US 2015.005O270A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0050270 A1 Li et al. (43) Pub. Date: Feb. 19, 2015

(54) ANTIBODIES TO B1 Publication Classification RECEPTOR LIGANDS (51) Int. Cl. (71) Applicant: Sanofi, Paris (FR) C07K 6/28 (2006.01) A647/48 (2006.01) (72) Inventors: Han Li, Yardley, PA (US); Dorothea Kominos, Millington, NJ (US); Jie (52) U.S. Cl. Zhang, Cambridge, MA (US); Alla CPC ...... C07K 16/28 (2013.01); A61K 47/48007 Pritzker, Cambridge, MA (US); (2013.01); C07K 2317/92 (2013.01); C07K Matthew Davison, Cambridge, MA 2317/565 (2013.01); C07K 2317/76 (2013.01); (US); Nicolas Baurin, Arpajon (FR); C07K 231 7/24 (2013.01); A61K 2039/505 Govindan Subramanian, Belle Mead, (2013.01) NJ (US); Xin Chen, Edison, NJ (US) USPC ...... 424/133.1; 530/387.3; 530/3917; 536/23.53; 435/320.1; 435/334: 435/69.6 (73) Assignee: SANOFI, Paris (FR) (21) Appl. No.: 14/382,798 (57) ABSTRACT (22) PCT Filed: Mar. 15, 2013 The invention provides antibodies that specifically bind to (86). PCT No.: PCT/US13A31836 Kallidin ordes-Arg10-Kallidin. The invention also provides S371 (c)(1), pharmaceutical compositions, as well as nucleic acids encod (2) Date: Sep. 4, 2014 ing anti-Kallidin ordes-Arg10-Kallidin antibodies, recombi nant expression vectors and host cells for making such anti Related U.S. Application Data bodies, or fragments thereof. Methods of using antibodies of (60) Provisional application No. 61/616,845, filed on Mar. the invention to modulate Kallidin or des-Arg10-Kallidin 28, 2012. activity or detect Kallidin ordes-Arg10-Kallidin or, either in vitro or in vivo, are also provided by the invention. The (30) Foreign Application Priority Data invention further provides methods of making antibodies that specifically bind to des-Arg-Bradykinin and des-Argo-Kal Feb. 4, 2013 (FR) ...... 1350953 lidin-like . Patent Application Publication Feb. 19, 2015 Sheet 1 of 17 US 2015/0050270 A1

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ANTIBODIES TO BRADYKINNB1 0010 c) specifically binds to Kallidin ordes-Argo-Kalli RECEPTOR LIGANDS din with a Kofless than 1x10's"; or 00.11 d) specifically binds to Kallidin ordes-Argo-Kalli RELATED APPLICATIONS din and inhibits binding to the bradykinin B1 receptor. 0001. This application claims priority to U.S. Provisional 0012. In one embodiment, the antibody or antigen binding Application No. 61/616,845, filed Mar. 28, 2012, and French fragment thereof binds to the N-terminal residue of Patent Application Number 1350953, filed Feb. 4, 2013. The Kallidin ordes-Argo-Kallidin. contents of these applications are each hereby incorporated 0013. In another embodiment, the antibody or antigen binding fragment thereof inhibits the binding of Kallidin or by reference in their entireties. des-Argo-Kallidin to a bradykinin-1 receptor. 0014. In another embodiment, the antibody or antigen SEQUENCE LISTING binding fragment thereof binds specifically to mouse Kalli 0002 The instant application contains a Sequence Listing din-like peptide (KLP). which has been submitted in ASCII format via EFS-Web and 0015. In another embodiment, the antibody or antigen is hereby incorporated by reference in its entirety. Said ASCII binding fragment thereof comprises a heavy chain variable copy, created on Mar. 15, 2013, is named 543895 SA9 domain comprising an HCDR3 amino acid sequence selected 029PC Seq List.txt and is 89,291 bytes in size. from the group consisting of 10016 a) SEQ ID NO: 7 DXY X, XD XHAMX,Y), BACKGROUND OF THE INVENTION wherein 0003. The bradykinin B1 receptor has been implicated in 0017 X is Y. For H, pathogenesis of inflammatory disease and chronic pain. By (0018 X is R, D, A, V. L., I, M, FY or W. modulating tissue inflammation and renal fibrosis, the B1 (0.019 X is Y. F. W or H, receptor has also been associated with pathogenesis of acute 0020 X is D, E or Y, and, kidney injury as well as chronic kidney diseases which are the (0021 X is D or E: main causes of end-stage renal failure. 0022 b) SEQ ID NO: 63 X EYDGXYXXLDX), 0004. In humans, the major agonists of the bradykinin B1 wherein receptor are the . Kinins are bioactive pro 0023 X is W or F, duced from the proteolytic cleavage of proteins. 0024 X is N or no amino acid; The major agonists of bradykinin B1 receptor are the 0025 X is Y or S, decapeptide Kallidin, and the nonapeptide des-Argo-Kalli (0026 X is D or P. and din (formed by the proteolytic cleavage the c-terminal argin (0027 X is ForY. ine form Kallidin). Therefore, agents that can inhibit the 0028 c) SEQ ID NO: 13; binding of Kallidin and des-Argo-Kallidin to the bradykinin 0029 d) SEQID NO:32: B1 receptor have the potential to treat or prevent bradykinin 0030 e) SEQ ID NO: 40; B1 receptor-mediated pathologies. 0031 f) SEQID NO: 47; and 0005 Accordingly, there is a need in the art for novel 0032 g) SEQID NO. 55. agents that inhibit the binding of Kallidin and des-Argo 0033. In another embodiment, the antibody or antigen Kallidinto the bradykinin B1 receptor for use in the treatment binding fragment thereof comprises an HCDR2 amino acid of bradykinin B1 receptor-mediated human pathologies. sequence selected from the group consisting of SUMMARY OF THE INVENTION 0034) a) SEQID NO: 8 (YFXPXNGNTGYNQKFRG), wherein 0006. The present invention provides antibodies, or anti 0035 X is D, R, A, V. L. I. M. F. Y or W, and gen binding fragments thereof, that specifically bind Kallidin 003.6 X is Y, D, E, N, or Q: and des-Argo-Kallidin and prevent binding to the bradykinin 0037 b) SEQ ID NO: 64 B1 receptor. Such antibodies are particularly useful for treat ing Kallidin and des-Argo-Kallidin-associated diseases or |WXDPENGDXXYAPKFQG), wherein disorders (e.g., pain or fibrosis). The invention also provides 0038 X is I, or V. pharmaceutical compositions, as well as nucleic acids encod 0039 X is T, or S, and ing anti-Kallidin and des-Argo-Kallidin antibodies, recom 0040 X is G, or D: binant expression vectors and host cells for making Such 0041 c) SEQ ID NO: 14 antibodies, or fragments thereof. Methods of using antibod 0.042 d) SEQID NO:33; ies, or fragments thereof, of the invention to detect Kallidin 0043 e) SEQ ID NO:41: and des-Argo-Kallidin or to modulate Kallidin and des 0044 f) SEQID NO: 48; and Argo-Kallidin activity, either in vitro or in vivo, are also 0045 g) SEQID NO: 56. encompassed by the invention. The invention also provides 0046. In another embodiment, the antibody or antigen methods of making antibodies that specifically bind to des binding fragment thereof comprises an HCDR1 amino acid Argo-Bradykinin and des-Argo-Kallidin-like peptide. sequence selected from the group consisting of 0007 Accordingly, in one aspect the invention provides an 0047 a) SEQID NO: 9 (GYSFTDYXIY). In wherein X isolated monoclonal antibody or antigen binding fragment is N, W or Y: thereof that: (0.048 b) SEQID NO: 65 IGFNIKDYYXH), wherein X 0008 a) specifically binds to Kallidin ordes-Argo-Kalli is L, or M: din but not to Bradykinin ordes-Argo-Bradykinin; 0049 c) SEQ ID NO: 15; 0009 b) specifically binds to Kallidin ordes-Argo-Kalli 0050 d) SEQID NO:34: din with a KD of less than 1x10' M: 0051 e) SEQ ID NO: 42: US 2015/005O270 A1 Feb. 19, 2015

0.052 f) SEQID NO: 49; and 0101 X is Y. F., T or H, and, 0053 g) SEQID NO:57. 01.02 X is W.Y. F., H or L: 0054. In another embodiment, the antibody or antigen (0103) b) SEQID NO: 66 QXXXSXPXT, wherein binding fragment thereof comprises a light chain variable 0104 X is Q or N. domain comprising an LCDR3 amino acid sequence selected 01.05 X is Y. F. D or H, from the group consisting of 01.06 X, is Y, F, Hor W. 0055 a) SEQID NO: 10 QQXXS XPXT, wherein 01.07 X is Y. F., T or H, and 0056 X is Y. For H, 0108 Xs is W.Y. F., H or L: 0057 X is Y, F, H or W. 0109 c) SEQID NO: 69XQGTHFPYT), wherein X is 0058 X is Y. F., T or H, and, L or M: 0059 X is W, Y, F, Hor L: 0110 d) SEQID NO: 16; 0060 b) SEQID NO: 66 QXXXSXPXT, wherein 0111 e) SEQID NO:35; 0061 X is Q or N, O112 f) SEQID NO:43: 0062 X is Y, F, D or H, 0113 g) SEQID NO:50; and 0063 X is Y, F, H or W. 0114 h) SEQID NO: 58. 0064 X is Y. F., T or H, and 0.115. In another embodiment, the antibody or antigen 0065 X is W, Y, F, H or L; binding fragment thereof comprises an LCDR2 amino acid 0066 c) SEQID NO: 69XQGTHFPYTI, wherein X is sequence selected from the group consisting of L or M: (0.116) a) SEQID NO: 11 WASTRX, whereinX is E, D, 0067 d) SEQID NO: 16; Q or N: 0068 e) SEQID NO:35; 0117 b) SEQID NO: 67 XASTRX), wherein 0069 f) SEQID NO:43: 0118 X is W or G, and 0070 g) SEQID NO:50; and 0119) X is E. D. Q or N: (0071 h) SEQID NO:58. 0120 c) SEQID NO: 17; 0072. In another embodiment, the antibody or antigen 0121 d) SEQID NO:36: binding fragment thereof comprises an LCDR2 amino acid 0122) e) SEQID NO: 51; and sequence selected from the group consisting of (0123 f) SEQID NO. 59. 0073 a) SEQID NO: 11 WASTRX, wherein X is E, D, 0.124. In another embodiment, the antibody or antigen Q or N: binding fragment thereof comprises an LCDR1 amino acid 0074 b) SEQID NO: 67 XASTRX), wherein sequence selected from the group consisting of 0075) X is W or G, and 0.125 a) SEQID NO: 12KSSQSLLXSSNQKNXLA), 0076 X is E. D. Q or N: wherein 0.077 c) SEQID NO: 17; I012.6 X is W. H. Y or F, and 0078 d) SEQID NO:36: O127 X is H or Y: 0079 e) SEQID NO: 51; and 0128 b) SEQ ID NO: 68 0080 f) SEQID NO. 59. KSSQSLLXXSXQXNXSLA, wherein 0081. In another embodiment, the antibody or antigen I0129 X is W. H. Y or F, binding fragment thereof comprises an LCDR1 amino acid I0130 X is S or G, sequence selected from the group consisting of I0131 X is N or D, 0082 a) SEQID NO: 12KSSQSLLXSSNQKNXLA), (0132 X is Kor R, wherein 0.133 X is HorY. 0.083 X is W. H. Y or F, and 0.134 c) SEQ ID NO: 70 KSSQSLLYSNGXTYLN, I0084. X is HorY: wherein X is K or E: 0085 b) SEQ ID NO: 68 0135 b) SEQID NO: 18; KSSQSLLXXSXQXNXSLA, wherein 0.136 c) SEQ ID NO:37; I0086 X is W. H. Y or F, 0.137 d) SEQID NO:44: 0087 X, is S or G, 0.138 e) SEQ ID NO. 52; and I0088 X is N or D, 0139 f) SEQID NO: 60. 0089 X is Kor R, 0140. In another embodiment, the antibody or antigen 0090 X is HorY. binding fragment thereof comprises a heavy chain variable 0091 c) SEQ ID NO: 70 KSSQSLLYSNGXTYLN), region comprising the HCDR3, HCDR2 and HCDR1 region wherein X is K or E: amino sequences set forth in SEQ ID NOS 13, 14, and 15, 0092 b) SEQID NO: 18; respectively, and one or more amino acid Substitutions at 0093 c) SEQ ID NO:37; positions selected from the group consisting of H1, H5, H9. 0094 d) SEQID NO:44: H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, 0.095 e) SEQ ID NO. 52; and H81, H82A, H83, H87, and H108 according to Kabat. 0096 f) SEQID NO: 60. 0.141. In another embodiment, the antibody or antigen 0097. In another embodiment, the antibody or antigen binding fragment thereof comprises a light chain variable binding fragment comprises a light chain variable domain region comprising the LCDR3, LCDR2 and LCDR1 region comprising an LCDR3 amino acid sequence selected from amino sequences set forth in SEQ ID NOS 16, 17, and 18, the group consisting of respectively, and one or more amino acid Substitution at posi 0.098 a) SEQID NO: 10 QQXXS XPXT, wherein tions selected from the group consisting of L5, L9, L15, L18, 0099 X is Y. For H, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 and 0100 X, is Y, F, H or W. L104, according to Kabat. US 2015/005O270 A1 Feb. 19, 2015

0142. In another embodiment, the antibody or antigen with a KD of less than 1x10" M; c) specifically binds to binding fragment thereof comprises a heavy chain variable Kallidin ordes-Argo-Kallidin with a K of less than 1x10 region amino acid sequence with at least 90% identity to an s'; ord) specifically binds to Kallidin ordes-Argo-Kallidin amino acid sequence selected from the group consisting of and inhibits binding to the bradykinin B1 receptor. SEQID NOs: 19, 20, 21, 22, 24, 25, 38, 45, 53, and 61. 0152. In another aspect, the antibody or antigen binding 0143. In another embodiment, the antibody or antigen fragment of the invention is conjugated to a diagnostic or binding fragment thereof comprises a light chain variable therapeutic agent. domain amino acid sequence with at least 90% identity to an 0153. In another aspect, the invention provides isolated amino acid sequence selected from the group consisting of nucleic acid encoding the amino acid sequence of the anti SEQID NOs: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54, and 62. body, or antigen binding fragment thereof, of the invention. 0144. In another embodiment, the antibody or antigen 0154) In another aspect, the invention provides recombi binding fragment thereof comprises a light chain variable nant expression vector comprising the nucleic acid of the region amino acid sequence with at least 90% identity to an invention. amino acid sequence selected from the group consisting of 0.155. In another aspect, the invention provides a host cell SEQID NOs: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54, and 62. comprising the recombinant expression vector of the inven 0145. In another embodiment, the antibody or antigen tion. binding fragment thereof comprises a heavy chain variable 0156. In another aspect, the invention provides a method domain comprising an amino acid sequence selected from the of producing an antibody that binds specifically to Kallidin group consisting of: SEQID NO: 19, 20, 21, 22, 24, 25, 38. and des-Argo-Kallidin, comprising culturing the host cell of 45, 53, and 61. the invention under conditions such that an antibody that 0146 In another embodiment, the antibody or antigen binds specifically to Kallidin and des-Argo-Kallidin is pro binding fragment thereof comprises a light chain variable duced by the host cell. domain amino acid sequence selected from the group con 0157. In another aspect, the invention provides a pharma sisting of: SEQID NO:26, 27, 28, 29, 29, 30, 31, 39, 46, 54, ceutical composition comprising the antibody, or antigen and 62. binding fragment thereof, of the invention and one or more 0147 In another embodiment, the antibody or antigen pharmaceutically acceptable carriers. binding fragment thereof comprises an amino acid sequence 0158. In another aspect, the invention provides a method selected from the group consisting of: SEQID NO:26, 27, 28. for treating a disease or disorder Kallidin ordes-Argo-Kal 29, 29, 30, 31, 39, 46, 54, and 62. lidin-associated disease or disorder, the method comprising 0148. In another embodiment, the antibody or antigen administering to a Subject in need of thereof the pharmaceu binding fragment thereof comprises the heavy chain and light tical composition of the invention. chain variable region amino acid sequences set forth in SEQ 0159. In one embodiment, the disease or disorder is ID NO: 19 and 26, SEQID NO: 20 and 27, SEQID NO: 21 chronic pain. and 28; SEQID NO:22 and 28; SEQID NO. 23 and 29; SEQ 0160. In another aspect, the invention provides a method ID NO: 24 and 30; SEQID NO: 25 and 31; SEQID NO:38 of generating an antibody that specifically binds to des-Argo and 39, SEQID NO: 45 and 46, SEQID NO: 53 and 54, or Bradykinin and des-Argo-Kallidin-like peptide comprising: SEQ ID NO: 61 and 62, respectively. immunizing an animal with an immunogen comprising a 0149. In another aspect, the invention provides an anti peptide, wherein the peptide consists of the amino acid body, or antigen binding fragment thereof, that specifically sequence set forth in SEQID No. 11, and wherein the amino binds to Kallidin and des-Argo-Kallidin, wherein the anti terminal arginine of the peptide is indirectly coupled to a body, orantigenbinding fragment thereof, competes forbind carrier moiety through a linker moiety, Such that an antibody ing to Kallidin and des-Argo-Kallidin with an antibody com that specifically binds to des-Argo-Bradykinin, des-Argo prising the heavy chain and light chain variable region amino Kallidin and des-Argo-Kallidin-like peptide is produced by acid sequences set forth in SEQID NO: 19 and 26, SEQID the immune system of the animal. NO:38 and 39, SEQID NO:45 and 46, SEQID NO:53 and 0.161. In another embodiment, the method further com 54, or SEQID NO: 61 and 62, respectively. prises isolating from the animal, the antibody, a nucleic iso 0150. In another aspect, the invention provides an isolated lating encoding the antibody, or an immune cell expressing monoclonal antibody or antigen binding fragment thereof the antibody. that competes for binding to Kallidin ordes-Argo-Kallidin 0162. In one embodiment, the carrier moiety is a protein. with the antibody of any one of the preceding claims, and does In another embodiment, the protein is Keyhole limpet not bind to Bradykinin or des Argo-Bradykinin. hemocyanin (KLH). In another embodiment, wherein the 0151. In another aspect, the invention provides an isolated linker moiety comprises Gly-Gly-Glyn, whereinn is at least monoclonal antibody or antigen binding fragment thereof 1. that specifically binds to a conformational epitope of kallidin (KD) or des Arg10-Kallidin (DAKD) which adopts a Pro4 BRIEF DESCRIPTION OF THE DRAWINGS kink conformation comprising a type II tight turn at Proline 4 of the KD or DAKD). In one embodiment, the Pro 4 kink 0163 FIG. 1 depicts the results of ELISA assays demon conformation of KD or DAKD further comprises amino acid strating binding of EE1 antibody to kinin peptides. repeats of a sigmoid shape which align the hydrophobic side 0164 FIG. 2 depicts the results of differential scanning chains of the amino acids in a spatially stacking mode. In calorimetry measurements of antibody F151. another embodiment, the antibody or antigen binding frag 0.165 FIG. 3 depicts amino acid sequence alignments of ment thereof comprises (a) specifically binds Kallidin ordes the variable regions of the murine and humanized F151 anti Argo-Kallidin but not to Bradykinin ordes-Argo-Bradyki body. All identical residues are listed in the alignment, while nin; b) specifically binds to Kallidin or des-Argo-Kallidin homologous residues are identified by '+' sign and non US 2015/005O270 A1 Feb. 19, 2015 homologous residues are left blank. Figure discloses SEQID also provides pharmaceutical compositions, as well as NOS 19, 24, 26, and 30, respectively, in order of appearance. nucleic acids encoding anti-Kallidin and des-Argo-Kallidin 0166 FIG.4 depicts an electron density map of the antigen antibodies, recombinant expression vectors and host cells for binding site of the F151 antibody/kallidin complex. making Such antibodies, or fragments thereof. Methods of 0167 FIG.5 depicts an electron density map of the antigen using antibodies of the invention to detect Kallidin and des binding site of the F151 antibody/des-Arg10-kallidin com Argo-Kallidin or to modulate Kallidin and des-Argo-Kalli plex. din activity, either in vitro or in vivo, are also encompassed by 0168 FIG. 6 depicts a ribbon and stick representation of the invention. the Fv Subunit of F151 bound to kallidin. 0169 FIG.7 depicts an amino acid sequence alignment of I. Definitions the light chain variable regions of exemplary murine anti 0181. In order that the present invention may be more kallidin antibodies of the invention. Amino acid residues that readily understood, certain terms are first defined. interact with kallidin are marked with asterisks. Figure dis 0182. As used herein, the term “Kallidin” refers to a pep closes SEQID NOS 134,125, 124,123, 126, and 131, respec tide comprising or consisting of the amino acid sequence tively, in order of appearance. KRPPGFSPFR (SEQ ID NO. 1). 0170 FIG. 8 depicts an amino acid sequence alignment of 0183. As used herein, the term “des-Argo-Kallidin' the heavy chain variable regions of exemplary murine anti refers to a peptide comprising or consisting of the amino acid kallidin antibodies of the invention. Amino acid residues that sequence KRPPGFSPF (SEQID NO. 2). interact with kallidin are marked with asterisks. Figure dis 0.184 As used herein, the term “mouse Kallidin' or “Kal closes SEQID NOS 135, 120, 119, 118, 121, and 122, respec lidin-like peptide' refers to a peptide comprising or consist tively, in order of appearance. ing of the amino acid sequence RRPPGFSPFR (SEQID NO. (0171 FIG. 9 depicts the results of in vivo experiments 3) determining the effect of EE1 antibody on formalin-induced 0185. As used herein, the term “mouse des-Argo-Kalli acute inflammatory pain. din” or “des-Argo Kallidin-like peptide” refers to a peptide 0172 FIG. 10 depicts the results of in vivo experiments comprising or consisting of the amino acid sequence RRP determining the effect of EE1 antibody on CFA-induced mechanical hypersensitivity. PGFSPF (SEQ ID NO. 4). 0173 FIG. 11 depicts the results of in vivo experiments 0186. As used herein, the term “Bradykinin” refers to a determining the effect of EE1 antibody on CFA-induced ther peptide comprising or consisting of the amino acid sequence mal hypersensitivity. RPPGFSPFR (SEQID NO. 5). 0.174 FIG. 12 depicts the results of in vivo experiments 0187. As used herein, the term “des-Argo-Bradykinin' determining the effect of EE1 antibody on CCI-induced refers to a peptide comprising or consisting of the amino acid mechanical hypersensitivity. sequence RPPGFSPF (SEQID NO. 6). (0175 FIG. 13 depicts the results of in vivo experiments 0188 As used herein, the term “antibody' refers to immu determining the effect of EE1 antibody on CCI-induced ther noglobulin molecules comprising four polypeptide chains, mal hypersensitivity. two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). 0176 FIG. 14 depicts schematic maps of VL and VH Each heavy chain comprises a heavy chain variable region expression constructs for generating humanized F151 variant (abbreviated V or VH)and aheavy chain constant region (C HC3a/LC3a with the restriction DNA endonuclease sites pre or CH). The heavy chain constant region comprises three sented as deduced sequences in bold and underlined. Panel A domains, C1, C2 and C3. Each light chain comprises a depicts the light chain and Panel B depicts the heavy chain. light chain variable region (abbreviated V.) and a light chain Figure discloses SEQID NOS 30, 24, and 136, respectively, constant region (C, or CL). The light chain constant region in order of appearance. comprises one domain (C1). The V and V regions can be (0177 FIG. 15 depicts an alignment of the F151 heavy further subdivided into regions of hypervariability, termed chain (A) and light chain (B) amino acid sequences with the complementarity determining regions (CDRS), interspersed closest human germline amino acid sequences. with regions that are more conserved, termed framework (0178 FIG. 16 depicts an alignment of the F151 heavy regions (FR). Each V and V, is composed of three CDRs and chain (A) and light chain (B) with a heavy chain locus (1-08 four FRS, arranged from amino-terminus to carboxy-termi & 1-18) and light chain (V IV-B3) locus of the VH1 sub nus in the following order: FR1, CDR1, FR2, CDR2, FR3, family. CDR regions and Vernier regions are indicated in CDR3, FR4. boldface and humanizing mutations are underlined. 0189 As used herein, the term “antigen-binding frag (0179 FIG. 17 depicts (A) the secondary and (B) tertiary ment of an antibody includes any naturally occurring, enzy structure of the main chain polypeptide backbone conforma matically obtainable, synthetic, or genetically engineered tion of kallidin (KD) as bound to F 151 antibody which com polypeptide or glycoprotein that specifically binds an antigen prises a type II tight turn at Proline 4 (C). Figure discloses to form a complex. Antigen-binding fragments of an antibody SEQID NOS 2, 2, and 133, respectively, in order of appear may be derived, e.g., from full antibody molecules using any aCC. Suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the DETAILED DESCRIPTION manipulation and expression of DNA encoding antibody 0180. The present invention provides antibodies that spe variable and optionally constant domains. Non-limiting cifically bind to Kallidin and des-Argo-Kallidin and prevent examples of antigen-binding portions include: (i) Fab frag binding to the bradykinin B1 receptor. Such antibodies are ments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) FV frag particularly useful for treating Kallidin and des-Argo-Kalli ments; (v) single-chain FV (ScPV) molecules; (vi) dAb frag din-associated disease or disorders (e.g., pain). The invention ments; and (vii) minimal recognition units consisting of the US 2015/005O270 A1 Feb. 19, 2015 amino acid residues that mimic the hyperVariable region of an terms, “plasmid' and “vector” may be used interchangeably. antibody (e.g., an isolated complementarity determining However, the invention is intended to include such other region (CDR)). Other engineered molecules, such as diabod forms of expression vectors, such as viral vectors (e.g., rep ies, triabodies, tetrabodies and minibodies, are also encom lication defective retroviruses, adenoviruses and adeno-asso passed within the expression “antigen-binding fragment.” ciated viruses), which serve equivalent functions. (0190. As used herein, the term “CDR' or “complementa (0195 As used herein, the term “host cell is intended to rity determining region” means the noncontiguous antigen refer to a cell into which a recombinant expression vector has combining sites found within the variable region of both been introduced. It should be understood that this term is heavy and light chain polypeptides. These particular regions intended to refer not only to the particular subject cell but to have been described by Kabat et al., J. Biol. Chem. 252, the progeny of such a cell. Because certain modifications may 6609-6616 (1977) and Kabat et al., Sequences of protein of occur in Succeeding generations due to either mutation or immunological interest. (1991), and by Chothia et al., J. Mol. environmental influences, such progeny may not, in fact, be Biol. 196:901-917 (1987) and by MacCallum et al., J. Mol. identical to the parent cell, but are still included within the Biol. 262:732-745 (1996) where the definitions include over scope of the term “host cell as used herein. lapping or Subsets of amino acid residues when compared 0196. As used herein, the term “treat,” “treating,” and against each other. The amino acid residues which encompass “treatment” refer to therapeutic or preventative measures the CDRs as defined by each of the above cited references are described herein. The methods of “treatment' employ admin set forth for comparison. In an embodiment of the invention, istration to a subject, an antibody orantigenbinding fragment the term “CDR' is a CDR as defined by Kabat, based on of the present invention, for example, a Subject having a sequence comparisons. Kallidin and des-Argo-Kallidin-associated disease or disor 0191 As used herein the term “framework (FR) amino der (e.g. an inflammatory disease) or predisposed to having acid residues' refers to those amino acids in the framework Such a disease or disorder, in order to prevent, cure, delay, region of an Ig chain. The term “framework region' or “FR reduce the severity of orameliorate one or more symptoms of region' as used herein, includes the amino acid residues that the disease or disorder or recurring disease or disorder, or in are part of the variable region, but are not part of the CDRs order to prolong the survival of a subject beyond that expected (e.g., using the Kabat definition of CDRs). Therefore, a vari in the absence of Such treatment. able region framework is between about 100-120 amino acids 0.197 As used herein, the term “Kallidin or des-Argo in length but includes only those amino acids outside of the Kallidin -associated disease or disorder” includes disease CDRS. states and/or symptoms associated with a disease state, where 0.192 As used herein, the term “specifically binds to altered levels or activity of Kallidin ordes-Argo-Kallidin are refers to the ability of an antibody or an antigen-binding found. Exemplary Kallidin ordes-Argo-Kallidin-associated fragment thereof to bind to an antigen with an Kd of at least diseases or disorders include, but are not limited to, pain and about 1x10M, 1x107M, 1x10M, 1x10M, 1x100M, fibrosis. 1x10' M, 1x10' M, or more. The term also encompasses 0198 As used herein, the term “effective amount refers to refers to the ability of an antibody or an antigen-binding that amount of an antibody or an antigen binding fragment fragment thereof to bind to an antigen with an affinity that is thereof that binds Kallidin or des-Argo-Kallidin, which is at least two-fold greater than its affinity for a nonspecific Sufficient to effect treatment, prognosis or diagnosis of a antigen. It shall be understood, however, that an antibody, or Kallidin or des-Argo-Kallidin-associated disease or disor an antigen-binding fragment thereof, is capable of specifi der, as described herein, when administered to a subject. A cally binding to two or more antigens which are related in therapeutically effective amount will vary depending upon sequence (e.g., Kallidin or des-Arg10-Kallidin and mouse the Subject and disease condition being treated, the weight Kallidin ordes-Arg10-Kallidin). and age of the Subject, the severity of the disease condition, 0193 As used herein, the term “antigen” refers to the the manner of administration and the like, which can readily binding site or epitope recognized by an antibody or antigen be determined by one of ordinary skill in the art. The dosages binding fragment thereof. for administration can range from, for example, about 1 ng to 0194 As used herein, the term “vector” is intended to refer about 10,000 mg, about 1 ug to about 5,000 mg, about 1 mg to to a nucleic acid molecule capable of transporting another about 1,000 mg, about 10 mg to about 100 mg. of an antibody nucleic acid to which it has been linked. One type of vector is or antigen binding fragment thereof, according to the inven a “plasmid, which refers to a circular double stranded DNA tion. Dosage regiments may be adjusted to provide the opti loop into which additional DNA segments may be ligated. mum therapeutic response. An effective amount is also one in Another type of vector is a viral vector, wherein additional which any toxic or detrimental effects (i.e., side effects) of an DNA segments may be ligated into the viral genome. Certain antibody or antigen binding fragment thereofare minimized vectors are capable of autonomous replication in a host cell or outweighed by the beneficial effects. into which they are introduced (e.g., bacterial vectors having 0199 As used herein, the term “subject' includes any a bacterial origin of replication and episomal mammalian human or non-human animal. vectors). Other vectors (e.g., non-episomal mammalian vec 0200. As used herein, the term "epitope” refers to an anti tors) can be integrated into the genome of a host cell upon genic determinant that interacts with a specific antigen bind introduction into the host cell, and thereby are replicated ing site in the variable region of an antibody molecule known along with the host genome. Moreover, certain vectors are as a paratope. A single antigen may have more than one capable of directing the expression of genes to which they are epitope. Thus, different antibodies may bind to different areas operatively linked. Such vectors are referred to herein as on an antigen and may have different biological effects. “recombinant expression vectors' (or simply, “expression Epitopes may be either conformational or linear. A confor vectors'). In general, expression vectors of utility in recom mational epitope is produced by spatially juxtaposed amino binant DNA techniques are often in the form of plasmids. The acids from different segments of the linear polypeptide chain. US 2015/005O270 A1 Feb. 19, 2015

A linear epitope is one produced by adjacent amino acid II. Anti-Kallidin ordes-Argo-Kallidin Antibodies residues in a polypeptide chain. 0202 In one aspect the invention provides antibodies, or 0201 It is noted here that, as used in this specification and antigen binding fragments thereof, that specifically bind to the appended claims, the singular forms 'a,” “an and the Kallidin ordes-Argo-Kallidin. Exemplary VH, VL and CDR include plural reference unless the context clearly dictates amino acid sequences of the antibodies of the invention are set otherwise. forth in Table 1.

