FGFR3 antibodies and methods of use

Information

  • Patent Grant
  • 11505611
  • Patent Number
    11,505,611
  • Date Filed
    Friday, August 20, 2021
    2 years ago
  • Date Issued
    Tuesday, November 22, 2022
    a year ago
Abstract
Anti-FGFR3 antigen-binding proteins and antigen-binding binding fragments thereof are provided. Methods of inhibiting FGFR3 activity and methods of treating FGFR3-mediated diseases and disorders are also provided.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 20, 2021, is named 720722_SA9-231_ST25.txt and is 545,959 bytes in size.


FIELD OF THE INVENTION

This disclosure relates to compositions of anti-fibroblast growth factor receptor 3 (anti-FGFR3) antigen-binding proteins or antigen-binding fragments thereof, such as antibodies and fragments thereof, and methods of using the same.


BACKGROUND

Fibroblast growth factor receptor 3 (FGFR3) is a protein involved, in part, in the negative regulation of bone development, being highly expressed in growth plate chondrocytes (Sarabipour et al. Biochim Biophys Acta. 1858(7 Pt A): 1436-1442. 2016). FGFR3 is a single-pass membrane receptor tyrosine kinase with 3 Ig-like domains (D1-D3). Binding of FGFR3 to an FGFR3-ligand, such as FGF18, triggers ligand-dependent receptor dimerization which leads to tyrosine kinase activation and downstream signal transduction. This signaling cascade regulates, among other things, chondrocyte proliferation and differentiation.


FGFR3 is a member of the fibroblast growth factor receptor family, which also includes FGFR1, FGFR2, and FGFR4. Each member of the receptor family is a single-pass membrane receptor tyrosine kinase and shares the feature of 3 Ig-like domains. Moreover, each member of the receptor family possesses a high degree of homology with the other members. Strategies to develop FGFR3-specific inhibitors have proven challenging for this reason. Nonetheless, it is important to develop FGFR3-specific inhibitors that do not cross-react with other fibroblast growth factor receptor family members to avoid unwanted side effects in the treatment of an FGFR3-mediate disease or disorder.


Accordingly, there is a need in the art to identify antigen-binding proteins or antigen-binding fragments thereof, that achieve effective inhibition of FGFR3 activity. Such antigen-binding proteins or antigen-binding fragments thereof may be useful in the treatment of FGFR3-mediated diseases and disorders.


SUMMARY

Disclosed herein are anti-FGFR3 antigen-binding proteins or antigen-binding fragments thereof, such as antibodies and antigen-binding fragments thereof. The antigen-binding proteins or antigen-binding fragments thereof, such as anti-FGFR3 antibodies and antigen-binding fragments thereof of the disclosure are suitable for treating FGFR3-mediated diseases and disorders.


In one aspect, the disclosure provides an antigen-binding protein or antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 3 (FGFR3), comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein: (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GX1TFTDX2E (SEQ ID NO: 157), wherein X1 comprises or consists of Y or D and X2 comprises or consists of F or Y; a CDR-H2 sequence comprising the amino acid sequence of IDPETGX3T (SEQ ID NO: 158), wherein X3 comprises or consists of G or S; or CDR-H2 sequence comprising the amino acid sequence of INPNNGX4T (SEQ ID NO: 159), wherein X4 comprises or consists of G or V; or CDR-H2 sequence comprising the amino acid sequence of VX5PETGGT (SEQ ID NO: 160), wherein X5 comprises or consists of D or E; a CDR-H3 sequence comprising the amino acid sequence of TRX6YX7GYX8X9X10X11DY (SEQ ID NO: 161), wherein X6 comprises or consists of T or N, X7 comprises D or E, X8 comprises or consists of S or P, X9 comprises or consists of Q, R, or Y, X10 comprises or consists of T or A, X11 comprises or consists of F or M; and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QS X12LYS X13N X14KNY (SEQ ID NO: 162), wherein X12 comprises or consists of L or V, X13 comprises or consists of N, D, or S, and X14 comprises or consists of Q or N; a CDR-L2 sequence comprising the amino acid sequence of X15AS (SEQ ID NO: 163), wherein X15 comprises or consists of W, Y, or F; a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75), LQYDNLLWT (SEQ ID NO: 81), or HQYLSX16YT (SEQ ID NO: 290) wherein X16 comprises or consists of P or S; (b) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGAMDY (SEQ ID NO: 78); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87); (c) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGSMDF (SEQ ID NO: 84); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87); (d) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTVTDYY (SEQ ID NO: 88); a CDR-H2 sequence comprising the amino acid sequence of INPNNGVT (SEQ ID NO: 89); a CDR-H3 sequence comprising the amino acid sequence of AREEDFDGFDY (SEQ ID NO: 90); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVSTG (SEQ ID NO: 91); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98 and 104); a CDR-L3 sequence comprising the amino acid sequence of QQHYSTPLT (SEQ ID NO: 93); (e) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFSDFE (SEQ ID NO: 94); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTX17DY (SEQ ID NO: 308), wherein X17 comprises M or F; and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSX18NQKNY (SEQ ID NO: 309), wherein X18 comprises S or D; a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98 and 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105); or (f) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFTDFE (SEQ ID NO: 100); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTX17DY (SEQ ID NO: 308), wherein X17 comprises M or F; and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSX18NQKNY (SEQ ID NO: 309), wherein X18 comprises S or D; a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98 and 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105).


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), GDTFTDYE (SEQ ID NO: 295), or GYTFTDFE (SEQ ID NO: 296); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101), VDPETGGT (SEQ ID NO: 297), IDPETGST (SEQ ID NO: 298), or VEPETGGT (SEQ ID NO: 299); a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAMDY (SEQ ID NO: 72), TRTYEGYPYAMDY (SEQ ID NO: 300), or TRTYDGYPYAFDY (SEQ ID NO: 301); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLLYSNNQKNY (SEQ ID NO: 73), QSVLYSNNNKNY (SEQ ID NO: 302), or QSVLYSDNQKNY (SEQ ID NO: 306); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104), YAS (SEQ ID NO: 303), or FAS (SEQ ID NO: 304); a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75); (b) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGAMDY (SEQ ID NO: 78); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87); (c) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGSMDF (SEQ ID NO: 84); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87); (d) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTVTDYY (SEQ ID NO: 88); a CDR-H2 sequence comprising the amino acid sequence of INPNNGVT (SEQ ID NO: 89); a CDR-H3 sequence comprising the amino acid sequence of AREEDFDGFDY (SEQ ID NO: 90); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVSTG (SEQ ID NO: 91); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of QQHYSTPLT (SEQ ID NO: 93); (e) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFSDFE (SEQ ID NO: 94); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTMDY (SEQ ID NO: 96) or TRNYDGYSQTFDY (SEQ ID NO: 305); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSSNQKNY(SEQ ID NOs: 97 and 103) or QSVLYSDNQKNY (SEQ ID NO: 306); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105); or (f) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFTDFE (SEQ ID NO: 100); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSRTMDY (SEQ ID NO: 102) or TRNYDGYSRTFDY (SEQ ID NO: 307); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSSNQKNY(SEQ ID NOs: 97 and 103) or QSVLYSDNQKNY (SEQ ID NO: 306); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105).


In certain embodiments, (a) the VH domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 10, SEQ ID NO: 110, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 57, and SEQ ID NO: 58; and the VL domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 60, and SEQ ID NO: 61; (b) the VH domain comprises the amino acid sequence of SEQ ID NO: 8; and the VL domain comprises the amino acid sequence of SEQ ID NO: 9; (c) the VH domain comprises the amino acid sequence of SEQ ID NO: 10; and the VL domain comprises the amino acid sequence of SEQ ID NO: 11; (d) the VH domain comprises the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises the amino acid sequence of SEQ ID NO: 13; (e) the VH domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; and the VL domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; (f) the VH domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; the VL domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.


In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 63 or 65, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67 or 69.


In certain embodiments, the VH domain is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and wherein the VL domain is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, or SEQ ID NO: 132.


