptides were synthesized on chemical scaffolds in order to reconstruct conformational epitopes, using Chemically Linked Peptides on Scaffolds (CLIPS) technology (Timmerman et al. (2007). For example, the single looped peptides were synthesized containing a dicysteine, which was cyclized by treating with alpha, alpha’-dibromoxylene and the size of the loop was varied by introducing cysteine residues at variable spacing. If other cysteines besides the newly introduced cysteines were present, they were replaced by alanine. The side-chains of the multiple cysteines in the peptides were coupled to CEIPS templates by reacting onto credit-card format polypropylene PEPSCAN cards (455 peptide formats/card) with a 0.5 mM solution of CEIPS template such as 1,3-bis (bromomethyl) benzene in ammonium bicarbonate (20 mM, pH 7.9)/acetonitrile (l:l(v/v)). The cards were gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the cards were washed extensively with excess of H2O and sonicated in distrupt-buffer containing 1 percent SDS/0.1 percent beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 minutes, followed by sonication in H2O for another 45 minutes. The binding of antibody to each peptide were tested in a PEPSCAN-based ELISA. The 455-well credit card format polypropylene cards containing the covalently linked peptides were incubated with primary antibody solution for example consisting of 1 micrograms/mL diluted in blocking solution called SQ (4% horse serum, 5% ovalbumin (w/v) in PBS/1% Tween or

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2019204295 19 Jun 2019 diluted in PBS e.g., 20%SQ) overnight. After washing, the peptides were incubated with a 1/1000 dilution of rabbit anti-human antibody peroxidase or goat-anti-human FAB peroxidase for one hour at 25° C. After washing, the peroxidase substrate 2,2’-azino-di3-ethylbenzthiazoline sulfonate (ABTS) and 2 microlitres of 3 percent H2O2 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD) - camera and an image processing system (as firstly described in Slootstra et al., 1996).

Data calculation

Raw Data: Optical density (Arbitrary OD units)

The raw data are optical values obtained by a CCD-camera. The values mostly range from 0 to 3000, a log scale similar to 1 to 3 of a standard 96-well plate elisa-reader. First the CCD-camera makes a picture of the card before peroxidase coloring and then again a picture after the peroxidase coloring. These two pictures are subtracted from each other which results in the data which is called raw-data. This is copied into the Peplab^tmdatabase. Then the values are copied to excel and this file is labeled as raw-data file. One follow-up manipulation is allowed. Sometimes a well contains an air-bubble resulting in a false-positive value, the cards are manually inspected and any values caused by an airbubble are scored as 0.

Normally assays are not done in replicate (only upon request client request). Replicate tests are usually very similar. In addition, the dataset of thousands of peptides contains many peptides that are similar, thus results are never based on recognition of one peptide but on families of similar peptides. If one or a few peptides do not bind, or exhibit lower binding, in a replicate experiment, a different epitope mapping is not normally attributed.

Timmerman et al. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. J. Mol. Recognit. 20:283-99

Slootstra et al. (1996). Structural aspects of antibody-antigen interaction revealed through small random peptide libraries, Molecular Diversity,!, 87-96.

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EXAMPLE 9: Analysis of pKal Sequences from Different Species

All available sequence of pKal were obtained from public databases and aligned using ClustalW and regions were highlighted based on solvent accessibility, contact with an active site Kunitz inhibitor, and those peptides identified by CLIPS analysis (FIGURES 10A-10C). Citrated plasma from each of these species was obtained and activated using a commercially available prekallikrein activator (from Enzyme Research Laboratories) according to the instructions of the manufacturer. Kallikrein activity was then measured in each of the samples in the presence or absence of X81-B01.

It was found that X81-B01 inhibited pKal from all the species except for pig pKal. Since the CLIPS analysis identified four peptides of pKal that X81-B01 binds to- C2 (positions 81-94), C3 (positions 101-108), C5 (positions 162-178) and C6 (positions 186197) - differences in the pig pKal sequence that correspond to these peptides were examined to identify potential amino acids changes that account for the lack of inhibition of pig pKal by X81-B01. Peptides C2 and C3 are close in the sequence and are both highly similar in sequence among the different species. However, there is a difference at position 479. All the species except pig, frog, and dog have a serine at position 479. The frog and dog pKal sequence has an alanine and a threonine at position 479, respectively; both of which are considered conservative substitutions for a serine. In contrast, the pig pKal sequence has a leucine at position 479, which is a considerably less conservative substitution for a serine. Peptide C5 in pig pKal is highly similar to the sequences from the other species. However, at position 563, only in the pig pKal is a histidine present (bold in FIGURE 10C). This position in all the other species, except frog, is a tyrosine. In the frog pKal, which is inhibited by X81-B01, this position is a threonine. Peptide C6 in pig pKal is again highly similar to the other sequences. However, only in the pig pKal sequence is position 585 a glutamate (in bold in FIGURE 10C). In all the other species this position is an aspartate. This analysis may indicate potentially critical residues in pKal that interact with X81-B01.

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EXAMPLE 10: In vitro and in vivo assays to assess efficacy of a plasma kallikrein binding protein.

Binding to prekallikrein vs. kallikrein: The advantage of an antibody inhibitor of pKal that does not bind prekallikrein over an antibody that binds prekallikrein can be demonstrated experimentally. For example, an in vitro experiment can be designed to compare the potency of a pKal antibody inhibitor that does not bind prekallikrein (e.g. DX-2922) with one that binds prekallikrein (e.g. M6-D09) using an activated partial thromboplastin time (APTT) plasma clotting time assay. The APTT assay induces clotting in plasma by the addition of a reagent that specifically activates the contact system component of the intrinsic coagulation pathway, of which the activity of pKal is involved. It is well known in the literature that the inhibition of pKal or that a genetic deficiency in pKal leads to prolonged aPPT (see e.g., Morishita, H., et al., Thromb Res, 1994. 73(3-4): p. 193-204 ; Wynne Jones, D., et al., Br J Haematol, 2004.127(2): p. 2203). An in vitro experiment can be performed to measure the effect of spiking citrated human plasma with different concentrations of either M6-D09 or DX-2922 on observed clotting times induced using commercially available APTT reagents and a coagulation analyzer (Table 13). It is expected that the observed EC50 for APTT prolongation of M6-D09 will be significantly higher than that of DX-2922 due to the binding of M6-D09 to the high concentration prekallikrein (~500 nM) in the normal plasma sample. Efficacy of the antibody inhibitor of pKal as demonstrated by prolonging APTT supports the potential therapeutic use of the antibody in treating or preventing cardiovascular disease associated with aberrant clot formation, such as may be observed in atherosclerosis, stroke, vasculitis, aneurism, and patients implanted with ventricular assist devices.

Table 13: Study Design to Measure Effect of Antibody Inhibitors of pKal on APTT

Condition	Observed Effect on APPT
No treatment, just plasma	Normal
Prekallikrein depleted plasma control (commercially available)	Maximum prolongation
M6-D09 at low concentration	Normal
M6-D09 at middle concentration	Normal

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M6-D09 at high concentration	Prolonged APTT
DX-2922 at low concentration	Prolonged APTT
DX-2922 at middle concentration	Prolonged APTT
DX-2922 at high concentration	Maximum prolongation

Efficacy in a rat model of edema: An in vivo experiment can also be conducted to demonstrate the increased potency of an antibody inhibitor of pKal that does not bind prekallikrein. The carrageenan-induced paw edema (CPE) model of edema in rats is a common pharmacology model. A group of rats will be treated with escalating doses of M6-D09 and DX-2922 by intraperitoneal (IP) injection prior to injecting carrageenan (e.g. 0.1 mL of a 10% w/v solution) in the paws of the rats (Table 14). It is expected that DX2922 will be more effective in reducing the observed paw swelling than M6-D09. Efficacy of the antibody supports the therapeutic use of the antibody in various inflammatory diseases that are associated with either swelling (e.g. hereditary angioedema, stroke induced edema, brain edema) or bradykinin mediated inflammation and pain (e.g. rheumatoid arthritis, inflammatory bowel disease).