TABLE 1. VH, VL and CDR amino acid sequences of exemplary anti-Kallidin or des-Argo-Kallidin antibodies.

SEO ID Antibody Clone Sequence NO.

F151 HCDR3 XYXXDXHAMXY 7 CCS eSS where: X is Y, F or H; X2 is R, D, A, V, L., I, M, F, Y or W; X is Y, F, W or H; X is D, E or Y; and Xs is D or E.

F151 HCDR2 YFXPXNGNTGYNOKFRG 8 CCS eSS where: X is D, R, A, V, L., I, M, F, Y or W; and X2 is Y, D, E, N, or Q.

F151 HCDR1 GYSFTDYXIY 9 CCS eSS Where X is N, W or Y.

F151 LCDR3 QQXXSXPXT 1O CCS eSS where: X is Y, F or H; X2 is Y, F, H or W; X is Y, F, T or H; and X is W, Y, F, H or L.

F151 LCDR2 WASTRX 11 CCS eSS where X is E, D, Q or N.

F151 LCDR1 KSSQSLLXSSNQKNXLA 12 CCS eSS where: X is W., H, Y or F; and

X2 is H or Y.

F151 HCDR3 YYRYDDHAMDY 13

F151 HCDR2 YFDPYNGNTGYNOKFRG 14

F151 HCDR1 GYSETDYNY 15

F151 LCDR3 OQYYSYPWT 16

F151 LCDR2 WASTRES 17

F151 LCDR1 KSSQSLLYSSNOKNYLA 18

F151 WH EIOLOOSGPELVKPGTSVKVSCKASGYSFTDYNIYWWKO 19 SHGKSLEWIGYFDPYNGNTGYNOKFRGKATLTVDKSSST AFMHLSSLTSDDSAVYYCANYYRYDDHAMDYWGOGTSVT WSS

F151 EIOLVOSGPEVKKPGASVKVSCKASGYSFTDYNIYWWKO 2O Humanized HC1 SPGKSLEWIGYFDPYNGNTGYNOKFRGKATLTVDKSSST AFMHLSSLTSEDSAVYYCANYYRYDDHAMDYWGOGTSVT WSS

US 2015/005O270 A1 Feb. 19, 2015

TABLE 1 - continued VH, VL and CDR amino acid sequences of exemplary anti-Kallidin or des-Argo-Kallidin antibodies. SEO ID Antibody Clone Sequence NO.

and Xs is F or Y. B21/I22/I54 WXDPENGDXXYAPKFOG 64 HCDR2 consensus where: X is I, or V; X2 is T, or S; and X is G, or D. B21/I22/I54 GFNIKDYYXH 65 HCDR1 consensus where X is L, or M.

66 LCDR3 consensus where: X is Q or N; X2 is Y, F, D or H; X is Y, F, H or W; X is Y, F, T or H; and X5 is W, Y, F151/C63/I22 XASTRX2 67 LCDR2 consensus where: X is W or G; and X2 is E, D, Q or N F151/C63/I22 KSSQSLLXX2SXQXNX5LA 68 LCDR1 consensus where: X1 is H, Y or F; X2 is o X is o X4 is o Xs is o : B21/I54 LCDR3 XQGTHFPYT 69 CCS eSS where: X is L. or M;

B21/I54 LCDR2 LWSKLDS 36 both identical

B21/I54 LCDR1 KSSQSLLYSNGXTYLN 70 CCS eSS where: X is K or E;

0203. In certain embodiments, the antibody, or antigen 0209 e) SEQID NO: 47, 48, and 49; binding fragment thereof, comprises one or more CDR region 0210 f) SEQID NO:55, 56, and 57; and amino acid sequence selected from the group consisting of 0211 g) SEQID NO: 63, 64, and 65, respectively. SEQID NO: 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 32,33, 0212. In other embodiments, the antibody, or antigen 34,35, 36, 37, 40, 41, 42, 43,44, 47, 48,49, 50, 5152, 55,56, binding fragment thereof, comprises the LCDR3, LCDR2 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. and LCDR1 region amino acid sequences selected from the 0204. In other embodiments, the antibody, or antigen group consisting of: binding fragment thereof, comprises HCDR3, HCDR2 and HCDR1 regionamino acid sequences selected from the group 0213 a) SEQ ID NO: 10, 11, and 12: consisting of: 0214 b) SEQID NO: 16, 17, and 18; 0205 a) SEQ ID NO: 7, 8, and 9: 0215 c) SEQ ID NO:35, 36, and 37; 0206 b) SEQID NO: 13, 14, and 15; 0216 d) SEQID NO:43, 17, and 44; 0207 c) SEQ ID NO:32, 33, and 34; 0217 e) SEQ ID NO: 50, 51, and 52: 0208 d) SEQID NO:40, 41, and 42: 0218 f) SEQID NO:58, 59, and 60: US 2015/005O270 A1 Feb. 19, 2015

0219 g) SEQID NO: 66, 67, and 68; and 17, 18, 32,33,34, 35,36, 37, 40, 41, 42, 43,44, 47, 48,49, 50, 0220 h) SEQID NO: 69, 25, and 70, respectively. 5152, 55, 56, 57, 58, 59 and 60) of the antibodies of the 0221. In other embodiments, the antibody, or antigen invention, i.e., amino acid sequence modifications which do binding fragment thereof, comprises the HCDR3, HCDR2, not abrogate the binding of the antibody to the antigen, e.g., HCDR1, LCDR3, LCDR2 and LCDR1 region amino acid Kallidin or des-Arg10-Kallidin. Conservative amino acid sequences selected from the group consisting of: Substitutions include the Substitution of an amino acid in one 0222 a) SEQ ID NO: 7, 8, 9, 10, 11, and 12: class by an amino acid of the same class, where a class is 0223 b) SEQID NO: 13, 14, 15, 16, 17, and 18: defined by common physicochemical amino acid side chain 0224 c) SEQ ID NO:32, 33, 34, 35, 36 and 37; properties and high Substitution frequencies in homologous 0225. d) SEQID NO:40, 41, 42, 43, 17, and 44; proteins found in nature, as determined, for example, by a 0226 e) SEQID NO: 47, 48, 49, 50, 51, and 52; and standard Dayhoff frequency exchange matrix or BLOSUM 0227 f) SEQID NO:55, 56, 57, 58, 59, and 60, respec matrix. Six general classes of amino acid side chains have tively been categorized and include: Class I (Cys); Class II (Ser, Thr, 0228. In other embodiment, the invention provides Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, humanized antibodies, or antigen binding fragments thereof, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, comprising one or more CDR regions (or conservatively Trp). For example, substitution of an Asp for another class HI modified variants thereof) from the murine antibodies dis residue such as ASn, Gin, or Glu, is a conservative Substitu closed herein. Any method of humanization can be employed tion. Thus, a predicted nonessential amino acid residue in an to generate the humanized antibodies of the invention. Suit anti-Kallidin or des-Arg10-Kallidin antibody is preferably able methods are disclosed herein and specifically exempli replaced with anotheramino acid residue from the same class. fied in Example 4. Methods of identifying amino acid conservative substitutions 0229. In a one particular embodiment, the humanized anti which do not eliminate antigenbinding are well-known in the body, or antigen binding fragment thereof comprises: art (see, e.g., Brummell et al., Biochem. 32:1180-1187 0230 a heavy chain variable region comprising the (1993); Kobayashietal. Protein Eng. 12(10):879-884 (1999); HCDR3, HCDR2 and HCDR1 region amino sequences set and Burks et al. Proc. Natl. Acad. Sci. USA 94.412-417 forth in 13, 14, and 15, respectively, and one or more amino (1997)). acid Substitution at positions selected from the group consist 0237. In another embodiment, the present invention pro ing of H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, vides anti-Kallidin ordes-Arg10-Kallidin antibodies, or anti H44, H66, H75, H79, H81, H82A, H83, H87, and H108; gen binding fragment thereof, that comprise a VHand/or VL and/or region amino acid sequence with about 80%, 85%. 86%, 0231 a light chain variable region comprising the 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, LCDR3, LCDR2 and LCDR1 region amino sequences set 97%, 98%, or 99%, identity to the VH region amino acid forth in 16, 17, and 18, respectively, and one or more amino sequence set forth in SEQID NO: 19, 20, 21, 22, 24, 25, 38, acid Substitution at positions selected from the group consist 45, 53, or 61, or the VL region amino acid sequence set forth ing of L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, in SEQID NO: 26, 27, 28, 29, 29, 30, 31, 39,46, 54, or 62, L83, L85, L100 and L104 (according to the Kabat numbering respectively. convention). 0232. In other embodiments, the antibody, or antigen 0238. In another embodiment, the present invention pro binding fragment thereof, comprises the VH region amino vides anti-Kallidin or des-Arg10-Kallidin antibodies that acid sequences set forth in SEQID NO: 19, 20, 21, 22, 24, 25, bind to the same epitope and/or cross compete with an anti 38, 45, 53, and/or 61. body, orantigenbinding fragment thereof comprising the VH 0233. In other embodiments, the antibody, or antigen and VL region amino acid sequences set forth in SEQID NO: binding fragment thereof, comprises the VL region amino 19 and 25, SEQID NO:38 and 39, SEQID NO: 45 and 46, acid sequences set forth in SEQID NO:26, 27, 28, 29, 29.30, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respec 31, 39, 46, 54, and/or 62. tively. Such antibodies can be identified using routine com 0234. In other embodiments, the antibody, or antigen petition binding assays including, for example, Surface plas binding fragment thereof, comprises the VH and VL region mon resonance (SPR)-based competition assays. amino acid sequences selected from the group consisting of 0239. In certain embodiments, the antibodies of the inven SEQID NO: 19 and 26, SEQID NO:20 and 27, SEQID NO: tion bind a conformational epitope of kallidin (KD) or 21 and 28; SEQ ID NO: 22 and 28: SEQID NO: 23 and 29: des Arg10-Kallidin (DAKD) which adopts a “Pro4kink’ con SEQID NO: 24 and 30; SEQID NO:25 and 31; SEQID NO: formation. As depicted in FIG. 17, a hallmark of the “Pro 4 38 and 39, SEQID NO:45 and 46, SEQID NO:53 and 54, or kink conformation is a type H tight turn in the main chain SEQ ID NO: 61 and 62, respectively. polypeptide backbone of KD or DAKD at Proline 4. As 0235. In certain embodiments, the antibody, or antigen known to those of skill in the art, a type H tight turn confor binding fragment thereof, comprises one or more CDR region mation comprises three residues (X1-X2-X3) with the carbo amino acid sequence selected from the group consisting of nyl of residue X1 forming a hydrogen bond with the amide N SEQID NO: 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 32,33, of residue X3, which is typically a glycine (see Richardson J 34,35, 36, 37, 40, 41, 42, 43,44, 47, 48,49, 50, 5152, 55,56, S. “The anatomy and taxonomy of protein structure.” Adv 57, 58, 59 and 60, wherein the one or more CDR region amino Protein Chem. 1981: 34:167-339, which is incorporated by acid sequences comprises at least one or more conservative reference herein). Accordingly, in certain embodiments, a amino acid Substitutions. type II tight turn conformation is formed by the Pro3-Pro4 0236. The present invention also encompasses “conserva Gly5 motif of KD or DADK. In more specific embodiments, tive amino acid substitutions' in the CDR amino acid the “Pro 4 kink’ conformation is further defined by all or sequences (e.g., SEQID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, substantially all of the remaining amino acids of KD (1-2 and US 2015/005O270 A1 Feb. 19, 2015

6-9) or DAKD adopting repeats of a sigmoid shape which Immunology 28(4/5):489-498 (1991); Studnicka et al., Pro align the hydrophobic side chains in a spatially stacking tein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS mode. 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565, III. Modified Anti-Kallidin ordes-Argo-Kallidin Antibodies 332). 0240. In certain embodiments, anti-Kallidin or des 0244. In a particular embodiment, a humanization method Arg10-Kallidin antibodies of the invention may comprise one is employed that is based on the impact of the molecular or more modifications. Modified forms of anti-Kallidin or flexibility of the antibody during and at immune recognition des-Arg10-Kallidin antibodies of the invention can be made (see WO2009/032661, which is incorporated herein by ref. using any techniques known in the art. erence in its entirety). Protein flexibility is related to the molecular motion of the protein molecule. Protein flexibility i) Reducing Immunogenicity is the ability of a whole protein, a part of a protein or a single 0241. In certain embodiments, anti-Kallidin or des amino acid residue to adopt an ensemble of conformations Arg10-Kallidin antibodies, or antigen binding fragments which differ significantly from each other. Information about thereof, of the invention are modified to reduce their immu protein flexibility can be obtained by performing protein nogenicity using art-recognized techniques. For example, X-ray crystallography experiments (see, for example, Kundu antibodies, or fragments thereof, can be chimericized, et al. 2002, Biophys J 83:723-732.), nuclear magnetic reso humanized, and/or deimmunized. nance experiments (see, for example, Freedberg et al., J. Am 0242. In one embodiment, an antibody, or antigenbinding Chem Soc 1998, 120(31):7916-7923) or by running molecu fragments thereof, of the invention may be chimeric. A chi lar dynamics (MD) simulations. An MD simulation of a pro meric antibody is an antibody in which different portions of tein is done on a computer and allows one to determine the the antibody are derived from different animal species, such motion of all protein atoms over a period of time by calculat as antibodies having a variable region derived from a murine ing the physical interactions of the atoms with each other. The monoclonal antibody and a human immunoglobulin constant output of a MD simulation is the trajectory of the studied region. Methods for producing chimeric antibodies, or frag protein over the period of time of the simulation. The trajec ments thereof, are known in the art. See, e.g., Morrison, tory is an ensemble of protein conformations, also called Science 229:1202 (1985); Oi et al., BioTechniques 4:214 Snapshots, which are periodically sampled over the period of (1986); Gillies et al., J. Immunol. Methods 125:191-202 the simulation, e.g. every 1 picosecond (ps). It is by analyzing (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, the ensemble of snapshots that one can quantify the flexibility which are incorporated herein by reference in their entireties. of the protein amino acid residues. Thus, a flexible residue is Techniques developed for the production of "chimeric anti one which adopts an ensemble of different conformations in bodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 the context of the polypeptide within which that residue (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda resides. MD methods are known in the art, see, e.g., Brooks et et al., Nature 314:452-454 (1985)) may be employed for the al. “Proteins: A Theoretical Perspective of Dynamics, Struc synthesis of said molecules. For example, a genetic sequence ture and Thermodynamics” (Wiley, New York, 1988). Several encoding a binding specificity of a mouse anti-Kallidin or software enable MD simulations, such as Amber (see Case et des-Arg10-Kallidinantibody molecule may be fused together al. (2005) J Comp Chem 26:1668-1688), Charmm (see with a sequence from a human antibody molecule of appro Brooks et al. (1983) J Comp Chem 4:187-217; and MacKerell priate biological activity. As used herein, a chimericantibody et al. (1998) in “The Encyclopedia of Computational Chem is a molecule in which different portions are derived from istry” vol. 1:271-177, Schleyer et al., eds. Chichester: John different animal species, such as those having a variable Wiley & Sons) or Impact (see Rizzo et al. JAm Chem Soc: region derived from a murine monoclonal antibody and a 2000: 122(51): 12898-12900.) human immunoglobulin constant region, e.g., humanized 0245 Most protein complexes share a relatively large and antibodies. planar buried surface and it has been shown that flexibility of 0243 In another embodiment, an antibody, or antigen binding partners provides the origin for their plasticity, binding fragment thereof, of the invention is humanized. enabling them to conformationally adapt to each other (Struc Humanized antibodies, have a binding specificity comprising ture (2000) 8. R137-R142). As such, examples of “induced one or more complementarity determining regions (CDRS) fit have been shown to play a dominant role in protein from a non-human antibody and framework regions from a protein interfaces. In addition, there is a steadily increasing human antibody molecule. Often, framework residues in the body of data showing that proteins actually bind ligands of human framework regions will be substituted with the corre diverse shapes sizes and composition (Protein Science (2002) sponding residue from the CDR donorantibody to alter, pref 11:184-187) and that the conformational diversity appears to erably improve, antigen binding. These framework Substitu be an essential component of the ability to recognize different tions are identified by methods well known in the art, e.g., by partners (Science (2003). 299, 1362-1367). Flexible residues modeling of the interactions of the CDR and framework resi are involved in the binding of protein-protein partners (Struc dues to identify framework residues important for antigen ture (2006) 14, 683-693). binding and sequence comparison to identify unusual frame 0246 The flexible residues can adopt a variety of confor work residues at particular positions. (See, e.g., Queen et al., mations that provide an ensemble of interaction areas that are U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 likely to be recognized by memory B cells and to trigger an (1988), which are incorporated herein by reference in their immunogenic response. Thus, an antibody can be humanized entireties.) Antibodies can be humanized using a variety of by modifying a number of residues from the framework so techniques known in the art including, for example, CDR that the ensemble of conformations and of recognition areas grafting (EP239,400; PCT publication WO 91/09967; U.S. displayed by the modified antibody resemble as much as Pat. Nos. 5.225,539; 5,530,101; and 5,585,089), veneering or possible those adopted by a human antibody. That can be resurfacing (EP 592,106; EP 519,596: Padlan, Molecular achieved by modifying a limited number of residues by: (1) US 2015/005O270 A1 Feb. 19, 2015 building a homology model of the parent mAb and running an antigen binding fragment thereof, to modify T cell epitopes MD simulation; (2) analyzing the flexible residues and iden (see, e.g., WO9852976A1, WO0034317A2). For example, tification of the most flexible residues of a non-human anti VH and VL sequences from the starting antibody may be body molecule, as well as identifying residues or motifs likely analyzed and a human T cell epitope 'map' may be generated to be a source of heterogeneity or of degradation reaction; (3) from each V region showing the location of epitopes in rela identifying a human antibody which displays the most similar tion to complementarity-determining regions (CDRS) and ensemble of recognition areas as the parent antibody; (4) other key residues within the sequence. Individual T cell determining the flexible residues to be mutated, residues or epitopes from the T cell epitope map are analyzed in order to motifs likely to be a source of heterogeneity and degradation identify alternative amino acid substitutions with a low risk of are also mutated; and (5) checking for the presence of known altering activity of the final antibody. A range of alternative T cell or B cell epitopes. The flexible residues can be found VHandVL sequences are designed comprising combinations using an MD calculation as taught herein using an implicit of amino acid Substitutions and these sequences are Subse solvent model, which accounts for the interaction of the water quently incorporated into a range of Kallidin ordes-Arg10 solvent with the protein atoms over the period of time of the Kallidin-specific antibodies or fragments thereof for use in simulation. the diagnostic and treatment methods disclosed herein, which 0247 Once the set of flexible residues has been identified are then tested for function. Typically, between 12 and 24 within the variable light and heavy chains, a set of human variant antibodies are generated and tested. Complete heavy heavy and light chain variable region frameworks that closely and light chain genes comprising modified V and human C resemble that of the antibody of interest are identified. That regions are then cloned into expression vectors and the Sub can be done, for example, using a BLAST search on the set of sequent plasmids introduced into cell lines for the production flexible residues against a database of antibody human germ of whole antibody. The antibodies are then compared in line sequence. It can also be done by comparing the dynamics appropriate biochemical and biological assays, and the opti of the parent mab with the dynamics of a library of germline mal variant is identified. canonical structures. The CDR residues and neighboring resi dues are excluded from the search to ensure high affinity for ii) Effector Functions and Fc Modifications the antigen is preserved. Flexible residues then are replaced. 0251 Anti-Kallidin or des-Arg10-Kallidin antibodies of 0248. When several human residues show similar homolo the invention may comprise an antibody constant region (e.g. gies, the selection is driven also by the nature of the residues an IgG constant region e.g., a human IgG constant region, that are likely to affect the solution behavior of the humanized e.g., a human IgG1 or IgG4 constant region) which mediates antibody. For instance, polar residues will be preferred in one or more effector functions. For example, binding of the Cl exposed flexible loops over hydrophobic residues. Residues component of complement to an antibody constant region which are a potential source of instability and heterogeneity may activate the complement system. Activation of comple are also mutated even if there are found in the CDRs. That will ment is important in the opsonisation and lysis of cell patho include exposed as Sulfoxide formation can gens. The activation of complement also stimulates the result from oxygen radicals, proteolytic cleavage of acid inflammatory response and may also be involved in autoim labile bonds such as those of the Asp-Pro dipeptide (Drug Dev mune hypersensitivity. Further, antibodies bind to receptors Res (2004) 61:137-154), deamidation sites found with an on various cells via the Fc region, with a Fc receptor binding exposed asparagine residue followed by a small amino acid, site on the antibody Fc region binding to a Fc receptor (FcR) such as Gly, Ser, Ala, H is, Asn or Cys (J Chromatog (2006) on a cell. There are a number of Fc receptors which are 837:35-43) and N-glycosylation sites, such as the Asn-X-Ser/ specific for different classes of antibody, including IgG Thr site. Typically, exposed methionines will be substituted (gamma receptors), IgE (epsilon receptors), IgA (alpha by a Leu, exposed asparagines will be replaced by a receptors) and IgM (mu receptors). Binding of antibody to Fc glutamine or by an aspartate, or the Subsequent residue will be receptors on cell Surfaces triggers a number of important and changed. For the glycosylation site (Asn-X-Ser/Thr), either diverse biological responses including engulfment and the Asin or the Ser/Thr residue will be changed. destruction of antibody-coated particles, clearance of 0249. The resulting composite antibody sequence is immune complexes, lysis of antibody-coated target cells by checked for the presence of known B cell or linear T-cell killer cells (called antibody-dependent cell-mediated cyto epitopes. A search is performed, for example, with the pub toxicity, or ADCC), release of inflammatory mediators, pla licly available Immune Epitope DataBase (IEDB) (PLoS Biol cental transfer and control of immunoglobulin production. In (2005) 3(3)e91). If a known epitope is found within the com preferred embodiments, the antibodies, or fragments thereof, posite sequence, another set of human sequences is retrieved of the invention bind to an Fc-gamma receptor. In alternative and substituted. Thus, unlike the resurfacing method of U.S. embodiments, anti-Kallidin ordes-Arg10-Kallidin antibod Pat. No. 5,639,641, both B-cell-mediated and T-cell-medi ies of the invention may comprise a constant region which is ated immunogenic responses are addressed by the method. devoid of one or more effector functions (e.g., ADCC activ The method also avoids the issue of loss of activity that is ity) and/or is unable to bind Fc receptor. sometimes observed with CDR grafting (U.S. Pat. No. 5,530, 0252 Certain embodiments of the invention include anti 101). In addition, stability and solubility issues also are con Kallidin ordes-Arg10-Kallidin antibodies in which at least sidered in the engineering and selection process, resulting in one amino acid in one or more of the constant region domains an antibody that is optimized for low immunogenicity, high has been deleted or otherwise altered so as to provide desired antigen affinity and improved biophysical properties. biochemical characteristics Such as reduced or enhanced 0250. In some embodiments, de-immunization can be effector functions, the ability to non-covalently dimerize, used to decrease the immunogenicity of and antibody, or increased ability to localize at the site of a tumor, reduced antigen binding fragment thereof. As used herein, the term serum half-life, or increased serum half-life when compared "de-immunization' includes alteration of an antibody, or with a whole, unaltered antibody of approximately the same US 2015/005O270 A1 Feb. 19, 2015