In certain embodiments, the antigen binding protein or fragment thereof of comprises an antibody heavy chain at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 63 or 65, and an antibody light chain at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 67 or 69.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NO: 71), and a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAMDY (SEQ ID NO: 72); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLLYSNNQKNY (SEQ ID NO: 73), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NO: 74), and a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75). In some embodiments thereof, the VH domain comprises the amino acid sequence of SEQ ID NO: 6, and the VL domain comprises the amino acid sequence of SEQ ID NO: 7.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 76), a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NO: 77), and a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGAMDY (SEQ ID NO: 78); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NO: 79), a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NO: 80), and a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NO: 81).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 8, and the VL domain comprises the amino acid sequence of SEQ ID NO: 9.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 82), a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NO: 83), and a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGSMDF (SEQ ID NO: 84); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NO: 85), a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NO: 86), and a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NO: 87).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 10, and the VL domain comprises the amino acid sequence of SEQ ID NO: 11.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTVTDYY (SEQ ID NO: 88), a CDR-H2 sequence comprising the amino acid sequence of INPNNGVT (SEQ ID NO: 89), and a CDR-H3 sequence comprising the amino acid sequence of AREEDFDGFDY (SEQ ID NO: 90); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVSTG (SEQ ID NO: 91), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NO: 92), and a CDR-L3 sequence comprising the amino acid sequence of QQHYSTPLT (SEQ ID NO: 93).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 12, and the VL domain comprises the amino acid sequence of SEQ ID NO: 13.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFSDFE (SEQ ID NO: 94), a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NO: 95), and a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTMDY (SEQ ID NO: 96); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSSNQKNY (SEQ ID NO: 97), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NO: 98), and a CDR-L3 sequence comprising the amino acid sequence of HQYLSSYT (SEQ ID NO: 99).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 14, and the VL domain comprises the amino acid sequence of SEQ ID NO: 15.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFTDFE (SEQ ID NO: 100), a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NO: 101), and a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSRTMDY (SEQ ID NO: 102); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSSNQKNY (SEQ ID NO: 103), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NO: 104), and a CDR-L3 sequence comprising the amino acid sequence of HQYLSSYT (SEQ ID NO: 105).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 17.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), a CDR-H2 sequence comprising the amino acid sequence of VDPETGGT (SEQ ID NO: 297), and a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAFDY (SEQ ID NO: 301); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSNNNKNY (SEQ ID NO: 302), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, 104), and a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 57, and the VL domain comprises the amino acid sequence of SEQ ID NO: 19 or 59.


In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 63, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), a CDR-H2 sequence comprising the amino acid sequence of VDPETGGT (SEQ ID NO: 297), and a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAFDY (SEQ ID NO: 301); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSDNQKNY (SEQ ID NO: 306), a CDR-L2 sequence comprising the amino acid sequence of FAS (SEQ ID NO: 304), and a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 57, and the VL domain comprises the amino acid sequence of SEQ ID NO: 61.


In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 65, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds a human FGFR3 polypeptide comprising the amino acid sequence of SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or antigen-binding fragment thereof binds a human FGFR3 polypeptide comprising the amino acid sequence of SEQ ID NO: 134.


In certain embodiments, the antigen binding protein or antigen-binding fragment thereof binds a region of human FGFR3 polypeptide comprising the amino acids D143 through L163 of SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or antigen-binding fragment thereof binds a region of human FGFR3 polypeptide comprising the amino acids D143 through N170 of SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or antigen-binding fragment thereof binds a region of human FGFR3 polypeptide comprising the amino acids D143 through D160 and G197 through L213 of SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is a chimeric or humanized antibody or antigen-binding binding fragment thereof.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is a human antibody or antigen-binding binding fragment thereof.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is a monoclonal antibody or antigen-binding binding fragment thereof.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises one or more full-length antibody heavy chains comprising an Fc region.


In certain embodiments, the Fc region is a human IgG1 Fc region.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises an antibody F(ab), F(ab′)2, Fab′-SH, Fv, or scFv fragment.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises an antibody F(ab) fragment.


In certain embodiments, the antibody F(ab) fragment comprises SEQ ID NO: 56 and the first about 100 amino acids of SEQ ID NO: 54.


In certain embodiments, the antibody F(ab) fragment comprises SEQ ID NO: 57 and the first about 100 amino acids of SEQ ID NO: 54.


In certain embodiments, the antibody F(ab) fragment comprises SEQ ID NO: 58 and the first about 100 amino acids of SEQ ID NO: 54.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 141 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 142.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 143 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 144.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 145 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 146.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 147 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 148.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 149 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 150.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 152.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises cross-reactivity to mouse and cynomolgus FGFR3.


In certain embodiments, the antigen binding or antigen-binding binding fragment thereof protein does not bind to one or more of FGFR1, FGFR2, and FGFR4.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof does not bind to each of FGFR1, FGFR2, and FGFR4.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds to each of FGFR1, FGFR2, and FGFR4 with an affinity of 100 μM or greater.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds human FGFR3 with an equilibrium dissociation constant (KD) of 10 nM or less.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds human FGFR3 with an off rate (Kd) of 10−4 or greater.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof inhibits ligand-induced FGFR3 dimerization with IC50 of 5 μg/ml or less.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof inhibits FGFR3 receptor activation and downstream signaling with IC50 of 5 μg/ml or less.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof inhibits the activity of an FGFR3G380R mutant.


In certain embodiments, the antigen binding protein or antigen-binding fragment thereof is capable of penetrating a bone growth plate.


In certain embodiments, the antigen binding protein or antigen-binding fragment thereof is capable of decreasing binding of FGFR3 and its ligand in a bone growth plate.


In another aspect, the disclosure provides a pharmaceutical composition comprising the antigen binding protein or antigen-binding fragment thereof recited above, and a pharmaceutically acceptable carrier.


In another aspect, the disclosure provides an isolated nucleic acid molecule encoding the antigen binding protein or antigen-binding fragment thereof recited above.


In another aspect, the disclosure provides an expression vector comprising the nucleic acid molecule recited above.


In another aspect, the disclosure provides a host cell comprising the expression vector recited above.


In another aspect, the disclosure provides a method for treating a FGFR3-mediated disease or disorder in a subject, comprising administering to a subject in need thereof the antigen binding protein or antigen-binding fragment thereof recited above.


In certain embodiments, the FGFR3-mediated disease or disorder is achondroplasia.


In certain embodiments, the achondroplasia is FGFR3G380R+ achondroplasia.


In certain embodiments, the subject suffering from achondroplasia comprises one or more symptoms selected from the group consisting of shortened proximal limbs, brachydactyly, large head with prominent forehead frontal bossing, small midface with a flattened nasal bridge, spinal kyphosis, spinal lordosis, varus, valgus, ear infections, sleep apnea, and hydrocephalus.


In certain embodiments, the FGFR3-mediated disease or disorder is cancer.


In certain embodiments, the cancer is bladder cancer melanoma, urothelial cancer, and endometrial cancer.


In one aspect, the disclosure provides a method for treating achondroplasia in a subject, comprising administering to a subject in need thereof an antigen-binding protein fragment that specifically binds to FGFR3, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4.


In one aspect, the disclosure provides a method for inhibiting one or both of FGFR3 activity and expression in a bone growth plate of a subject, comprising administering to a subject an antigen-binding protein fragment that specifically binds to FGFR3, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4.


In one aspect, the disclosure provides an antigen-binding protein or antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 3 (FGFR3), comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the antigen binding protein binds a human FGFR3 polypeptide comprising the amino acid sequence of SEQ ID NO: 134.


In certain embodiments, the antigen binding protein binds a region of human FGFR3 polypeptide comprising the amino acids D143 through L163 of SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds a region of human FGFR3 polypeptide comprising the amino acids D143 through N170 of SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds a region of human FGFR3 polypeptide comprising the amino acids D143 through D160 and G197 through L213 of SEQ ID NO: 133.


In one aspect, the disclosure provides an antigen-binding protein or antigen-binding fragment thereof with binding specificity to a fibroblast growth factor receptor 3 (FGFR3) epitope, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the antigen binding protein or antigen-binding binding fragment thereof competes for binding to FGFR3 with an antibody comprising VH/VL domain amino acid sequence pairs selected from the group consisting of: SEQ ID NO: 6/SEQ ID NO: 7, SEQ ID NO: 8/SEQ ID NO: 9, SEQ ID NO: 10/SEQ ID NO: 11, SEQ ID NO: 12/SEQ ID NO: 13, SEQ ID NO: 14/SEQ ID NO: 15, and SEQ ID NO: 16/SEQ ID NO: 17.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is a chimeric or humanized antibody or antigen-binding binding fragment thereof.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is a human antibody or antigen-binding binding fragment thereof.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is a monoclonal antibody or antigen-binding binding fragment thereof.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises one or more full-length antibody heavy chains comprising an Fc region.


In certain embodiments, the Fc region is a human IgG1 Fc region.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises an antibody F(ab), F(ab′)2, Fab′-SH, Fv, or scFv fragment.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises an antibody F(ab) fragment.


In certain embodiments, the antibody F(ab) fragment comprises SEQ ID NO: 56 and the first about 100 amino acids of SEQ ID NO: 54.


In certain embodiments, the antibody F(ab) fragment comprises a heavy chain comprising SEQ ID NO: 57 and the first about 100 amino acids of SEQ ID NO: 54.