Table 14: Study Design to Observe Effect of Antibody Inhibitors on CPE

Group	Treatment	Example Dose (mg/Kg)	Effect Expected
1	Vehicle	N/A	Maximum swelling
2	Indomethacin (positive control)	5	Maximum reduction of swelling
3	M6-D09	1	No effect on swelling
4	M6-D09	3	No effect on swelling
5	M6-D09	10	Intermediate reduction of swelling
6	DX-2922	1	No effect on swelling
7	DX-2922	3	Intermediate reduction of swelling
8	DX-2922	10	Maximum reduction of swelling

Measuring half-life: The pharmacokinetic properties of DX-2922 will be determined in rats and cynomolgus monkeys in a study design outlined in Table 15.

Serum will be collected at the times indicated following IV injection of DX-2922. The concentration of DX-2922 will be determined by ELISA and plotted versus time in order to obtain pharmacokinetic parameters (clearance, half life, volume of distribution, etc). It

237

2019204295 19 Jun 2019 is expected that DX-2922 will have a half-life greater than 5 days in cynomolgus monkeys that will scale to a half life greater than 7 days in humans.

Table 15: A Pharmacokinetic study of plasma kallikrein inhibitor pKal administered by intravenous and subcutaneous routes to cynomolgus monkeys.

Group	Treatment	Dosage Level (mg/kg/day)	Route	Number of Animals (sex)	Serum Collection
1	pKAL	20	IV	4 (M)	Pre-dose 0.08,0.25.0.5, 1,4, 24 hours Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
2	pKAL	20	IV	4 (M)	Pre-dose 0.08,0.25.0.5, 1,4, 24 hours Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

EXAMPLE 11: Epitope mapping using amino acid mutations of pKal

Based on the epitope mapping studies described herein in Example 8, we inspected the published 3 dimensional model in the RCSB Protein Data Bank (available on the world wide web at rcsb.org; pdb code 2ANY) and identified a collection of sets of amino acids in surface accessible loops near the enzyme active site that we reasoned could interact with the antibody binding resulting in enzyme inhibition. These amino acids were substituted for alanine and the catalytic domain of each of the mutant was expressed in Pichia pastoris with a His tag fusion and purified by IMAC. Four different mutant pKal mutants were synthesized and tested:

Mutant 1: Amino acids S478, N481, S525, and K526 of the human kallikrein sequence (Accession No. NP_00883.2) were mutated to alanine. These amino acids were determined to be involved in substrate recognition (S3 subsite).

Mutant 2: Amino acid residues R551, Q553, Y555, T558 and R560 of the human kallikrein sequence (Accession No. NP_00883.2) were mutated to alanine. It was determined that these residues are involved in the active site substrate recognition (S1 ’ subsite).

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2019204295 19 Jun 2019

Mutant 3: Amino acids D572, K575, and D577 of the human kallikrein sequence (Accession No. NP_00883.2) were mutated to alanine. These amino acid residues are involved in substrate recognition (St ’ subsite)

Mutant 4: Amino acids N395, S397 and S398 of the human kallikrein sequence (Accession No. NP_00883.2) were mutated to alanine. These residues are distal from the active site of plasma kallikrein.

Three of the 4 mutants (Mutant 1, 2, and 4) have similar activity to that of the wildtype catalytic domain of pKal. The amino acid substitutions in Mutant 3 yielded an inactive protein that was not recognized in SPR (Biacore) binding assays by any of the tested anti-pKal antibodies.

The antibodies tested for inhibition of mutants 1, 2 and 4 are shown herein in Table 16. Based on the measured Ki_iapp values for the antibodies in Group 1 (i.e., antibodies that inhibit human and mouse pKal but do not bind prekallikrein) it is evident that this group of antibodies binds an epitope on pKal that contains the amino acids that were mutated in Mutant 2 but were not dependent on residues mutated in Mutants 1 or 4. In addition, the interaction of plasma kallikrein binding proteins X81-B01/X101A01/DX-2922 and affinity matured derivative XI15-B07 to kallikrein is adversely affected by the substitutions in Mutant 1. For an example of the differences in the ability of the antibodies to bind prekallikrein see e.g., Figure 11A and 1 IB, which compares prekallikrein the binding of DX-2922 (Group 1) to that of M6-D09 (Group 3).

The antibodies in Group 2 (i.e., those that inhibit human pKal not mouse pkal and do not bind prekallikrein) were not significantly affected by the mutated amino acids indicating that they make contact with alternate amino acids. The Group 2 antibodies are likely to bind near the active site, as they were unable to bind pKal complexed with a Kunitz domain (EPI-KAL2), which are known to bind at the active site of a serine protease. Furthermore, one of the antibodies in Group 2 (M145-D11) is similar to those in Group 1 in that it is unable to bind pKal in a Biacore assay that is inactivated with the suicide inhibitor AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), which is a small molecule covalent inhibitor of trypsin-like serine proteases (Figure 12). However, the other antibody (M29-D09) assigned to Group 2 was able to bind AEBSF

239

2019204295 19 Jun 2019 inactivated pKal, indicating that it may bind a different epitope than M145-D11 despite sharing similar binding properties.

The antibodies in Group 3 inhibited human and mouse pKal but bound prekallikrein. One of these antibodies, M6-D09, was unable to bind pKal inactivated by either EPI-KAL2 or AEBSF, indicating that this group of pKal inhibitors interacts with alternative amino acids near the active site. The Ki_iapp for M6-D09 increased approximately 5-fold towards Mutant 2 (i.e., decreased potency of M6-D09).

EXAMPLE 12: Affinity Maturation

In addition to the affinity maturation described herein in Examples 4 and 5, which involved optimization of the light chain we attempted to further optimize affinity with libraries that vary amino acids in the CDR1, CDR2, and CDR3 regions of the variable heavy chain of two different parental anti-pKal antibodies. Both of the antibodies selectedfor further optimization (X63-G06 and M162-A04) exhibit desirable properties for further development as a therapeutic antibody inhibitor of plasma kallikrein; properties which include: a) complete inhibition of human and rodent plasma kallikrein and b) no binding to prekallikrein. In some embodiments, complete inhibition of human pKal is essential to block the activity of plasma kallikrein in disease uses. Inhibition of rodent pKal facilitates preclinical development including toxicity assessment. The lack of binding to prekallikrein is a highly desirable property for an antibody inhibitor of pKal to maximize the bioavailability of the antibody therapeutic towards active pKal target and to potentially reduce the dose required for efficacy.

Affinity maturation was performed using 4 different phage display libraries. For each parental antibody (e.g., I62-A04), a library was constructed that contained varied amino acid positions in both the CDR1 and the CDR2 of the heavy chain. An additional library was constructed for each of the two parental antibodies wherein positions in the CDR3 of the heavy chain were varied. Each of these 4 phage display libraries were selected (panned) with decreasing amounts of active pKal in each subsequent round in order to obtain high affinity antibodies. To minimize the appearance of prekallikrein binding in the selected antibody output libraries were initially depleted against immobilized prekallikrein. After screening as Fab fragments, we discovered the affinity

240

2019204295 19 Jun 2019 matured antibodies shown in Table 16 (i.e. the antibodies with the identification number starting with “XI15”).

Four discovered antibodies (XI15-B07, XI15-D05, XI15-E09, and XI15-H06) are derived from the DX-2922 parental antibody (also known as X63-G06 as a Fab fragment, X8f-B0f as an IgG produced in 293T cells, or X101-A01 as an IgG produced in CHO cells) were found to be potent pKal inhibitors. For comparison the amino acid sequence of DX-2922 is shown. It is evident that three of the affinity matured antibodies (XI15-B07, XI15-E09, and XI15-H06) contain mutations in Hv-CDR3; whereas XI15D05 has a different Hv-CDR1/CDR2. Four other discovered antibodies (XI15-F02, XI15-A03, XI15-D01, and XI15-G04) are derived from the Mf62-A04 parental antibody. All 8 affinity matured antibodies do not bind prekallikrein.