immunogenicity. For example, certain antibodies, or frag function and/or FcR binding. Said Fc variants may include, ments thereof, for use in the diagnostic and treatment meth for example, any one of the amino acid substitutions dis ods described herein are domain deleted antibodies which closed in International PCT Publications WO88/07089A1, comprise a polypeptide chain similar to an immunoglobulin WO96/14339A1, WO98/05787A1, WO98/23289A1, heavy chain, but which lack at least a portion of one or more WO99/51642A1, WO99/58572A1, WO00/09560A2, heavy chain domains. For instance, in certain antibodies, one WO00/32767A1, WO00/42072A2, WO02/44215A2, entire domain of the constant region of the modified antibody WO02/060919A2, WO03/074569A2, WOO4/016750A2, will be deleted, for example, all or part of the CH2 domain WOO4/029207A2, WOO4/035752A2, WOO4/063351A2, will be deleted. WOO4/074455A2, WOO4/099249A2, WO05/040217A2, 0253. In certain other embodiments, anti-Kallidin ordes WO05/070963A1, WO05/077981A2, WO05/092925A2, Arg10-Kallidin antibodies comprise constant regions derived WO05/123780A2, WO06/019447A1, WO06/047350A2, from different antibody isotypes (e.g., constant regions from and WO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739, two or more of a human IgG1, IgG2, IgG3, or IgG4). In other 277:5,834,250; 5,869,046; 6,096,871; 6,121,022: 6,194.551; embodiments, anti-Kallidin ordes-Arg10-Kallidin antibod 6.242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; ies comprises a chimeric hinge (i.e., a hinge comprising hinge 6,821,505; 6,998.253; and 7,083,784, each of which is incor portions derived from hinge domains of different antibody porated by reference herein. In one exemplary embodiment, isotypes, e.g., an upper hinge domain from an IgG4 molecule an antibody of the invention may comprise an Fc variant and an IgG1 middle hinge domain). In one embodiment, an comprising an amino acid Substitution at EU position 268 anti-Kallidin ordes-Arg10-Kallidin antibodies comprises an (e.g., H268D or H268E). In another exemplary embodiment, Fc region orportion thereoffrom a human IgG4 molecule and an antibody of the invention may comprise an amino acid a Ser228Pro mutation (EU numbering) in the core hinge substitution at EU position 239 (e.g., S239D or S239E) and/or region of the molecule. EU position 332 (e.g., 1332D or 13320). 0254. In certain anti-Kallidin ordes-Arg10-Kallidin anti 0258. In certain embodiments, an antibody of the inven bodies, the Fc portion may be mutated to increase or decrease tion may comprise an Fc variant comprising an amino acid effector function using techniques known in the art. For substitution which alters the antigen-independent effector example, the deletion or inactivation (through point muta functions of the antibody, in particular the circulating half-life tions or other means) of a constant region domain may reduce of the antibody. Such antibodies exhibit either increased or Fc receptor binding of the circulating modified antibody decreased binding to FcRn when compared to antibodies thereby increasing tumor localization. In other cases it may be lacking these substitutions, therefore, have an increased or that constant region modifications consistent with the instant decreased half-life in serum, respectively. Fc variants with invention moderate complement binding and thus reduce the improved affinity for FcRn are anticipated to have longer serum half life and nonspecific association of a conjugated serum half-lives, and Such molecules have useful applications cytotoxin. Yet other modifications of the constant region may in methods of treating mammals where long half-life of the be used to modify disulfide linkages or oligosaccharide moi administered antibody is desired, e.g., to treat a chronic dis eties that allow for enhanced localization due to increased ease or disorder. In contrast, Fc variants with decreased FcRn antigen specificity or flexibility. The resulting physiological binding affinity are expected to have shorter half-lives, and profile, bioavailability and other biochemical effects of the Such molecules are also useful, for example, for administra modifications. Such as tumor localization, biodistribution and tion to a mammal where a shortened circulation time may be serum half-life, may easily be measured and quantified using advantageous, e.g. for in vivo diagnostic imaging or in situ well know immunological techniques without undue experi ations where the starting antibody has toxic side effects when mentation. present in the circulation for prolonged periods. Fc variants 0255. In certain embodiments, an Fc domain employed in with decreased FcRn binding affinity are also less likely to an antibody of the invention is an Fc variant. As used herein, cross the placenta and, thus, are also useful in the treatment of the term “Fc variant” refers to an Fc domain having at least diseases or disorders in pregnant women. In addition, other one amino acid substitution relative to the wild-type Fc applications in which reduced FcRn binding affinity may be domain from which said Fc domain is derived. For example, desired include those applications in which localization the wherein the Fc domain is derived from a human IgG1 anti brain, kidney, and/or liver is desired. In one exemplary body, the Fc variant of said human IgG1 Fc domain comprises embodiment, the altered antibodies of the invention exhibit at least one amino acid substitution relative to said Fc domain. reduced transport across the epithelium of kidney glomeruli 0256 The amino acid substitution(s) of an Fc variant may from the vasculature. In another embodiment, the altered be located at any position (i.e., any EU convention amino acid antibodies of the invention exhibit reduced transport across position) within the Fc domain. In one embodiment, the Fc the blood brainbarrier (BBB) from the brain, into the vascular variant comprises a Substitution at an amino acid position space. In one embodiment, an antibody with altered FcRn located in a hinge domain or portion thereof. In another binding comprises an Fc domain having one or more amino embodiment, the Fc variant comprises a Substitution at an acid substitutions within the “FcRn binding loop' of an Fc amino acid position located in a CH2 domain or portion domain. The FcRn binding loop is comprised of amino acid thereof. In another embodiment, the Fc variant comprises a residues 280–299 (according to EU numbering). Exemplary Substitution at an amino acid position located in a CH3 amino acid substitutions which altered FcRn binding activity domain or portion thereof. In another embodiment, the Fc are disclosed in International PCT Publication No. WO05/ variant comprises a Substitution at an amino acid position 047327 which is incorporated by reference herein. In certain located in a CH4 domain or portion thereof. exemplary embodiments, the antibodies, or fragments 0257 The antibodies of the invention may employ any thereof, of the invention comprise an Fc domain having one or art-recognized Fc variant which is known to impart an more of the following substitutions: V284E, H285E, N286D, improvement (e.g., reduction or enhancement) in effector K290E and S304D (EU numbering). US 2015/005O270 A1 Feb. 19, 2015

0259. In other embodiments, antibodies, for use in the assays and effector molecules such as heterologous polypep diagnostic and treatment methods described herein have a tides, drugs, radionuclides, or toxins. See, e.g., PCT publica constant region, e.g., an IgG1 or IgG4 heavy chain constant tions WO92/08495; WO 91/14438: WO 89/12624; U.S. Pat. region, which is altered to reduce or eliminate glycosylation. No. 5,314,995; and EP 396,387. For example, an antibody of the invention may also comprise 0263 Anti-Kallidin or des-Arg10-Kallidin antibodies an Fc variant comprising an amino acid substitution which may be fused to heterologous polypeptides to increase the in alters the glycosylation of the antibody. For example, said Fc Vivo half life or for use in immunoassays using methods variant may have reduced glycosylation (e.g., N- or O-linked known in the art. For example, in one embodiment, PEG can glycosylation). In exemplary embodiments, the Fc variant be conjugated to the anti-Kallidin ordes-Arg10-Kallidin anti comprises reduced glycosylation of the N-linked glycan nor bodies of the invention to increase their half-life in vivo. mally found at amino acid position 297 (EU numbering). In Leong, S. R., et al., Cytokine 16:106 (2001); Adv. in Drug another embodiment, the antibody has an amino acid Substi Deliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. tution near or within a glycosylation motif, for example, an Transactions 30:512 (2002). N-linked glycosylation motif that contains the amino acid 0264. Moreover, anti-Kallidin ordes-Arg10-Kallidinanti sequence NXT or NXS. In a particular embodiment, the anti bodies of the invention can be fused to marker sequences, body comprises an Fc variant with an amino acid Substitution Such as a peptide to facilitate their purification or detection. In at amino acid position 228 or 299 (EU numbering). In more preferred embodiments, the marker amino acid sequence is a particular embodiments, the antibody comprises an IgG1 or hexa-histidine peptide (SEQ ID NO: 137), such as the tag IgG4 constant region comprising an S228P and a T299A provided inapGEvector (QIAGEN, Inc., 9259 Eton Avenue, mutation (EU numbering). Chatsworth, Calif., 91311), among others, many of which are 0260 Exemplary amino acid substitutions which confer commercially available. As described in Gentz et al., Proc. reduce or altered glycosylation are disclosed in International Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa PCT Publication No. WO05/018572, which is incorporated histidine (SEQ ID NO: 137) provides for convenient purifi by reference herein. In preferred embodiments, the antibod cation of the fusion protein. Other peptide tags useful for ies, or fragments thereof, of the invention are modified to purification include, but are not limited to, the “HA' tag, eliminate glycosylation. Such antibodies, or fragments which corresponds to an epitope derived from the influenza thereof, may be referred to as “agly’ antibodies, or fragments hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and thereof (e.g. "agly’ antibodies). While not being bound by the “flag” tag. theory, it is believed that “agly’ antibodies, or fragments 0265 Anti-Kallidin or des-Arg10-Kallidin antibodies of thereof, may have an improved safety and stability profile in the invention may be used in non-conjugated form or may be vivo. Exemplary agly antibodies, or fragments thereof, com conjugated to at least one of a variety of molecules, e.g., to prise anaglycosylated Fc region of an IgG4 antibody which is improve the therapeutic properties of the molecule, to facili devoid of Fc-effector function thereby eliminating the poten tate target detection, or for imaging or therapy of the patient. tial for Fc mediated toxicity to the normal vital organs that Anti-Kallidin ordes-Arg10-Kallidin antibodies of the inven express Kallidin ordes-Arg10-Kallidin. In yet other embodi tion can be labeled or conjugated either before or after puri ments, antibodies, or fragments thereof, of the invention com fication, when purification is performed. In particular, anti prise an altered glycan. For example, the antibody may have Kallidin or des-Arg10-Kallidin antibodies of the invention a reduced number of fucose residues on an N-glycan at may be conjugated to therapeutic agents, prodrugs, peptides, Asn297 of the Fc region, i.e., is afucosylated. In another proteins, enzymes, viruses, lipids, biological response modi embodiment, the antibody may have an altered number of fiers, pharmaceutical agents, or PEG. sialic acid residues on the N-glycan at Asn297 of the Fc 0266 The present invention further encompasses anti region. Kallidin or des-Arg10-Kallidin antibodies of the invention iii) Covalent Attachment conjugated to a diagnostic or therapeutic agent. The anti 0261) Anti-Kallidin or des-Arg10-Kallidin antibodies of Kallidin ordes-Arg10-Kallidin antibodies can be used diag the invention may be modified, e.g., by the covalent attach nostically to, for example, monitor the development or pro ment of a molecule to the antibody such that covalent attach gression of a immune cell disorder (e.g., CLL) as part of a ment does not prevent the antibody from specifically binding clinical testing procedure to, e.g., determine the efficacy of a to its cognate epitope. For example, but not by way of limi given treatment and/or prevention regimen. Detection can be tation, the antibodies, or fragments thereof, of the invention facilitated by coupling the anti-Kallidin ordes-Arg10-Kalli may be modified by glycosylation, acetylation, pegylation, din antibodies to a detectable substance. Examples of detect phosphorylation, amidation, derivatization by known protect able Substances include various enzymes, prosthetic groups, ing/blocking groups, proteolytic cleavage, linkage to a cellu fluorescent materials, luminescent materials, bioluminescent lar ligand or other protein, etc. Any of numerous chemical materials, radioactive materials, positron emitting metals modifications may be carried out by known techniques, using various positron emission tomographies, and nonradio including, but not limited to specific chemical cleavage, active paramagnetic metal ions. See, for example, U.S. Pat. acetylation, formylation, etc. Additionally, the derivative may No. 4,741,900 for metal ions which can be conjugated to contain one or more non-classical amino acids. antibodies for use as diagnostics according to the present 0262 Antibodies, or fragments thereof, of the invention invention. Examples of suitable enzymes include horseradish may further be recombinantly fused to a heterologous peroxidase, alkaline phosphatase, beta.-galactosidase, or polypeptide at the N- or C-terminus or chemically conjugated acetylcholinesterase; examples of Suitable prosthetic group (including covalent and non-covalent conjugations) to complexes include streptavidin/biotin and avidin/biotin: polypeptides or other compositions. For example, anti-Kalli examples of suitable fluorescent materials include umbellif din ordes-Arg10-Kallidin antibodies may be recombinantly erone, fluorescein, fluorescein isothiocyanate, rhodamine, fused or conjugated to molecules useful as labels in detection dichlorotriazinylamine fluorescein, dansyl chloride or phy US 2015/005O270 A1 Feb. 19, 2015 coerythrin; an example of a luminescent material includes 0272) Numerous expression vector systems may be luminol; examples of bioluminescent materials include employed for the purposes of this invention. For example, one luciferase, luciferin, and aequorin; and examples of Suitable class of vector utilizes DNA elements which are derived from radioactive material include 125I, 131I, 111 In or 99Tc. animal viruses such as bovine papilloma virus, polyoma 0267 Anti-Kallidin ordes-Arg10-Kallidin antibodies for virus, adenovirus, vaccinia virus, baculovirus, retroviruses use in the diagnostic and treatment methods disclosed herein (RSV. MMTV or MOMLV) or SV40 virus. Others involve the may be conjugated to cytotoxins (such as radioisotopes, cyto use of polycistronic systems with internal ribosome binding toxic drugs, or toxins) therapeutic agents, cytostatic agents, sites. Additionally, cells which have integrated the DNA into biological toxins, prodrugs, peptides, proteins, enzymes, their chromosomes may be selected by introducing one or viruses, lipids, biological response modifiers, pharmaceutical more markers which allow selection of transfected host cells. agents, immunologically active ligands (e.g., lymphokines or The marker may provide for prototrophy to an auxotrophic other antibodies wherein the resulting molecule binds to both host, biocide resistance (e.g., antibiotics) or resistance to the neoplastic cell and an effector cell such as a T cell), or heavy metals such as copper. The selectable marker gene can PEG. either be directly linked to the DNA sequences to be 0268. In another embodiment, an anti-Kallidin or des expressed, or introduced into the same cell by cotransforma Arg10-Kallidin antibody for use in the diagnostic and treat tion. Additional elements may also be needed for optimal ment methods disclosed herein can be conjugated to a mol synthesis of mRNA. These elements may include signal ecule that decreases tumor cell growth. In other sequences, splice signals, as well as transcriptional promot embodiments, the disclosed compositions may comprise ers, enhancers, and termination signals. In particularly pre antibodies, or fragments thereof, coupled to drugs or pro ferred embodiments the cloned variable region genes are drugs. Still other embodiments of the present invention com inserted into an expression vector along with the heavy and prise the use of antibodies, or fragments thereof, conjugated light chain constant region genes (preferably human) Syn to specific biotoxins or their cytotoxic fragments such as thetic as discussed above. ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin. 0273. In other preferred embodiments the anti-Kallidin or The selection of which conjugated or unconjugated antibody des-Arg10-Kallidin antibodies, or fragments thereof, of the to use will depend on the type and stage of cancer, use of invention may be expressed using polycistronic constructs. In adjunct treatment (e.g., chemotherapy or external radiation) Such expression systems, multiple gene products of interest and patient condition. It will be appreciated that one skilled in Such as heavy and light chains of antibodies may be produced the art could readily make such a selection in view of the from a single polycistronic construct. These systems advan teachings herein. tageously use an internal ribosome entry site (IRES) to pro 0269. It will be appreciated that, in previous studies, anti vide relatively high levels of polypeptides of the invention in tumor antibodies labeled with isotopes have been used suc eukaryotic host cells. Compatible IRES sequences are dis cessfully to destroy tumor cells in animal models, and in some closed in U.S. Pat. No. 6,193.980, which is incorporated by cases in humans. Exemplary radioisotopes include: 90Y. reference herein. Those skilled in the art will appreciate that 125I, 131I, 123I, 111 In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, Such expression systems may be used to effectively produce 177Lu, 186Re and 188Re. The radionuclides act by produc the full range of polypeptides disclosed in the instant appli ing ionizing radiation which causes multiple strand breaks in cation. nuclear DNA, leading to cell death. The isotopes used to 0274 More generally, once a vector or DNA sequence produce therapeutic conjugates typically produce high encoding an antibody, or fragment thereof, has been prepared, energy alpha- or beta-particles which have a short path length. the expression vector may be introduced into an appropriate Such radionuclides kill cells to which they are in close prox host cell. That is, the host cells may be transformed. Intro imity, for example neoplastic cells to which the conjugate has duction of the plasmid into the host cell can be accomplished attached or has entered. They have little or no effect on non by various techniques well known to those of skill in the art. localized cells. Radionuclides are essentially non-immuno These include, but are not limited to, transfection (including genic. electrophoresis and electroporation), protoplast fusion, cal IV. Expression of Anti-Kallidin ordes-Arg10-Kallidin Anti cium phosphate precipitation, cell fusion with enveloped bodies, or Antigen Binding Fragments Thereof DNA, microinjection, and infection with intact virus. See, 0270. Following manipulation of the isolated genetic Ridgway, A. A. G. "Mammalian Expression Vectors' Chapter material to provide anti-Kallidin ordes-Arg10-Kallidin anti 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. bodies of the invention as set forth above, the genes are (Butterworths, Boston, Mass. 1988). Most preferably, plas typically inserted in an expression vector for introduction into mid introduction into the host is via electroporation. The host cells that may be used to produce the desired quantity of transformed cells are grown under conditions appropriate to the claimed antibodies, or fragments thereof. the production of the light chains and heavy chains, and (0271 The term “vector” or “expression vector” is used assayed for heavy and/or light chain protein synthesis. Exem herein for the purposes of the specification and claims, to plary assay techniques include enzyme-linked immunosor mean vectors used in accordance with the present invention as bent assay (ELISA), radioimmunoassay (RIA), or floures a vehicle for introducing into and expressing a desired gene in cence-activated cell SOrter analysis (FACS), a cell. As known to those skilled in the art, such vectors may immunohistochemistry and the like. easily be selected from the group consisting of plasmids, 0275. As used herein, the term “transformation' shall be phages, viruses and retroviruses. In general, vectors compat used in abroad sense to refer to the introduction of DNA into ible with the instant invention will comprise a selection a recipient host cell that changes the genotype and conse marker, appropriate restriction sites to facilitate cloning of the quently results in a change in the recipient cell. desired gene and the ability to enter and/or replicate in 0276 Along those same lines, “host cells' refers to cells eukaryotic or prokaryotic cells. that have been transformed with vectors constructed using US 2015/005O270 A1 Feb. 19, 2015 recombinant DNA techniques and encoding at least one het marker for a mutant strain of yeast lacking the ability to grow erologous gene. In descriptions of processes for isolation of in tryptophan, for example ATCC No. 44076 or PEP4-1 polypeptides from recombinant hosts, the terms “cell' and (Jones, Genetics, 85:12 (1977)). The presence of the trpl “cell culture' are used interchangeably to denote the source lesion as a characteristic of the yeast host cell genome then of antibody unless it is clearly specified otherwise. In other provides an effective environment for detecting transforma words, recovery of polypeptide from the “cells' may mean tion by growth in the absence of tryptophan. either from spun down whole cells, or from the cell culture V. Pharmaceutical Formulations and Methods of Administra containing both the medium and the Suspended cells. tion of Anti-Kallidin ordes-Arg10-Kallidin Antibodies. 0277. In one embodiment, the host cell line used for anti 0281. In another aspect, the invention provides pharma body expression is of mammalian origin; those skilled in the ceutical compositions comprising an anti-Kallidin or des art can determine particular host cell lines which are best Arg10-Kallidin antibody, or fragment thereof. suited for the desired gene product to be expressed therein. 0282 Methods of preparing and administering antibodies, Exemplary host cell lines include, but are not limited to, or fragments thereof, of the invention to a subject are well DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR known to or are readily determined by those skilled in the art. minus), HELA (human cervical carcinoma). CVI (monkey The route of administration of the antibodies, or fragments kidney line), COS (a derivative of CVI with SV40 Tantigen), thereof, of the invention may be oral, parenteral, by inhalation R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse or topical. The term parenteral as used herein includes intra fibroblast), HAK (hamster kidney line), SP2/O (mouse venous, intraarterial, intraperitoneal, intramuscular, Subcuta myeloma), BFA-1c1 BPT (bovine endothelial cells), RAJI neous, rectal or vaginal administration. The intravenous, (human lymphocyte), 293 (human kidney). In one embodi intraarterial, Subcutaneous and intramuscular forms of ment, the cell line provides for altered glycosylation, e.g., parenteral administration are generally preferred. While all afucosylation, of the antibody expressed therefrom (e.g., these forms of administration are clearly contemplated as PER.C6(R) (Crucell) or FUT8-knock-out CHO cell lines (Po being within the scope of the invention, a form for adminis telligent(R) Cells) (Biowa, Princeton, N.J.)). In one embodi tration would be a solution for injection, in particular for ment NS0 cells may be used. CHO cells are particularly intravenous or intraarterial injection or drip. Usually, a Suit preferred. Host cell lines are typically available from com able pharmaceutical composition for injection may comprise mercial services, the American Tissue Culture Collection or a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant from published literature. (e.g. polysorbate), optionally a stabilizer agent (e.g. human 0278. In vitro production allows scale-up to give large albumin), etc. However, in other methods compatible with the amounts of the desired polypeptides. Techniques for mam teachings herein, the polypeptides can be delivered directly to malian cell cultivation under tissue culture conditions are the site of the adverse cellular population thereby increasing known in the art and include homogeneous Suspension cul the exposure of the diseased tissue to the therapeutic agent. ture, e.g. in an airlift reactor or in a continuous stirrer reactor, 0283 Preparations for parenteral administration include or immobilized or entrapped cell culture, e.g. in hollow fibers, sterile aqueous or non-aqueous solutions, Suspensions, and microcapsules, on agarose microbeads or ceramic cartridges. emulsions. Examples of non-aqueous solvents are propylene If necessary and/or desired, the Solutions of polypeptides can glycol, polyethylene glycol, vegetable oils such as olive oil, be purified by the customary chromatography methods, for and injectable organic esters such as ethyl oleate. Aqueous example gel filtration, ion-exchange chromatography, chro carriers include water, alcoholic/aqueous Solutions, emul matography over DEAF-cellulose and/or (immuno-)affinity sions or Suspensions, including saline and buffered media. In chromatography. the Subject invention, pharmaceutically acceptable carriers 0279 Genes encoding the anti-Kallidin or des-Arg10 include, but are not limited to, 0.01-0.1 M and preferably Kallidin antibodies, or fragments thereof, of the invention can 0.05M phosphate buffer or 0.8% saline. Other common also be expressed non-mammalian cells such as bacteria or parenteral vehicles include Sodium phosphate Solutions, yeast or plant cells. In this regard it will be appreciated that Ringer's dextrose, dextrose and sodium chloride, lactated various unicellular non-mammalian microorganisms such as Ringer's, or fixed oils. Intravenous vehicles include fluid and bacteria can also be transformed; i.e. those capable of being nutrient replenishers, electrolyte replenishers, such as those grown in cultures or fermentation. Bacteria, which are sus based on Ringer's dextrose, and the like. Preservatives and ceptible to transformation, include members of the entero other additives may also be present such as for example, bacteriaceae, such as strains of Escherichia coli or Salmo antimicrobials, antioxidants, chelating agents, and inert gases nella, Bacillaceae. Such as Bacillus subtilis, Pneumococcus, and the like. More particularly, pharmaceutical compositions Streptococcus, and Haemophilus influenzae. It will further be Suitable for injectable use include sterile aqueous solutions appreciated that, when expressed in bacteria, the polypep (where water soluble) or dispersions and sterile powders for tides can become part of inclusion bodies. The polypeptides the extemporaneous preparation of sterile injectable solutions must be isolated, purified and then assembled into functional or dispersions. In Such cases, the composition must be sterile molecules. and should be fluid to the extent that easy syringability exists. 0280. In addition to prokaryotes, eukaryotic microbes It should be stable under the conditions of manufacture and may also be used. Saccharomyces cerevisiae, or common storage and will preferably be preserved against the contami baker's yeast, is the most commonly used among eukaryotic nating action of microorganisms, such as bacteria and fungi. microorganisms although a number of other strains are com The carrier can be a solvent or dispersion medium containing, monly available. For expression in Saccharomyces, the plas for example, water, ethanol, polyol (e.g., glycerol, propylene mid YRp7, for example, (Stinchcomb et al., Nature, 282:39 glycol, and liquid polyethylene glycol, and the like), and (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et suitable mixtures thereof. The proper fluidity can be main al., Gene, 10:157 (1980)) is commonly used. This plasmid tained, for example, by the use of a coating Such as lecithin, by already contains the TRP1 gene which provides a selection the maintenance of the required particle size in the case of US 2015/005O270 A1 Feb. 19, 2015

dispersion and by the use of surfactants. Prevention of the weekly. In some methods, two or more monoclonal antibod action of microorganisms can beachieved by various antibac ies with different binding specificities are administered terial and antifungal agents, for example, parabens, chlorobu simultaneously, in which case the dosage of each antibody tanol, phenol, ascorbic acid, thimerosal and the like. In many administered may fall within the ranges indicated. cases, it will be preferable to include isotonic agents, for 0288 Antibodies, or fragments thereof, of the invention example, Sugars, polyalcohols, such as mannitol, Sorbitol, or can be administered on multiple occasions. Intervals between Sodium chloride in the composition. Prolonged absorption of single dosages can be, e.g., daily, weekly, monthly or yearly. the injectable compositions can be brought about by includ Intervals can also be irregular as indicated by measuring ing in the composition an agent which delays absorption, for blood levels of polypeptide or target molecule in the patient. example, aluminum monostearate and gelatin. In some methods, dosage is adjusted to achieve a certain 0284. In any case, sterile injectable solutions can be pre plasma antibody or toxin concentration, e.g., 1-1000 ug/ml or pared by incorporating an active compound (e.g., an antibody 25-300 ug/ml. Alternatively, antibodies, or fragments thereof, by itself or in combination with other active agents) in the can be administered as a Sustained release formulation, in required amount in an appropriate solvent with one or a which case less frequent administration is required. Dosage combination of ingredients enumerated herein, as required, and frequency vary depending on the half-life of the antibody followed by filtered sterilization. Generally, dispersions are in the patient. In general, humanized antibodies show the prepared by incorporating the active compound into a sterile longest half-life, followed by chimeric antibodies and non vehicle, which contains a basic dispersion medium and the human antibodies. In one embodiment, the antibodies, or required other ingredients from those enumerated above. In fragments thereof, of the invention can be administered in the case of sterile powders for the preparation of sterile inject unconjugated form. In another embodiment, the antibodies of able solutions, the preferred methods of preparation are the invention can be administered multiple times in conju vacuum drying and freeze-drying, which yields a powder of gated form. In still another embodiment, the antibodies, or an active ingredient plus any additional desired ingredient fragments thereof, of the invention can be administered in from a previously sterile-filtered solution thereof. The prepa unconjugated form, then in conjugated form, or vise versa. rations for injections are processed, filled into containers such 0289. The dosage and frequency of administration can as ampoules, bags, bottles, Syringes or vials, and sealed under vary depending on whether the treatment is prophylactic or aseptic conditions according to methods known in the art. therapeutic. In prophylactic applications, compositions con Further, the preparations may be packaged and sold in the taining the present antibodies or a cocktail thereofare admin form of a kit such as those described in co-pending U.S. Ser. istered to a patient not already in the disease state to enhance No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which the patients resistance. Such an amount is defined to be a is incorporated herein by reference. Such articles of manu “prophylactic effective dose.” In this use, the precise amounts facture will preferably have labels or package inserts indicat again depend upon the patients state of health and general ing that the associated compositions are useful for treating a immunity, but generally range from 0.1 to 25 mg per dose, Subject Suffering from, or predisposed to autoimmune or neo especially 0.5 to 2.5 mg per dose. A relatively low dosage is plastic disorders. administered at relatively infrequent intervals over a long 0285 Effective doses of the stabilized antibodies, or frag period of time. Some patients continue to receive treatment ments thereof, of the present invention, for the treatment of for the rest of their lives. the above described conditions vary depending upon many different factors, including means of administration, target 0290. In therapeutic applications, a relatively high dosage site, physiological state of the patient, whether the patient is (e.g., from about 1 to 400 mg/kg of antibody per dose, with human or an animal, other medications administered, and dosages of from 5 to 25 mg being more commonly used for whether treatment is prophylactic or therapeutic. Usually, the radioimmunoconjugates and higher doses for cytotoxin-drug patient is a human, but non-human mammals including trans conjugated molecules) at relatively short intervals is some genic mammals can also be treated. Treatment dosages may times required until progression of the disease is reduced or betitrated using routine methods known to those of skill in the terminated, and preferably until the patient shows partial or art to optimize safety and efficacy. complete amelioration of symptoms of disease. Thereafter, 0286 For passive immunization with an antibody of the the patent can be administered a prophylactic regime. invention, the dosage may range, e.g., from about 0.0001 to 0291. In one embodiment, a subject can be treated with a 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 nucleic acid molecule encoding a polypeptide of the inven mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 tion (e.g., in a vector). Doses for nucleic acids encoding mg/kg, etc.), of the host body weight. For example dosages polypeptides range from about 10ng to 1 g, 100ng to 100 mg. can be 1 mg/kg body weight or 10 mg/kg body weight or 1 ug to 10 mg. or 30-300 ug DNA per patient. Doses for within the range of 1-10 mg/kg, preferably at least 1 mg/kg. infectious viral vectors vary from 10-100, or more, virions per Doses intermediate in the above ranges are also intended to be dose. within the scope of the invention. 0292. Therapeutic agents can be administered by 0287 Subjects can be administered such doses daily, on parenteral, topical, intravenous, oral, Subcutaneous, intraar alternative days, weekly or according to any other schedule terial, intracranial, intraperitoneal, intranasal or intramuscu determined by empirical analysis. An exemplary treatment lar means for prophylactic and/or therapeutic treatment. entails administration in multiple dosages over a prolonged Intramuscular injection or intravenous infusion are preferred period, for example, of at least six months. Additional exem for administration of a antibody of the invention. In some plary treatment regimes entail administration once per every methods, therapeutic antibodies, or fragments thereof, are two weeks or once a month or once every 3 to 6 months. injected directly into the cranium. In some methods, antibod Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg ies, or fragments thereof, are administered as a Sustained on consecutive days, 30 mg/kg on alternate days or 60 mg/kg release composition or device, such as a MedipadTM device. US 2015/005O270 A1 Feb. 19, 2015