In certain embodiments, the antibody F(ab) fragment comprises a heavy chain comprising SEQ ID NO: 58 and the first about 100 amino acids of SEQ ID NO: 54.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 141, 164, 173, 182, 191, 200, 209, 218, 227, 236, 245, 254, 263, 272, or 281, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 142.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 143, 165, 174, 183, 192, 201, 210, 219, 228, 237, 246, 255, 264, 273, or 282, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 144.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 145, 166, 175, 184, 193, 202, 211, 220, 229, 238, 247, 256, 265, 274, or 283, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 146.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 147, 167, 176, 185, 194, 203, 212, 221, 230, 239, 248, 257, 266, 275, or 284, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 148.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 149, 168, 177, 186, 195, 204, 213, 222, 231, 240, 249, 258, 267, 276, or 285, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 150.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 151, 169, 178, 187, 196, 205, 214, 223, 232, 241, 250, 259, 268, 277, or 286, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 152.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, or 287, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, or 287, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, or 287, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, or 289, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, or 289, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, or 289, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof comprises has cross-reactivity to mouse and cynomolgus FGFR3.


In certain embodiments, the antigen binding or antigen-binding binding fragment thereof protein does not bind to one or more of FGFR1, FGFR2, and FGFR4.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof does not bind to each of FGFR1, FGFR2, and FGFR4.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds to each of FGFR1, FGFR2, and FGFR4 with an affinity equilibrium dissociation constant (KD) of 100 μM or greater.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds human FGFR3 with an equilibrium dissociation constant (KD) of 10 nM or less.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof binds human FGFR3 with an off rate (Kd) of 10′ or greater.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof inhibits ligand-induced FGFR3 dimerization with IC50 of 5 μg/ml or less.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof inhibits FGFR3 receptor activation and downstream signaling with IC50 of 5 μg/ml or less.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof inhibits the activity of an FGFR3G380R mutant.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is capable of penetrating a bone growth plate.


In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is capable of decreasing the binding of FGFR3 with its ligand in a bone growth plate. In certain embodiments, the antigen binding protein or antigen-binding binding fragment thereof is capable of decreasing the kinase activity of FGFR3 in a bone growth plate.


Also provided is a pharmaceutical composition comprising the antigen binding protein or antigen-binding fragment thereof as described herein. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.


Also provided is an isolated nucleic acid molecule encoding the antigen binding protein or antigen-binding fragment thereof as described herein. Also provided is an expression cassette comprising said nucleic acid molecule. Also provided is an expression vector comprising the isolated nucleic acid molecule. Further provided is a host cell comprising the expression vector, the expression cassette or the nucleic acid molecule.


Further provided is a method for treating a FGFR3-mediated disease or disorder in a subject. In certain embodiments, the method comprises administering to a subject in need thereof the antigen binding protein or antigen-binding fragment thereof as described herein. In certain embodiments, the FGFR3-mediated disease or disorder is achondroplasia. In certain embodiments, the achondroplasia is FGFR3G380R+ achondroplasia.


In certain embodiments, the FGFR3-mediated disease or disorder is cancer.


In certain embodiments, the cancer is bladder cancer melanoma, urothelial cancer, and endometrial cancer.


Also provided is a method for treating achondroplasia in a subject. In certain embodiments, the method comprises administering to a subject in need thereof an antigen-binding protein fragment that specifically binds to FGFR3, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4.


Also provided is a method for inhibiting one or both of FGFR3 activity and expression in a bone growth plate of a subject. In certain embodiments, the method comprises administering to a subject an antigen-binding protein fragment that specifically binds to FGFR3, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4.


In certain embodiments, the subject is a child. In certain embodiments, the child is an infant. In certain embodiments, the infant is a newborn.


Also provided is a method for preventing or alleviating one or more symptoms of achondroplasia in a subject. In certain embodiments, the method comprises administering to the subject an antigen-binding protein or an antigen-binding protein fragment thereof that specifically binds to FGFR3, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4.





BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided to the Office upon request and payment of the necessary fee.



FIG. 1 schematically depicts fibroblast growth factor receptor 3 (FGFR3) isoform IIIc.



FIG. 2 depicts heat maps generated with hydrogen deuterium exchange (HDX) mass spectrometry used to determine the epitopes on FGFR3 for select antibodies by measuring the amide hydrogen deuterium exchange on FGFR3.



FIG. 3 depicts fluorescent images from internalization assays to detect internalization of anti-FGFR3 antibodies in KMS-11 cells.



FIG. 4 graphically depicts the average amount of anti-FGFR3 antibody internalization relative to the total amount of antibody.



FIG. 5A-FIG. 5C graphically depict inhibition of FGFR3 dimerization by anti-FGFR3 antibody KC18 (FIG. 5A), KE58 (FIG. 5B), KE94 (FIG. 5C). Inhibition was evaluated by a chemiluminescent assay in U2OS cells co-expressing fusion protein of 0-galactosidase-prolink (PK) and FGFR3 (FGFR3-PK) and β-galactosidase-enzyme acceptor (EA) and FGFR3 (FGFR3-EA).



FIG. 6 depicts an alignment of anti-FGFR3 antibodies KC18, KE63, and KE94.



FIG. 7 depicts an alignment of anti-FGFR3 antibody KC18_Hu18 with variants. Mutations of Hu18 residues are highlighted green or red.



FIG. 8A-FIG. 8C depict graphs demonstrating IC50 values for the inhibition of Erk phosphorylation by humanized anti-FGFR3 antibodies Hu18 (FIG. 8A), Hu44 (FIG. 8B), and Hu46 (FIG. 8C). Inhibition of Erk phosphorylation was determined by a homogenous time-resolved fluorescence (HTRF) assay.



FIG. 9A-FIG. 9B graphically depict relative Erk phosphorylation inhibition by anti-FGFR3 antibodies KC18 (FIG. 9A), KE63 (FIG. 9B), and KE94 (FIG. 9B) and their corresponding Fab fragments.



FIG. 10A-FIG. 10C graphically depict relative Erk phosphorylation inhibition by anti-FGFR3 antibody KC18 in different formats. FIG. 10A depicts inhibition effect of KC18 as a full-length antibody (KC18), a Fab fragment (Kc18 Fab), a one-armed, monovalent antibody (MetMab), and a PEGylated Fab fragment (PEG) in WT cells. FIG. 10B depicts inhibition effect of KC18 as a full-length antibody (IgG), a Fab fragment, a one-armed, monovalent antibody (MetMab), and a PEGylated Fab fragment (PEG) in Ach cells. FIG. 10C depicts KC18 fab fragments with half-life extension moieties using a human albumin nanobody conjugate (KC18 Fab-HLE), with and without human serum albumin (HSA) or mouse serum albumin (MSA). Erk phosphorylation was measured in an HTRF assay and compared to an isotype control (Iso).



FIG. 11 graphically depicts inhibition of Erk phosphorylation by humanized anti-FGFR3 antibodies Hu18, Hu44, and Hu46. Inhibition of Erk phosphorylation was determined using an HTRF assay.



FIG. 12A-FIG. 12B graphically depict femur (FIG. 12A) and tibia (FIG. 12B) length in an achondroplasia mouse model (Ach), which is a transgenic mouse that overexpresses the mouse FGFR3 protein with the G380R mutation under the collagen II promoter. Mice were administered the KC18 fab fragment subcutaneously from 3 days of age to 20 days of age.



FIG. 13A-FIG. 13B graphically depict vertebrae (FIG. 13A) and skull (FIG. 13B) length in the Ach mouse model. For skull measurements, NL=nasal length; FL=frontal length; and PL=parietal length.



FIG. 14 graphically depicts brain volume in the WT and Ach mouse model treated by saline or KC18 fab fragment.



FIG. 15A graphically depicts Kyphosis Index in the WT and Ach mouse model treated by saline or KC18 fab fragment. FIG. 15B depicts the calculation of Kyphotic Index using a mouse model.



FIG. 16A graphically depicts femur growth plate (GP) volume in Ach mouse model treated by vehicle or KC18 fab fragment. FIG. 16B depicts femur diameter in Ach mouse model treated by vehicle or KC18 fab fragment.



FIG. 17 graphically depicts increased secondary ossification center in tibia in Ach mouse model compared to those treated with vehicle.





DETAILED DESCRIPTION

Antigen-binding proteins or antigen-binding fragments thereof are provided. Methods of inhibiting one or more FGFR3 activities, and methods of treating FGFR3-mediated diseases and disorders are also provided.


Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.


Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.


So that the disclosure may be more readily understood, certain terms are first defined.