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Table 16: Summary of Affinity Matured Anti-pKal Antibodies Inhibition Constants (Ki,app) on Wild Type pKal Cataly Domain and Mutants 1,2, and 4^a.	Characteristics	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein	inhibits human and mouse pKal; does not bind pre-kallikrein
Competes with epika!2	>	nd	nd	nd	nd	nd	nd	>	>
Competes with AEBSF	>	>	>	>	>		>	>	>
Mutant 4 Ki, app (nM)	0.25	0.14	nd	nd	nd	0.13	0.12	ό	0.3
Mutant 2 Ki, app (nM)	20	47	nd	nd	nd	3.7	2.5	5.9	2.2
Mutant 1 Ki, app (nM)		4.7	nd	nd	nd	0.23	0.26	0.14	0.4
WT cat. Domain Ki, app (nM)	0.22	0.13	nd	nd	nd	0.16	0.18	60Ό	0.3
Isolate	DX-2922	559A-X115-B07 (aff mat; X101-A01 parent)	559A-X115-D05 (aff mat; X101-A01 parent)	559A-X115-E09 (aff mat; X101-A01 parent)	559A-X115-H06 (aff mat; X101-A01 parent)	559A-X115-A03 (aff mat; M162-A04 parent)	559A-X115-D01 (aff mat; M162-A04 parent)	559A-X115-F02 (aff mat; M162-A04 parent)	559A-X115-G04 (aff mat; M162-A04

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2019204295 19 Jun 2019

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2019204295 19 Jun 2019

Equilibrium K^_app measurements. Apparent Inhibition constants (Ki_iapp values) were measured by pre-incubating enzyme and inhibitor solutions prior to initiating the reactions with substrate. Enzyme and inhibitor were pre-incubated for 2 hours at 30°C in a 96-well plate by adding 10 pL of a 10X enzyme solution and 10 pL of 10X inhibitor solutions to 70 pL of reaction buffer. Reactions were initiated by the addition of 10 pL of a 10X concentrated stock of substrate, and were monitored at 30°C in a fluorescence plate reader with the excitation and emission wavelengths set at 360 nm/460 nm, respectively. Kinetic data were acquired by the increase in fluorescence, and initial rates for each condition were plotted against the total inhibitor concentration. The data was fit to the following equation for tight binding inhibitors:

A = Ao,_app +Inh + e)~/κ._αρρ + Inh + e)²-4-Inh e

2-E

Eqn. 1

Where A = initial rate observed at each inhibitor concentration; A_o = initial rate observed in the absence of inhibitor; Ainh = initial rate observed for the enzyme inhibitor complex; Inh = concentration of inhibitor; E = total enzyme concentration (treated as a floated parameter); and K_papp = apparent equilibrium inhibition constant.

Groups of Antibody Inhibitors. Antibodies in Group 1 inhibit human and mouse pKal but do not bind prekallikrein. Antibodies in Group 2 inhibit human but not mouse pKal and do not bind prekallikrein. Antibodies in Group 3 inhibit human and mouse pKal but bind prekallikrein.

Biacore competition analysis with an exemplary kallikrein antibody, epi-Kal2.

Epi-Kal2 is an antibody inhibitor of kallikrein that acts by binding to the active site of kallikrein (for sequence see Example 8). The Biacore competition analysis is used herein as an assay to determine whether a test kallikrein antibody binds to the same site as epi-Kal2 and is assessed by measuring the competition (e.g., displacement) between epi-Kal2 and the test antibody for binding to the active site.

Goat anti-human Fc fragment specific IgG or anti-human Fab IgG was immobilized by amine coupling on a CM5 sensor chip at immobilization densities of

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2019204295 19 Jun 2019 approximately 5000 RU. Anti-pKal antibodies or sFabs were captured on their respective surfaces by injecting a 50 nM solution of IgG/sFab for 1-2 minutes at 5 at μΐ/min. Human pKal (100 nM) or human pKal-ep-kal2 complex (100 nM hpKal that had been pre-incubated with 1 μΜ epi-kal2 for 1 hour at room temperature) were injected over the captured IgGs or sFabs for 5 minutes at 20-50 μΐ/min followed by a 5-10 minute dissociation phase. Binding responses were recorded at the end of the association phase. Anti-pKal IgGs or sFabs were considered to compete with epi-kal2 for binding to human pKal if binding of the pKal-epi-kal2 complex to anti-pKal antibodies was significantly reduced (> 70%) as compared to an injection of hpKal only. The sensor chip surface was regenerated with a pulse of 10 mM glycine pH 1.5 at a flow rate of 100 μΐ/min. Measurements were performed at 25°C using HBS-P (10 mM HEPES pH 7.4, 150 mM NaCI and 0.005% surfactant P20) as the running buffer. Results from the Biacore competition analysis for epi-Kal2 are shown herein in Figures 11A and 1 IB.

Biacore competition analysis with the small molecule kallikrein inhibitor, AEBSF.

AEBSF (i.e., 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride) is a small molecule inhibitor of kallikrein. The Biacore competition analysis is used herein to determine whether a test antibody binds to the same site (or an overlapping site) utilized by AEBSF for kallikrein inhibition.

Goat anti-human Fc fragment specific IgG or anti-human Fab IgG was immobilized by amine coupling on a CM5 sensor chip at immobilization densities of approximately 5000 RU. Anti-pKal IgGs or sFabs were captured on their respective surfaces by injecting a 50 nM solution of IgG/sFab for 1-2 minutes at 5 at μΐ/min. Human pKal (100 nM) or human pKal-AEBSF complex (100 nM hpKal that had been pre-treated with 1 mM AEBSF for 1 hour at room temperature) were injected over the captured IgGs or sFabs for 5 minutes at 20-50 μΐ/min followed by a 5-10 minute dissociation phase. Binding responses were recorded at the end of the association phase. Anti-pKal IgGs or sFabs were considered to compete with AEBSF for binding to human pKal if binding of the pKal-AEBSF complex to anti-pKal antibodies was significantly reduced (> 70%) as compared to an injection of hpKal only. The sensor chip surface was

246 regenerated with a pulse of 10 mM glycine pH 1.5 at a flow rate of 100 μΙ/min.

Measurements were performed at 25°C using HBS-P (10 mM HEPES pH 7.4, 150 mM

NaCl and 0.005% surfactant P20) as the running buffer. Results from the Biacore competition analysis for AEBSF are shown herein in Figure 12.

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The following are Sequences for the light chain variable regions (LV), and heavy chain variable regions (HV) regions for 8 Exemplary Affinity Matured Anti-pKal Antibodies: 559A-M0029-DO9-LV

QSALTQPPTVSVSPGQTARITCSGNKLGDKYVAWYQQKPGQSPMLVIYQDTKRPSRVSERFSGSNSANTAT

LSISGTQALDEADYYCQAWDSSIVIFGGGTRLTVL

559A-M0145-DI1-LV

QSVLTQPPSVSVSPGQTASITCSGDKLGDKYTSWYQQRPGQSPVLVIYQDIKRPSGIPERFSGSNSGNTAT

LTISGTQAMDEADYYCQAWDSPNARVFGSGTKVTVL

5 9A-M0162-A0 4-LV

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPNLLIYKASTLESGVPSRFSGSGSGTEF

TLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK

559A-X0101-A01-LV

EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD

FTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK

559A-X0115-A03-LV

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEF

TLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK

559A-X0115-B07-LV

EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD

FTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK

559A-X0115-DO1-LV

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEF

TLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK

559A-X0115-DO5-LV

EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD

FTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK

559A-X0115-E09-LV

EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD

FTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK

559A-X0115-F02-LV

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEF

TLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK

559A-X0115-GO 4-LV

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEF

TLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK

559A-X0115-HO6-LV

EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD

FTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK

559A-M0006-DO9-LV

DIQMTQSPSSLSASVGDRVTITCRASQSIRNYLNWYQQKPGKAPNLLIYAASTLQSGVPARFSGSGSGTDF

TLTISSLQPEDFATYYCQQLSGYPHTFGQGTKLEIK

559A-M0035-G04-LV

QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD

FTLTISSLEPEDFAVYYCQQRSNWPRGFTFGPGTKVDIK

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559A-M0029-DO9-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMVWVRQAPGKGLEWVSYIYPSGGATFYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAMGSYDYIWGFYSDHWGQGTLVTVSS

559A-M0145-DI1-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYRMSWVRQAPGKGLEWVSSIYPSGGRTVYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAKDKFEWRLLFRGIGNDAFDIWGQGTMVTVSS

5 9A-M0162-AO 4-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAYRRTGIPRRDAFDIWGQGTMVTVSS

559A-X0101-AO1-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCARVARGIAARSRTSYFDYWGQGTLVTVSS

559A-X0115-AO3-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAWRRIGVPRRDSFDMWGQGTMVTVSS

559A-X0115-BO7-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAMVGQGIRGRSRTSYFAQWGQGTLVTVSS

559A-X0115-DO1-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYSMHWVRQAPGKGLEWVSSIYPSRGMTWYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAYRRTGIPRRDAFDIWGQGTMVTVSS

559A-X0115-DO5-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYMMAWVRQAPGKGLEWVSSIVPSGGHTHYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCARVARGIAARSRTSYFDYWGQGTLVTVSS

559A-X0115-EO9-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCARVAQGIAARSRTSSVDQWGQGTLVTVSS

559A-X0115-F02-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSS

559A-X0115-GO 4-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAYRRTGVPRRDEFDIWGQGTMVTVSS

559A-X0115-HO6-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCARVAQGISARSRTSYFDYWGQGTLVTVSS 559A-M0006-D09-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYYMVWVRQAPGKGLEWVSVIYPSGGITVYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCARDKWAVMPPYYYYAMDVWGQGTTVTVSS 559A-M0035-G04-HV

EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYHMSWVRQAPGKGLEWVSVISPSGGSTKYADSVKGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCARGGSSDYAWGSYRRPYYFDYWGQGTLVTVSS

559A-M0029-D09 LV

CAGAGCGCTTTGACTCAGCCACCCACAGTGTCTGTGTCCCCAGGACAGACAGCCAGGATCACCTGCTCTGG AAATAAATTGGGGGATAAATATGTTGCCTGGTATCAGCAGAAGCCAGGCCAGTCCCCTATGTTGGTCATCT ATCAAGATACTAAGCGCCCCTCAAGAGTTTCTGAGCGATTCTCTGGCTCCAACTCTGCGAATACAGCCACT CTGTCCATCAGCGGGACCCAGGCTCTGGATGAGGCTGACTATTACTGTCAGGCGTGGGACAGCAGCATTGT GATCTTCGGCGGAGGGACCAGGCTGACCGTCCTA

559A-M0145-DI1 LV

CAGAGCGTCTTGACTCAGCCACCCTCAGTGTCCGTGTCTCCAGGACAGACAGCCAGCATCACCTGCTCTGG

AGATAAATTGGGGGATAAATATACTTCCTGGTATCAGCAGAGGCCAGGCCAGTCCCCTGTATTGGTCATCT

ATCAAGATATCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACT

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CTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCAGGCGTGGGACAGTCCCAATGC

GAGGGTCTTCGGATCTGGGACCAAGGTCACCGTCCTA

559A-M0162-AO 4 LV

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCG GGCCAGTCAGAGTATCAGTAGTTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGA TCTATAAGGCGTCTACTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTC ACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATACTTATTG GACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

559A-X0101-AO 1 LV

GAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGTCCTGCCG GACCTCCCAGTTCGTGAACTCCAACTACCTGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGC TGATCTACGGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGAC TTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCCGGAC CCCTTGGACCTTTGGCCAGGGCACCAAGGTGGAGATCAAG

559A-X0115-AO3 LV

GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCG GGCCTCCCAGTCCATCTCCAGCTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA TCTACAAGGCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTC ACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACTG GACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG

559A-X0115-BO7 LV

559A-X0115-DO 1 LV

559A-X0115-DO5 LV

559A-X0115-EO9 LV

559A-X0115-F02 LV

GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCG

GGCCTCCCAGTCCATCTCCAGCTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA

TCTACAAGGCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTC

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ACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACTG

GACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG

559A-X0115-GO 4 LV

559A-X0115-HO 6 LV

559A-M0006-DO9 LV

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCG GGCAAGTCAGAGTATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGA TCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCAGCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACTATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAGTGGTTACCC CCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

559A-M0035-G04 LV

CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG CAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAC TTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTG GCCTCGCGGATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

559A-M0029-DO9 HV

GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGC TTCCGGATTCACTTTCTCTTGGTACACTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGG TTTCTTATATCTATCCTTCTGGTGGCGCTACTTTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCT AGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTA CTGTGCGATGGGTTCATATGATTACATTTGGGGATTTTATAGTGACCACTGGGGCCAGGGAACCCTGGTCA CCGTCTCAAGC

559A-M0145-DI1 HV

GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGC TTCCGGATTCACTTTCTCTCATTACCGTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGG TTTCTTCTATCTATCCTTCTGGTGGCCGTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCT AGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTA CTGTGCGAAAGATAAGTTCGAGTGGAGGTTATTATTTCGCGGGATTGGAAATGATGCTTTTGATATCTGGG GCCAAGGGACAATGGTCACCGTCTCAAGC

559A-M0162-AO 4 HV

GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGC

TTCCGGATTCACTTTCTCTCATTACATTATGATGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGG

TTTCTGGTATCTATTCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCT

AGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTA

CTGTGCGTACCGCCGGACTGGGATTCCAAGAAGAGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCA

CCGTCTCAAGC

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559A-X0101-AO 1 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGTCTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGCCAGGCTCCTGGCAAGGGCCTCGAATGGG TGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCC CGGGACAACTCCAAGAACACCCTGTATCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTA CTGCGCCAGGGTGGCCAGAGGAATCGCCGCCAGGTCCCGGACCTCCTACTTCGACTACTGGGGCCAGGGCA CCCTGGTGACCGTGTCCTCC

559A-X0115-AO3 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTTACCTTCTCCCACTACATCATGATGTGGGTGCGACAGGCTCCAGGCAAGGGCCTGGAATGGG TGTCCGGCATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGTGCCTGGCGGAGAATCGGCGTGCCCAGACGGGACTCCTTCGACATGTGGGGACAGGGCACCATGGTGA CAGTGTCCTCC

559A-X0115-BO7 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGG TGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTTACCATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGTGCCATGGTCGGCCAGGGAATCCGGGGCAGATCCCGGACCTCCTACTTCGCCCAGTGGGGCCAGGGCA CCCTGGTGACAGTGTCCTCT

559A-X0115-DO 1 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCATCTACTCCATGCACTGGGTGCGACAGGCTCCAGGCAAGGGCCTGGAATGGG TGTCCTCCATCTACCCCTCCCGGGGCATGACTTGGTACGCCGACTCCGTGAAGGGCCGGTTCACAATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGCGCCTACCGGCGGACCGGCATCCCTAGACGGGACGCCTTCGACATCTGGGGGCAGGGCACCATGGTGA CAGTGTCCTCC

559A-X0115-DO5 HV

GAGGTGCAATTGCTGGAATCCGGCGGTGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCGACTACATGATGGCCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGG TGTCCTCCATCGTGCCCTCTGGCGGCCACACCCACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGCGCCAGAGTGGCCAGAGGAATCGCCGCCAGATCCCGGACCTCCTACTTCGACTACTGGGGCCAGGGCA CCCTGGTGACAGTGTCCTCC