0293 Agents of the invention can optionally be adminis In the case of tumor cells, the polypeptide will be preferably tered in combination with other agents that are effective in be capable of interacting with selected immunoreactive anti treating the disorder or condition in need of treatment (e.g., gens on neoplastic or immunoreactive cells and provide for an prophylactic or therapeutic). Preferred additional agents are increase in the death of those cells. Of course, the pharma those which are art recognized and are standardly adminis ceutical compositions of the present invention may be admin tered for a particular disorder. istered in single or multiple doses to provide for a pharma 0294 Effective single treatment dosages (i.e., therapeuti ceutically effective amount of the polypeptide. cally effective amounts) of 90Y-labeled antibodies of the 0297. In keeping with the scope of the present disclosure, invention range from between about 5 and about 75 mCi. the antibodies of the invention may be administered to a more preferably between about 10 and about 40 mCi. Effec human or other animal in accordance with the aforemen tive single treatment non-marrow ablative dosages of 131 I tioned methods of treatment in an amount Sufficient to pro labeled antibodies range from between about 5 and about 70 duce atherapeutic or prophylactic effect. The polypeptides of mCi, more preferably between about 5 and about 40 mCi. the invention can be administered to such human or other Effective single treatment ablative dosages (i.e., may require animal in a conventional dosage form prepared by combining autologous bone marrow transplantation) of 131I-labeled the antibody of the invention with a conventional pharmaceu antibodies range from between about 30 and about 600 mCi, tically acceptable carrier or diluent according to known tech more preferably between about 50 and less than about 500 niques. It will be recognized by one of skill in the art that the mCi. In conjunction with a chimeric modified antibody, form and character of the pharmaceutically acceptable carrier owing to the longer circulating half life vis-a-vis murine or diluent is dictated by the amount of active ingredient with antibodies, an effective single treatment non-marrow ablative which it is to be combined, the route of administration and dosages of iodine-131 labeled chimeric antibodies range other well-known variables. Those skilled in the art will fur from between about 5 and about 40 mCi, more preferably less ther appreciate that a cocktail comprising one or more species than about 30 mCi. Imaging criteria for, e.g., the 111 In label, of polypeptides according to the present invention may prove are typically less than about 5 mCi. to be particularly effective. 0295 While a great deal of clinical experience has been gained with 131I and 0.90Y, other radiolabels are known in VI. Methods of Treating Kallidin ordes-Arg10-Kallidin-As the art and have been used for similar purposes. Still other sociated Disease or Disorders radioisotopes are used for imaging. For example, additional 0298. The anti-Kallidin ordes-Arg10-Kallidin antibodies, radioisotopes which are compatible with the scope of the or fragments thereof, of the invention are useful for antago instant invention include, but are not limited to, 123I, 125I, nizing Kallidin ordes-Arg10-Kallidin activity. Accordingly, 32P 57Co., 64Cu, 67Cu, 77Br, 81Rb, 81 Kr, 87Sr, 113In, in another aspect, the invention provides methods for treating 127Cs, 129Cs, 1321, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, Kallidin ordes-Arg10-Kallidin-associated diseases or disor 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, ders by administering to a Subject in need of thereof a phar 225Ac, 21 1A 213Bi. In this respect alpha, gamma and beta maceutical composition comprising one or more anti-Kalli emitters are all compatible with in the instant invention. Fur din or des-Arg10-Kallidin antibody, or antigen binding ther, in view of the instant disclosure it is submitted that one fragment thereof of the invention. skilled in the art could readily determine which radionuclides are compatible with a selected course of treatment without 0299 Kallidin ordes-Arg10-Kallidin-associated diseases undue experimentation. To this end, additional radionuclides or disorders amenable to treatment include, without limita which have already been used in clinical diagnosis include tion, pathophysiologic conditions such as inflammation, 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as 111 In. trauma, burns, shock, allergy, acute or chronic pain, and fibro Antibodies have also been labeled with a variety of radionu sis, e.g., renal fibrosis. In certain exemplary, embodiments, clides for potential use in targeted immunotherapy (PeirerSZ antibodies of the invention may be issued to treat renal fibro et al. Immunol. Cell Biol. 65: 111-125 (1987)). These radio sis and associated acute kidney injury as well as chronic nuclides include 188Re and 186Re as well as 199Au and kidney diseases which are the main causes of end-stage renal 67Cu to a lesser extent. U.S. Pat. No. 5,460,785 provides failure. additional data regarding Such radioisotopes and is incorpo (0300. One skilled in the art would be able, by routine rated herein by reference. experimentation, to determine what an effective, non-toxic 0296. As previously discussed, the antibodies, or frag amount of antibody (or additional therapeutic agent) would ments thereof, of the invention, can be administered in a be for the purpose of treating a Kallidin ordes-Arg10-Kalli pharmaceutically effective amount for the in vivo treatment din-associated disease or disorder. For example, a therapeu of mammalian disorders. In this regard, it will be appreciated tically active amount of a polypeptide may vary according to that the disclosed antibodies, or fragments thereof, will be factors such as the disease stage (e.g., stage I versus stage IV), formulated so as to facilitate administration and promote age, sex, medical complications (e.g., immunosuppressed stability of the active agent. Preferably, pharmaceutical com conditions or diseases) and weight of the Subject, and the positions in accordance with the present invention comprise a ability of the antibody to elicit a desired response in the pharmaceutically acceptable, non-toxic, sterile carrier Such Subject. The dosage regimen may be adjusted to provide the as physiological Saline, non-toxic buffers, preservatives and optimum therapeutic response. For example, several divided the like. For the purposes of the instant application, a phar doses may be administered daily, or the dose may be propor maceutically effective amount of a antibody of the invention, tionally reduced as indicated by the exigencies of the thera conjugated or unconjugated to a therapeutic agent, shall be peutic situation. Generally, however, an effective dosage is held to mean an amount sufficient to achieve effective binding expected to be in the range of about 0.05 to 100 milligrams per to a target and to achieve a benefit, e.g., to ameliorate symp kilogram body weight per day and more preferably from toms of a disease or disorder or to detect a substance or a cell. about 0.5 to 10, milligrams per kilogram body weight per day. US 2015/005O270 A1 Feb. 19, 2015 19

VII. Exemplification ul per mouse (day 0). On day 21, mice were boosted with a 0301 The present invention is further illustrated by the mixture of even amounts of KLH-KD and KD-KLH in PBS following examples which should not be construed as further as an antigen total of 50 ug per mouse mixed at 1:1 ratio of limiting. The contents of Sequence Listing, figures and all Sigma Adjuvant System (Sigma cat #6322) in a total volume references, patents and published patent applications cited of 200 ul per mouse. On day 30, blood samples were har throughout this application are expressly incorporated herein vested for KD specific antibody titer evaluation. On day 51, by reference. mice were boosted for fusion with a mixture of even amounts 0302 Furthermore, in accordance with the present inven of KLH-KD and KD-KLH in PBS as an antigen total of 50 ug tion there may be employed conventional molecular biology, per mouse mixed at 1:1 ratio of Sigma Adjuvant System microbiology, and recombinant DNA techniques within the (Sigma cat #6322) in a total volume of 200 ul per mouse. At skill of the art. Such techniques are explained fully in the day 55 mice were sacrificed by CO2 chamber, blood was literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecu collected through the cardiac puncture and spleen was har lar Cloning: A Laboratory Manual, Second Edition (1989) vested for hybridoma production. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrooket al., 1989''); DNA Cloning: A Prac 0305 Hybridomas were made by fusing mouse myeloma tical Approach, Volumes I and II (D. N. Glover ed. 1985); cells that are deficient in adenosine phosphoribosyltrans Oligonucleotide Synthesis (M.J. Gaited. 1984); Nucleic Acid ferase (APRT) with spleen cells from mice immunized with Hybridization B. D. Hames & S. J. Higgins eds. (1985); specific antigens. A selection system using HAT (hypoxan Transcription And Translation B. D. Hames & S.J. Higgins, thine, azaserine, and thymidine) medium eliminates all but eds. (1984); Animal Cell Culture R.I. Freshney, ed. (1986); the fusion cells that are APRT+. Successful hybridomas must Immobilized Cells And Enzymes IRL Press, (1986); B. Per also retain the immunoglobulin (Igh) heavy chain, one of the bal, A Practical Guide To Molecular Cloning (1984); F. M. immunoglobulin light chain loci and secrete functional anti Ausubeletal. (eds.), Current Protocols in Molecular Biology, body. John Wiley & Sons, Inc. (1994). (0306 Hybridoma Production Medium (IMDM) was made by combining the following: 500 ml Iscove’s Modified Dul EXAMPLE1 becco's Medium (HyClone SH30259.01), 50 ml fetal bovine serum (HyClone SH30070.03), 5 ml L-glutamine (Gibco Hybridoma Production: Immunization of Mice with Invitrogen cat #25030), 5 ml non-essential amino acids Kallidin Peptide Conjugated to KLH and Antibody (Gibco Invitrogen cat #11 140050), 5 ml sodium pyruvate Generation against Human BKR1 Ligands (Gibco Invitrogen cat #11360070), 5 ml 0.1% penicillin 0303. The objective was to develop cross-reactive anti streptomycin (Gibco Invitrogen cat #15140148). The bodies against Kallidin (KD; SEQ ID NO: 1) and des-arg medium was filtered before use. Expansion medium was Kallidin (DAKD; SEQ ID NO:2) that would inhibit these made by combining the following: 1000 ml serum free ligands binding to the human BKR1. Generally, immuniza medium (Gibco Hybridoma SFM #12045), 100 ml 10% tion of mice with KLH conjugated KD through additional HyClone SuperLow IgG Defined FBS #SH30898.03 and 10 cysteines on either the C- or N-terminus of the peptide was ml penicillin/streptomycin. Freezing medium was 45 ml heat used to obtain mouse splenocytes for fusion with mouse inactivated FBS (HyClone SH30070.03) and 5 ml DMSO, myeloma cell lines as a fusion partner to produce the hybri filter sterilized. Other materials included the following: HAT domas. (50x) was obtained from Sigma-Aldrich (#HO262): Hybri 0304 Briefly, the immunization protocol was as follows: doma Fusion and Cloning Supplement (50x) (Roche Diag BALB/c Mice (8-20 week-old naive female) were immunized nostics 11363735 001); Trypan Blue Stain 0.4% (Invitrogen intraperitoneally with a mixture of even amounts of KLH-KD cat #15250-061 or T10282); PEG 1500 in 75 mM Hepes 50% and KD-KLH in phosphate buffered saline (PBS) as an anti w/v (Roche cat #783641(10783641001). All the reagents gen total of 100 ug per mouse mixed at 1:1 ratio of Sigma except HAT and Hybridoma Fusion and cloning Supplement Adjuvant System (Sigma cat #6322) in a total volume of 200 were used at 37° C. TABLE 2 Peptide Reagents Used in Immunization and Screening Peptide SEQ ID Peptide Peptide Alternative No. NO. Sequence Name Name

1. 5 RPPGFSPFR bradykinin BK 2 117 biotin-RPPGFSPFR lo-BK 3 71. RPPGFSPFR-biotin BK-b 4. 72 KLH-RPPGFSPFR KLH-BK 5 73 RPPGFSPFR-KLH BK-KLH

6 1. KRPPGFSPFR kallidin KD 7 74 biotin-KRPPGFSPFR o-KD 8 7s KRPPGFSPFR-biotin KD-lo 9 76 KLH-KRPPGFSPFR KLH-KD 10 77 KRPPGFSPFR-KLH KD-KLH

11 6 RPPGFSPF desArg9bradykinin DABK 12 78 biotin-RPPGFSPF lo-DABK US 2015/005O270 A1 Feb. 19, 2015 20

TABLE 2 - continued Peptide Reagents Used in Immunization and Screening Peptide SEQ ID Peptide Peptide Alternative No. NO. Sequence Name Name

13 79 RPPGFSPF-biotin DABK-lo KLHDABK 15 81 RPPGFSPF-KLH DABK-KLH

16 2 KRPPGFSPF desArg1 Okallidin DAKD 17 82 biotin-KRPPGFSPF lo-DAKD 18 83 KRPPGFSPF-biotin DAKD -o 19 84 KLH-KRPPGFSPF KLH-DAKD 2O 85 KRPPGFSPF-KLH DAKD-KLH

21 3 RRPPGFSPFR Kallidin like peptide 22 86 biotin-RRPPGFSPFR 23 87 RRPPGFSPFR-biotin 24 88 KLH-RRPPGFSPFR 25 89 RRPPGFSPFR-KLH

26 9 O RRPPGFSPF desArg10kallidin like DAKLP peptide 27 91 biotin-RRPPGFSPF lo-DAKLP 28 92 RRPPGFSPF-biotin DAKLP-lo 29 93 KLH-RRPPGFSPF KLH-DAKLP 3 O 94 RRPPGFSPF-KLH DAKLP-lo

31 95 RPPGF bradykinin1-5 BK15 32 96 biotin-RPPGF lo-BK15

0307 Briefly, three or four days before the fusion, the following was added: 70 ml IMDM with 10% FBS, 2 ml mouse was boosted with an antigen of interest either intrap HATand 2 ml Hybridoma and Fusion Cloning Supplement. eritonealy or intravenously. On the day of the fusion, the Cells were resuspended in 10 ml IMDM with 10% FBS and mouse was sacrificed in CO chamber, blood was collected by split into (2)50 ml tubes (5 ml cells/tube) and 25 ml IMDM cardiac puncture and the spleen was taken out and placed into with 10% FBS was added. The resulting 30 ml was trans 10 ml of serum free IMDM in a Petridish. Fusion partner cells ferred to the troughs containing 70 ml HBSS/HAT/cloning myeloma: FO (ATCC ref CRL-1646)/X63 Ag8.653 (ATCC supplement and 200 ul cells/well were pipetted into (10) ref CRL1580) were grown at a log phase, then split one day 96-well plates. Fusion was ready for screening by ELISA (50 ul) about 10 to 14 days later, or when medium in the wells before the fusion (1:2 and 1:5), and collected into 20 ml turns yellow. After the primary screening, positive clones are centrifuge tubes, spun and resuspended the pellet in 10 ml selected, numbered and moved to a 24-well plate in 500ul per IMDM. The pellet was washed two times with serum free well of IMDM with 10% FBSHI. Hybridoma supernatants IMDM medium. All the centrifugations are performed at were screened by ELISA on streptavidin plated coated with 1570 rpm for 5 min. Final resuspension was in 10 ml serum N- and C-term biotinylated peptides (see below). free IMDM. The connective tissue was dissected away from the spleen. The spleen was injected with 1 ml of serum free EXAMPLE 2 IMDM preheated to 37° C. by 1 ml syringe and 25-gauge Characterization and Selection of Hybridomas needle. Splenocytes are squeezed out of the fibroelastic coat Expressing Antibodies Against Human BKR1 by forceps and washed two times in 10 ml of serum free Ligands IMDM (including initial spin) and were resuspended in 10 ml 0309 Hybridoma Supernatants were screened by ELISA serum free IMDM. Cells were counted in Countess Auto on streptavidin plated coated with N- and C-term biotinylated mated Cell Counter. peptides (see e.g., those set forth in Table 2) and thenantibody 0308 Fusion partner cells and splenocytes were combined binding kinetics were determined for confirmed positive in one 50 ml tube at ratio of 1:2 to 1:10 (by cell number) and hybridoma clones. spun down at 970 rpm for 10 min (slow spin) to form a loose 0310. The ability of the antibodies in hybridoma superna pellet. After the “slow spin, supernatant was taken out with tants to bind to BKR1 ligand peptide was evaluated with an the precaution not to disturb the pellet, but minimize the ELISA assay. DAKD-biotin or KD-biotin peptides was coated on a 96-well SA plate in phosphate buffered saline amount of liquid over the cells in order not to dilute PEG (PBS) buffer for an hour at room temperature at 5ug/ml, and 1500. The last remaining medium was reserved and added the nonspecific binding sites were blocked with 1% bovine back after the PEG is added (below). Preheated PEG 1500 serum albumin (BSA) in PBS buffer. This plate was used to (37°C., total 1 ml) was added drop by drop to the cell pellet perform primary and secondary screening of the crude hybri over 1 minute period of time and cells were mixed after every doma Supernatants. drop of PEG was added. Pellet was incubated with PEG for 0311 Hybridoma Supernatants were added to the plates another 1 minute followed by addition of 10 ml of serum-free for binding to the coated KD or DAKD peptides. After 1 hour IMDM medium over 1 minute, so that the first 1 ml out of 10 incubation, the plate was washed and bound antibodies were is added over 30 sec. Cells underwent slow spin at 970 rpm for detected using horseradish peroxidase (HRP) conjugated sec 10 min and supernatant decanted. Into (2) 100 ml troughs, the ondary antibody (HRP-goat anti-mouse IgG (H+L): Jackson US 2015/005O270 A1 Feb. 19, 2015 21

ImmunoResearch Labs #115-035-166) and developed using After subtracting signals of Fc 1 and blank buffer runs, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) dissociation rate of the antibodies from the Supernatants to (ABTS) substrate (Roche diagnostics #11 204521 001). Data each peptide was analyzed and ranked using BIAevaluation was analyzed using Excel. The antibodies showing positive software. Only the antibody clones that demonstrated supe signals (2 fold higher than 1:10000 serum dilution ELISA rior (kd.<10-41/s) binding dissociation rate were selected for signal) were selected and re-screened in duplicates for con Subcloning and further characterization. In kinetics analysis, firmation. Confirmed positive hybridoma clones were the corresponding biotin-peptides identified in screening for selected and Subjected to binding dissociation rate ranking by the testing antibody were immobilized in Fc2 to Fc4 while Biacore. Fc1 with a random peptide used as reference cell. Each puri 0312 For antibody binding kinetics, the instruments used fied antibody selected from screenings were made into a were the BIACORE 2000 or BIACORE 3000 (GE Health series of two fold dilutions in running buffer (1x HBS-EP care), designed for biomolecular interaction analysis (BIA) in buffer, GE Healthcare) between 0.1 to 10 nM. Binding asso real time. The sensor chip used was SA chip (GE Healthcare) ciation rate, dissociation rate and the overall affinity were with streptavidin covalently immobilized on a carboxym calculated in BIAevaluation. Antibody binding kinetics for ethylated dextran matrix. Each sensor chip has four parallel eachantibody was always confirmed in triplicate assays using flow cells (Fc). Every biotinylated BKR1 or BKR2 ligand Biacore. peptides were immobilized to one of the flow cells 2 to 4 (Fc2 0313 A total of 8 mice were immunized with mixed KLH to Fc4) in the SA chip for binding dissociation rate screening KD/KD-KLH and KLH-DAKD/DAKD-KLH and the and selectivity screening. Flow cell 1 (Fc 1) was reserved and spleens were fused using the above protocols. After primary immobilized with a random peptide (biotinylated at one ter screening of about 7680 hybridoma clones in ELISA with minus) with equal or close peptide length in comparison to the DAKD-biotin and KD-biotin, only 76 clones were confirmed testing ligand peptides as the negative control. In screening positive and selected for binding dissociation rate ranking in assays, cell culture Supernatants of the hybridoma clones Biacore 3000/2000 over the immobilized DAKD-biotin and selected through primary screening or of transiently KD-biotin on Streptavidin (SA) chips. Among those, 8 hybri expressed humanized variants were injected over immobi doma clones with binding dissociation rate < of 10' were lized peptides. Hybridoma cell culture media was also Subcloned, sequenced, purified and further characterized (see injected over the chip surface as blank to establish a baseline. Table 3). TABLE 3

Immunization Results with KLH-KDKD-KLH and KLH-DAKDDAKD-KLH Peptide Osed in Clone ID

Ligand Assay Assay B21 C63 F151 F306 I2 I8 I22** I54* *

DAKD b-DAKD ELISA ------Biacore NS NS (KD, M) DAKD-b ELISA ------Biacore 4.15E-11 1.42E-10 1.6OE-10 1.6OE-10 11 OE-10 6.25E-10 NTD NTD (KD, M) DAKD FLIPR 22-25, 9 6.9 6.9 9.4 8.1 (50 nM) (nM) DABK b-DABK ELISA +f- +f------Biacore NS NS (KD, M) DABK-b ELISA -- +f------Biacore * NS NS (KD, M) DABK FLIPR (nM) BK - ELISA BK Biacore NS NS (KD, M) BK-b ELISA ------Biacore 8.57E-09 NS NS (KD, M) BK FLIPR (nM) KD b-KD ELISA -- Biacore NS NS (KD, M) KD-b ELISA ------Biacore 8.4E-11 2.21E-10 2.9E-11 2.9E-11 7.8E-11 7.53E-10 NTD NTD (KD, M) KD FLIPR 12 3.0 3.0 7.3 8.3 25 30 (15 nM)M) (nM) NA = not applicable, negative in ELISA NS = nonspecific binding NTD = not to be determined -* = residual binding (low RU in Biacore)

US 2015/005O270 A1 Feb. 19, 2015

TABLE 5- continued Heavy and Light Chain Sequences of Antibodies

154 122 GDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDF WGOGTSVTVSSAKTTPPS Antibody Isotype SEQ ID NO. Light ChainSequence B21 IgG1/k O2 EL DIVMTQTTLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLI YL VSKLDSGVPDRFTGSGSGTDFTL IRVEAEDLGVYYCLQGTHEPYTFGGGTK LEIKRADAAPTWSIFPPSKLELY DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSDQRNYLAWYQQRSGQSPKLL WASTRESGVPDRFTGSGSGTDFT TISSVKAEDLAVYYCQQYYSYPYTFGGGT : EIKRADAAPTWSIFPPSKLELY F151 O4 WMTOTPSSLAVSVGEKVTMSCKSSQSLLYTSNOKNYLAWYQQKPGQSPKPL ASTRESGVPDRFTGSGSGTDFT TISSVKAEDLAIYYCQQYYSYPWTEGGGT ; KRADAAPTWSIFPPSKLELY I22 OS D VITQTTLSLSVPIGQPASISCKSRQSLLYSNGETYLNWLLQRPGQSPKRLI VSKLDSGVPDRFTGSRSGTDFTL RVESEDLGVYYCMQGTHEPYTFGGGTK : I KRADAAPTWSIFPPSKLELY 154 O6 LDIVITQSTLTLSVPIGQPASISCKSSQSLLYSNGETYLNWLLQRPGQSPKRQI WSKLDSGWPDRFTGSRSGTDFTL RVESEDLGVYYCMOGTHEPYT FGGGTK LEIKRADAAPTWSIFPPSKLELY

B21 mIgG1/K 23 DVVMTOTPLTLSVTIGOPASISCKSSOS VSKLDSGVPDRFTGSGSGTDFTLK IRVEAEDLGVYYCLQGTHEPYTFGGGTKLE IKRADAAPT

C63 24 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSDQRNYLAWYQQRSGQSPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKL EIKRADAAPT

F151 25 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKPLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYCQQYYSYPWTFGGGTKL EIKRADAAPT

I22 26 DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIY GASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKL ELKRADAAPT

154 31 DVVMTOTPLTLSWPIGQPASISCKSSQSLLYSNGETYLNWLLQRPGQSPKRLIYL WSKLDSGWPDRFTGSRSGTDFTLK SRVESEDLGVYYCMOGTHEPYTFGGGTKLE IKRADAAPT

Single underscore = CDR region; double underscore = Signature amino acids for identifying CDRS

EXAMPLE 3 ing System (FDSS) (Hamamatsu Photonics K.K., Japan). Moreover, the biotin-DABK and biotin-DAKD completely Generation of Surrogate Antibody for Murine lost bioactivity in the FDSS assay in comparison to the native, Animal Studies unmodified DABK and DAKD peptides (data not shown). 0316 A surrogate antibody to be used in murine animal 0317. It was hypothesized that the direct N-terminus con studies needed to be able to bind and neutralize rodent BKR1 jugation of KLH and biotin prevented the native confirmation ligands, DABK and DAKLP (mouse equivalent of DAKD). of DABK and DAKD to form. With the aim to restore the In order to generate the required Surrogate antibody, mice native conformation in KLH- and biotin-conjugated peptides, were first immunized with DABK and/or DAKD with KLH linkers were designed and added to the N-terminus of DABK directly conjugated to the N-terminals of the peptides. Biotin and/or DAKD with the intention to “cushion' the KLH/biotin DABK/biotin-DAKD (biotinylation directly on N-terminus conjugation effects on peptide conformation. Poly-glycine of the peptide) positive hybridoma clones from ELISA linkers were first attempted and tested because of their screening were selected for Scaling up and purification. The simple, non-polar and neutral properties based on modeling antibodies listed in Family 7 (see Table 12) that demonstrated results. The FDSS assay results indicated that the gly-gly-gly high binding affinities to biotin-DABK, biotin-DAKLP and (3G) linker was the best according to its ability to restore the biotin-DAKD were selected based on Biacore direct binding bioactivities of KLH and biotin conjugated DABK and assay (Table 10). However, these Family 7 antibodies showed DAKD peptides (data not shown). Therefore, KLH-3G no binding to the native, unmodified DABK and DAKD pep DABK was chosen to immunize mice. And biotin-3G-DABK tides in competitive ELISA, and lacked neutralizing function and biotin-3G-KD were used in binding based screening ality in a calcium influx assay with Functional DrugScreen assays (ELISA and Biacore). Several DABK/DAKD specific US 2015/005O270 A1 Feb. 19, 2015 24 antibodies (Family 3, see Table 13) were identified in this new (Ahx) linker (a 6-carbon inert linker). All linker peptides were round of surrogate antibody hybridoma selection. EE1 was custom synthesized by Abgent (Can Diego, Calif.). All tested selected as the lead Surrogate antibody based on its Superior biotinylated peptides with linkers (biotin-linker-DABK/ binding affinity and neutralization activity against native DAKD) bound well to EE 1, indicating that any inert N-ter DABK/DAKD and lack of cross-reactivity to other peptides minus linkers helped DABK and DAKD peptides to retain (see Tables 6-12) their native bioactive conformation when conjugated with 0318 Antibodies with different specificities were gener biotin and other molecules. In contrast, no binding or poor ated when using the different immunogens listed in Table 13. binding to EE1 was observed with biotin-DABK and biotin Family 4 antibodies were specific to the BKR2 receptor DAKD, peptides that have direct N-terminal biotin conjuga ligands, BK and KD. Family 5 antibodies specifically bind to tion (see FIG. 1). the C terminus of BK and DABK. Family 6 antibodies bind 0320. The binding kinetics of generated antibodies are BK, DABK and DAKD but do not bind to KD. summarized in Tables 5-11. Then, all antibodies generated 0319. Additional linkers were evaluated for binding to the were sorted into families and their binding specificities are surrogate EE1 antibody for their ability to fit into the DABK/ summarized below in Table 12. Table 13 provides the heavy DAKD binding pocket in EE1, including longer poly-glycine and light chain sequences of antibodies that were placed into linkers, poly-alanine linkers and preexisting linkers such as family 1 and family 2 based on their binding specificity (see polyethylene glycol (PEG2) linker and aminohexanoic acid Table 12). TABLE 6