Fibroblast Growth Factor Receptor 3 (FGFR3)


As used herein, the term “FGFR3” or “fibroblast growth factor receptor 3” or “CD333” refers to the FGFR3 protein encoded by the FGFR3 gene. FGFR3 belongs to a family of fibroblast growth factor receptors that also includes FGFR1, FGFR2, and FGFR4. Like the other fibroblast growth factor receptor family members, FGFR3 is a single-pass membrane receptor tyrosine kinase with 3 Ig-like domains (D1, D2, and D3). Ligand dependent receptor dimerization leads to tyrosine kinase activation and downstream signal transduction. FGFR3 undergoes alternative splicing leading to several isoforms, including isoform IIIb and isoform IIIc. IIIb and IIIc arise from alternative splicing of exons 8 and 9. IIb and IIc have identical Ig1 (D1) and Ig2 (D2) domains, but vary in the Ig3 (D3) domain. It is FGFR3 IIc that is the major FGFR3 isoform in chondrocytes and mediates the anabolic effects of the FGFR3 ligand, FGF18, in articular cartilage. The structure and function of FGFR3 is described in further detail in Olsen et al. (PNAS. 101(4): 935-940. 2004), incorporated herein by reference in its entirety for all purposes.


The human FGFR3 isoform IIIc amino acid sequence is recited below.









(SEQ ID NO: 135)


MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV





FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS





HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA





PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR





IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP





HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP





DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH





SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS





PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT





LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA





KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP





LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ





VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK





TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV





EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR





VLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPS





SGGSRT






Mutations in FGFR3 can lead to certain undesired conditions. A G380R mutation in the transmembrane domain of FGFR3 is associated with 98% of all achondroplasia cases. FGFR3 with a G380R mutation can be referred to as FGFR3G380R and the sequence of the human FGFR3G380R isoform IIIc is recited below.









(SEQ ID NO: 133)


MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV





FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS





HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA





PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR





IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP





HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP





DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH





SAWLVVLPAEEELVEADEAGSVYAGILSYRVGFFLFILVVAAVTLCRLRS





PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT





LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA





KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP





LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ





VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK





TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV





EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR





VLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPS





SGGSRT







Antigen-Binding Proteins


As used herein, the term “antibody” or “antigen-binding protein” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with an antigen or epitope (e.g., a FGFR3 antigen or epitope), and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments thereof, including but not limited to fragment antigen-binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain variable fragments (scFv) and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term “antibody” includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, meditope-enabled antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv) and the like. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. As used herein, the term “functional antibody fragment” refers to an antibody fragment having at least 80%, at least 85%, at least 90%, or at least 95% affinity as the antibody of interest from which the fragment is derived from.


As used herein, the term “complementarity determining region” or “CDR” refers to sequences of amino acids within antibody variable regions, which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” or “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).


The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme), Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme), and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme).


The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.


A “CDR” or “complementary determining region,” or individual specified CDRs (e.g., “CDR-H1,” “CDR-H2,” “CDR-H3”), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the known schemes. Likewise, an “FR” or “framework region,” or individual specified FRs (e.g., “FR-H1,” “FR-H2”) of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR or FR is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, AbM, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given. Unless otherwise specified, all particular CDR amino acid sequences mentioned in the disclosure are IMGT CDRs. However, alternative CDRs defined by other schemes are also encompassed by the present disclosure, such as those determined by abYsis Key Annotation (Website: abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi).


As used herein, the term “specifically binds,” “specifically binding,” “binding specificity” or “specifically recognized” refers that an antigen binding protein or antigen-binding fragment thereof that exhibits appreciable affinity for an antigen (e.g., an FGFR3 antigen) and does not exhibit significant cross reactivity to a target that is not an FGFR3 protein. As used herein, the term “affinity” refers to the strength of the interaction between an antigen binding protein or antigen-binding fragment thereof antigen binding site and the epitope to which it binds. As readily understood by those skilled in the art, an antigen binding protein affinity may be reported as a dissociation constant (KD) in molarity (M). The antigen binding protein or antigen-binding fragment thereof of the disclosure have KD values in the range of about 10−6 M to about 10−12 M (i.e., low micromolar to picomolar range), about 10−7 M to 10−11 M, about 10−8 M to about 10−10 M, about 10−9 M. In certain embodiments, the antigen binding protein or antigen-binding fragment thereof has a binding affinity of about 10−6 M, 10−7 M, 10−8 M, 10−9 M, 10−10 M, 10−11 M, or 10−12 M. In certain embodiments, the antigen binding protein or antigen-binding fragment thereof has a binding affinity of about 10−7 M to about 10−9 M (nanomolar range).


Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined by competitive binding assays (e.g. ELISA) or Biacore assays. In certain embodiments, the assay is conducted at about 20° C., 25° C., 30° C., or 37° C.


Anti-FGFR3 Antigen-Binding Proteins


In one aspect, the disclosure provides antigen binding proteins and antigen-binding fragments thereof with binding specificity to FGFR3.


Exemplary anti-FGFR3 antigen binding protein and antigen-binding fragment thereof CDRs are recited below in Table 1 and Table 4. Exemplary anti-FGFR3 antigen binding protein and antigen-binding fragment thereof variable heavy (VH) and variable light (VL) domains are recited below in Table 3, Table 8, Table 9, and Table 13. Exemplary anti-FGFR3 antigen binding protein full length heavy and light chains are recited below in Table 14.









TABLE 1





Antibody Heavy chain and Light chain CDR regions







HEAVY Chain










VH Chain ID
HCDR1-IMGT
HCDR2-IMGT
HCDR3-IMGT





KC18_VH
GDTFTDFE
IDPETGGT
TRTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 71)
(SEQ ID NO: 72)





KE35_VH
GYTFTDYN
INPNNGGT
ARERDYDGAMDY



(SEQ ID NO: 76)
(SEQ ID NO: 77)
(SEQ ID NO: 78)





KE42_VH
GYTFTDYN
INPNNGGT
ARERDYDGSMDF



(SEQ ID NO: 82)
(SEQ ID NO: 83)
(SEQ ID NO: 84)





KE58_VH
GYTVTDYY
INPNNGVT
AREEDFDGFDY



(SEQ ID NO: 88)
(SEQ ID NO: 89)
(SEQ ID NO: 90)





KE63_VH
GSTFSDEE
IDPETGGT
TRNYDGYSQTMDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)





KE94_VH
GSTFTDEE
IDPETGGT
TRNYDGYSRTMDY



(SEQ ID NO: 100)
(SEQ ID NO: 101)
(SEQ ID NO: 102)





KC18 Hu18 VH
GDTFTDFE
VDPETGGT
TRTYDGYPYAFDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 301)





KC18Hrw1
GDTFTDFE
IDPETGST
TRTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 298)
(SEQ ID NO: 72)





KC18Hrw2
GDTFTDYE
IDPETGST
TRTYDGYPYAMDY



(SEQ ID NO: 295)
(SEQ ID NO: 298)
(SEQ ID NO: 72)





KC18Hrw3
GDTFTDYE
IDPETGST
TRTYDGYPYAMDY



(SEQ ID NO: 295)
(SEQ ID NO: 298)
(SEQ ID NO: 72)





KC18HV1-69rw2
GDTFTDFE
VDPETGGT
TRTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 72)


KC18HV1-69rw3
GDTFTDFE
VDPETGGT
TRTYEGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 300)





KC18HV1-69rw4
GDTFTDFE
VDPETGGT
TRTYDGYPYAFDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 301)





KC18_CL_VH1
GDTFTDFE
IDPETGGT
ARTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 95)
(SEQ ID NO: 310)





KC18_CL_VH1b
GYTFTDFE
VDPETGGT
ARTYDGYPYAMDY



(SEQ ID NO: 296)
(SEQ ID NO: 297)
(SEQ ID NO: 310)





KC18_CL_VH1c
GYTFTDFE
VEPETGGT
ARTYDGYPYAMDV



(SEQ ID NO: 296)
(SEQ ID NO: 299)
(SEQ ID NO: 311)





KC18_CL_VH2
GDTFTDFE
IDPETGGT
TRTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 95)
(SEQ ID NO: 72)





KC18_CL_VH3
GDTFTDFE
IDPESGGT
ARTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 312)
(SEQ ID NO: 310)





KC18_CL_VH3b
GYTFTDFE
IDPESGGT
ARTYDGYPYAMDY



(SEQ ID NO: 296)
(SEQ ID NO: 312)
(SEQ ID NO: 310)





KC18_CL_VH3c
GYTFTDFE
IEPESGGT
ARTYDGYPYAMDV



(SEQ ID NO: 296)
(SEQ ID NO: 313)
(SEQ ID NO: 311)





KC18_CL_VH4
GDTFTDFE
IDPETGGT
TRTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 95)
(SEQ ID NO: 72)





KC18_VH_6
GDTFTDFE
VDPETGGT
TRTYDGYPYAFDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 301)





KC18_VH_15
GDTFTDFE
VDPETGGT
TRTYDGYPYAFDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 301)





KC18_VH_16
GDTFTDFE
VDPETGGT
TRTYDGYPYAFDY



(SEQ ID NO: 70)
(SEQ ID NO: 297)
(SEQ ID NO: 301)





KE63 Hu01 VH
GSTFSDEE
IDPETGGT
TRNYDGYSQTFDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 305)