559A-X0115-EO9 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGG TGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTTACCATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGTGCCCGGGTGGCCCAGGGAATCGCCGCCAGATCCCGGACCTCCTCTGTGGATCAGTGGGGCCAGGGCA CCCTGGTGACAGTGTCCTCT

559A-X0115-F02 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC

CTCCGGCTTCACCTTCTCCCACTACATCATGATGTGGGTGCGACAGGCTCCTGGCAAGGGGCTGGAATGGG

TGTCCGGCATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCT

CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA

CTGCGCCTACCGGCGGATCGGCGTGCCCAGACGGGACGAGTTCGACATCTGGGGGCAGGGCACCATGGTGA

CAGTGTCCTCC

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559A-X0115-GO 4 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCTCACTACATTATGATGTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGG TGTCCGGCATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGCGCCTACAGACGGACCGGCGTGCCCAGACGGGACGAGTTCGATATCTGGGGGCAGGGCACCATGGTGA CAGTGTCCTCC

559A-X0115-HO 6 HV

GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGC CTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGG TGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTTACCATCTCC CGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGTGCCCGGGTGGCCCAGGGAATCTCCGCCAGATCCCGGACCTCCTACTTCGATTACTGGGGCCAGGGCA CCCTGGTGACAGTGTCCTCT

559A-M0006-DO9 HV

GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGC TTCCGGATTCACTTTCTCTTTTTACTATATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGG TTTCTGTTATCTATCCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCT AGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTA CTGTGCGAGAGATAAATGGGCGGTGATGCCCCCCTACTACTACTACGCTATGGACGTCTGGGGCCAAGGGA CCACGGTCACCGTCTCAAGC

559A-M0035-G04 HV

GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGC TTCCGGATTCACTTTCTCTTATTACCATATGTCTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGG TTTCTGTTATCTCTCCTTCTGGTGGCTCTACTAAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCT AGAGACAAC T C TAAGAATAC TC T C TAC T T GCAGAT GAACAGC T TAAGGGC T GAGGACAC TGCAGT C TAC TA TTGTGCGAGAGGCGGTTCGAGCGATTACGCTTGGGGGAGTTATCGTCGACCCTACTACTTTGACTACTGGG GCCAGGGAACCCTGGTCACCGTCTCAAGC

Claims

1. An antibody or functional fragment thereof that binds to the active form of human plasma kallikrein and does not bind human prekallikrein.

2. An antibody or antigen binding fragment thereof that binds the same epitope or competes for binding with an antibody or antibody fragment selected from the group consisting of X81-B01, X63-G06, X101-A01, M162-A04, M160-G12, M142-H08, X63G06, X101-A01, X67-D03, X67-G04, XI15-B07, XI15-D05, XI15-E09, XI15-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

3. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen binding fragment thereof competes with or binds the same epitope as X81-B01.

4. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen binding fragment thereof competes with or binds the same epitope as X67-D03.

5. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen binding fragment thereof competes with or binds the same epitope as X101-A01.

6. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen binding fragment thereof competes with or binds the same epitope as M162-A04.

7. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen binding fragment thereof competes with or binds the same epitope as X63-G06.

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8. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof binds human plasma kallikrein and murine plasma kallikrein but does not bind human prekallikrein.

9. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment binds one or more of amino acids of: Arg551, Gln553, Tyr555, Thr558, and Arg560 of human plasma kallikrein.

10. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof has an apparent inhibition constant (Kj_!app) of plasma kallikrein of less than 100 nM.

11. The antibody or antigen binding fragment thereof of claim 10, wherein the antibody or antigen binding fragment thereof has an apparent inhibition constant (Ki,_app) of plasma kallikrein of less than 10 nM.

12. The antibody or antigen binding fragment thereof of claim 11, wherein the antibody or antigen binding fragment thereof has an apparent inhibition constant (Kj_!app) of plasma kallikrein of less than 1 nM.

13. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody is an IgG or the antigen binding fragment thereof is a soluble Fab (sFab).

14. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen binding fragment thereof comprises a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein:

the heavy chain immunoglobulin variable domain sequence comprises three CDR regions from the heavy chain variable domain of an antibody or antibody fragment 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, X115254

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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 three CDR regions from the light chain variable domain of an antibody or antibody fragment selected from the group consisting of Mf62-A04, Mf60-Gf2, Mf42-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, XI

15-B07, X115-D05, XI15-E09, X115H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

f5. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof comprises three CDR regions from the heavy chain variable domain of X81-B01 and/or three CDR regions from the light chain variable domain ofX81-B01.

16. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof comprises three CDR regions from the heavy chain variable domain of X67-D03 and/or three CDR regions from the light chain variable domain of X67-D03.

17. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof comprises three CDR regions from the heavy chain variable domain of X101-A01 and/or three CDR regions from the light chain variable domain ofX101-A01.

18. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof comprises three CDR regions from the heavy chain variable domain of X63-G06 and/or three CDR regions from the light chain variable domain of X63-G06.

19. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof comprises three CDR regions from the

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2019204295 19 Jun 2019 heavy chain variable domain of M162-A04 and/or three CDR regions from the light chain variable domain of M162-A04.

20. The antibody or antigen binding fragment thereof of claim 14, wherein the heavy chain variable region comprises the heavy chain variable domain of X81-B01, X63-G06, X101-A01, M162-A04, M160-G12, M142-H08, X67-D03, X67-G04, X115B07, XI15-D05, X115-E09, XI15-H06, XI15-A03, X115-D01, XI15-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chain variable region comprises the light chain variable domain ofX81-B01, X63-G06, X101-A01, Ml 62A04, M160-G12, M142-H08, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06D09 and M35-G04.

21. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of claim 1 and a pharmaceutically acceptable carrier.

22. A method of treating or preventing a plasma kallikrein associated disorder in a subject, the method comprising:

administering an antibody or antigen binding fragment thereof of claim 1 to the subject.

23. The method of claim 22, wherein the plasma kallikrein associated disorder is selected from the group consisting of edema rheumatoid arthritis, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, post operative ileus, aortic aneurysm, osteoarthritis, vasculitis, hereditary angioedema, cerebral edema, pulmonary embolism, stroke, clotting on ventricular assistance devices or stents, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, and burn injury.

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24. A method of detecting plasma kallikrein in a subject, the method comprising: administering an antibody or antigen binding fragment thereof of claim 2 to a subject; and detecting an interaction between the protein and the plasma kallikrein in the subject, if present.