Summary of Antibody Kinetics to b-3G-DABK and b-3G-DAKD Peptides

b-3G-DABK b-3G-DAKD

Clone Ka (1/Ms) Kd (1/s) KD (M) Ka (1/Ms) Kd (1/s) KD (M)

DD2O 15E-06 2.3E-04 16E-10 4.7E--OS 4.OE-04 8.8E-10 UR11 2.OE-05 3.OE-04 15E-09 3.OE--OS 16E-03 S2E-09 DD7 2.3E--OS 6.OE-04 2.7E-09 2.1E--OS 14E-03 6.6E-09 EE1 4.4E--05 1.2E-04 2.8E-10 4.4E--OS 2.OE-04 4.5E-10 EE36 4.3E-03 5.3E-04 1.2E-O7 n.b. nfb nfb UR29 nb : n.b. nfb n.b. nfb nfb JK3 3.44E--OS 3.91E-OS 1.14E-10 3.18E--OS S.O7E-OS 1.6OE-10 LR4 nfb n.b. nfb n.b. nfb nfb LR16 nfb n.b. nfb n.b. nfb nfb LR6 nfb n.b. nfb n.b. nfb nfb LR12 nfb n.b. nfb n.b. nfb nfb NR1 nfb n.b. nfb n.b. nfb nfb NR1S nfb n.b. nfb n.b. nfb nfb

n.b. = non-specific binding, nb = no binding

TABLE 7 Summary of Antibody Kinetics to b-3G-DAKLP and b-3G-BK Peptides

Clone

nb = no binding; ND = not determined

US 2015/005O270 A1 Feb. 19, 2015 27

- Continued (SEQ ID NO: 108) GCCTTCCTGGATTTCCTGGTCCAGGTGAGAGTGATCCAGGACTGCT 5 - CAAGAAAGCTGGGTCGGATCCTTATAAAGTTCCCAG

AACCCTGGTC-3' TCTGGAAGGAGCTCACAGACCTGGGCCTGCAGCTGGCCAACTTCTT 0324 and Pfu Polymerase (Agilent Technologies, Cat #600264) and cloned into pl)ONR201 using BP clonase TGCTTTTGTCAACAGCTGCCTGAACCCACTGATTTATGTCTTTGCA enzyme mix (Invitrogen, Cat #11789-020). In parallel, the pEAK8 expression vector (EDGE Biosystems) was modified GGCCGGCTCTTTAAGACCAGGGTTCTGGGAACTTTATAA by inserting a N-terminal HA tag (GCATACCCATAC GACGTCCCAGACTACGCT, GenBank SEQ ID NO:109 0326 HEKmBKR1 or MRC5 cells were plated into 384 CY100443) into pEAK8 linearized with EcoRI and Hind III (vector pEAK8-nHA) and subsequent insertion of the Gate well clear bottom plates in growth medium, and allowed to way cassette B (Invitrogen, Cat #11828-029) into pEAK8 attach overnight. Then growth media was removed, cells were nHA digested with EcoRI and NotI and blunt-ended with washed in assay buffer (HBSS, 20 mM HEPES, 2.5 mM Klenow polymerase (NEB, cat #M0210S) resulting in vector probenecid), then dye-loaded with 0.5uM Fluo-4AM, a cell pEAK8 nHA DEST. Next mouse Bakrb1 was subcloned permeable calcium sensing dye, with 0.04% Pluronic Acid into pEAK8 nHA DEST using LR clonase (Invitrogen, Cat for 1 hr at 37 C. The AM ester is cleaved, and the calcium dye #11791-100). 293-PSC cells were then transfected with pEAK8-Bdkrb1 plasmid using Fugene 6 transfection reagent. is retained in the cytoplasm. After 1 hr., the cells were washed The cells were put under antibiotic () selection 24 to remove excess dye, and 20 ul of residual buffer remained hours after transfection, and selection was maintained togen on the cells. Treatments were added as 2x solutions on the erate a stable cell line. Presence of the Bdkrb1 gene in the Functional Drug Screening System from Hamamatsu resultant stable cell lines was confirmed using real time RT (FDSS), and the calcium mobilization was monitored kineti PCR, and by agarose gel electrophoresis. Cell surface expres cally for at least 4 minutes. B1 R or B2R receptor activation sion of the Bradykinin B1 receptor was performed by using an results in Galpha q mediated activation of phospholipase C antibody against the N-terminal-HA tag (Covance, Cat #MMS-101P) on the Bradykinin B1R on a FACS instrument. and IP3 mediated calcium mobilization. The Fluo-4 dye che Functional activity of the Bradykinin B1 receptor was dem lates the released calcium, and a robust change in fluores onstrated in calcium mobilization assay with selective ago cence is observed. The results were exported as max-min nists. relative fluorescence units to normalize for differences 0325 Bdkrb1 gene subcloned into cells: between cell density or dye loading across the plate. 0327 Ligand potency was determined each day by run (GenBank NM_007539; SEQ ID NO: 110) ning concentration response curves of ligand, and an approxi ATGGCGTCCCAGGCCTCGCTGAAGCTACAGCCTTCTAACCAAAGCC mate EC70-80 concentration of ligand was selected for incu bation with antibodies. An EC80 concentration was selected AGCAGGCCCCTCCCAACATCACCTCCTGCGAGGGCGCCCCGGAAGC because it is on the linear range of the detection curve and CTGGGATCTGCTGTGTCGGGTGCTGCCAGGGTTTGTCATCACTGTC there was ample window to see a decrease with antagonists or ligand depleting antibodies. Dose response curve of antibod TGTTTCTTTGGCCTCC TCGGGAACCTTTTAGTCCTGTCCTTC: TTCC ies were allowed to binda EC80 concentration ligand, and the TTTTGCCTTGGCGACGATGGTGGCAGCAGCGGCGGCAGCGCCTAAC extent of ligand depletion was monitored using change in fluorescence. Results were normalized to buffer and EC80 CATAGCAGAAATCTACCTGGCTAACTTGGCAGCTTCTGATCTGGTG ligand response, and an EC50 for ligand depletion was cal TTTGTGCTGGGCCTGCCCTTCTGGGCAGAGAACGTTGGGAACCGTT culated. The results were then reported as molar ratio which corresponds to the Antibody concentration that reduces TCAACTGGCCCTTTGGAAGTGACCTCTGCCGGGTGGTCAGCGGGGT depletes 50% of the ligand response (i.e., EC50 of Ab) CA CAAGGCCAACCTGTTCATCAGCATCTTCCTGGTGGTGGCCATC divided by the ligand concentration used. The theoretical max should be 0.5 because one unit of antibody should be able to AGTCAGGACCGCTACAGGTTGCTGGTATACCCCATGACCAGCTGGG deplete 2 units of ligand, but we have seen lower values in GGAACCGGCGGCGACGGCAAGCCCAAGTGACCTGCCTGCTCATCTG practice but that may be a reflection of the insensitivity of the detection method for low ligand concentrations, rather than a GGTAGCTGGGGGCCTCTTGAGCACCCCCACGTTCCTTCTGCGTTCC stochiometric constraint for the antibody. The results of these GTCAAAGTCGTCCCTGATCTGAACATCTCTGCCTGCATCCTGCTTT experiments are set forth in Tables 14-16.

TCCCCCACGAAGCTTGGCACTTTGTAAGGATGGTGGAGTTGAACGT 0328. All family 1 and family 2 antibodies (see Table 13) demonstrated superior binding kinetics by Biacore (Table 3) TTTGGGTTTCCTCC TOCCATTGGCTGCCATCCTCTACTTCAACTTT and neutralization activity as measured by calcium mobiliza CACATCCTGGCCTCCCTGAGAGGACAGAAGGAGGCCAGCAGAACCC tion against DAKD and KD peptides (Tables 14 and 15). The antibodies were further analyzed for their thermal stability GGTGTGGGGGACCCAAGGACAGCAAGACAATGGGGCTGATCCTCAC and sequence suitability for humanization. F 151 was ACTGGTAGCCTCCTTCCTGGTCTGCTGGGCCCCTTACCACTTCTTT advanced for humanization because it was thermally stable, there were no problematic residues in the CDR regions and it was cross-reactive to the mouse ligand KLP and DAKLP. US 2015/005O270 A1 Feb. 19, 2015 28

TABLE 1.4

Characterization of des-arg-Kinin Ligand Depletion using Calcium Mobilization in HEKmBKR1 cells

Depletion of DABK Depletion of DAKD Depletion of DAKLP

DABK DAKD DAKLP (Mean SD (Mean SD (Mean SD Molar Molar Molar Molar Molar Molar Family Antibody Ratio) Ratio in Ratio) Ratio in Ratio) Ratio in

1 F151 IA1OO 5 O.08 O.O4 7 O.15 O.04 4 1 B21 IA1OO 1 O.15 O.04 3 0.67 1 1 I22 IA1OO 1 O.O7 O.O2 3 O.21 1 1 IS4 IA1OO 1 O.15 O.OS 3 O.35 1 2 C63 IA1OO 1 O.08 O.O2 3 5.85 1 3 EE1 1.03 O.S2 5 O.86 O.S2 3 0.57 O.36 4 3 DD2O 3.45 1.34 3 1.82 O.76 3 1.31 O.86 3 DD7 2.18 O45 3 4.22 O.9S 3 S.34 122 2 3 JK3 186 O.O3 2 ND 1.44 O.O3 2 4 MBK3 ND ND ND 4 NR1S ND ND ND 4 NR1 ND ND ND S UR29 O.60 O.12 S IA2OO 3 IA300 4 6 UR11 6.99 1.61 3 19.65 14.95 3 11.09 3.13 7 LR4 IA1OO 1 IA400 1 IA400 1 7 LR6 IA1OO 1 IA100 1 ND 7 LR12 IA1OO 1 IA100 1 ND 7 LR16 IA1OO 1 IA100 1 ND

Antibodies were pre-incubated with a set concentration of ligand, usually an EC70-80 for activating calcium mobilization at the Bradykinin B1 Receptor. The antibody-ligand mixture was added to H pre-loaded with a calcium sensing dye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium mobilization was monitored. Data was exported as a max-min relative fluorescence of the biological response, and IC50 for ligand depletion was calculated using sigmoidal curve fit in GraphPad Prism V4.03. Data reported as molar ratio for ligand depletion by the antibody to standardize the different concentration of ligand that was used for the various experiments, Molar Ratio for ligand depletion = IC50 of Antibody Ligand SD = Standard Deviation; ND = not determined; IA100 = Inactive at 100 nM; IA200 = Inactive at 200 nM; IA300 = Inactive at 300 nM; IA400 = Inactive at 400 nM

TABLE 1.5 Characterization of Kinin Ligand Depletion using Calcium Mobilization in MRCS Fetal Lung Fibroblasts cells Depletion of BK Depletion of KD Depletion of KLP

BK KD KLP (Mean SD (Mean SD (Mean SD Molar Molar Molar Molar Molar Molar Family Antibody Ratio) Ratio in Ratio) Ratio in Ratio) Ratio in 1 F151 IA1OO 5 O.14 0.05 5 O.15 O.O2 3 1 B21 IA1OO 1 O.33 1 ND 1 I22 IA1OO 1 O.22 1 ND 1 IS4 IA1OO 1 O.30 1 ND 2 C63 IA1OO 1 O.23 1 ND 3 EE1 IA3OO 4 IA3OO 4 IA150 3 DD2O IA6OO 4 IA6OO S IA1SO DD7 7.11 3.62. 3 17.37 12.11 3 4.27 1 3 JK3 IA3OO 2 IA300 2 ND 4 MBK3 22.11 14.10 9 3.46 2.64 6 9.45 4 NR1S 15.26 11.51 S 4.34 2.SS S 11.18 4 NR1 39.31 1 42.15 1. 32.58 S UR29 1.15 O.86 S O.30 O.O8 2 O41 US 2015/005O270 A1 Feb. 19, 2015 29

TABLE 15-continued Characterization of Kinin Ligand Depletion using Calcium Mobilization in MRCS Fetal Lung Fibroblasts cells Depletion of BK Depletion of KD Depletion of KLP

BK KD KLP (Mean SD (Mean SD (Mean SD Molar Molar Molar Molar Molar Molar Family Antibody Ratio) Ratio in Ratio) Ratio in Ratio) Ratio in 6 UR11 5.41 O.8O 2 25.21 4.54 2 1.53 1 7 LR4 IA1OO 1 IA100 1 ND 7 LR6 IA1OO 1 IA100 1 ND 7 LR12 IA1OO 1 IA100 1 ND 7 LR16 IA1OO 1 IA100 1 ND Antibodies were pre-incubated with a set concentration of ligand, usually an EC70-80 for activating calcium mobilization at the Bradykinin B2 Receptor. The antibody-ligand mixture was added to MRC5 Fetal Lung Fibro blasts (ATCC CCL-171) pre-loaded with a calcium sensing dye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium mobilization was monitored. Data was exported as a max-min relative fluores cence of the biological response, and IC50 for ligand depletion was calculated using sigmoidal curve fitin GraphPad PrismV4.03. Data reported as molar ratio for ligand depletion by the antibody to standardize the different concen tration of ligand that was used for the various experiments, Molar Ratio for ligand depletion = IC50 of Antibody Ligand SD = Standard Deviation; ND = not determined; IA100 = Inactive at 100 nM; IA150 = Inactive at 150 nM; IA300 = Inactive at 300 nM; IA400 = Inactive at 400 nM; IA600 = Inactive at 600 nM

EXAMPLE 5 rmsd of all F151 murine amino-acids, one decides if the amino-acid is flexible enough, as seen during the MD to be Engineering of F151: Humanization, Stabilization considered as likely to interact with T-cell receptors and and Mutation of Unwanted Sequence Motifs responsible for activation of the immune response. 62 amino acids were identified as flexible in the murine F151 antibody, 1. Humanization excluding the CDR and its immediate 5A vicinity. 0330. The motion of the 28 most flexible murine F151 0329. The humanization protocol used has been described amino acids, during the 20 ns (10x2 ns), were then compared in PCT/US08/74381 (US20110027266), herein incorporated to the motion of the corresponding flexible amino-acids of 49 by reference in its entirety. The variable light (VL) and vari human germline homology models, for each of which were able heavy (VH) sequences of murine F151 were used to run the 10x2 ns MD simulations. The 49 human germline build a homology model of anti-DAKD/KDF151 LC and HC models were built by systematically combining the 7 most in Molecular Operating Environment (MOE: v. 2009.10; common human germline light chains (vkl, vk2, Vk3, Vk4. Chemical Computing Group). The following templates were Vlambda1, Vlambda2, Vlambda3) and 7 most common human used: light chain framework—1SBS (93% identity in the germline heavy chains (Vh1a, Vh1b, wh2, wh3, wh4, whi5, whé). framework regions), heavy chain framework 2VXT (84% The vk1-vh1b human germline antibody showed 0.80 4D identity in the framework regions), L1-1 LVE (93% identity), similarity of its flexible amino-acids compared to the flexible L2-1 EEU (100% identity), L3-2R56 (93% identity), amino-acids of the murine F151 antibody; the vk1-vh1b ger H1-1NJ9 (95% identity), H2-2VXU (76% identity) and mline antibody was therefore used to humanize F151 anti H3-1HIL (49% identity). Templates were available at the body focusing on the flexible amino-acids. For the pair wise RCSB Protein Data Bank found on the world wide web at amino-acid association between murine F 151 vk1-vh1b rcsb.org, a website managed by Rutgers and the University of amino-acids, the 2 sequences were aligned based on the opti California San Diego (Berman, H. M. Westbrook J.; Feng. Z.: mal 3D superposition of the alpha carbons of the 2 corre Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.: sponding homology models (see FIG. 15 for an alignment of Bourne, P. E. The Protein Data Bank, Nucleic Acids F151 LC and F151 HC with vk1 and vh1b, respectively). Research, 2000, 28, 235-242.). The homology model was Subsequently energy minimized using the standard proce 2. Stabilization dures implemented in MOE. A molecular dynamics (MD) 0331 Two approaches were used to improve the stability simulation of the minimized 3D homology model of the of the antibody. murine F151 was subsequently performed, with constraints on the protein backbone at 500 K temperature for 1.1 nano a) Knowledge-Based Approach seconds (ns) in Generalized Born implicit solvent. Ten diverse conformations were extracted from this first MD run 0332 The amino acids of the light and heavy chains with every 100 picoseconds (ps) for the last 1 ns. These diverse low frequency of occurrence vs. their respective canonical conformations were then each submitted to a MD simulation, sequences, excluding the CDRs, were proposed to be mutated with no constraints on the protein backbone and at 300 K into the most frequently found amino acids (AAGth-0.5 kcal/ temperature, for 2.3 ns. For each of the 10 MD runs, the last mol: E. Monsellier, H. Bedouelle. J. Mol. Biol. 362, 2006, p. 2,000 snapshots, one every ps, from the MD trajectory were 580-593). This first list of consensus mutations for the light then used to calculate, for each murine F 151 amino acid, its chain (LC) and heavy chain (HC) was restricted to the amino root mean square deviations (rmsd) compared to a reference acids found in the closest human germline (vk1-Vh1b). Sug medoid position. By comparing the average rmsd on the 10 gested changes in the immediate vicinity of the CDRs (5 separate MD runs of a given amino-acid to the overall average Angstroms “Vernier Zone, J. Mol. Biol. 224, 1992, p. 487 US 2015/005O270 A1 Feb. 19, 2015 30

499) were removed from consideration. This resulted in 5 exposed areas). Among other criteria, the VL & VH domains stabilizing mutations in the LC (see Table 19) and 4 stabiliz of murine F 151 was selected from other murine antibodies ing mutations in the HC (see Table 20). Other criteria were because murine F 151 did not have exposed unwanted taken into account to consider these mutations for potentially sequence motifs, but they are introduced in Some humanized stabilizing the anti-DAKD/KD F151 antibody. These criteria variants. were a favorable change of hydropathy at the Surface or a 0337 LC3a, LC3b, HC3a and HC3b each have potentially molecular mechanics based predicted stabilization of the problematic succinimide sites that were identified. These mutant. Also, additional stabilizing mutations reported to be sites were not modified in the proposed sequences as the successful in the literature (E. Monsellier & H. Bedouelle, J. residues involved are potentially involved in H-bond network Mol. Biol. 362, 2006, p. 580-593: B. J. Steipe et al. J. Mol. (visual inspection of the homology model). These positions Biol, 1994, 240, 188-192) were considered (see Tables are also found in a number of other antibody structures. 16-22). One of these changes was incorporated as a stabiliz Additionally, in both HC3a and HC3b, a strict humanization ing mutation (D89E) in sequences HC2a, HC2b and HC2c by grafting would include a substitution of Ser115 to Met. below. Another Suggested change (Q62E) was incorporated This is exposed. A substitution to at this in variant HC2b. position is suggested as a humanizing mutation as it is a common residue among many close human germline b) 3D and MD-Based Approaches Sequences. 0333 3D and MD-based approaches have been previously 0338. The resulting humanized sequences were blasted for reported (Seco J, Luque FJ, Barril X., JMed Chem. 2009 Apr. sequence similarity against the International Epitope Data 23: 52(8):2363-71; Malin Jonsson et al., J. Phys. Chem. B 2003, 107,551 1-5518). Hydrophobic regions of the antibody base (IEDB) database (found on the worldwide web at immu were explicitly identified by analyzing the molecular dynam neepitope.com; version June 2009; Vita R, Zarebski L. ics simulation of the Fab in a binary solvent (20% isopropanol Greenbaum JA, Emami H, Hoof I, Salimi N. Damle R, Sette in water, 20 ns production simulation). Lysine mutations were A. Peters B. The immune epitope database 2.0. Nucleic Acids then introduced in the vicinity of these regions as an attempt Res. 2010 January: 38 (Database issue):D854-62. Epub 2009 to prevent the aggregation. Additional analysis using a hydro Nov. 11) to ensure that none of the sequences contain any phobic surface map within Schrodinger's maestro software known human B- or T-cell epitopes (sequence identity of 70% (v. 8.5.207) was completed. Using a combination of these two used as cut-off for the results obtained through BLAST search techniques, 2 Lys mutations, 1 in the heavy chain and 1 in the and considering only the results from human species). light chain, are Suggested. 5. Original Sequences of Murine F 151 Variable Domains 3. Humanization by Grafting 0339) CDRs are highlighted in bold and Vernier regions 0334 Humanization using grafting grafting techniques (as defined in Foote & Winter, J. Mol. Biol., 1992, 224, has previously been reported (Peter T. Jones, Paul H. Dear, 487-499) are underlined. Jefferson Foote, Michael S. Neuberger & Greg Winter Nature, 1986, 321, 522-525). The humanization process which was used started by identifying the closest human Light Chain germlines to anti-DAKD/KD light and heavy chains. This is (SEQ ID NO: 26) done by performing a BLAST search vs. all the human ger DIVMSQSPSS LAVSVGEKVTMSCKSSQSLLYSSNQKNYLA mlines which were systematically enumerated (all possible WYOQKPGQSP KPLIYWASTRESGVPDRFTGSGSGTDFTLT combinations of the V & J domains for the kappa and lambda chains: V, D and J domains for the heavy chains). ISSVKAEDLA IYYCQQYYSYPWTFGGGTKLEIK 0335 The following closest human germlines were iden Germinality index = 8.3% with tified with 83% and 62% sequence identity to anti-DAKD/ Z46615 1. V X67858 1 J V IV-B3) KD F151 light chains (LC) and heavy chains (HC), respec Heavy Chain (SEQ ID NO: 19) : tively (see FIG.16). Using the internal VBASE germline, the EIOLOOSGPELVKPGTSVKVSCKASGYSETDYNIYWWKOS light chain is found to be close to V IV-B3 (-83% identity) locus and the heavy chain close to 1-08 & 1-18 (~62% iden HGKSLEWIGY FDPYNGNTGYNQKFRGKATLTVDKSSSTAF tity) locus of the VH1 sub-family. CDR regions (as defined by MHLSSLTSDDSAVYYCANYYRYDDHAMDYWGQGTSVTVSS MOE), and Vernier regions (as defined in Foote & Winter, J. Germinality index = 62% with Mol. Biol., 1992,224, 487-499) are indicated in boldface The Z12316. 1 VX97051 4 D X97051, 5 J VH1 1-18) humanizing mutations in underlining were obtained by per forming a pairwise comparison of the 2 aligned sequences, excluding the CDR & Vernier Zone residues as defined above. 6. Engineered Sequences In another variant of the humanization, only the CDRs were excluded in the comparison. a) Background 4. Mutation of Unwanted Sequence MOTIFS 0340 5 versions for the light chain (LC1, LC2a, LC2b. 0336. The following motifs of sequences were considered: LC3a, and LC3b) and 5 versions of the heavy chain (HC1, Asp-Pro (acid labile bond), Asn-X-Ser/Thr (glycosylation, HC2a, HC2b, HC3a, and HC3b) were proposed. X=any amino-acid but Pro), Asp-Gly/Ser/Thr (succinimide/ 0341 LC1 contains 5 humanizing mutations identified iso-asp formation in flexible regions), Asn-Gly/His/Ser/Ala/ using the 4D humanization protocol. LC2a introduced an CyS (exposed deamidation sites), and Met (oxidation in additional 5 stabilizing mutations. LC2b added 1 Lysine US 2015/005O270 A1 Feb. 19, 2015 31 mutations to help prevent aggregation. LC3a contains 15 0343 6 combinations in total were proposed (summarized mutations derived from grafting to the closest human germ in Table 16): line sequence and retaining the murine CDR and Vernier Zone 0344 LC1 xHC1 (mutations addressing humanization residues. LC3b contained 16 mutations derived from CDR only) grafting with one additional humanizing mutation. 0345 LC2axHC2a (mutations addressing humaniza 0342. HC1 has 6 humanizing mutations identified by the tion and stabilization) in-house protocol. HC2a introduced 5 additional stabilizing 0346 LC2axHC2b (mutations addressing humaniza mutations while HC2b contains 6 additional stabilizing muta tion and stabilization) tions as compared to HC1. HC2c contains 1 Lys mutation, in 0347 LC2bxHC2c (mutations addressing humaniza addition to the stabilizing mutations of HC2a, to help prevent tion, stabilization and “anti-aggregation') aggregation. HC3a contains 19 mutations derived from graft 0348 LC3axHC3a (mutations addressing mostly ing to the closest human germline sequence and retaining the humanization by grafting--Vernier) murine CDR and Vernier Zone residues. HC3b contains 25 0349 LC3bxHC3b (mutations addressing humaniza mutations derived from CDR grafting. tion by grafting) TABLE 16 Summary of the 6 LCXHC combinations proposed (LC2b) (LC3a) (LC2a) Humanizing + Grafting (LC1) Humanizing + stabilizing + anti- With Vernier (LC3b) Humanizing stabilizing aggregation Regions Grafting (HC1) X Humanizing (HC2a) X Humanizing + stabilizing (HC2b) X Humanizing + stabilizing (HC2c) X Humanizing + stabilizing + “anti aggregation (HC3a) X grafting (HC3b) X grafting

TABLE 17 Mutations of the SLC variants of the anti-DAKDKD F1.51 antibody (LC2b) Humanizing + (LC3a) Light Light (LC2a) stabilizing Grafting (LC3b) Chain Chain (LC1) Humanizing + mutations + CDRs + Grafting Sequential Kabat Humanizing stabilizing anti-aggregation Vernier CDRS numbering Numbering mutations mutations mutations residues only Ser5 Ser5 Thr Thr Thr Thr Ser Ser Asp Asp Ala12 Ala12 Ser Ser Wal13 Wal13 Ala Ala Ala Wa15 Wa15 Leu Leu Glu17 Glu17 Asp Asp Asp Lys18 Lys18 Arg Arg Arg Arg Arg Val19 Val19 Ala Ala Met21 Met21 Ile Ile Ile Ile Ser22 Ser22 ASn ASn Glm48 Glm42 Lys Lys Lys Ser49 Ser43 Pro Pro ProS2 Pro46 Leu Thr69 Thr63 Ser Ser Ser Ser Wal84 Waf3 Leu Leu Lys85 Lys79 Gln Gln Gln Gln Gln Leu89 Leu83 Lys Wall Wall US 2015/005O270 A1 Feb. 19, 2015 32

TABLE 17-continued Mutations of the SLC variants of the anti-DAKDKD F151 antibody (LC2b) Humanizing + (LC3a) Light Light (LC2a) stabilizing Grafting (LC3b) Chain Chain (LC1) Humanizing + mutations + CDRs + Grafting Sequential Kabat Humanizing stabilizing anti-aggregation Vernier CDRS numbering Numbering mutations mutations mutations residues only Ile91 Ile85 Thr Thr Wall Wal Gly106 Gly100 Gln Gln Leu110 Leu104 Wall Wal Mutations: 5 10 11 15 16

TABLE 1.8 Mutations of the 6 HC variants of the anti-DAKDKD F151 antibody

humanizing + Heavy Heavy (HC2a) (HC2b) stabilizing Grafting (HC3b) Chain Chain (HC1) Humanizing + Humanizing mutations + CDRS + Grafting Sequential Kabat Humanizing stabilizing stabilizing antiaggregation Vernier CDRS numbering numbering mutations mutations mutations mutations residue only Glu1 Glu1 Gln Gln Gn Gln Gln le2 le2 Wall GlnS GlnS Wall Wall Wall Wall Wal Wall Pro9 Pro9 Ala Ala Ala Ala Ala Leu11 Leu11 Wall Wall Wall Wall Wal Wall Wal12 Wal12 Lys Lys Lys Lys Lys Lys Thr16 Thr16 Ala Ala Ala Ala Ala Ala Lys38 Lys38 Arg Arg Ser40 Ser40 Ala Ala His41 His41 Pro Pro Pro Pro Pro Pro Lys43 Lys43 Gln Gln Ser44 Ser44 Gly Gly Gly Gly Gly le48 le48 Met Ginó2 Ginó1 Glu Lys67 Lys66 Arg Arg Ala88 Ala67 Wall Leu70 Leu69 Met Waf2 Walf1 Thr Lys74 Lys73 Thr Ser76 Ser75 Thr Thr Pe3O Phe79 Tyr Tyr Tyr Tyr Tyr His82 His81 Glu Glu Ser84 Ser82A Arg Arg Leu86 Leu82C Lys Thr37 Thr33 Arg Arg Asp89 Asp85 Glu Glu Glu Glu Asp90 Asp86 Glu Glu Glu SerC1 Ser87 Thr Thr Ser115 Ser108 Leu Leu Mutations: 6 11 12 12 19 25 a) Engineered Light Chain Sequences: mutations are in italics (T at position 5, Sat position 12, I at 0350 No potentially problematic known T-cell or B-cell position 21, Sat position 69, Tat position 91 shown below): epitopes were found in all the variants proposed. 0351 LC1 (SEQ ID NO:27), humanizing mutations are DIVMTQSPSSLSASVGDRVTISCKSSQSLLYSSNQKNYLA underlined, CDRs and vernier Zones are in bold: WYOOKPGKSPKPLIY WASTRESGVPDRFSGSGSGTDFTLT