KE63 Hu02 VH
GSTFSDEE
IDPETGGT
TRNYDGYSQTFDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 305)





KE63 Hu03 VH
GSTFSDEE
IDPETGGT
TRNYDGYSQTFDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 305)





KE63 Hu04 VH
GSTFSDEE
IDPETGGT
TRNYDGYSQTFDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 305)





KE94 Hu01 VH
GSTFTDEE
IDPETGGT
TRNYDGYSRTFDY



(SEQ ID NO: 100)
(SEQ ID NO: 101)
(SEQ ID NO: 307)





KE94 Hu02 VH
GSTFTDEE
IDPETGGT
TRNYDGYSRTFDY



(SEQ ID NO: 100)
(SEQ ID NO: 101)
(SEQ ID NO: 307)





KE94 Hu03 VH
GSTFTDEE
IDPETGGT
TRNYDGYSRTFDY



(SEQ ID NO: 100)
(SEQ ID NO: 101)
(SEQ ID NO: 307)





KE94 Hu04 VH
GSTFTDEE
IDPETGGT
TRNYDGYSRTFDY



(SEQ ID NO: 100)
(SEQ ID NO: 101)
(SEQ ID NO: 307)










LIGHT Chain










VL Chain ID
LCDR1-IMGT
LCDR2-IMGT
LCDR3-IMGT





KC18_VL
QSLLYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KE35_VL
QDINKF
YTS
LQYDNLLWT



(SEQ ID NO: 79)
(SEQ ID NO: 80)
(SEQ ID NO: 81)





KE42_VL
QDINKF
YTS
LQYDNLLWT



(SEQ ID NO: 85)
(SEQ ID NO: 86)
(SEQ ID NO: 87)





KE58_VL
QDVSTG
WAS
QQHYSTPLT



(SEQ ID NO: 91)
(SEQ ID NO: 92)
(SEQ ID NO: 93)





KE63_VL
QSVLYSSNQKNY
WAS
HQYLSSYT



(SEQ ID NO: 97)
(SEQ ID NO: 98)
(SEQ ID NO: 99)





KE94_VL
QSVLYSSNQKNY
WAS
HQYLSSYT



(SEQ ID NO: 103)
(SEQ ID NO: 104)
(SEQ ID NO: 105)





KC18 Hu18 VL
QSVLYSNNNKNY
WAS
QQYYSYRT



(SEQ ID NO: 302)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18Lrw1
QSLLYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18Lrw2
QSVLYSSNNKNY
WAS
QQYYSYRT



(SEQ ID NO: X)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18Lrw3
QSVLYSNNNKNY
WAS
QQYYSYRT



(SEQ ID NO: 302)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_CL_VL1
QSVLYSSNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 97)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_CL_VL1b
QSVLYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 315)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_CL_VL1c
QSVLYSSNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 97)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_CL_VL1d
QSVLYSSNQKNY
YAS
QQYYSYRT



(SEQ ID NO: 97)
(SEQ ID NO: 303)
(SEQ ID NO: 75)





KC18_CL_VL2
QSLLYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_CL_VL3
QSLLHSNNQKNY
WGS
QQYYSYRT



(SEQ ID NO: 317)
(SEQ ID NO: 316)
(SEQ ID NO: 75)





KC18_CL_VL3b
QSLLYSNNQKNY
WGS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 316)
(SEQ ID NO: 75)





KC18_CL_VL4
QSLLYSNNQKNY
WGS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 316)
(SEQ ID NO: 75)





KC18_CL_VL5
QGISYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: X)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_CL_VL6
QSLLYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_VL_3
QSVLYSNNNKNY
WAS
QQYYSYRT



(SEQ ID NO: 302)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KC18_VL_14
QSVLYSDNQKNY
YAS
QQYYSYRT



(SEQ ID NO: 306)
(SEQ ID NO: 303)
(SEQ ID NO: 75)





KC18_VL_15
QSVLYSDNQKNY
FAS
QQYYSYRT



(SEQ ID NO: 306)
(SEQ ID NO: 304)
(SEQ ID NO: 75)





KE63 Hu01 VL
QSVLYSSNQKNY
WAS
HQYLSSYT



(SEQ ID NO: 97)
(SEQ ID NO: 98)
(SEQ ID NO: 99)





KE63 Hu02 VL
QSVLYSSNQKNY
WAS
HQYLSPYT



(SEQ ID NO: 97)
(SEQ ID NO: 98)
(SEQ ID NO: 314)





KE63 Hu03 VL
QSVLYSDNQKNY
YAS
HQYLSPYT



(SEQ ID NO: 306)
(SEQ ID NO: 303)
(SEQ ID NO: 314)





KE63 Hu04 VL
QSVLYSDNQKNY
FAS
HQYLSPYT



(SEQ ID NO: 306)
(SEQ ID NO: 304)
(SEQ ID NO: 314)





KE94 Hu01 VL
QSVLYSSNQKNY
WAS
HQYLSSYT



(SEQ ID NO: 97)
(SEQ ID NO: 104)
(SEQ ID NO: 105)





KE94 Hu02 VL
QSVLYSSNQKNY
WAS
HQYLSPYT



(SEQ ID NO: 97)
(SEQ ID NO: 104)
(SEQ ID NO: 314)





KE94 Hu03 VL
QSVLYSDNQKNY
YAS
HQYLSPYT



(SEQ ID NO: 306)
(SEQ ID NO: 303)
(SEQ ID NO: 314)





KE94 Hu04 VL
QSVLYSDNQKNY
FAS
HQYLSPYT



(SEQ ID NO: 306)
(SEQ ID NO: 304)
(SEQ ID NO: 314)









In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof comprise an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence, a CDR-H2 sequence, and a CDR-H3 sequence. The CDR-H1 sequence comprises the amino acid sequence of GX1TFTDX2E (SEQ ID NO: 157), wherein X1 comprises Y or D and X2 comprises F or Y; the CDR-H2 sequence comprises the amino acid sequence of IDPETGX3T (SEQ ID NO: 158), wherein X3 comprises G or S; or CDR-H2 sequence comprising the amino acid sequence of INPNNGX4T (SEQ ID NO: 159), wherein X4 comprises G or V; or CDR-H2 sequence comprising the amino acid sequence of VX5PETGGT (SEQ ID NO: 160), wherein X5 comprises D or E; and the CDR-H3 sequence comprises the amino acid sequence of TRX6YX7GYX8X9X10X11DY (SEQ ID NO: 161), wherein X6 comprises T or N, X7 comprises D or E, X8 comprises S or P, X9 comprises Q, R, or Y, X10 comprises T or A, X11 comprises F or M.


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof comprise an antibody light chain variable (VL) domain comprising a CDR-L1 sequence, a CDR-L2 sequence, and a CDR-L3 sequence. The CDR-L1 sequence comprises the amino acid sequence of QS X12LYS X13N X14KNY (SEQ ID NO: 162), wherein X12 comprises L or V, X13 comprises N, D, or S, and X14 comprises Q or N; the CDR-L2 sequence comprises the amino acid sequence of X15AS (SEQ ID NO: 163), wherein X15 comprises W, Y, or F; and the CDR-L3 sequence comprises the amino acid sequence of QQYYSYRT (SEQ ID NO: 75), LQYDNLLWT (SEQ ID NO: 81), or HQYLSX16YT (SEQ ID NO: 290), wherein X16 comprises P or S.


In one aspect, the disclosure provides an antigen-binding protein or fragment thereof that specifically binds to fibroblast growth factor receptor 3 (FGFR3), comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein:


(a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GX1TFTDX2E (SEQ ID NO: 157), wherein X1 comprises Y or D and X2 comprises F or Y; a CDR-H2 sequence comprising the amino acid sequence of IDPETGX3T (SEQ ID NO: 158), wherein X3 comprises G or S; or CDR-H2 sequence comprising the amino acid sequence of INPNNGX4T (SEQ ID NO: 159), wherein X4 comprises G or V; or CDR-H2 sequence comprising the amino acid sequence of VX5PETGGT (SEQ ID NO: 160), wherein X5 comprises D or E; a CDR-H3 sequence comprising the amino acid sequence of TRX6YX7GYX8X9X10X11DY (SEQ ID NO: 161), wherein X6 comprises T or N, X7 comprises D or E, X8 comprises S or P, X9 comprises Q, R, or Y, X10 comprises T or A, X11 comprises F or M; and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QS X12LYS X13N X14KNY (SEQ ID NO: 162), wherein X12 comprises L or V, X13 comprises N, D, or S, and X14 comprises Q or N; a CDR-L2 sequence comprising the amino acid sequence of X15AS (SEQ ID NO: 163), wherein X15 comprises W, Y, or F; a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75), LQYDNLLWT (SEQ ID NO: 81), or HQYLSX16YT (SEQ ID NO: 290) wherein X16 comprises P or S;


(b) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGAMDY (SEQ ID NO: 78); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87);


(c) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGSMDF (SEQ ID NO: 84); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87);


(d) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTVTDYY (SEQ ID NO: 88); a CDR-H2 sequence comprising the amino acid sequence of INPNNGVT (SEQ ID NO: 89); a CDR-H3 sequence comprising the amino acid sequence of AREEDFDGFDY (SEQ ID NO: 90); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVSTG (SEQ ID NO: 91); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of QQHYSTPLT (SEQ ID NO: 93);


(e) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFSDFE (SEQ ID NO: 94); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTX17DY (SEQ ID NO: 308), wherein X17 comprises M or F; and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSX18NQKNY (SEQ ID NO: 309), wherein X18 comprises S or D; a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105); or


(f) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFTDFE (SEQ ID NO: 100); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTX17DY (SEQ ID NO: 308), wherein X17 comprises M or F; and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSX18NQKNY (SEQ ID NO: 309), wherein X18 comprises S or D; a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of HQYLSSYT (SEQ ID NOs: 99 and 105).