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CN CN CN CN o CN LO CN CN CN CN o CN LO CN CN CN CN o CN LO CN CN CN CN o CN CN LO ,-1 t—1 t—1 t—1 CN t—1 t—1 o (to o- (to o- co o- o- to to to to to to CN LO LO LO LO <© LO LO CN CN CN CN CN CN CN CN Η E-4 E-4 E-4 E-4 E-4 E-4 E-4 * x to to to to to x to to to to to to to to E-4 E-4 E-4 E-4 E-4 E-4 E-4 E-4 4c O O O O O O O o 4c o o o o o o o o 4c to to to to to to to to O O O O O O O O 4c to s s s s > s s > >4 to x to to >4 to to 1—1 1—1 1—1 > 1—1 1—1 1—1 1—1 S to to to s to s s to to to co to to to to to to to to to to to to 4c E-4 E-4 E-4 E-4 E-4 to E-4 E-4 ο oi Oi Oi oi Oi oi Oi 4c 1—1 1—1 1—1 to 1—1 1—1 1—1 1—1 to to to to to to to to to to to to to Oi to Oi O o o o o o o O 4c Oi Oi Oi oi Oi to Oi Oi to to to to to >4 to to O O O o O O O O 4c >4 >4 >4 x to >4 to x to to to to to to to to o o o o o o o o 4c > > > > > E-4 > > oi oi Oi oi to ω Oi to o o o o o to o o to E-4 to to to to E-4 to to to to to to to to to > o o o o o o o o 4c to to to to to to to to > to Q Q Q to Q to to oi to oi oi ο to oi ο to to to to to to to to 4c Q Q Q Q Q Q Q Q 4c to to to to to to to to 4c to to to to to to to to 4c >4 >4 >4 >4 >4 to >4 >4 to to to to to to to to 4c Η E-4 E-4 E-4 E-4 E-4 E-4 to to to to to to to to 4c ω ω ω ω ω oi ω ω E-4 E-4 E-4 E-4 >4 >4 >4 >4 >4 to >4 >4 to to to to to to to to 4c to to > to 1—1 1—1 £> 1—1 to to £> S 1—1 1—1 > s to to to to to to to to 4c to to to to to to to to > 1—1 > > > > > > co % 3 to to o o o o o o o o 4c to to >4 to to x to >4 to to Oi Oi to to to to to to to E-4 to to to to to to to to 4c to to to to to to to to 4c to to E-4 > > to to > to > to > 1—1 1—1 1—1 1—1 1—1 1—1 1—1 E-4 E-4 ω E-4 E-4 > > > > > to > > Q Q Q Q Q Q Q Q 4c > ο to to to oi oi Oi ω Q Q to to Q E-4 Q Q to to to to to to to to 4c o to to to to to to to E-4 E-4 E-4 E-4 E-4 E-4 E-4 E-4 4c > > to to > ω > > to to to to to to to to 4c E-4 E-4 E-4 E-4 ω E-4 E-4 > Q Q Q Q Q to Q Q to to to to to oi to to to to to to to to to 3 >4 >4 >4 >4 >4 >4 >4 >4 4c to to to to to to Ito to to to to 3 to to to to to to to E-4 to to to E-4 E-4 E-4 E-4 E-4 to E-4 to to to to to to to to to to to to to >4 to to > > 1—1 1—1 > > > S to to to to to to to to 4c ω o 1—1 E-4 E-4 E-4 1—1 E-4 to to to to to to to to to to to to to >4 to to to to to to to S to to to 2 to to to Q to to to to to to to to to to 4c ο ο ο ο to Oi ο ο to to to to to to to to ω x to to Oi ω to to to to to to to to to 4c o O o o o o o O 4c oi oi oi oi oi oi oi oi 4c >4 >4 >4 >4 to >4 >4 >4 E-4 E-4 E-4 E-4 S s E-4 E-4 to x to to to >4 to x to to to to to to to to 4c to to to to to to to to 4c to to to to o to to ω 1—1 1—1 to 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 to 1—1 1—1 oi oi Oi ο oi ω oi ο E-4 E-4 E-4 E-4 E-4 E-4 E-4 E-4 4c to to to to to Q to to to Q Q Q Q Q to to P E-4 E-4 E-4 to to to E-4 E-4 > > 1—1 1—1 > > > to to to to to to to to 4c S > s s S to S S to to s to to 1—1 to to to to to to to to to to 4c E-4 E-4 E-4 E-4 E-4 E-4 E-4 E-4 4c to to to to to oi to to ϋ O o O o o O O o 4c Q Q ω ω Q ω Q Q O O O O o O O o 4c o o O O o o o o 4c to to to to o to to to 4c to to to to to to to to 4c to to to to to to to to o to to to o u o o 4c O o O o o to O Oi to to s s to to to to to ω to to ω ω to to 1—1 1—1 1—1 1—1 1—1 to 1—1 1—1 to to to to to to to to 4c to to to to to >4 to to oi oi Oi Oi oi to oi oi ω to ω ω ω co ω ω > > > > > > > to 4c O O o o o o O o 4c o o o o o o o o 4c to to to to to to to to 4c E-4 E-4 E-4 E-4 to E-4 s to to to to to to to ω ω ω ω ω oi ω ω to to to to Q to to to E-4 to to to to to to to to to 4c ω to ω ω ω oi ω ω o o to to to to to to to to to to to to to to to to 4c > 1—1 1—1 > > > > o o o o O o o o 4c to to to to to to to to to to to to ω to to to to to to E-4 to to to to to to to to to to O to to o to to ω Q to Q Q to ω to to to Q Q to to to to to to to to to to to 4c 1—1 1—1 > > s to 1—1 s E-4 E-4 E-4 ω s > > > E-4 E-4 > E-4 > > to to to to to to to to 4c to to to to to to to Q Q ω ω > Q Q to to to to to to to to to to to to to to to to 4c >4 >4 >4 >4 >4 to >4 to to to to to to 2 to to to to to to E-4 to to to to to to to to to to 4c E-4 to to > to E-4 1—1 E-4 to E-4 E-4 to 1—1 E-4 > 1—1 1—1 > > > > o to o o o to o o 4c > > to to to to to to to 3 to to to to to to to to to to 4c to to to to to to to to 4c oi Oi to ο ο to oi to to 2 E-4 to Q to to to to to to to to >4 to to ω ω > > ω 1—1 ω to to to to to >4 to to ω > > ω to to to to to to to to 4c > > to to > > > to to to to to >4 to 1—1 to to to to to to > > to to > E-4 > > to Q to to to to to Q to to to to O to to to to to to to to to to 4c o o o o o to o o 4c to to Q Q Q to 1—1 1—1 1—1 1—1 1—1 1—1 S to to to to to >4 to to to to to to to 1—1 to to to to > to to to to s S S S 1—1 2? s to to CO to to to to to 4c s s s s s S s s 4c > > to to > to > > to to to to to to to to to 4c Q Q Q Q Q Q Q Q 4c to to to to to to to to to to to to to to s to to > 1—1 to to 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 4c to to to to to to to to to 4c to to to to to to to to 4c to to to to to E-4 to > O o O O O o O o 4c to to to to to to to to E-4 E-4 E-4 E-4 E-4 E-4 E-4 to 1—1 to to to to to to to to 4c >4 >4 >4 >4 >4 to >4 x to to to to to to to to 4c > to to to to to to to to 4c 1—1 1—1 1—1 1—1 1—1 to 1—1 1—1 E-4 E-4 E-4 E-4 E-4 s E-4 E-4 ω χ x χ x x to x x Q to Q Q Q to Q Q to to to to to to to to 4c S to to to to to to to to * to to to Oi to Q to to to Oi to to to to to to

d o to <υ O to <υ O to <υ O cd ω to to ω to to ω to to ω to £ 3 to to to o o to £ 3 to to to o o to £ 3 to to to o o to £ 3 to to to o o to to O o to •1—1 to o to O o to •1—1 to o to O o to •1—1 to o to O o to •1—1 to o to o S to to to s Q to o S to to to S Q to o S to to to S Q to o S to to to S Q