DIVMSQSPSSLAASVGDRVTMSCKSSQSLLYSSNQKNYLA ISSVQAEDLA TYYCQQYYSYPWTFGGGTKLEIK WYOOKPGKSP KPLIY WASTRESGVPDRFTGSGSGTDFTLT 0353 LC2b (SEQ ID NO:29) humanizing mutations are underlined, CDRs and vernier Zones are in bold, stabilization ISSVQAEDLAIYYCQQYYSYPWTFGGGTKLEIK mutations are in italics (T at position 5, Sat position 12, I at 0352 LC2a (SEQ ID NO:28), humanizing mutations are position 21, S at position 69, Tat position 91 shown below) underlined, CDRs and vernier Zones are in bold, stabilization and an anti-aggregation mutation is Kat position 89: US 2015/005O270 A1 Feb. 19, 2015

position 44, Y at position 80 and E at position 90 shown DIVMTQSPSSLSASVGDRVTISCKSSQSLLYSSNQKNYLA below) and an anti-aggregation mutation at Kat position 86: WYOOKPGKSPKPLIY WASTRESGVPDRFSGSGSGTDFTLT ISSVQAEDKA TYYCQQYYSYPWTFGGGTKLEIK QIQLVOSGAEvKKPGASVKVSCKASGYSFTDYNIYWVKQS 0354) LC3a (SEQID NO:30), grafted mutations shown in PGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAY underline and CDRs and vernier Zones shown in bold: MHLSSKTSEESAVYYCANYYRYDDHAMDYWGQGTSVTVSS 0362. HC3a (SEQID NO:24), grafted mutations shown in DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLA underline and CDRs and vernier Zones shown in bold: WYOOKPGQPPKPLIY WASTRESGVRDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK QIQLVOSGAEvKKPGASVKVSCKASGYSFTDYNIYWVRQA 0355 LC3b (SEQID NO:31), grafted mutations shown in PGQGLEWIGYFDPYNGNTGYNQKFRGRATLTVDKSTSTAY underline and CDRs and vernier Zones shown in bold: MELRSLRSDDTAVYYCANYYRYDDHAMDYWGQGTLVTVSS 0363 LC3b (SEQID NO:25), grafted mutations shown in DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLA underline and CDRs and vernier Zones shown in bold: WYOOKPGQPPKLLIY WASTRESGVPDRFSGSGSGTDFTLT QVOLVOSGAEWKKPGASVKVSCKASGYSFTDYNIYWVRQA 0356. Note that L at position 52 is a vernier residue that is mutated to human. MELRSLRSDDTAVYYCANYYRYDDHAMDYW c) Engineered Heavy Chain Sequences 0357 HC1 (SEQ ID NO:20), humanizing mutations are 0364. Note that the following Vernier Residue are mutated underlined, CDRs and vernier Zones are in bold: to human: V at position 2, Mat position 48, V at position 68, Mat position 70 and T at position 74. 0365 No human epitopes were identified for sequence EIQLVOSGPEvKKPGASVKVSCKASGYSFTDYNIYWVKQS HC3b in IEDB database. PGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAF 0366 HC3b germinality index=83% with Z12316 1 V J00235 1. D U42590 1 J1-18/DP-14. MHLSSLTSEDSAVYYCANYYRYDDHAMDYWGQGTSVTVSS 0358 HC2a (SEQID NO:21), humanizing mutations are TABLE 19 underlined, CDRs and vernier Zones are in bold, stabilization mutations are in italics (Qat position 1, A at position 9, G at Stabilizing Changes Proposed in Light Chain position 44, Y at position 80 and E at position 90 shown Residue Proposed Change Calculated Gth Accept Change below): Ser-5 Thr 2.32286 Yes Ala-12 Ser 0.75228 Yes Met-21 Ile O.768959 Yes QIQLVQSGAEvKKPGASVKVSCKASGYSFTDYNIYWVKQS Pro-52 Leu 1.70059 No - Vernier region Thr-69 Ser 1.10843 Yes PGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAY Lys-86 Glu 2.00115 No - changed to Gln during humanization MHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVTVSS Ile-91 Thr 127255 Yes 0359 HC2b (SEQ ID NO:22), humanizing mutations are underlined, CDRs and vernier Zones are in bold, stabilization mutations are in italics (Qat position 1, A at position 9, G at TABLE 20 position 44, Eat position 62, Yat position 80 and Eat position 90 shown below): Stabilizing Changes Proposed in Heavy Chain Residue Proposed Change Calculated Gth Accept Change QIQLVOSGAEvKKPGASVKVSCKASGYSFTDYNIYWVKQS Glu-1 Gln O.S62423 Yes Ile-2 Wall 2.15882 No - Vernier region PGKGLEWIGYFDPYNGNTGYNEKFRGKATLTVDKSSSTAY Pro-9 Ala O.SOS324 Yes Thr-16 Ala 1.50552 Already changed to Ala MHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVTVSS in humanization Wal-20 Leu 2.21586 No - not in germline 0360 No human epitopes were identified for sequence Sequence HC2b in IEDB database. Ser-40 Arg 1.03643 No - not in germline Sequence 0361 HC2c (SEQID NO:23), humanizing mutations are His-41 Pro 167738 Already changed to Pro underlined, CDRs and vernier Zones are in bold, stabilization in humanization mutations are in italics (Qat position 1, A at position 9, G at US 2015/005O270 A1 Feb. 19, 2015 34

TABLE 20-continued TABLE 22-continued Stabilizing Changes Proposed in Heavy Chain Potential Stabilizing Mutations Residue Proposed Change Calculated Gth Accept Change Light Chain Ser-44 Gly 1.5068 Yes Residue Additional changes suggested Accept Change Gln-62 Glu O.74934 No - not in germline Sequence 112->I None - already I112 None Arg-65 Lys 2.32314 No - not in germline 69->S G69->S No - G69 is in Wernier Sequence Region Phe-80 Tyr 1.30935 Yes 21->I M21->I Already changed (see His-82 Gln 2.24674 No - not in germline Table 19) Sequence Asp-89 Glu 1.65409 Already changed to Glu *Note: in humanization Sequential numbering used to refer to residues ASn-98 Arg 3.65643 No - Vernier region

EXAMPLE 6 TABLE 21 Characterization of Humanization Variants Combinations of stabilizing mutations evaluated 0367 Based on the in silico modeling presented in Table Additional changes 16, the variable region of the light chain (VL) and heavy chain Combination* Suggested Accept Change (VH) DNA of humanized F151 were codon optimized for HEK293 expression and gene synthesized by GeneArt (sub L1 (46->P & 48->Q) K48->Q No - K48 humanizing sidiary of Life Technologies). The synthesized DNA frag mutation L2 (51->K) None - already None ments were cloned into the constant region of the light chain KS1 (CL) encoding vectors, pFF0362 (A. Human Kappa LC vec L3 (80->T) None - already None tor) at Apal I/BsiWI sites and the constant regions of the T8O heavy chain (CH1, CH2 and CH3) encoding vectors, L4 (82->S) None - already None S82 pFF0363 (B. Human IgG1 HC vector) at Apal I/ApaI sites L5 (90->A,91->T) None - already None respectively. The resulted plasmids pFF0460 containing the A90, T91 full sequence of LC and pFF0466 containing the full length of Suggested HC of humanized F151 variants were co-transfected and above (Table 1) H1 (15->G) None - already None transiently expressed in FreeStyleTM 293 Expression System G15 (Invitrogen/Life Technologies, catalog no. K9000-01). H2 (62->E, 63->K, 64->F) Q62->E, Yes - considered in HC2b already 0368. The six humanized variants shown in Table 16 were K63 and F64 characterized by various parameters such as binding kinetics H3 (87->T, 88->S, 89->D) D89->E, Yes - potential salt bridges (discussed above) as well as chemical and physical properties already with K63 and K43 such as thermostability that are routinely used in the art. T87 and S88 S1 (L1 & L5) K48->Q No - K48 humanizing 0369. The characterization was done in two tiers. Tier I mutation included differential scanning calorimetry (DSC) shown in S2 (H1 & H3) D89->E No (see H3) Table 24 and FIG. 2. Briefly, for the DCS experiments, the *Note: antibodies were dialyzed against phosphate-buffered saline Sequential numbering used to refer to residues solution. Antibody concentrations were measured by UV absorbance. The antibodies were diluted to 1 mg/mL using TABLE 22 PBS. Scans were performed using a Calorimetry Sciences Potential Stabilizing Mutations Corporation N-DSC II instrument using a 0.3268 mL capil Light lary cell with PBS in the reference cell. The scan rate was 2 Chain C./min and the samples were scanned from 20°C. to 100° C. Residue Additional changes suggested Accept Change 0370 All variants, except for HC3b/LC3b showed com 15->L W15->L No - V15 in Vk1 germline parable binding affinities to the parental antibody. Variant 96->Q None - already Q96 None HC3a/LC3a was selected over the other variants based on 38->Y None - already Y38 None other physiochemical properties such as SEC data, stability and lack of aggregation (see Tables 23-25). TABLE 23 Comparison of Kinetics of the Humanized F151 Variants

HC1 LC1 HC2a LC2a

Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)

DAKD-b 4.16E--O5 6.OOE-06 1.45E-11 6.66E--OS 1.22E-OS 1.83E-11 KD-b 4.24E--OS 1.74E-07 3.94E-13 7.03E--OS 6.12E-06 8.71E-12

US 2015/005O270 A1 Feb. 19, 2015 37

TABLE 26 - continued Comparison of Parental F151 and humanized variant F151

Variable Heavy Chain Variable Light Chain

ACCGCCTACATGGAACTGCGGAGCCTGAGAAGCATAGATTTTCCGGCAGCGGCTCCGGCAC GACGACACCGCCGTGTACTACTGCGCCAACTAC CGACTTCACCCTGACCATCAGCAGCCTG TACAGATACGACGACCACGCCATGGACTACTGG CAGGCCGAGGACGTGGCCGTGTACTACT GGCCAGGGCACCCTGGTCACCGTGTCCTCT GCCAGCAGTACTACAGCTACCCCTGGAC (SEQ ID NO: 129) CTTCGGCCAGGGCACCAAGGTGGAAATC AAG (SEQ ID NO: 13 O)

Protein QIQLVQSGAEVKKPGASVKVScKASGYSFTDYN DIVMTQSPDSLAVSLGERATINCKSSQS IYWVRQAPGQGLEWIGYFDPYNGNTGYNQKFRG LLYSSNQKNYLAWYQQKPGQPPKPLIYW RATLTVDKSTSTAYMELRSLRSDDTAVYYCANYASTRESGVPDRFSGSGSGTDFTLTISSL YRYDDHAMDYWGQGTLVTVSS (SEQ ID NO: 24) K (SEQ ID NO: 30) Single underscore = CDR region; double underscore = Signature amino acids for identifying CDRS

0371 For alignment of light and heavy chains of parental KD complex is 8.9% and I/s(I)=20.2, while those for the F151 to humanized F151 variant (HC3a/LC3a), see FIG. 3. F151-DAKD are 7.7% and 18.5, respectively. The F151-KD structure was solved by molecular replacement in Phaser EXAMPLE 7 using Fab coordinates from PDB entry 3OOS, treating the V-V and C-Cl domains as independent units. The struc Crystal Structure of Humanized Antibody F151 ture was refined in autoBuster at 2.07 A resolution in space against BRK1 Ligand Kallidan and group P222 to an Rfactor of 0.205 and an Rfree of 0.228. des-arg'-Kallidin The F151-DAKD structure was solved using the F151-KD coordinates. The structure was refined in autoBuster at 1.86 A 0372. The crystal structures of humanized F151 (HC3a/ LC3a) Fab bound to kallidan ordes-arg'-kallidin was deter resolution in space group P212121 to an Rfactor of 0.232 and mined and the molecular interactions analyzed. an Rfree of 0.238. 0373) Kallidin powder was purchased from Phoenix Phar 0374. The electron density maps shown in FIGS. 4 and 5 maceuticals (Cat. No. 009-37). For Fab protein generation, depict the binding of kallidin (KD) and Des-Arg'-kallidin the DNA of heavy chain (HC) VH region from humanized (DAKD) to the F151 Fab and unambiguously determine the F151 HC3a was cloned into 6xHis tagged CH1 vector positions of each amino acid. For kallidin, the electron den pFF0366 (“6xHis' disclosed as SEQID NO: 137). The light sity for the extreme C-terminal residue Arg' is not present. chain (LC) plasmid used here was the same as of the original This is in agreement with the observation that DAKD, which F151 LC3a used in F151 humanization (see Example 5). The is missing the C-terminal residue arginine (shown in Table 27 two plasmids were co-transfected into free style HEK293 below), binds equally well to F151 as KD. The 1050 values of cells for Fab expression. The Fab protein was purified using F151 in the neutralization FDSS cellular assay towards KD cobalt-resin, bufferexchanged to 50 mMMES pH6.0, 50 mM and DAKD are 0.12 nM and 0.09 nM, respectively. In both NaCl before being concentrated to about 9 mg/mL. Purified cases the electron density is weaker towards the C-termini of F151 Fab protein was mixed with kallidin in a molar ratio of the peptides. Since Phe in KD has slightly better electron 1:2 and set up for crystallization screening. Crystallization density than that in DAKD, it is possible that the presence of screening was done with a wide range of conditions. The best the additional arginine at the C-terminus of KD stabilizes the crystal was observed under condition B10, B12 and G10 of C-terminus of this peptide when binding to F 151 although Hampton Research screening kit PEG/ION HT. The crystals this arginine itself is not stable enough to be observed by were cryo-protected with 20% glycerol in well buffer and X-ray. Since the two structures are essentially identical (rms frozen for diffraction data collection. The X-ray diffraction between KD and DAKD is 0.139 for C atoms and 0.328 for all data for both complexes were collected at Canadian Light atoms), all the following discussions are based on the F151 Source, beamline CMCF-08ID. The Rmerge for the F151 KD structure. TABL E 27 A selected list of kinin peptides SEQ Peptide ID Sequence ale NO : 1 2 3 4 5 6 7 8 9 10 KD (Kallidin) 1. Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg DAKD (des 2 Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg'- Kallidin) US 2015/005O270 A1 Feb. 19, 2015

TABLE 27-continued A selected list of kinin peptides SEQ Peptide ID Sequence ale NO : 1 2 3 4 5 6 7 8 9 10 KLP (rodent 3 Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg KD ortholog) BK 5 Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Bradykinin)

0375 KD is bound with its N-terminus buried in the inter minus is essentially solvent exposed. Except for the first 4 face between Fv subunits of the light and heavy chains, as residues (Lys-Arg-Pro-Pro) (SEQ ID NO: 132), the other shown in FIG. 6. The interface between light and heavy residues of KD gradually extend into the bulk solvent. The chains are packed with aromatic amino acids, including Tyr amidinium group of Lys1 sidechain is anchored by Glu-L61 L42, Tyr-L93, Tyr-L100, Trp L102, Phe-L104 and Tyr-H35, (L: light chain) through salt bridges, while the amino terminal Trp-H47, Tyr-H50, Tyr-H99, Trp-H1 10, stabilizing each amino group Lys 1 forms a salt bridge with Asp-H108 (H: other through stacking and hydrophobic interactions. Resi heavy chain). The amidinium group of Lys1 sidechain also dues from each of the CDR's of light and heavy chains con hangs over the aromatic ring of Tyr-L55, involved in cation tribute to the binding. The residues along the light and heavy interactions. Such intensive interactions involved with Lys1 chains that are involved in interactions with KD as mapped on tightly anchor the amino terminus of KD in the Fab. This also the CDRs are shown in FIGS. 7 and 8. CDR H3 of the heavy accounts for the importance of Lys1 in the binding of KD to chain is the longest loop and the one most frequently used in F151. Without it (i.e. bradykinin), no detectable binding to the interactions with KD, forming a side cover for KD. The hF151 or F151 can be measured. Like Lys1, Arg2 interacts loop was stabilized mostly through interactions with the other with the Fab through a salt bridge. The guanidium group of two CDRs, H1 and H2 of the heavy chain, namely, Salt bridge Arg2 interacts with the sidechain of Asp-H104. The sidechain between Asp-H101 and Arg-H52 (stabilizing H1 and H3), of Arg2 is also H-bonded with the mainchain carbonyl oxy arene-H interaction between Tyr-H102 and Tyr-H54 (stabi gen of Arg-H101. Also, the mainchain oxygen of Pro8 is lizing H2 and H3), H-bond between Asp-H108 and Tyr-H35 H-bonded to the sidechain of Arg-H101. Tyr-H102 is half and H-bond between His-H105 and Tyr-L55 (stabilizing H3 way intercalated into Phe8 and Pro9, involving in hydropho and L2). bic interactions with KD. In addition to direct interaction, 0376 Comparing the KD interacting residues among the numerous water-mediated H-bonds between KD and the Fab antibodies generated, it can be seen that there is similarity are also seen. It is also interesting to notice that tyrosine among the antibodies, and some were more related in use of residues are most frequently used in the interaction compared particular amino acids for KD-interaction than others. For to other amino acids; 9 out of the 16 residues marked with example, in the light chain F151, C63 and 122 use more asterisks in FIGS. 7 and 8 are tyrosines. All the residues in similar amino acids in their CDRs to bind KD, while B21 and F151 Surrounding KD appear to play a role in ligand binding, I54 were more similar. In the heavy chain, F151 and C63 were except for Asn-H33, which is close to Phe6 sidechain but Surprisingly unique from each other and from B21, I22 and incompatible in polarity and lack of other important interac I54. The latter three appear to form a group in similarity. C63 tions. Substitution with aromatic/hydrophobic residues, such is particularly interesting in its heavy chain, that the loop Trp or Tyr to interaction with PheB appears to be a quick pick length in H2 and H3 are more different from all others. Con if affinity maturation is considered. These two aromatic sidering the Fab as a whole, B21 and I54 were most closely amino acids are in fact seen in other antibodies (Trp in C63, related. and Tyr in B21, I22. I54). Table 28 below provides a detailed 0377. In the crystal structure, we found that KD is analysis of 16 KD-interacting amino acid residues marked in involved in Systematic hydrogenbond and hydrophobic inter FIGS. 7 and 8 and sets forth functional substitutions that can actions with the Fab. The N-terminus of KD is buried in the be made in the CDR regions that should not disrupt antigen Fab and harbors more intensive interactions, while the C-ter binding. TABLE 28 A list of amino acid residues found around the KD binding pocket, and their roles in KD binding and potential functional Substitutions (light chain residues in grey-colored cells and heavy chain residues in unshaded cells Residue Role in KD Binding or CDR Stabilization Functional Substitution Tyr-L31 Edge-on hydrophobic interactions with Pro4 His to add an H-bond with amide N of Along with Tyr-L38 and Tyr-L98 form three ProS orthogonal planes that Surround the 90 deg turn of other aromatic a.a. Such as Trp, and KD at Pro4 Phe Tyr-L38 hydrophobic stacking with Pro4 His to add an H-bond with carbonyl O Along with Tyr-L31 and Tyr-L98 form three of Arg2 orthogonal planes that Surround the 90 deg turn of KD at Pro4 US 2015/005O270 A1 Feb. 19, 2015 39

TABLE 28-continued A list of amino acid residues found around the KD binding pocket, and their roles in KD binding and potential functional Substitutions (light chain residues in grey-colored cells and heavy chain residues in unshaded cells Residue Role in KD Binding or CDR Stabilization Functional Substitution Tyr-L55 cation-interaction with amidinium ion of Lys1 Trp to pair with H105 mutations of Gln, sidechain ASn, Glu or Asp (maintaining H-bond); H-bond with His-H105: Tyr-L55--His-H105 pairing Other variants of His H105 are Tyr adds stabilization between L2 and H3 loops and Ser. Other aromatic a.a., such as Trp, His and Phe Glu-L61 Forming key salt bridges with Lys1 sidechain Asp, Gln, ASN (Asp is seen in B21 and I54 already, FIG. 7) Tyr-L97 H-bond with amide N of Trp-L56 Aromatic a.a. Such as Phe or His (too Forming pocket for Arg1 extended sidechain tight space for Trp) Tyr-L98 Along with Tyr-L31 and Tyr-L38 form three Other aromatic a.a, such as Phe, Trp orthogonal planes that Surround the 90 deg turn of or His KD at Pro4 Tyr- Forming pocket surface for Pro3 Aromatic a.a., Such as Phe (better L100 Part of the aromatic a.a. cluster interface between hydrophobic interactions with Pro3) LH chains, further including Tyr-LA2, Tyr-L93, Other variants seen are Thr (in B21 Trp L102, Phe-L104 and Tyr-H35, Trp-H47, Tyr- and I54) and His (in I22) H50, Tyr-H99, Trp-H110 Partial stacking with Tyr-H50 Trp- Part of the aromatic a.a. cluster interface between Other aromatic a.a, such as Tyr (in L102 LH chains C63 and B21), Phe and His Stacking with Trp-H47 Other hydrophobic residue, such as L (in I22) ASn-H33 Close to Pheósidechain but incompatible in Replace with aromatic/hydrophobic polarity, no other roles seen either; can be a target a.a., such as Trp (seen in C63) or Tyr or affinity maturation (seen inB21, I22, I54) Asp-H52 Salt bridge with Arg-H101, stabilizing H1 and H3 Mutate as a pair with Arg-H101 to Oops reversely charged a.a., such Arg H52. Asp-H101, or a pair of hydrophobic a.a. (Leu, Ile, Val, Met, Phe, Tyr, Trp, Ala) to form a cluster with Phe6 of KD Tyr-H54 Close to Pro8 but no specific interactions Mutated to negatively charged a.a. to Close to Arg-H101, but no charge interactions stabilize Arg-H101, such D or E (seen in B21, I22 and I54) or N or Q, also provide H-bond with carbonyl O of Pro8 (A Lys in C63, which can be charge-reversed to Glu) Tyr-H99 Part of aromatic interface between H and L chains Mutate to Small aromatic a.a. except H-bond with Asn-H33 W, such as Phe and His Tight space Arg- H-bond with amide of Pro8 Mutate as a pair with Asp-H52 to H101 Supported by Asp-H52 (salt bridge) reversely charged a.a., Such Arg H52. Asp-H101, or a pair of hydrophobic a.a. to form a cluster with Phe6 of KD Tyr- Half-way intercalating into Phe9 and Pro8, Phe can be better, Trp or His may be H102 hydrophobic interactions with KD OK too Asp- Key residue to salt-bridge with Arg2 Glu to maintain Salt bridge with Arg2 H104 Bigger a.a. to fill the gap from Pro3, Such as Tyr as seen in B21, I22 and IS4 Asp- Key residue to salt bridge with N-term —NH3+ of KD Glu H108 H-bond with Tyr-H33, stabilizing H3 loop Conserved residue. Not in CDR

0378 Analysis of the conformational epitope of kallidin EXAMPLE 8 (KD) or desArg10-Kallidin (DAKD) revealed that it adopts a “Pro4kink’ conformation. As depicted in FIG. 17, a hallmark In vivo Pharmacology of Anti-BKR1-Ligand of the “Pro 4 kink’ conformation is a type II tight turn in the Antibodies in Pain Models main chain polypeptide backbone of KD or DAKD at Proline 4 (see Richardson J S.“The anatomy and taxonomy of protein 0379 The examples of the present invention illustrate the structure.” Adv Protein Chem. 1981: 34:167-339, which is in vivo efficacy of anti-BKR1 receptor-ligand antibodies in incorporated by reference herein). The “Pro 4 kink’ confor different preclinical models of acute and chronic pain accord mation may further defined by all or substantially all of the ing to modified procedures described in (a) Saddi G M and remaining amino acids of KD (1-2 and 6-9) or DAKD adopt Abbott FV., Pain (2000), 89:53-63; (b) Chen et al., Molecular ing repeats of a sigmoid shape which align the hydrophobic Pain (2010), 2:6-13 and (c) Bennett GJ and Xie Y K. Pain side chains in a spatially stacking mode. (1988), 33:87-107. US 2015/005O270 A1 Feb. 19, 2015 40

Animals TABLE 29 0380 Experiments were carried out using adult male OF1 Effect of EE1 and F151 antibodies on formalin-induced mice (20-30 gr) for formalin studies and adult male C57BI/6J pain-like behavior in male OF1 mice mice (25-30 gr) for both CFA and CCI studies. The mice were Reversal of pain-like behavior Dose A.U.C. SEM (in %) + SEM kept in a controlled temperature room under a 12-hlight-dark Group (mg/kg, i.v.) (15-45 min) (15-45 min) cycle. Food and water were provided ad libitum. For all of the Isotype- 30 63.62.9 O 5 experiments, mice were acclimatized to the laboratory room control for at least 2 hours before testing. No randomization was (EE1) performed for the studies. Experimenters performing the EE1 2.5 41.6+3.4 (***) 3SS behavioral tests were not blind to treatment; however they 10 42.9 + 2.9 (***) 335 30 36.5 +4.3 (***) 45 - 7 were not aware of the study hypothesis. All procedures have Isotype- 10 57.33 O 5 been approved by the “Comité d'Expérimentation pour la control Protection de I Animal de Laboratoire' (Animal Care and (F151) Use Committee) of Sanofi-aventis recherche & developpe F151 2.5 48.9 +3.8 (NS) 1S 7 ment and were carried out in accordance with French legis 10 45.0 + 2.8 (*) 215 lation (Decree n°87-848-19 Oct. 1987- and decision-19 Apr. (*), p < 0.05; 1988) implementing European directive 86/609/EEC. (***), p<0.001: Student's t-test versus adequate control was used. NS: non significant A. Formalin-Induced Acute Inflammatory Pain B. CFA (Complete Freund's Adjuvant)-Induced Chronic 0381. The formalin test was used to measure nociceptive Inflammatory Pain and inflammatory pain. Indeed, intraplantar injection of for 0386 Chronic inflammation was induced under brief malin induces an initial acute nociceptive behavioral anesthesia (Isoflurane, 3%) by an intraplantar administration response (0-12 minutes), followed by a second inflammatory of 25uL of Complete Freund's Adjuvant (CFA) containing 1 mediated response (15-45 minutes), which is attributed to ug/uL heat-killed Mycobacterium tuberculosis in mineral oil spinal cord excitability. and mannide monooleate (Sigma). Group sizes were 8 male 0382 Formaldehyde (37%, Sigma) was diluted in saline C57BL/6 mice. (v/v) to obtain a 2.5% formaldehyde concentration (i.e. 0387 EE1 antibody was administered intravenously 22 s6.25% formalin concentration). Mice were gently restrained hours after intraplantar CFA injection at 2.5 and 30 mg/kg and and 20 uL of this solution was injected Subcutaneously into mechanical and thermal hypersensitivities were assessed at the dorsal part of one hind paw. Behavioral responses were Day 1 (D1), Day 4 (D4) and Day 7 (D7) post-CFA intraplantar scored immediately after formalin injection, then at 3 minutes administration. intervals over 45 minutes as follows: (0): normal weight bearing of the injected paw; (1): injected paw resting lightly B1. Mechanical Hypersensitivity on floor; (2): lifting-elevation of the injected paw; (3): licking 0388 Mechanical hypersensitivity was assessed by mea orbiting the injected paw. Group sizes were 11-12 male OF1 suring the Frequency of withdrawal Response (FR, in %) 1CC. following 10 applications of a 0.6 g. Von Frey filament (Bio 0383 Scores were plotted versus time and areas under the Seb, France) onto the plantar Surface of the injected paw. curves (AUC) were calculated from the mean scores (SEM) 0389. To investigate the efficacy of EE1 antibody on pain for both the early (0-12 min) and the late (15-45 min) phases. like behavior, we calculated the reversal of mechanical hyper Reversal of pain-like behaviors was expressed as change in sensitivity (in 96) as follows: AUC in 96. Percent reversals were calculated as(Mean FR-iso type-controlese-FR-Ipsise)/(Mean 0384 EE1 antibody inhibited the pain-like behavior in the FR-isotype-control-Mean FR-sham late phase of the formalin test in male OF1 mice. EE1 anti dose)for each mouse. body, when administered intravenously 48 hours before intra 0390 At D1, D4 and D7 after intraplantar injection of plantar injection of formalin, showed a dose dependent rever CFA, a significant increase of FR to the Von Frey filaments sal of the pain-like behavior only in the late phase with a was observed in the isotype-control 1 B7.11-treated group in Minimal Effective Dose (MED)=2.5 mg/kg, as depicted in comparison with the naive group, demonstrating the devel FIG. 9. Indeed, when administered at 2.5, 10 and 30 mg/kg, opment of mechanical hypersensitivity. EE1 antibody, when EE1 reversed the late phase by 35+5%, 33+5% and 45+7%, administered intravenously 22 hours after intraplantar CFA, respectively, as depicted in Table 29. was able to significantly decrease this FR at the different times studied compared with that obtained in the isotype 0385. In contrast, F151 weakly inhibits the pain-like control 1B7.11-treated group. (FIG. 10). behavior in the late phase of the formalin test when adminis 0391 Reversal of mechanical hypersensitivity was tered 48 hours before intraplantar injection of formalin. 41+8% and 22.8% at D1, 36.9% and 32.9% at D4 and Indeed, when administered at 2.5 and 10 mg/kg, F 151 27+10% and 50+9% at D7 for a 2.5 mg/kg and 30 mg/kg reversed the late phase by 15+7% and 21+5%, respectively, as intravenous administration of EE1 antibody, respectively depicted in Table 29. (Table 30). US 2015/005O270 A1 Feb. 19, 2015 41