In certain embodiments, the antigen binding protein or fragment thereof comprises a VH domain and a VL domain, wherein:


(a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), GDTFTDYE (SEQ ID NO: 295), or GYTFTDFE (SEQ ID NO: 296); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101), VDPETGGT (SEQ ID NO: 297), IDPETGST (SEQ ID NO: 298), or VEPETGGT (SEQ ID NO: 299); a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAMDY (SEQ ID NO: 72), TRTYEGYPYAMDY (SEQ ID NO: 300), or TRTYDGYPYAFDY (SEQ ID NO: 301); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLLYSNNQKNY (SEQ ID NO: 73), QSVLYSNNNKNY (SEQ ID NO: 302), or QSVLYSDNQKNY (SEQ ID NO: 306); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104), YAS (SEQ ID NO: 303), or FAS (SEQ ID NO: 304); a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75);


(b) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGAMDY (SEQ ID NO: 78); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87);


(c) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTDYN (SEQ ID NOs: 76 and 82); a CDR-H2 sequence comprising the amino acid sequence of INPNNGGT (SEQ ID NOs: 77 and 83); a CDR-H3 sequence comprising the amino acid sequence of ARERDYDGSMDF (SEQ ID NO: 84); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDINKF (SEQ ID NOs: 79 and 85); a CDR-L2 sequence comprising the amino acid sequence of YTS (SEQ ID NOs: 80 and 86); a CDR-L3 sequence comprising the amino acid sequence of LQYDNLLWT (SEQ ID NOs: 81 and 87);


(d) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTVTDYY (SEQ ID NO: 88); a CDR-H2 sequence comprising the amino acid sequence of INPNNGVT (SEQ ID NO: 89); a CDR-H3 sequence comprising the amino acid sequence of AREEDFDGFDY (SEQ ID NO: 90); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVSTG (SEQ ID NO: 91); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, 104); a CDR-L3 sequence comprising the amino acid sequence of QQHYSTPLT (SEQ ID NO: 93);


(e) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFSDFE (SEQ ID NO: 94); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSQTMDY (SEQ ID NO: 96) or TRNYDGYSQTFDY (SEQ ID NO: 305); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSSNQKNY(SEQ ID NOs: 97 and 103) or QSVLYSDNQKNY (SEQ ID NO: 306); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105); or


(f) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GSTFTDFE (SEQ ID NO: 100); a CDR-H2 sequence comprising the amino acid sequence of IDPETGGT (SEQ ID NOs: 71, 95, and 101); a CDR-H3 sequence comprising the amino acid sequence of TRNYDGYSRTMDY (SEQ ID NO: 102) or TRNYDGYSRTFDY (SEQ ID NO: 307); and the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSSNQKNY(SEQ ID NOs: 97 and 103) or QSVLYSDNQKNY (SEQ ID NO: 306); a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, 104); a CDR-L3 sequence comprising the amino acid sequence of or HQYLSSYT (SEQ ID NOs: 99 and 105).


In certain embodiments, the FGFR3 antigen binding proteins and antigen-binding fragments thereof comprise one VH domain and one VL domain recited in Table 3, 8, 9, or 13. In certain embodiments, exemplary antigen-binding proteins or antigen-binding fragments thereof are provided:









TABLE 1.1







Heavy chains and light chains of exemplary


antigen-binding proteins or antigen-binding fragments









No.
Heavy Chain
Light Chain












1
SEQ ID NO: 6
SEQ ID NO: 7


2
SEQ ID NO: 6
SEQ ID NO: 9


3
SEQ ID NO: 6
SEQ ID NO: 11


4
SEQ ID NO: 6
SEQ ID NO: 13


5
SEQ ID NO: 6
SEQ ID NO: 15


6
SEQ ID NO: 6
SEQ ID NO: 17


7
SEQ ID NO: 8
SEQ ID NO: 7


8
SEQ ID NO: 8
SEQ ID NO: 9


9
SEQ ID NO: 8
SEQ ID NO: 11


10
SEQ ID NO: 8
SEQ ID NO: 13


11
SEQ ID NO: 8
SEQ ID NO: 15


12
SEQ ID NO: 8
SEQ ID NO: 17


13
SEQ ID NO: 10
SEQ ID NO: 7


14
SEQ ID NO: 10
SEQ ID NO: 9


15
SEQ ID NO: 10
SEQ ID NO: 11


16
SEQ ID NO: 10
SEQ ID NO: 13


17
SEQ ID NO: 10
SEQ ID NO: 15


18
SEQ ID NO: 10
SEQ ID NO: 17


19
SEQ ID NO: 12
SEQ ID NO: 7


20
SEQ ID NO: 12
SEQ ID NO: 9


21
SEQ ID NO: 12
SEQ ID NO: 11


22
SEQ ID NO: 12
SEQ ID NO: 13


23
SEQ ID NO: 12
SEQ ID NO: 15


24
SEQ ID NO: 12
SEQ ID NO: 17


25
SEQ ID NO: 14
SEQ ID NO: 7


26
SEQ ID NO: 14
SEQ ID NO: 9


27
SEQ ID NO: 14
SEQ ID NO: 11


28
SEQ ID NO: 14
SEQ ID NO: 13


29
SEQ ID NO: 14
SEQ ID NO: 15


30
SEQ ID NO: 14
SEQ ID NO: 17


31
SEQ ID NO: 18
SEQ ID NO: 19


32
SEQ ID NO: 18
SEQ ID NO: 24


33
SEQ ID NO: 18
SEQ ID NO: 25


34
SEQ ID NO: 18
SEQ ID NO: 26


35
SEQ ID NO: 18
SEQ ID NO: 27


36
SEQ ID NO: 18
SEQ ID NO: 32


37
SEQ ID NO: 18
SEQ ID NO: 33


38
SEQ ID NO: 18
SEQ ID NO: 34


39
SEQ ID NO: 18
SEQ ID NO: 35


40
SEQ ID NO: 18
SEQ ID NO: 59


41
SEQ ID NO: 18
SEQ ID NO: 60


42
SEQ ID NO: 18
SEQ ID NO: 61


43
SEQ ID NO: 18
SEQ ID NO: 67


44
SEQ ID NO: 18
SEQ ID NO: 69


45
SEQ ID NO: 18
SEQ ID NO: 112


46
SEQ ID NO: 18
SEQ ID NO: 113


47
SEQ ID NO: 18
SEQ ID NO: 114


48
SEQ ID NO: 18
SEQ ID NO: 123


49
SEQ ID NO: 18
SEQ ID NO: 124


50
SEQ ID NO: 18
SEQ ID NO: 125


51
SEQ ID NO: 18
SEQ ID NO: 126


52
SEQ ID NO: 18
SEQ ID NO: 127


53
SEQ ID NO: 18
SEQ ID NO: 128


54
SEQ ID NO: 18
SEQ ID NO: 129


55
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SEQ ID NO: 110
SEQ ID NO: 125


537
SEQ ID NO: 110
SEQ ID NO: 126


538
SEQ ID NO: 110
SEQ ID NO: 127


539
SEQ ID NO: 110
SEQ ID NO: 128


540
SEQ ID NO: 110
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541
SEQ ID NO: 110
SEQ ID NO: 130


542
SEQ ID NO: 110
SEQ ID NO: 131


543
SEQ ID NO: 111
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544
SEQ ID NO: 111
SEQ ID NO: 19


545
SEQ ID NO: 111
SEQ ID NO: 24


546
SEQ ID NO: 111
SEQ ID NO: 25


547
SEQ ID NO: 111
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548
SEQ ID NO: 111
SEQ ID NO: 27


549
SEQ ID NO: 111
SEQ ID NO: 32


550
SEQ ID NO: 111
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551
SEQ ID NO: 111
SEQ ID NO: 34


552
SEQ ID NO: 111
SEQ ID NO: 35


553
SEQ ID NO: 111
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554
SEQ ID NO: 111
SEQ ID NO: 60


555
SEQ ID NO: 111
SEQ ID NO: 61


556
SEQ ID NO: 111
SEQ ID NO: 67


557
SEQ ID NO: 111
SEQ ID NO: 69


558
SEQ ID NO: 111
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559
SEQ ID NO: 111
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560
SEQ ID NO: 111
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561
SEQ ID NO: 111
SEQ ID NO: 123