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2019204295 19 Jun 2019 d (0

07 07 07 O co CN CN o 07 o o LO LO o ο ο LO LO LO o LO co co o 0 0 ,—1 LO CO CO CO CO 07 CO 07 co LO LO U ο ο o o ,-1 o o 07 O LO CN CN CN CN CN CN CN CN o O o o O o o ο υ Η o Q-i Q-i Q-i Q-i Q-i CO Q-i Q-i o o o o o O o ο * οι οι Ol Ol Ol Ol Ol Ol * [xj [xj co [xj [XJ [xj [xj [XJ J J Ol Ol J LG J J -κ μ :¾ :¾ :¾ :¾ :¾ :¾ :¾ :¾ -K + Q-i CO Q-i Q-i Q-i Q-i Q-i Q-i H H H H CO H o O O O O O O Ο -Κ >1 LG μ-ι LG LG LG LG LG LG -K + H H H H H H H H -K O', CX til til O< O O O <—ι -K ΓΝ ΓΝ ΓΝ CO ΓΝ ΓΝ til til i r. i r. til til til ΓτΊ ΓτΊ It | It | ΓτΊ ΓτΊ ΓτΊ nJ |τ I |τ I |T | |T | |T | It | |T | |T | -K H e, C C C C C O til C ω O ΓτΊ <! O ΓτΊ ΓτΊ Ο μ ο ο o o o o o o -K H H H H H H H H -K H H H H H CO H H i r. i r. J ί r. nJ S S 1—1 1—1 1—1 1—1 s s 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 -K H-l H-l H-l lxj ι—1 J <_X 1—1 o C c ( ) C C c C -κ U co co co co [XJ > co co Q ω ω ω ω Q ω J J J J J h4 til J (—1 (—1 (—1 (—1 (—1 O (—1 (—1 co «: «: co CO CO co co co co co co co J co co CO CO CO CO CO CO 'fo CO ΓτΊ ΓτΊ Ol Ol ΓτΊ CO ΓτΊ ΓτΊ μ 'fo 'fo 'fo ( 1 ( 1 'fo 'fo 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 >H >H >H >H >H O til >H Q-i Q-i Q-i Q-i Ο-ι Η Η Η H H H H H H -K co 01 O O O O O O O J [XJ J J X > J nJ ο ο o u u U u u -K J J 1—1 J J > J J CO CO CO CO CO CO til CO h4 J h4 h4 J Q-i μ Ο ο o u u U u u -K ,—1 1—1 1—1 1—1 1—1 1—1 Ο 1—1 1—1 1—1 1—1 1—1 > 1—1 1—1 <_X >: CO CO CO CO CO O CO CO > > > > > > > > -K o u 0 0 0 0 O 0 0 -K H H til >H >H >H >H >H >H >H >Η -κ — 1—I Η 1—1 1—1 1—1 1—1 1—1 1—1 -K — co 0 O 0 0 CO co co X J H H 1—1 H H H H Η (fo (fo (fo (fo -K + >H >H >H >H >H >H >H -K | | | | | | Ol | [xj It | Γτ 1 It | It | It | It | |τ ι -κ Η Η <: <: H 'fo co H < J) 1 1 1 1 1 1 1 1 1 1 1 1—1 1—1 1—1 -K I I I I I I J I [xj [XJ [XJ [XJ [XJ [XJ [XJ [XJ -κ co | co co <' Ί 1 Ol Οι 10 10 10 1—1 01 01 | | | | | | Ol | Ol Ol Ol Ol Ol Ol Ol οι -κ Η 1—1 1—1 X H H < J) 1 S S -K M M M ΓτΊ ΓτΊ ω «: O O O O O O O Ο -κ ω ' Ί 1 > 1 1 > > 1 1 >: > > Ol Ol O Ol O til (—1 O -κ Η Η H H H co H H ' Ί 1 1 1 : ·, 1 1 1 1 1 1 ( X (—1 (—1 co o~i o~i o~i 1—1 0~i rn CO 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 -Κ | | | | | LG | | 1· ' > 1 < X 1 1 1 1 1 1 1 1 1 LG Q-i Q-i H H 1—1 1—1 H 1—1 1—1 H S H H H I | I I I ΓτΊ I I ' '> 1 1 1 1 1 ,> 1 ,> 1 1 1 O J J CO o~i Q-i Q-I o~i > CO 'fo Η Η H H O H H ' '> 1 1 1 1 1 1 1 1 1 1 1 1 LG Q-i CO CO CO Q-i Q-i [XJ It | CO CO H H H H H H H Η -κ Ο Ο ( > ( > o o o o -K ' '> 1 : 1 1 1 1 1 1 1 1 >H J J CO CO CO CO CO Q X CO O ( ) ( ) ( ) ( ) O ( ) ( ) -κ > > (—1 (—1 «: u > > ' '> 1 :¾¾ ι ·> I ' > I ¹ > C D J O ω pu 0~i 0~i LG LG LG Q-i <! H H H H H <: H Η CO Q-I co co co co 1· ' > 1 : 1 1 1 1 1 1 1 1 ( 1 n H CO 01 01 H 1—1 H It | It | It | It | It | o It | |τ I 'fo CO co co X > co co ' '> 1 : In In In In > [xj [XJ kb O O O O O O o O -K Ol Ol Ol Ol Ol < « Ol οι -κ η ( 1 (—1 ΓτΊ n co ( 1 ( 1 < J) 1 : O O 0 -K P CO CO CO CO CO til (—1 CO o o o o o O o ο -κ ο ο > > u u u kl 1 : _::^:j X|X|X|X|X|X|X| -K ω CO <_X CO til CO til CO X CO H CO O CO CO * > O Q > Q Q 1—1 N > Η J £ co H co + rtji<di<di<di<di<di<di<d 1<C1<C1<C1<C1<C1<C1<C1<C * ϋ H H H H H H o H > > «: ΓτΊ > > C c c c c ( ) c c -K H H H H H H H H -K til til til til til CZ O O O O o O O ο -κ ο ο 1—1 1—1 1—1 1—1 1—1 1—1 -K 1—1 1—1 1—1 1—1 1—1 > 1—1 1—1 J J J J J > J J O O o CO |τ I -K III 1—1 1—1 11 [xj [xj [xj [xj [xj > > > > > > > «: J J J J J J J -K < -K O O O O O O O O -K [XJ O O O O It | O ο co co co CO CO co co co -K Ol Ο O O O O 0 0 -K > > > > > > > > -K H H H H H ΓτΊ H Η >Η >H >H >H lxj >H >H X 0Ί 0Ί 0Ί X X 01 (fo > > > > > > > ο o o U u u u u -K 0 O O O 0 CO 0 0 -K 'fo X 'fo 'fo J 'fo 'Ρ co co co CO co co co co -K 1—1 l—l 1—1 1—1 1—1 1—1 1—1 1—1 -K <_X <_X <_X o o o o o -K J J J J J h4 J -κ co co co co rn 1—1 co co 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CO CO CO CO CO 1—1 CO m ΓτΊ ΓτΊ ΓτΊ ΓτΊ ΓτΊ ΓτΊ ΓτΊ ΓτΊ -κ ο «: o o <! o o > -K N (—1 |T | |T | ti-l Q-i ti-l ΓτΊ ΓτΊ ΓτΊ ΓτΊ ΓτΊ ΓτΊ (—1 ΓτΊ ΓτΊ ο o o <_X j_q u <_X O O O O O O O O -K 1—1 H H H 1—1 Q 1—1 J Ο Ο Ο Ο Ο o Ο Ο -κ Η H >: >: 'fo H H ( U (') (') (') ( u (') (') -K til til It | It | til til til X Ο |τ 1 |τ 1 |τ 1 |τ 1 1—1 Ο |τ I ο o o lxj u u u u -K :¾ :¾ :¾ :¾ CO :¾ :¾ [XJ [xj [xj [xj [xj [xj ο [XJ -κ >Η X X 1 1 1 1 ·: 1 1 «: 1 1 1 1 > It | It | (—1 <: <! Q Q Q ΓτΊ Q Q H H H H cz H H X X X X X X X -K til i4 > > > > > CO > > 1—1 1—1 1—1 1—1 1—1 1—1 1—1 >: W 1 1 : ·, : ·, til 10 H H H H H til H H Ο ( X O o ΓτΊ (—1 Ο Ο o o < X < X < X < X til < X < X O Q-ι μ-ι CO CO CO CO LG CO Η H H H H H H H -K I I I I I I I [xj [XJ [XJ [XJ [XJ [XJ [XJ [XJ -K >H >H >H J >Η >Η LG LG LG LG LG LG LG LG -K | | | | | ro | | H H H H H H H H -K 1—1 1—1 1—1 1—1 1—1 1—1 I—I CO co co CO co J co co ΓΟ ro <: O Lxj lxj O O O O O -K H Ο o o o o u o o -K 10 > > 10 [xj [XJ [XJ [XJ [XJ [XJ [XJ [XJ -K CO CO CO [xj CO 1—1 CO co ( 1 ( 1 ( 1 ( 1 (—1 'fo (—1 ( 1 < X 1 1 1 1 1 1 1 1 1 1 J J J J J J J J J -K X X X X X Q-i X X >: J H H co co co J 10 til O O O O O O O O -K O C C C C O C C -κ co co co CO co co co co -K > > > > «: 1 1 > > 'N CO O O O O CO e, Q-i CG CG CG CG > LG LG ο 1—1 1—1 1—1 1—1 1—1 1—1 1—1 X 0 Q-i Q-i Q-i Q-i Q-i Q-i Q-i Q-i -K ω ΓτΊ ω ω ω ω ω <2J 1 1 1 1 1 1 1 1 1 1 ·: 1 1 1—1 X X X X X >H X X I H I I I I | | J J J J J J co CO CO co co L/J co CO -K [XJ [XJ [XJ 1 O 1 1 1 1 1 1 [XJ co co co co [xj co > > > > > > > > *