TABLE 30 Effect of EE1 antibody on CFA-induced mechanical hyperSensitivity in male C57BL/6 mice Dose Day 1 post-CFA Day 4 post-CFA Day 7 post-CFA (mg/kg, FR (%) FR(%) FR(%) Group i.v.) % effect % effect % effect

Naive Ila. 41.3 44 100 11 43.8 2.6 1OO 8 45 - 5 1OO 13 Isotype- 30 813 - 4.4 O 11 78.83 O 8 825 - 2.5 O7 control 1B7.11 EE1 2.5 65 33 418 66.3 + 3.2 (*) 369 72.53.7 27 10 30 (**) 228 67.5 +3.1 (*) 329 (*) SO 9 72.53.1 63.8 - 3.2 (*) (***) FR: Frequency of Response (in %) + SEM, 96 effect SEM, n.a. not applicable (*) p < 0.05, (**) p < 0.01 and (***) p<0.001, Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time

B2. Thermal Hypersensitivity Withdrawal Latency at D1, whatever the dose tested (FIG. 11). However, EE1 significantly increased the Paw With 0392 For thermal hypersensitivity, measures of Paw drawal Latency at D4 and this effect was also present at D7 Withdrawal Latencies (PWL, in seconds) in response to a (FIG. 11). radiant heat using a plantar apparatus (IITC. Woodland Hills, USA) were assessed. 0397 Reversal of thermal hypersensitivity was 41+15% 0393 To investigate the efficacy of EE1 antibody on pain and 58-21% at D4 and 46-10% and 52.17% at D7 for a 2.5 like behavior, we calculated the reversal of thermal hypersen mg/kg and 30 mg/kg intravenous administration of EE1. sitivity (in 96) as follows: respectively (Table 31). TABLE 31

Effect of EE1 antibody on CFA-induced thermal hypersensitivity in male C57BL/6 mice

Dose Day 1 post-CFA Day 4 post-CFA Day 7 post-CFA (mg/kg, PWL (sec) PWL (sec) PWL (sec) Group i.v.) % effect % effect % effect

Isotype- 30 3.30.3 Ila. 4.1 + 0.1 Ila. 3.6 O.3 Ila. control 1B7.11 EE1 2.5 3.10.3 - 7 - 10 5.204 41 + 15 5.0 + 0.3 (*) 46 + 10 30 2.60.2 -205 5.7 + 0.6 (*) 58 + 21 5.2 + 0.6 (*) 52 + 17

PWL: Paw withdrawal latency + SEM, 96 effect + SEM, n.a. not applicable (*) p<0.05, Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time

Percent reversals were calculated as(PWL C. CCI (Chronic Constriction Injury)-Induced Mean isotype-controlae.)/(Mean isotype Neuropathic-Like Pain (Bennett's Model) controlese-Mean isotype-controlsee) for each mouse. 0398. CCI model was used as a model of peripheral nerve injury. Briefly, mice were anesthetized with Isoflurane (3%), 0394 Thermal hypersensitivities were not different and the right sciatic nerve was exposed at mid thigh level between all groups at baseline, before intraplantar injection of through a small incision. Three loose ligatures of 6.0 chromic CFA (data not shown). gut (Ethicon) at 1 mm space were placed around the Sciatic nerve. The Surgical procedure was completed by closing the 0395. At D1, D4 and D7 after intraplantar injection of muscles and skin. The day of CCI Surgery was considered as CFA, a significant decrease in paw withdrawal latency of the Day 0. Group sizes were 6-10 male C57BL/6 mice. injected paw was observed in isotype-control 1 B7.11-treated 0399 EE1 antibody was administered intravenously at 2.5 group of mice, demonstrating that CFA induced a thermal and 30 mg/kg on Day 11 post Surgery and mechanical and hypersensitivity (data not shown). thermal hypersensitivities were assessed on Day 12 (D12), 0396 EE1 antibody, administered intravenously 22 hours Day 14 (D14) and Day 18 (D18) post-surgery which corre after intraplantar CFA injection (i.e. on Day 1 post-intraplan sponded to Day 1 (D1), Day 3 (D3) and Day 7 (D7) post tar CFA injection), and was not able to increase the Paw treatment. US 2015/005O270 A1 Feb. 19, 2015 42

C1. Mechanical Hypersensitivity TABLE 32-continued 0400 Mechanical hypersensitivity was assessed by mea Effect of EE1 antibody on CCI-induced mechanical suring hind paw withdrawal thresholds (on both injured i.e. hyperSensitivity in male C57BL/6 mice Ipsi and non-injured i.e. Contra paws) to an increasing Dose Day 18 pressure (in g) stimulus using a Dynamic Plantar Aesthesi (mg/kg, Day 12 post-CCI Day 14 post-CCI post-CCI% ometer (Ugo-Basile, Italy); a steel rod was applied to the hind Group i.v.) % effect SEM 96 effect SEM effect SEM paws of the mice with an increasing force (5 grams in 10 EE1 2.5 1524.9 26.8 +5.7 (**) 3O3 + 7.1 seconds). 30 15.25.7 25.7 + 4.5 (*) 20.8 - 5.9 04.01 To investigate the efficacy of EE1 antibody on pain (*), p < 0.05, and like behavior, we determined the reversal of mechanical (**), p < 0.01 Two-Way ANOVA with time as repeated measure followed by Dunnett's test hypersensitivity as follows: percent reversals were calculated for factor group for each level of factor time (statistics performed on Delta ipsi values) as(Ipsiestos-lpsite)/(Contratose-lpsites)for each C2. Thermal Hypersensitivity OUS. 0404 For thermal hypersensitivity, measures of Paw 0402 Following surgery, operated mice developed a Withdrawal Latencies (in seconds) in response to a radiant robust sensitization to mechanical stimulus on the injured heat using a plantar apparatus (IITC. Woodland Hills, USA) paw, whereas the non-injured paw was not affected. At Day were assessed on the injected hind paw. 11, the mechanical sensitization on the injured paw reached a 04.05 To investigate the efficacy of EE1 antibody on pain plateau (data not shown). like behavior, we calculated the reversal of thermal hypersen sitivity (in 96) as follows: 0403. EE1 antibody, administered intravenously on Day Percent reversals were calculated as(Ipsi'postdose -Mean 11 demonstrated a slight tendency to reverse CCI-induced isotype-control.)/(Mean naive postdose mechanical hypersensitivity on D12, D14 and D18 with Mean isotype-control)for each mouse. 15.2+4.9% and 15.2+5.7% on D12, 26.8+5.7% and 25.7+4. 0406 Following surgery, operated mice developed a 5% on D14 and 30.3+7.1% and 20.8+5.9% on D18, at 2.5 and robust sensitization to thermal stimulus on the injured paw, 30 mg/kg respectively (FIG. 12 and Table 32). whereas the non-injured paw was not affected. At Day 11, the thermal sensitization on the injured paw reached a plateau TABLE 32 (data not shown). 04.07 EE1 antibody, administered intravenously on Day Effect of EE1 antibody on CCI-induced mechanical 11 did not significantly increase Paw Withdrawal Latency of hypersensitivity in male C57BL/6 mice the injured paw on D12, even if a trend was observed. How Dose Day 18 ever, from D14, EE1 antibody significantly increased the Paw (mg/kg, Day 12 post-CCI Day 14 post-CCI post-CCI% Withdrawal (FIG. 13). Group i.v.) 9% effect + SEM 9% effect + SEM effect + SEM 0408 Reversal of thermal hypersensitivity was 41.16% Isotype- 30 O.23.O 1.84.3 18.1 6.6 and 56-24% at D12 and 51.16% and 98.48% at D14 and control 78+19% and 84+22% at D18, for a 2.5 mg/kg and 30 mg/kg 1B7.11 intravenous administration of EE1 antibody, respectively (Table 33). TABLE 33

Effect of EE1 antibody on CCI-induced thermal hypersensitivity in male C57BIf 6 mice Kinetic evaluation

Dose Day 12 post-CCI Day 14 post-CCI Day 18 post-CCI (mg/kg, PWL (sec) PWL (sec) PWL (sec) Group i.v.) % effect % effect % effect

naive Ila. 6.3 OS 100 19 6.2. O.4 1OO 14 6.1 - O.S 100 17 Isotype- 30 3.8 O2 O7 3.4 O2 O 5 3.40.2 O7 control 1B7.11 EE1 2.5 4.8 + 0.4 41 - 16 4.8 - O.S 51 16 5.5 + 0.5 (*) 78.19 30 5.20.6 56 24 6.1 + 1.3 (**) 9848 5.7+ 0.6 (**) 84 - 22

PWL: Paw withdrawal latency + SEM, 9% effect + SEM, n.a. non applicable (*) p < 0.05, and (**) p < 0.01 Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time US 2015/005O270 A1 Feb. 19, 2015 43

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS : 137

<210s, SEQ ID NO 1 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 1 Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 2 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 2 Lys Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 3 &211s LENGTH: 10 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 3 Arg Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 4 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 4 Arg Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 5 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Unknown : Bradykinin peptide'

<4 OOs, SEQUENCE: 5 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5

<210s, SEQ ID NO 6 &211s LENGTH: 8 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Unknown: Des-Arg9 Bradykinin peptide'

<4 OOs, SEQUENCE: 6 Arg Pro Pro Gly Phe Ser Pro Phe 1. 5 US 2015/005O270 A1 Feb. 19, 2015 44

- Continued

SEO ID NO 7 LENGTH: 11 TYPE PRT ORGANISM: Artificial Sequence FEATURE: NAMEAKEY: source OTHER INFORMA CION: /note=Description of Artificial Sequence: Synthetic consensus peptide' FEATURE: NAME/KEY: WAR ANT LOCATION: (1) . . (1) OTHER INFORMA CION: /replace=Phe" or His" FEATURE: NAMEAKEY: misc feature LOCATION: (1) . . (1) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' FEATURE: NAME/KEY: WAR ANT LOCATION: (3) . . (3) OTHER INFORMA CION: /replace="Asp" or Ala" or Val" or Lieu" or ve' or Met or Phe" or Tyr" or Trp" FEATURE: NAME/KEY: WAR ANT LOCATION: (4) . . (4) OTHER INFORMA CION: /replace=Phe" or Trip" or His" FEATURE: NAMEAKEY: misc feature LOCATION: (3) . . (4) OTHER INFORMA CION: /note= Residues given in the sequence have no preference with respect to those in the annotations for said positions" FEATURE: NAME/KEY: WAR ANT LOCATION: (6) . . (6) OTHER INFORMA CION: /replace=" Glu" or Tyr" FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' FEATURE: NAME/KEY: VARIANT LOCATION: (10) . . (10) OTHER INFORMA CION: /replace=" Glu" FEATURE: NAMEAKEY: misc feature LOCATION: (10) . . (10) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

SEQUENCE: 7 Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr 1. 5

SEQ ID NO 8 LENGTH: 17 TYPE PRT ORGANISM: Artificial Sequence FEATURE: NAMEAKEY: source OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' FEATURE: NAME/KEY: VARIANT LOCATION: (3) . . (3) OTHER INFORMATION: /replace=Arg" or 'Ala' or val" or Lieu" or Ile" or Met" or Phe" or Tyr" or Trp" FEATURE: NAMEAKEY: misc feature US 2015/005O270 A1 Feb. 19, 2015 45

- Continued

<222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: /replace="Asp" or 'Glu" or 'Asn" or 'Glin' 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position'

<4 OOs, SEQUENCE: 8 Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Gln Llys Phe Arg 1. 5 1O 15 Gly

<210s, SEQ ID NO 9 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222 LOCATION: (8) ... (8) <223> OTHER INFORMATION: /replace= Trip" or Tyr" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position'

<4 OOs, SEQUENCE: 9 Gly Tyr Ser Phe Thr Asp Tyr Asn Ile Tyr 1. 5 1O

<210s, SEQ ID NO 10 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: /replace=Phe" or His' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: /replace=Phe" or His" or Trip" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (4) <223> OTHER INFORMATION: /note= Residues given in the sequence have no preference with respect to those in the annotations for said positions' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: /replace=Phe" or Thr' or His" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature US 2015/005O270 A1 Feb. 19, 2015 46

- Continued

<222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: /replace= Tyr' or Phe" or His" or Lieu" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position'

<4 OOs, SEQUENCE: 10 Gln Glin Tyr Tyr Ser Tyr Pro Trp Thr 1. 5

<210s, SEQ ID NO 11 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: /replace="Asp" or Gln" or Asn" & 22 O FEATURE; <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position'

<4 OOs, SEQUENCE: 11 Trp Ala Ser Thr Arg Glu 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: /replace=His" or Tyr' or Phe" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: /replace= Tyr" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

<4 OOs, SEQUENCE: 12 US 2015/005O270 A1 Feb. 19, 2015 47

- Continued

Llys Ser Ser Glin Ser Lieu Lleu Trip Ser Ser Asn. Glin Lys Asn His Lieu. 1. 5 15

Ala

<210s, SEQ ID NO 13 &211s LENGTH: 11 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 13 Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr 1. 5

<210s, SEQ ID NO 14 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 14 Tyr Phe Asp Pro Tyr Asn Gly Asn. Thr Gly Tyr Asn Gln Llys Phe Arg 1. 5 15 Gly

<210s, SEQ ID NO 15 &211s LENGTH: 10 212. TYPE PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 15 Gly Tyr Ser Phe Thr Asp Tyr Asn. Ile Tyr 1. 5 1O

<210s, SEQ ID NO 16 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 16 Gln Glin Tyr Tyr Ser Tyr Pro Trp Thr 1. 5

<210s, SEQ ID NO 17 &211s LENGTH: 7 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 17 Trp Ala Ser Thr Arg Glu Ser 1. 5

<210s, SEQ ID NO 18 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 18 Lys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser Ser Asn Gln Lys Asn Tyr Lieu. 1. 5 15

Ala US 2015/005O270 A1 Feb. 19, 2015 48

- Continued

<210s, SEQ ID NO 19 &211s LENGTH: 120 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 19 Glu Ile Gln Leu Gln Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Thr 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Lys Glin Ser His Gly Lys Ser Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Phe 65 70 7s 8O Met His Leu Ser Ser Lieu. Thir Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210 SEQ ID NO 20 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 2O Glu Ile Glin Lieu Val Glin Ser Gly Pro Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn Ile Tyr Trp Val Lys Glin Ser Pro Gly Lys Ser Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Phe 65 70 7s 8O Met His Leu Ser Ser Lieu. Thir Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O

Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 21 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: US 2015/005O270 A1 Feb. 19, 2015 49

- Continued Synthetic polypeptide'

<4 OOs, SEQUENCE: 21 Glin Ile Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Lys Glin Ser Pro Gly Lys Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Tyr 65 70 7s 8O Met His Leu Ser Ser Lieu. Thir Ser Glu Glu Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 22 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 22 Glin Ile Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Lys Glin Ser Pro Gly Lys Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glu Lys Phe SO 55 6 O Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Tyr 65 70 7s 8O Met His Leu Ser Ser Lieu. Thir Ser Glu Glu Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 23 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 23 US 2015/005O270 A1 Feb. 19, 2015 50

- Continued

Glin Ile Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Lys Glin Ser Pro Gly Lys Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Tyr 65 70 7s 8O Met His Leu Ser Ser Lys Thir Ser Glu Glu Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 24 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 24 Glin Ile Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Arg Glin Ala Pro Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Arg Ala Thr Lieu. Thr Val Asp Llys Ser Thr Ser Thr Ala Tyr 65 70 7s 8O Met Glu Lieu. Arg Ser Lieu. Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 25 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 25 Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15

Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr US 2015/005O270 A1 Feb. 19, 2015 51

- Continued

2O 25 3O Asn Ile Tyr Trp Val Arg Glin Ala Pro Gly Glin Gly Lieu. Glu Trp Met 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Arg Val Thr Met Thir Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 7s 8O Met Glu Lieu. Arg Ser Lieu. Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 26 &211s LENGTH: 113 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 26 Asp Ile Val Met Ser Glin Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1. 5 1O 15 Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Ser Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O Ile Ser Ser Wall Lys Ala Glu Asp Lieu Ala Ile Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 27 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 27 Asp Ile Val Met Ser Glin Ser Pro Ser Ser Leu Ala Ala Ser Val Gly 1. 5 1O 15

Asp Arg Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O

Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Lys 35 4 O 45

Ser Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr US 2015/005O270 A1 Feb. 19, 2015 52

- Continued

Ile Ser Ser Val Glin Ala Glu Asp Lieu Ala Ile Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O Lys

<210s, SEQ ID NO 28 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 28 Asp Ile Val Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Ser Cys Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Lys 35 4 O 45 Ser Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O Ile Ser Ser Val Glin Ala Glu Asp Leu Ala Thr Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 29 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 29 Asp Ile Val Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Ser Cys Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser 2O 25 3O

Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Lys 35 4 O 45

Ser Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O

Ile Ser Ser Val Glin Ala Glu Asp Lys Ala Thr Tyr Tyr Cys Glin Glin 85 90 95

Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile US 2015/005O270 A1 Feb. 19, 2015 53

- Continued

1OO 105 11 O

<210s, SEQ ID NO 3 O &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 30 Asp Ile Val Met Thr Glin Ser Pro Asp Ser Lieu Ala Val Ser Lieu. Gly 1. 5 1O 15 Glu Arg Ala Thir Ile Asn. Cys Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Pro Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O Ile Ser Ser Lieu. Glin Ala Glu Asp Val Ala Val Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Glin Gly Thr Lys Val Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 31 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 31 Asp Ile Val Met Thr Glin Ser Pro Asp Ser Lieu Ala Val Ser Lieu. Gly 1. 5 1O 15 Glu Arg Ala Thir Ile Asn. Cys Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Pro Pro Llys Lieu. Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O

Ile Ser Ser Lieu. Glin Ala Glu Asp Val Ala Val Tyr Tyr Cys Glin Glin 85 90 95

Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Glin Gly Thr Lys Val Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 32 US 2015/005O270 A1 Feb. 19, 2015 54

- Continued

&211s LENGTH: 11 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 32 Trp Glu Tyr Asp Gly Tyr Tyr Asp Lieu. Asp Tyr 1. 5 1O

<210s, SEQ ID NO 33 &211s LENGTH: 17 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 33 Trp Ile Asp Pro Glu Asn Gly Asp Thr Gly Tyr Ala Arg Llys Phe Glin 1. 5 1O 15 Gly

<210s, SEQ ID NO 34 &211s LENGTH: 10 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 34 Gly Phe Asn. Ile Lys Asp Tyr Tyr Lieu. His 1. 5 1O

<210s, SEQ ID NO 35 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 35 Lieu. Glin Gly Thr His Phe Pro Tyr Thr 1. 5

<210s, SEQ ID NO 36 &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 36 Lieu Val Ser Lys Lieu. Asp Ser 1. 5

<210s, SEQ ID NO 37 &211s LENGTH: 16 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 37 Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser Asn Gly Llys Thr Tyr Lieu. Asn 1. 5 1O 15

<210s, SEQ ID NO 38 &211s LENGTH: 120 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 38 Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Val Arg Ser Gly Ala 1. 5 1O 15 US 2015/005O270 A1 Feb. 19, 2015 55

- Continued

Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn. Ile Lys Asp Tyr 2O 25 3O Tyr Lieu. His Trp Wall Lys Glin Arg Pro Glu Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Gly Tyr Ala Arg Llys Phe SO 55 6 O Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 7s 8O Lieu. His Leu Ser Ser Lieu. Thir Ser Glu Asp Thr Ala Val Tyr Tyr Phe 85 90 95 Asn Ala Trp Glu Tyr Asp Gly Tyr Tyr Asp Lieu. Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Ser Val Thr Val Ser Ser 115 12O

<210s, SEQ ID NO 39 &211s LENGTH: 112 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 39 Asp Val Val Met Thr Glin Thr Pro Leu. Thir Leu Ser Val Thir Ile Gly 1. 5 1O 15 Gln Pro Ala Ser Ile Ser Cys Llys Ser Ser Glin Ser Lieu Lieu. Tyr Ser 2O 25 3O Asn Gly Lys Thr Tyr Lieu. Asn Trp Lieu. Lieu. Glin Arg Pro Gly Glin Ser 35 4 O 45 Pro Lys Arg Lieu. Ile Tyr Lieu Val Ser Lys Lieu. Asp Ser Gly Val Pro SO 55 6 O Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O Ile Arg Val Glu Ala Glu Asp Lieu. Gly Val Tyr Tyr Cys Lieu. Glin Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys 1OO 105 11 O

<210s, SEQ ID NO 4 O &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 4 O Glu Asp Tyr Gly Gly Asp Tyr 1. 5

<210s, SEQ ID NO 41 &211s LENGTH: 19 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 41 Glu Ile Arg Ser Lys Ser Asn. Asn Tyr Ala Thr His Tyr Ala Glu Ser 1. 5 1O 15 Val Lys Gly US 2015/005O270 A1 Feb. 19, 2015 56

- Continued

<210s, SEQ ID NO 42 &211s LENGTH: 10 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 42 Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn 1. 5 1O

<210s, SEQ ID NO 43 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 43 Gln Glin Tyr Tyr Ser Tyr Pro Tyr Thr 1. 5

<210s, SEQ ID NO 44 &211s LENGTH: 17 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 44 Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser Ser Asp Glin Arg Asn Tyr Lieu. 1. 5 1O 15

Ala

<210s, SEQ ID NO 45 &211s LENGTH: 118 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 45 Glu Val Lys Lieu. Glu Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Met Lys Lieu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 2O 25 3O Trp Met Asn Trp Val Arg Glin Ser Pro Glu Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ala Glu Ile Arg Ser Lys Ser Asn. Asn Tyr Ala Thr His Tyr Ala Glu SO 55 6 O Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 7s 8O Val Tyr Lieu. Glin Met Asn. Asn Lieu. Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Ile Gly Glu Asp Tyr Gly Gly Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O

Ser Wall. Thir Wal Ser Ser 115

<210s, SEQ ID NO 46 &211s LENGTH: 113 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 46 Asp Ile Val Met Ser Glin Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1. 5 1O 15 US 2015/005O270 A1 Feb. 19, 2015 57

- Continued

Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O Ser Asp Glin Arg Asn Tyr Lieu Ala Trp Tyr Glin Glin Arg Ser Gly Glin 35 4 O 45 Ser Pro Llys Lieu. Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O Ile Ser Ser Wall Lys Ala Glu Asp Lieu Ala Val Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O Lys

<210s, SEQ ID NO 47 &211s LENGTH: 12 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 47 Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Leu Asp Phe 1. 5 1O

<210 SEQ ID NO 48 &211s LENGTH: 16 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 48 Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Llys Phe Glin 1. 5 1O 15

<210s, SEQ ID NO 49 &211s LENGTH: 10 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 49 Gly Phe Asin Ile Lys Asp Tyr Tyr Met His 1. 5 1O

<210s, SEQ ID NO 50 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 50 Gln Asn Asp His Ser Tyr Pro Leu. Thr 1. 5

<210s, SEQ ID NO 51 &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 51 Gly Ala Ser Thr Arg Glu Ser 1. 5 US 2015/005O270 A1 Feb. 19, 2015 58

- Continued

<210s, SEQ ID NO 52 &211s LENGTH: 17 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 52 Llys Ser Ser Glin Ser Lieu. Lieu. Asn. Ser Gly Asn. Glin Lys Asn Tyr Lieu. 1. 5 1O 15

Ala

<210s, SEQ ID NO 53 &211s LENGTH: 121 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 53 Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Val Arg Ser Gly Ala 1. 5 1O 15 Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn. Ile Lys Asp Tyr 2O 25 3O Tyr Met His Trp Val Lys Glin Arg Pro Glu Gln Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Llys Phe SO 55 6 O Gln Gly Lys Ala Thr Met Thr Ala Asp Thir Ser Ser ASn Thr Val Tyr 65 70 7s 8O Lieu. Glin Phe Ser Ser Lieu. Thir Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Lieu. Asp Phe Trp Gly 1OO 105 11 O Gln Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 54 &211s LENGTH: 113 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 54 Asp Ile Val Met Thr Glin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1. 5 1O 15 Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Lieu. Asn Ser 2O 25 3O Gly Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45

Pro Pro Llys Lieu. Lieu. Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O

Ile Ser Ser Val Glin Ala Glu Asp Lieu Ala Val Tyr Tyr Cys Glin Asn 85 90 95 Asp His Ser Tyr Pro Lieu. Thir Phe Gly Ala Gly. Thir Lys Lieu. Glu Lieu. 1OO 105 11 O US 2015/005O270 A1 Feb. 19, 2015 59

- Continued

<210s, SEQ ID NO 55 &211s LENGTH: 12 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 55 Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Leu Asp Phe 1. 5 1O

<210s, SEQ ID NO 56 &211s LENGTH: 17 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 56 Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Llys Phe Glin 1. 5 1O 15 Gly

<210s, SEQ ID NO 57 &211s LENGTH: 10 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 57 Gly Phe Asin Ile Lys Asp Tyr Tyr Met His 1. 5 1O

<210s, SEQ ID NO 58 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 58 Met Glin Gly Thr His Phe Pro Tyr Thr 1. 5

<210s, SEQ ID NO 59 &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 59 Lieu Val Ser Lys Lieu. Asp Ser 1. 5

<210s, SEQ ID NO 60 &211s LENGTH: 16 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 60 Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser Asn Gly Glu Thir Tyr Lieu. Asn 1. 5 1O 15

<210s, SEQ ID NO 61 &211s LENGTH: 121 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 61 US 2015/005O270 A1 Feb. 19, 2015 60

- Continued

Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Val Arg Ser Gly Ala 1. 5 1O 15 Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn. Ile Lys Asp Tyr 2O 25 3O Tyr Met His Trp Val Lys Glin Arg Pro Glu Gln Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Llys Phe SO 55 6 O Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 7s 8O Lieu. Glin Phe Ser Ser Lieu. Thir Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Lieu. Asp Phe Trp Gly 1OO 105 11 O Gln Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 62 &211s LENGTH: 112 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 62 Asp Val Val Met Thr Glin Thr Pro Leu. Thir Leu Ser Val Pro Ile Gly 1. 5 1O 15 Gln Pro Ala Ser Ile Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O Asn Gly Glu Thir Tyr Lieu. Asn Trp Lieu. Lieu. Glin Arg Pro Gly Glin Ser 35 4 O 45 Pro Lys Arg Lieu. Ile Tyr Lieu Val Ser Lys Lieu. Asp Ser Gly Val Pro SO 55 6 O Asp Arg Phe Thr Gly Ser Arg Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O Ser Arg Val Glu Ser Glu Asp Leu Gly Val Tyr Tyr Cys Met Glin Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys 1OO 105 11 O