562
SEQ ID NO: 111
SEQ ID NO: 124


563
SEQ ID NO: 111
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564
SEQ ID NO: 111
SEQ ID NO: 126


565
SEQ ID NO: 111
SEQ ID NO: 127


566
SEQ ID NO: 111
SEQ ID NO: 128


567
SEQ ID NO: 111
SEQ ID NO: 129


568
SEQ ID NO: 111
SEQ ID NO: 130


569
SEQ ID NO: 111
SEQ ID NO: 131


570
SEQ ID NO: 111
SEQ ID NO: 132


571
SEQ ID NO: 115
SEQ ID NO: 19


572
SEQ ID NO: 115
SEQ ID NO: 24


573
SEQ ID NO: 115
SEQ ID NO: 25


574
SEQ ID NO: 115
SEQ ID NO: 26


575
SEQ ID NO: 115
SEQ ID NO: 27


576
SEQ ID NO: 115
SEQ ID NO: 32


577
SEQ ID NO: 115
SEQ ID NO: 33


578
SEQ ID NO: 115
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579
SEQ ID NO: 115
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580
SEQ ID NO: 115
SEQ ID NO: 59


581
SEQ ID NO: 115
SEQ ID NO: 60


582
SEQ ID NO: 115
SEQ ID NO: 61


583
SEQ ID NO: 115
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584
SEQ ID NO: 115
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585
SEQ ID NO: 115
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586
SEQ ID NO: 115
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587
SEQ ID NO: 115
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588
SEQ ID NO: 115
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SEQ ID NO: 115
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590
SEQ ID NO: 115
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591
SEQ ID NO: 115
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592
SEQ ID NO: 115
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593
SEQ ID NO: 115
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594
SEQ ID NO: 115
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595
SEQ ID NO: 115
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596
SEQ ID NO: 115
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597
SEQ ID NO: 115
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598
SEQ ID NO: 116
SEQ ID NO: 19


599
SEQ ID NO: 116
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600
SEQ ID NO: 116
SEQ ID NO: 25


601
SEQ ID NO: 116
SEQ ID NO: 26


602
SEQ ID NO: 116
SEQ ID NO: 27


603
SEQ ID NO: 116
SEQ ID NO: 32


604
SEQ ID NO: 116
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605
SEQ ID NO: 116
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606
SEQ ID NO: 116
SEQ ID NO: 35