Θ O Θ O Θ O Θ O (ft ω O (ft ω O (ft ω O (ft ω O £ 3 P O O O £ 3 P O O O £ 3 P O O O £ 3 P O O O O 0 (ft •r4 til cC O O 0 (ft •r4 til cC O O 0 (ft •r4 til cC O O 0 (ft •r4 til cC O X O S (fo Q-i [XJ 2 Q X O S (fo Q-i [XJ 2 Q X O S (fo Q-i [XJ 2 Q X O S (fo Q-i [XJ 2 Q

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2019204295 19 Jun 2019

CN CN CO CN CN CN CN CN o (to CN LO CN CN CO (to co CN CO CN co o ΟΊ CO LO co o- 73 Er LO LO LO LO LO LO LO LO o LO LO LO LO LO LO LO LO σ3 CT Q Q + 3 3 3 3 3 3 3 3 4c + to to to to to to to to 4C CO co co to to to CZl + O o O O O o O O 4c + to to to to to to to to 4c (Ό co co to CN CN Q + H H H H H H H H 4c + o O o o O o o o 4c 43 Ch + 2> 1—1 2> 2> 2> 1—1 2> 2> + O O O o o O o o 4c 03 + s 3 s s s 3 s s 4c + :&5i to to to to to to to 4c g O O O O O O O O 4c + Q Q Q Q ω Q Q Q to to to to g Ch to Q to to to co to to + O O o o O O O 4c CO to to H H H H H to H H + to to to to to to to 4c o to H H to o O 42 >H >H >H to >H >H + o o o o o o o 4c S to ω ω s s to 1—1 > 1—1 1—1 > to 1—1 H + :g: 4c O to to to 3 to O Q H to H H > > H H Q Q Q Q Q Q Q 4c to <1 to 44 CO to to to to to to to to' to H 1—1 to 1—1 to to to o to to o to to +-> H H H H H H H H 4c O o O o o to o o O o > ω o o o 4= Q Q Q Q Q Q Q O o O o o o o o 4c Q to Q to Q Q Q C O Q <] <] Q ω O Q ω ω ω ω ω co ω ω CO co to to Q to o o to to to to to co to to to to to to to to to to 4c co Q to to Q to to > CO CO co co CO to CO CO >H >H >H >H >H >H 4c O O O Oi O to o o toD Ό to to to to to to to to 4c O o o o o o o o 4c H H H 1—1 H H H H Q to to to to to to to to 4c 4c to to to to to to to to CZl O O O O O o o o -K LO o o o o o o o o -K ω ω ω ω ω ω ω ω -K 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 4c O 1—1 1—1 1—1 1—1 to 1—1 to to to to to to to to 4c u Φ 4^ to to to to to to s s s S > s 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 4c to to to to to to to to 4c <2J + o O o o O to to O 3 3 3 3 3 3 3 3 4c o 2i) O O o o O o o o 4c + o to to to to to o to Q Q Q Q Q to Q Q f to to to to to χ to to + H H to H co Q H to S > S 1—1 s H S > g Ch ω Q ω ω Q Q ω ω + 1—1 1—1 1—1 1—1 1—1 1—1 1—1 > >H to σ3 *Ch H H H H H to H H + to. to > > to to to ω ω ω ω ω to H ω ω Q to >H to + :fe% ffi H to 1—1 to Hm o _Q to to to to to > to to + 'Q Q Q Q Q ω Q Q > > > > > > > > 4c fl Th to to to to to 1—1 to to + O to to to to > o to to to to to to to to to ¹ ' g CO to to to to to to to to 4c + >H >H >H >H 4c H H H H H H H H 4c C*5 If O H H H H + to to to to co CO to to 4c w Q 3 -— Oi ω o o ω to o ω + to to to to to o to to > > > > > > > 4c M S-H Ch + to to to to to to to to 4c + o o o o o O o o 4c ;:£& o o o O O o o 4c σΰ + to to to to to to to to 4c + o o o o o o o o 4c (2J to to to to to to to to 4c oo + 1—1 1—1 1—1 1—1 to to 1—1 1—1 + ω ω ω ω ω ω ω ω 4c (2J o o o Oi O to o o CH r—, + to to to to to to to to 4c + ω ω ω ω ω ω ω ω 4c (2J ω ω Q ω ω o ω ω Ch l + 4c + to to to to to H to to (2J to to to to to to to to o ι/Ί + 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 4c + H to to to CO co H to to 4c f Ch + Q 1 Q 1 Q I Q Q I Q 1 Q 1 Q 4c + > 1—1 > > > 1—1 > > O + X X x co to to to to to to to to to 4c CZ1 CO to co to co o O to co to to to to to to to to 4c -ύξ® 4c o O 1—1 1—1 1—1 1—1 1—1 > 1—1 1—1 4c o, 0. o o o co to o O to to H H to ω to to 4c WH c :S5^: ω ω ω ω o ω Q > > > -K Ό CN co co co to CO H to to to to to to to to 4c 4c Q O > 1—1 > 1—1 o 1—1 1—1 o o o o o o o o 4c 4c — to to to s to H to to to to to to to to to to 4c 1—1 1—1 1—1 1—1 H 1—1 1—1 1—1 C CD + >H >H >H >H >H 4c 1—1 H 1—1 1—1 1—1 > 1—1 co O O O O o O O O 4c OO + to ω to to to to to o to to to to to to Q to > > > > > H > > Ch + o to o o o to o o o o o o o o O o to to to to to to to to 4c <υ + X X X X to to to x 4c + ^:H+: 1—1 H H 1—1 to 1—1 1—1 to to O O to >H to to Ό Ph <2J + 1—1 > 1—1 1—1 1—1 1—1 1—1 1—1 + ω to ω ω ω co ω ω 3 3 3 3 3 3 3 3 4c f CZ) + 1—1 1—1 1—1 1—1 1—1 1—1 1—1 1—1 4c + SS: o o o o o o o 4c s H to to 1—1 1—1 s to Ch < + 1—1 1—1 to to 1—1 1—1 1—1 1—1 + :i*£ to o to to H to to o ω o o O ω o o £5 + ω ω ω ω ω to ω ω + ω ω ω ω ω ω ω ω 4c to ω to to to Q to to + to to to to to ω to to + to ω to to ω ω to to x to to to >H > to to + 1—1 1—1 1—1 1—1 > to 1—1 1—1 + co H H H H H co H to o to to to ω to to 42 cc + O o to co o ω o o + •toi: to to to to o o o o o o o o 4c H + co co co co co co co co + <2J + O o o o o o o o + > > > > > > > CZ) w H Ϊ O to d Θ O d Θ O d <υ o ω o ω o (Ό ω o £ 3 2 to O o O O £ 3 to to o o o o g 3 to to o o o o O 0 to Li O to O o o Ή L o to O o (Ό •H L o X o S to to to s Q to o s to to to s Q to o S to to to s Q

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2019204295 19 Jun 2019