<210s, SEQ ID NO 63 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: /replace=Phe" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: /replace= " US 2015/005O270 A1 Feb. 19, 2015 61

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: /replace= Ser" 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: /replace="Pro" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (9) <223> OTHER INFORMATION: /note= Residues given in the sequence have no preference with respect to those in the annotations for said positions' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: /replace= Tyr" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

<4 OOs, SEQUENCE: 63 Trp. Glu Tyr Asp Gly ASn Tyr Tyr Asp Lieu. Asp Phe 1. 5 1O

<210s, SEQ ID NO 64 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: /replace= Val" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: /replace= Ser" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: /replace="Asp" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' US 2015/005O270 A1 Feb. 19, 2015 62

- Continued

SEQUENCE: 64 Trp Ile Asp Pro Glu Asn Gly Asp Thr Gly Tyr Ala Pro Llys Phe Glin 1. 5 1O 15 Gly

SEO ID NO 65 LENGTH: 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE: NAMEAKEY: source OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' FEATURE: NAME/KEY: VARIANT LOCATION: (9) . . (9) OTHER INFORMATION: /replace=Met" FEATURE: NAMEAKEY: misc feature LOCATION: (9) . . (9) OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

SEQUENCE: 65 Gly Phe Asn. Ile Lys Asp Tyr Tyr Lieu. His 1. 5

SEQ ID NO 66 LENGTH: 9 TYPE PRT <213> ORGANISM: Artificial Sequence FEATURE: NAMEAKEY: source OTHER INFORMA CION: /note=Description of Artificial Sequence: Synthetic consensus peptide' FEATURE: NAME/KEY: WAR ANT LOCATION: (2) .. (2) OTHER INFORMA CION: /replace= Asn." FEATURE: NAME/KEY: WAR ANT LOCATION: (3) . . (3) OTHER INFORMA CION: /replace=Phe" or Asp" or His" FEATURE: NAMEAKEY: misc feature LOCATION: (2) .. (3) OTHER INFORMA CION: /note= Residues given in the sequence have no preference with respect to those in the annotations for said positions' FEATURE: NAME/KEY: WAR ANT LOCATION: (4) . . (4) OTHER INFORMA CION: /replace=Phe" or His" or Trip" FEATURE: NAMEAKEY: misc feature LOCATION: (4) . . (4) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' FEATURE: NAME/KEY: WAR ANT LOCATION: (6) . . (6) OTHER INFORMA CION: /replace=Phe" or Thr' or His" FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position' FEATURE: US 2015/005O270 A1 Feb. 19, 2015 63

- Continued NAME/KEY: VARIANT LOCATION: (8) ... (8) OTHER INFORMATION: /replace= Tyr" or Phe" or His" or Lieu" FEATURE: NAMEAKEY: misc feature LOCATION: (8) ... (8) OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position'

SEQUENCE: 66 Gln Glin Tyr Tyr Ser Tyr Pro Trp Thr 1. 5

SEO ID NO 67 LENGTH: 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: NAME/KEY: Solice OTHER INFORMA CION: /note=Description of Artificial Sequence: Synthetic consensus peptide' FEATURE: NAME/KEY: WAR ANT LOCATION: (1) . . (1) OTHER INFORMA CION: /replace=" Gly" FEATURE: NA EAKEY: misc feature LOCATION: (1) . . (1) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position" FEATURE: NAME/KEY: WAR ANT LOCATION: (6) . . (6) OTHER INFORMA CION: /replace="Asp" or Gln" or Asn." FEATURE: NA EAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMA CION: /note= Residue given in the sequence has no preference with respect to those in the annotations for said position'

SEQUENCE: 67 Trp Ala Ser Thr Arg Glu 1. 5

<210s, SEQ ID NO 68 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source 223 OTHER INFORMA CION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221 > NAMEAKEY: WAR ANT <222s. LOCATION: (8) . . (8) 223 OTHER INFORMA CION: /replace=His" or Tyr' or Phe" 22 Os. FEATURE: <221 > NAMEAKEY: WAR ANT <222s. LOCATION: (9) . . (9) 223 OTHER INFORMA CION: /replace=" Gly" 22 Os. FEATURE: <221 > NAMEA KEY: misc feature <222s. LOCATION: (8) . . (9) 223 OTHER INFORMA CION: /note= Residues given in the sequence have no preference with respect to those in the annotations for said positions' 22 Os. FEATURE: <221 > NAMEAKEY: WAR ANT <222s. LOCATION: (11) . . (11) US 2015/005O270 A1 Feb. 19, 2015 64

- Continued <223> OTHER INFORMATION: /replace="Asp" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: /replace= Arg" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: /replace= Tyr" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

<4 OOs, SEQUENCE: 68 Llys Ser Ser Glin Ser Lieu Lleu Trip Ser Ser Asn. Glin Lys Asn His Lieu. 1. 5 1O 15

Ala

<210s, SEQ ID NO 69 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: /replace=Met" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

<4 OOs, SEQUENCE: 69 Lieu. Glin Gly Thr His Phe Pro Tyr Thr 1. 5

<210s, SEQ ID NO 70 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic consensus peptide' 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: /replace=" Glu" 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) US 2015/005O270 A1 Feb. 19, 2015 65

- Continued <223> OTHER INFORMATION: /note= Residue given in the sequence has no preference with respect to that in the annotation for said position'

<4 OO > SEQUENCE: 7 O Llys Ser Ser Glin Ser Lieu. Lieu. Tyr Ser Asn Gly Llys Thr Tyr Lieu. Asn 1. 5 1O 15

<210s, SEQ ID NO 71 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term biotin' <4 OOs, SEQUENCE: 71 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5

<210s, SEQ ID NO 72 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term KLH'

<4 OOs, SEQUENCE: 72 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5

<210s, SEQ ID NO 73 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term KLH'

<4 OO > SEQUENCE: 73 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5

<210s, SEQ ID NO 74 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin' US 2015/005O270 A1 Feb. 19, 2015 66

- Continued

<4 OOs, SEQUENCE: 74 Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 75 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term biotin' <4 OO > SEQUENCE: 75 Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 76 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note=N-term KLH'

<4 OO > SEQUENCE: 76 Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 77 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term KLH'

<4 OO > SEQUENCE: 77 Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 78 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin'

<4 OO > SEQUENCE: 78 Arg Pro Pro Gly Phe Ser Pro Phe 1. 5 US 2015/005O270 A1 Feb. 19, 2015 67

- Continued

<210s, SEQ ID NO 79 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term biotin' <4 OO > SEQUENCE: 79 Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 8O &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term KLH'

<4 OOs, SEQUENCE: 80 Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 81 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term KLH'

<4 OOs, SEQUENCE: 81 Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 82 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin'

<4 OOs, SEQUENCE: 82 Lys Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 83 &211s LENGTH: 9 US 2015/005O270 A1 Feb. 19, 2015 68

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term biotin' <4 OOs, SEQUENCE: 83 Lys Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 84 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term KLH'

<4 OOs, SEQUENCE: 84 Lys Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 85 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term KLH' <4 OOs, SEQUENCE: 85 Lys Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 86 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin'

<4 OOs, SEQUENCE: 86 Arg Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 87 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source US 2015/005O270 A1 Feb. 19, 2015 69

- Continued <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term biotin' <4 OO > SEQUENCE: 87 Arg Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 88 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term KLH'

<4 OOs, SEQUENCE: 88 Arg Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 89 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term KLH' <4 OOs, SEQUENCE: 89 Arg Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5 1O

<210s, SEQ ID NO 90 &211s LENGTH: 9 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 90 Arg Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 91 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin'

<4 OOs, SEQUENCE: 91 Arg Arg Pro Pro Gly Phe Ser Pro Phe 1. 5 US 2015/005O270 A1 Feb. 19, 2015 70

- Continued

<210s, SEQ ID NO 92 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term biotin'

<4 OOs, SEQUENCE: 92 Arg Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 93 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term KLH'

<4 OOs, SEQUENCE: 93 Arg Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 94 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note= C-term KLH' <4 OOs, SEQUENCE: 94 Arg Arg Pro Pro Gly Phe Ser Pro Phe 1. 5

<210s, SEQ ID NO 95 &211s LENGTH: 5 212. TYPE: PRT <213s ORGANISM: Unknown 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Unknown : Bradykinin 1-5 peptide"

<4 OO > SEQUENCE: 95 Arg Pro Pro Gly Phe 1. 5

<210s, SEQ ID NO 96 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source US 2015/005O270 A1 Feb. 19, 2015 71

- Continued <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin' <4 OOs, SEQUENCE: 96 Arg Pro Pro Gly Phe 1. 5

<210s, SEQ ID NO 97 &211s LENGTH: 136 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 97 Lieu Pro Glu Phe Glin Val Llys Lieu. Glu Glu Ser Gly Ala Glu Lieu Val 1. 5 1O 15 Arg Ser Gly Ala Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn 2O 25 3O Ile Lys Asp Tyr Tyr Lieu. His Trp Val Lys Glin Arg Pro Glu Glin Gly 35 4 O 45 Lieu. Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Gly Tyr SO 55 6 O Ala Arg Llys Phe Glin Gly Lys Ala Thr Met Thr Ala Asp Thir Ser Ser 65 70 7s 8O Asn Thr Val Tyr Lieu. His Leu Ser Ser Lieu. Thir Ser Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Phe Asn Ala Trp Glu Tyr Asp Gly Tyr Tyr Asp Lieu. Asp 1OO 105 11 O Tyr Trp Gly Glin Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thir Thr 115 12 O 125 Pro Pro Ser Val Tyr Gly Ser Ser 13 O 135

<210s, SEQ ID NO 98 &211s LENGTH: 134 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 98 Lieu Pro Glu Phe Glin Val Glin Lieu. Glu Glu Ser Gly Gly Gly Lieu Val 1. 5 1O 15 Gln Pro Gly Gly Ser Met Lys Lieu Ser Cys Val Ala Ser Gly Phe Thr 2O 25 3O Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Glin Ser Pro Glu Lys Gly 35 4 O 45

Lieu. Glu Trp Val Ala Glu Ile Arg Ser Lys Ser Asn. Asn Tyr Ala Thr SO 55 6 O His Tyr Ala Glu Ser Wall Lys Gly Arg Phe Thir Ile Ser Arg Asp Asp 65 70 7s 8O

Ser Lys Ser Ser Val Tyr Lieu Gln Met Asn. Asn Lieu. Arg Ala Glu Asp 85 90 95 Thr Gly Ile Tyr Tyr Cys Ile Gly Glu Asp Tyr Gly Gly Asp Tyr Trp 1OO 105 11 O

Gly Glin Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro US 2015/005O270 A1 Feb. 19, 2015 72

- Continued

115 12 O 125 Ser Val Tyr Gly Ser Ser 13 O

<210s, SEQ ID NO 99 &211s LENGTH: 136 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 99 Lieu Pro Glu Phe Glu Val Glin Leu Glu Glu Ser Gly Pro Glu Lieu Val 1. 5 1O 15 Llys Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser 2O 25 3O Phe Thr Asp Tyr Asn Ile Tyr Trp Val Lys Glin Ser His Gly Lys Ser 35 4 O 45 Lieu. Glu Trp Ile Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr SO 55 6 O Asn Glin Llys Phe Arg Gly Lys Ala Thir Lieu. Thr Val Asp Llys Ser Ser 65 70 7s 8O Ser Thr Ala Phe Met His Leu Ser Ser Lieu. Thir Ser Asp Asp Ser Ala 85 90 95 Val Tyr Tyr Cys Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp 1OO 105 11 O Tyr Trp Gly Glin Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thir Thr 115 12 O 125 Pro Pro Ser Val Tyr Gly Ser Ser 13 O 135

<210s, SEQ ID NO 100 &211s LENGTH: 137 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 1.OO Lieu Pro Glu Phe Glu Val Llys Lieu. Glin Glu Ser Gly Ala Glu Lieu Val 1. 5 1O 15 Arg Ser Gly Ala Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn 2O 25 3O Ile Lys Asp Tyr Tyr Met His Trp Val Lys Glin Arg Pro Glu Gln Gly 35 4 O 45 Lieu. Glu Trp Ile Gly Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr SO 55 6 O Ala Pro Llys Phe Glin Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser 65 70 7s 8O

Asn Thr Val Tyr Lieu. Glin Phe Ser Ser Lieu. Thir Ser Glu Asp Thr Ala 85 90 95

Val Tyr Tyr Cys Asn Ala Phe Glu Tyr Asp Gly Asn Tyr Ser Ser Leu 1OO 105 11 O

Asp Phe Trp Gly Glin Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thr 115 12 O 125

Thr Pro Pro Ser Val Tyr Gly Ser Ser 13 O 135 US 2015/005O270 A1 Feb. 19, 2015 73

- Continued

<210s, SEQ ID NO 101 &211s LENGTH: 137 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 101 Lieu Pro Glu Phe Glu Val Llys Lieu. Glu Glin Ser Gly Ala Glu Lieu Val 1. 5 1O 15 Arg Ser Gly Ala Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn 2O 25 3O Ile Lys Asp Tyr Tyr Met His Trp Val Lys Glin Arg Pro Glu Gln Gly 35 4 O 45 Lieu. Glu Trp Ile Gly Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr SO 55 6 O Ala Pro Llys Phe Glin Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser 65 70 7s 8O Asn Thr Val Tyr Lieu. Glin Phe Ser Ser Lieu. Thir Ser Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Asn Ala Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Leu 1OO 105 11 O Asp Phe Trp Gly Glin Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thr 115 12 O 125 Thr Pro Pro Ser Val Tyr Gly Ser Ser 13 O 135

<210s, SEQ ID NO 102 &211s LENGTH: 133 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 102 Glu Lieu. Asp Ile Val Met Thr Glin Thr Thr Lieu. Thir Lieu Ser Val Thr 1. 5 1O 15 Ile Gly Glin Pro Ala Ser Ile Ser Cys Llys Ser Ser Glin Ser Lieu. Lieu. 2O 25 3O Tyr Ser Asn Gly Llys Thr Tyr Lieu. Asn Trp Lieu. Lieu. Glin Arg Pro Gly 35 4 O 45 Glin Ser Pro Lys Arg Lieu. Ile Tyr Lieu Val Ser Llys Lieu. Asp Ser Gly SO 55 6 O Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. 65 70 7s 8O Lys Ile Ile Arg Val Glu Ala Glu Asp Lieu. Gly Val Tyr Tyr Cys Lieu. 85 90 95 Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu 1OO 105 11 O

Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 115 12 O 125

Llys Lieu. Glu Lieu. Tyr 13 O

<210s, SEQ ID NO 103 &211s LENGTH: 134 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 103 US 2015/005O270 A1 Feb. 19, 2015 74

- Continued

Glu Lieu. Asp Ile Val Lell Thir Glin Ser Pro Ser Ser Lell Ala Wall Ser 1O 15

Val Gly Glu, Llys Val Thir Met Ser Cys Lys Ser Ser Glin Ser Luell Luell 2O 25

Tyr Ser Ser Asp Gln Arg Asn Tyr Luell Ala Trp Glin Glin Arg Ser 35 4 O 45

Gly Glin Ser Pro Llys Lell Lell Ile Trp Ala Ser Thir Arg Glu Ser SO 55 6 O

Gly Val Pro Asp Arg Phe Thir Gly Ser Gly Ser Gly Thir Asp Phe Thir 65 70

Lieu. Thir Ile Ser Ser Wall Ala Glu Asp Luell Ala Wall Tyr 85 90 95

Gln Glin Tyr Tyr Ser Pro Thir Phe Gly Gly Gly Thir Luell 1OO 105 11 O

Glu Ile Lys Arg Ala Ala Ala Pro Thir Wall Ser Ile Phe Pro Pro 115 12 O 125 Ser Lys Lieu. Glu Lieu 13 O

<210s, SEQ ID NO 104 &211s LENGTH: 134 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 104

Glu Lieu. Asp Ile Val Met Thir Glin Thir Pro Ser Ser Lell Ala Wall Ser 1. 5 1O 15

Val Gly Glu, Llys Val Thir Met Ser Cys Lys Ser Ser Glin Ser Luell Luell 2O 25

Tyr Thr Ser Asn Glin Asn Tyr Luell Ala Trp Glin Glin Pro 35 4 O 45

Gly Glin Ser Pro Llys Pro Lell Ile Trp Ala Ser Thir Arg Glu Ser SO 55 6 O

Gly Val Pro Asp Arg Phe Thir Gly Ser Gly Ser Gly Thir Asp Phe Thir 65 70

Lieu. Thir Ile Ser Ser Wall Ala Glu Asp Luell Ala Ile Tyr 85 90 95

Gln Glin Tyr Tyr Ser Pro Trp Thir Phe Gly Gly Gly Thir Luell 1OO 105 11 O

Glu Ile Lys Arg Ala Ala Ala Pro Thir Wall Ser Ile Phe Pro Pro 115 12 O 125 Ser Lys Lieu. Glu Lieu 13 O

<210s, SEQ ID NO 105 &211s LENGTH: 133 212. TYPE: PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 105

Glu Lieu. Asp Ile Val Ile Thr Glin. Thir Thir Lieu. Ser Leu Ser Wall Pro 1. 5 15

Ile Gly Glin Pro Ala Ser Ile Ser Cys Llys Ser Arg Glin Ser Lieu. Lieu. 2O 25 US 2015/005O270 A1 Feb. 19, 2015 75

- Continued

Tyr Ser Asn Gly Glu Thir Tyr Lieu. Asn Trp Lieu. Lieu. Glin Arg Pro Gly 35 4 O 45 Glin Ser Pro Lys Arg Lieu. Ile Tyr Lieu Val Ser Llys Lieu. Asp Ser Gly SO 55 6 O Val Pro Asp Arg Phe Thr Gly Ser Arg Ser Gly Thr Asp Phe Thr Lieu. 65 70 7s 8O Lys Ile Ser Arg Val Glu Ser Glu Asp Lieu. Gly Val Tyr Tyr Cys Met 85 90 95 Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu 1OO 105 11 O Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 115 12 O 125 Llys Lieu. Glu Lieu. Tyr 13 O

<210s, SEQ ID NO 106 &211s LENGTH: 133 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 106 Glu Lieu. Asp Ile Val Ile Thr Glin Ser Thr Lieu. Thir Lieu Ser Val Pro 1. 5 1O 15 Ile Gly Gln Pro Ala Ser Ile Ser Cys Llys Ser Ser Glin Ser Lieu Lieu 2O 25 3O Tyr Ser Asn Gly Glu Thir Tyr Lieu. Asn Trp Lieu. Lieu. Glin Arg Pro Gly 35 4 O 45 Glin Ser Pro Lys Arg Glin Ile Tyr Lieu Val Ser Llys Lieu. Asp Ser Gly SO 55 6 O Val Pro Asp Arg Phe Thr Gly Ser Arg Ser Gly Thr Asp Phe Thr Lieu. 65 70 7s 8O Lys Ile Ser Arg Val Glu Ser Glu Asp Lieu. Gly Val Tyr Tyr Cys Met 85 90 95 Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu 1OO 105 11 O Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 115 12 O 125 Llys Lieu. Glu Lieu. Tyr 13 O

<210s, SEQ ID NO 107 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic primer"

<4 OOs, SEQUENCE: 107 aaaagcaggc titaggagcgg cc.gc.catggc gtc.cc aggcc ticgctg 46

<210s, SEQ ID NO 108 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence

US 2015/005O270 A1 Feb. 19, 2015 77

- Continued

Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Ser Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O Ile Ser Ser Wall Lys Ala Glu Asp Lieu Ala Ile Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. Glu 1OO 105 11 O

<210s, SEQ ID NO 112 &211s LENGTH: 105 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 112 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Ile Ser Ser Tyr 2O 25 3O Lieu. Asn Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O Glu Asp Leu Ala Thr Tyr Tyr Cys Glin Glin Ser Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Glin Gly Thr Lys Val Glu 1OO 105

<210s, SEQ ID NO 113 &211s LENGTH: 120 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 113 Glu Ile Gln Leu Gln Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Thr 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Lys Glin Ser His Gly Lys Ser Lieu. Glu Trp Ile 35 4 O 45

Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O

Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Phe 65 70 7s 8O Met His Leu Ser Ser Lieu. Thir Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O US 2015/005O270 A1 Feb. 19, 2015 78

- Continued

Gly. Thir Ser Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 114 &211s LENGTH: 123 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 114 Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 2O 25 3O Tyr Met His Trp Val Arg Glin Ala Pro Gly Glin Gly Lieu. Glu Trp Met 35 4 O 45 Gly Trp Ile Asin Pro Asn Ser Gly Gly Thr Asn Tyr Ala Glin Llys Phe SO 55 6 O Gln Gly Arg Val Thr Met Thr Arg Asp Llys Ser Ser Ser Thr Ala Tyr 65 70 7s 8O Met Glu Lieu Ser Ser Lieu. Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Tyr Asp Tyr Asp Val Phe Tyr Tyr Ala Met Asp Tyr 1OO 105 11 O Trp Gly Glin Gly Thr Lieu Val Thr Val Ser Ser 115 120

<210s, SEQ ID NO 115 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 115 Asp Ile Val Met Thr Glin Ser Pro Asp Ser Lieu Ala Val Ser Lieu. Gly 1. 5 1O 15 Glu Arg Ala Thir Ile Asn. Cys Llys Ser Ser Glin Ser Val Lieu. Tyr Ser 2O 25 3O Ser Asn. Asn Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Pro Pro Llys Lieu. Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O

Ile Ser Ser Lieu. Glin Ala Glu Asp Val Ala Val Tyr Tyr Cys Glin Glin 85 90 95

Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Glin Gly Thr Lys Val Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 116 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2015/005O270 A1 Feb. 19, 2015 79

- Continued <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic polypeptide'

<4 OOs, SEQUENCE: 116 Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 2O 25 3O Gly Ile Ser Trp Val Arg Glin Ala Pro Gly Glin Gly Lieu. Glu Trp Met 35 4 O 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn. Thir Asn Tyr Ala Glin Llys Lieu. SO 55 6 O Gln Gly Arg Val Thr Met Thir Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 7s 8O Met Glu Lieu. Arg Ser Lieu. Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Ser Ser Ser Ser Asp Ala Phe Asp Ile Trp Gly Glin 1OO 105 11 O Gly Thr Met Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 117 &211s LENGTH: 9 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=Description of Artificial Sequence: Synthetic peptide" 22 Os. FEATURE: <221 > NAMEAKEY: source <223> OTHER INFORMATION: /note=N-term biotin'

<4 OOs, SEQUENCE: 117 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1. 5

<210s, SEQ ID NO 118 &211s LENGTH: 127 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 118 Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Val Arg Ser Gly Ala 1. 5 1O 15 Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn. Ile Lys Asp Tyr 2O 25 3O Tyr Lieu. His Trp Wall Lys Glin Arg Pro Glu Glin Gly Lieu. Glu Trp Ile 35 4 O 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Gly Tyr Ala Arg Llys Phe SO 55 6 O

Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 7s 8O

Lieu. His Leu Ser Ser Lieu. Thir Ser Glu Asp Thr Ala Val Tyr Tyr Phe 85 90 95

Asn Ala Trp Glu Tyr Asp Gly Tyr Tyr Asp Lieu. Asp Tyr Trp Gly Glin 1OO 105 11 O US 2015/005O270 A1 Feb. 19, 2015 80

- Continued

Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thir Thr Pro Pro Ser 115 12 O 125

<210s, SEQ ID NO 119 &211s LENGTH: 125 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 119 Glu Val Lys Lieu. Glu Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Met Lys Lieu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 2O 25 3O Trp Met Asn Trp Val Arg Glin Ser Pro Glu Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ala Glu Ile Arg Ser Lys Ser Asn. Asn Tyr Ala Thr His Tyr Ala Glu SO 55 6 O Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 7s 8O Val Tyr Lieu. Glin Met Asn. Asn Lieu. Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Ile Gly Glu Asp Tyr Gly Gly Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O Ser Val Thr Val Ser Ser Ala Lys Thir Thr Pro Pro Ser 115 12 O 125

<210s, SEQ ID NO 120 &211s LENGTH: 127 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 120 Glu Ile Gln Leu Gln Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Thr 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 2O 25 3O Asn. Ile Tyr Trp Val Lys Glin Ser His Gly Lys Ser Lieu. Glu Trp Ile 35 4 O 45 Gly Tyr Phe Asp Pro Tyr Asn Gly Asn Thr Gly Tyr Asn Glin Llys Phe SO 55 6 O Arg Gly Lys Ala Thr Lieu. Thr Val Asp Llys Ser Ser Ser Thr Ala Phe 65 70 7s 8O Met His Leu Ser Ser Lieu. Thir Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Arg Tyr Asp Asp His Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O

Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thir Thr Pro Pro Ser 115 12 O 125

<210s, SEQ ID NO 121 &211s LENGTH: 128 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 121 US 2015/005O270 A1 Feb. 19, 2015 81

- Continued

Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Val Arg Ser Gly Ala 1. 5 1O 15 Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn. Ile Lys Asp Tyr 2O 25 3O Tyr Met His Trp Val Lys Glin Arg Pro Glu Gln Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Llys Phe SO 55 6 O Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 7s 8O Lieu. Glin Phe Ser Ser Lieu. Thir Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Lieu. Asp Phe Trp Gly 1OO 105 11 O Gln Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 115 12 O 125

<210s, SEQ ID NO 122 &211s LENGTH: 128 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 122 Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Val Arg Ser Gly Ala 1. 5 1O 15 Ser Val Llys Lieu. Ser Cys Thr Ala Ser Gly Phe Asn. Ile Lys Asp Tyr 2O 25 3O Tyr Met His Trp Val Lys Glin Arg Pro Glu Gln Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Trp Val Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala Pro Llys Phe SO 55 6 O Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 7s 8O Lieu. Glin Phe Ser Ser Lieu. Thir Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Phe Glu Tyr Asp Gly Asn Tyr Ser Pro Lieu. Asp Phe Trp Gly 1OO 105 11 O Gln Gly. Thir Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 115 12 O 125

<210s, SEQ ID NO 123 &211s LENGTH: 119 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 123 Asp Val Val Met Thr Glin Thr Pro Leu. Thir Leu Ser Val Thir Ile Gly 1. 5 1O 15

Gln Pro Ala Ser Ile Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O

Asn Gly Lys Thr Tyr Lieu. Asn Trp Lieu. Lieu. Glin Arg Pro Gly Glin Ser 35 4 O 45

Pro Lys Arg Lieu. Ile Tyr Lieu Val Ser Lys Lieu. Asp Ser Gly Val Pro SO 55 6 O US 2015/005O270 A1 Feb. 19, 2015 82

- Continued Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O Ile Arg Val Glu Ala Glu Asp Lieu. Gly Val Tyr Tyr Cys Lieu. Glin Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys 1OO 105 11 O Arg Ala Asp Ala Ala Pro Thr 115

<210s, SEQ ID NO 124 &211s LENGTH: 120 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 124 Asp Ile Val Met Ser Glin Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1. 5 1O 15 Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O Ser Asp Glin Arg Asn Tyr Lieu Ala Trp Tyr Glin Glin Arg Ser Gly Glin 35 4 O 45 Ser Pro Llys Lieu. Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 75 8O Ile Ser Ser Wall Lys Ala Glu Asp Lieu Ala Val Tyr Tyr Cys Glin Glin 85 90 95 Tyr Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O Lys Arg Ala Asp Ala Ala Pro Thr 115 12 O

<210s, SEQ ID NO 125 &211s LENGTH: 120 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 125 Asp Ile Val Met Ser Glin Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1. 5 1O 15 Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Leu Tyr Ser 2O 25 3O Ser Asn Gln Lys Asn Tyr Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin 35 4 O 45 Ser Pro Llys Pro Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val SO 55 6 O

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thr 65 70 7s 8O

Ile Ser Ser Wall Lys Ala Glu Asp Lieu Ala Ile Tyr Tyr Cys Glin Glin 85 90 95

Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O

Lys Arg Ala Asp Ala Ala Pro Thr 115 12 O