607
SEQ ID NO: 116
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608
SEQ ID NO: 116
SEQ ID NO: 60


609
SEQ ID NO: 116
SEQ ID NO: 61


610
SEQ ID NO: 116
SEQ ID NO: 67


611
SEQ ID NO: 116
SEQ ID NO: 69


612
SEQ ID NO: 116
SEQ ID NO: 112


613
SEQ ID NO: 116
SEQ ID NO: 113


614
SEQ ID NO: 116
SEQ ID NO: 114


615
SEQ ID NO: 116
SEQ ID NO: 123


616
SEQ ID NO: 116
SEQ ID NO: 124


617
SEQ ID NO: 116
SEQ ID NO: 125


618
SEQ ID NO: 116
SEQ ID NO: 126


619
SEQ ID NO: 116
SEQ ID NO: 127


620
SEQ ID NO: 116
SEQ ID NO: 128


621
SEQ ID NO: 116
SEQ ID NO: 129


622
SEQ ID NO: 116
SEQ ID NO: 130


623
SEQ ID NO: 116
SEQ ID NO: 131


624
SEQ ID NO: 116
SEQ ID NO: 132


625
SEQ ID NO: 117
SEQ ID NO: 19


626
SEQ ID NO: 117
SEQ ID NO: 24


627
SEQ ID NO: 117
SEQ ID NO: 25


628
SEQ ID NO: 117
SEQ ID NO: 26


629
SEQ ID NO: 117
SEQ ID NO: 27


630
SEQ ID NO: 117
SEQ ID NO: 32


631
SEQ ID NO: 117
SEQ ID NO: 33


632
SEQ ID NO: 117
SEQ ID NO: 34


633
SEQ ID NO: 117
SEQ ID NO: 35


634
SEQ ID NO: 117
SEQ ID NO: 59


635
SEQ ID NO: 117
SEQ ID NO: 60


636
SEQ ID NO: 117
SEQ ID NO: 61


637
SEQ ID NO: 117
SEQ ID NO: 67


638
SEQ ID NO: 117
SEQ ID NO: 69


639
SEQ ID NO: 117
SEQ ID NO: 112


640
SEQ ID NO: 117
SEQ ID NO: 113


641
SEQ ID NO: 117
SEQ ID NO: 114


642
SEQ ID NO: 117
SEQ ID NO: 123


643
SEQ ID NO: 117
SEQ ID NO: 124


644
SEQ ID NO: 117
SEQ ID NO: 125


645
SEQ ID NO: 117
SEQ ID NO: 126


646
SEQ ID NO: 117
SEQ ID NO: 127


647
SEQ ID NO: 117
SEQ ID NO: 128


648
SEQ ID NO: 117
SEQ ID NO: 129


649
SEQ ID NO: 117
SEQ ID NO: 130


650
SEQ ID NO: 117
SEQ ID NO: 131


651
SEQ ID NO: 117
SEQ ID NO: 132


652
SEQ ID NO: 118
SEQ ID NO: 19


653
SEQ ID NO: 118
SEQ ID NO: 24


654
SEQ ID NO: 118
SEQ ID NO: 25


655
SEQ ID NO: 118
SEQ ID NO: 26


656
SEQ ID NO: 118
SEQ ID NO: 27


657
SEQ ID NO: 118
SEQ ID NO: 32


658
SEQ ID NO: 118
SEQ ID NO: 33


659
SEQ ID NO: 118
SEQ ID NO: 34


660
SEQ ID NO: 118
SEQ ID NO: 35


661
SEQ ID NO: 118
SEQ ID NO: 59


662
SEQ ID NO: 118
SEQ ID NO: 60


663
SEQ ID NO: 118
SEQ ID NO: 61


664
SEQ ID NO: 118
SEQ ID NO: 67


665
SEQ ID NO: 118
SEQ ID NO: 69


666
SEQ ID NO: 118
SEQ ID NO: 112


667
SEQ ID NO: 118
SEQ ID NO: 113


668
SEQ ID NO: 118
SEQ ID NO: 114


669
SEQ ID NO: 118
SEQ ID NO: 123


670
SEQ ID NO: 118
SEQ ID NO: 124


671
SEQ ID NO: 118
SEQ ID NO: 125


672
SEQ ID NO: 118
SEQ ID NO: 126


673
SEQ ID NO: 118
SEQ ID NO: 127


674
SEQ ID NO: 118
SEQ ID NO: 128


675
SEQ ID NO: 118
SEQ ID NO: 129


676
SEQ ID NO: 118
SEQ ID NO: 130


677
SEQ ID NO: 118
SEQ ID NO: 131


678
SEQ ID NO: 118
SEQ ID NO: 132


679
SEQ ID NO: 119
SEQ ID NO: 19


680
SEQ ID NO: 119
SEQ ID NO: 24


681
SEQ ID NO: 119
SEQ ID NO: 25


682
SEQ ID NO: 119
SEQ ID NO: 26


683
SEQ ID NO: 119
SEQ ID NO: 27


684
SEQ ID NO: 119
SEQ ID NO: 32


685
SEQ ID NO: 119
SEQ ID NO: 33


686
SEQ ID NO: 119
SEQ ID NO: 34


687
SEQ ID NO: 119
SEQ ID NO: 35


688
SEQ ID NO: 119
SEQ ID NO: 59


689
SEQ ID NO: 119
SEQ ID NO: 60


690
SEQ ID NO: 119
SEQ ID NO: 61


691
SEQ ID NO: 119
SEQ ID NO: 67


692
SEQ ID NO: 119
SEQ ID NO: 69


693
SEQ ID NO: 119
SEQ ID NO: 112


694
SEQ ID NO: 119
SEQ ID NO: 113


695
SEQ ID NO: 119
SEQ ID NO: 114


696
SEQ ID NO: 119
SEQ ID NO: 123


697
SEQ ID NO: 119
SEQ ID NO: 124


698
SEQ ID NO: 119
SEQ ID NO: 125


699
SEQ ID NO: 119
SEQ ID NO: 126


700
SEQ ID NO: 119
SEQ ID NO: 127


701
SEQ ID NO: 119
SEQ ID NO: 128


702
SEQ ID NO: 119
SEQ ID NO: 129


703
SEQ ID NO: 119
SEQ ID NO: 130


704
SEQ ID NO: 119
SEQ ID NO: 131


705
SEQ ID NO: 119
SEQ ID NO: 132


706
SEQ ID NO: 120
SEQ ID NO: 19


707
SEQ ID NO: 120
SEQ ID NO: 24


708
SEQ ID NO: 120
SEQ ID NO: 25


709
SEQ ID NO: 120
SEQ ID NO: 26


710
SEQ ID NO: 120
SEQ ID NO: 27


711
SEQ ID NO: 120
SEQ ID NO: 32


712
SEQ ID NO: 120
SEQ ID NO: 33


713
SEQ ID NO: 120
SEQ ID NO: 34


714
SEQ ID NO: 120
SEQ ID NO: 35


715
SEQ ID NO: 120
SEQ ID NO: 59


716
SEQ ID NO: 120
SEQ ID NO: 60


717
SEQ ID NO: 120
SEQ ID NO: 61


718
SEQ ID NO: 120
SEQ ID NO: 67


719
SEQ ID NO: 120
SEQ ID NO: 69


720
SEQ ID NO: 120
SEQ ID NO: 112


721
SEQ ID NO: 120
SEQ ID NO: 113


722
SEQ ID NO: 120
SEQ ID NO: 114


723
SEQ ID NO: 120
SEQ ID NO: 123


724
SEQ ID NO: 120
SEQ ID NO: 124


725
SEQ ID NO: 120
SEQ ID NO: 125


726
SEQ ID NO: 120
SEQ ID NO: 126


727
SEQ ID NO: 120
SEQ ID NO: 127


728
SEQ ID NO: 120
SEQ ID NO: 128


729
SEQ ID NO: 120
SEQ ID NO: 129


730
SEQ ID NO: 120
SEQ ID NO: 130


731
SEQ ID NO: 120
SEQ ID NO: 131


732
SEQ ID NO: 120
SEQ ID NO: 132


733
SEQ ID NO: 121
SEQ ID NO: 19


734
SEQ ID NO: 121
SEQ ID NO: 24


735
SEQ ID NO: 121
SEQ ID NO: 25


736
SEQ ID NO: 121
SEQ ID NO: 26


737
SEQ ID NO: 121
SEQ ID NO: 27


738
SEQ ID NO: 121
SEQ ID NO: 32


739
SEQ ID NO: 121
SEQ ID NO: 33


740
SEQ ID NO: 121
SEQ ID NO: 34


741
SEQ ID NO: 121
SEQ ID NO: 35


742
SEQ ID NO: 121
SEQ ID NO: 59


743
SEQ ID NO: 121
SEQ ID NO: 60


744
SEQ ID NO: 121
SEQ ID NO: 61


745
SEQ ID NO: 121
SEQ ID NO: 67


746
SEQ ID NO: 121
SEQ ID NO: 69


747
SEQ ID NO: 121
SEQ ID NO: 112


748
SEQ ID NO: 121
SEQ ID NO: 113


749
SEQ ID NO: 121
SEQ ID NO: 114


750
SEQ ID NO: 121
SEQ ID NO: 123


751
SEQ ID NO: 121
SEQ ID NO: 124


752
SEQ ID NO: 121
SEQ ID NO: 125


753
SEQ ID NO: 121
SEQ ID NO: 126


754
SEQ ID NO: 121
SEQ ID NO: 127


755
SEQ ID NO: 121
SEQ ID NO: 128


756
SEQ ID NO: 121
SEQ ID NO: 129


757
SEQ ID NO: 121
SEQ ID NO: 130


758
SEQ ID NO: 121
SEQ ID NO: 131


759
SEQ ID NO: 121
SEQ ID NO: 132


760
SEQ ID NO: 122
SEQ ID NO: 19


761
SEQ ID NO: 122
SEQ ID NO: 24


762
SEQ ID NO: 122
SEQ ID NO: 25


763
SEQ ID NO: 122
SEQ ID NO: 26


764
SEQ ID NO: 122
SEQ ID NO: 27


765
SEQ ID NO: 122
SEQ ID NO: 32


766
SEQ ID NO: 122
SEQ ID NO: 33


767
SEQ ID NO: 122
SEQ ID NO: 34


768
SEQ ID NO: 122
SEQ ID NO: 35


769
SEQ ID NO: 122
SEQ ID NO: 59


770
SEQ ID NO: 122
SEQ ID NO: 60


771
SEQ ID NO: 122
SEQ ID NO: 61


772
SEQ ID NO: 122
SEQ ID NO: 67


773
SEQ ID NO: 122
SEQ ID NO: 69


774
SEQ ID NO: 122
SEQ ID NO: 112


775
SEQ ID NO: 122
SEQ ID NO: 113


776
SEQ ID NO: 122
SEQ ID NO: 114


777
SEQ ID NO: 122
SEQ ID NO: 123


778
SEQ ID NO: 122
SEQ ID NO: 124


779
SEQ ID NO: 122
SEQ ID NO: 125


780
SEQ ID NO: 122
SEQ ID NO: 126


781
SEQ ID NO: 122
SEQ ID NO: 127


782
SEQ ID NO: 122
SEQ ID NO: 128


783
SEQ ID NO: 122
SEQ ID NO: 129


784
SEQ ID NO: 122
SEQ ID NO: 130


785
SEQ ID NO: 122
SEQ ID NO: 131


786
SEQ ID NO: 122
SEQ ID NO: 132


787
SEQ ID NO: 141
SEQ ID NO: 142


788
SEQ ID NO: 143
SEQ ID NO: 142


789
SEQ ID NO: 145
SEQ ID NO: 142


790
SEQ ID NO: 147
SEQ ID NO: 142


791
SEQ ID NO: 149
SEQ ID NO: 142


792
SEQ ID NO: 151
SEQ ID NO: 142


793
SEQ ID NO: 153
SEQ ID NO: 142


794
SEQ ID NO: 155
SEQ ID NO: 142


795
SEQ ID NO: 156
SEQ ID NO: 142


796
SEQ ID NO: 164
SEQ ID NO: 142


797
SEQ ID NO: 165
SEQ ID NO: 142


798
SEQ ID NO: 166
SEQ ID NO: 142


799
SEQ ID NO: 167
SEQ ID NO: 142


800
SEQ ID NO: 168
SEQ ID NO: 142


801
SEQ ID NO: 169
SEQ ID NO: 142


802
SEQ ID NO: 170
SEQ ID NO: 142


803
SEQ ID NO: 171
SEQ ID NO: 142


804
SEQ ID NO: 172
SEQ ID NO: 142


805
SEQ ID NO: 173
SEQ ID NO: 142


806
SEQ ID NO: 174
SEQ ID NO: 142


807
SEQ ID NO: 175
SEQ ID NO: 142


808
SEQ ID NO: 176
SEQ ID NO: 142


809
SEQ ID NO: 177
SEQ ID NO: 142


810
SEQ ID NO: 178
SEQ ID NO: 142


811
SEQ ID NO: 179
SEQ ID NO: 142


812
SEQ ID NO: 180
SEQ ID NO: 142


813
SEQ ID NO: 181
SEQ ID NO: 142


814
SEQ ID NO: 182
SEQ ID NO: 142


815
SEQ ID NO: 183
SEQ ID NO: 142


816
SEQ ID NO: 184
SEQ ID NO: 142


817
SEQ ID NO: 185
SEQ ID NO: 142


818
SEQ ID NO: 186
SEQ ID NO: 142


819
SEQ ID NO: 187
SEQ ID NO: 142


820
SEQ ID NO: 188
SEQ ID NO: 142


821
SEQ ID NO: 189
SEQ ID NO: 142


822
SEQ ID NO: 190
SEQ ID NO: 142


823
SEQ ID NO: 191
SEQ ID NO: 142


824
SEQ ID NO: 192
SEQ ID NO: 142


825
SEQ ID NO: 193
SEQ ID NO: 142


826
SEQ ID NO: 194
SEQ ID NO: 142


827
SEQ ID NO: 195
SEQ ID NO: 142


828
SEQ ID NO: 196
SEQ ID NO: 142


829
SEQ ID NO: 197
SEQ ID NO: 142


830
SEQ ID NO: 198
SEQ ID NO: 142


831
SEQ ID NO: 199
SEQ ID NO: 142


832
SEQ ID NO: 200
SEQ ID NO: 142


833
SEQ ID NO: 201
SEQ ID NO: 142


834
SEQ ID NO: 202
SEQ ID NO: 142


835
SEQ ID NO: 203
SEQ ID NO: 142


836
SEQ ID NO: 204
SEQ ID NO: 142


837
SEQ ID NO: 205
SEQ ID NO: 142


838
SEQ ID NO: 206
SEQ ID NO: 142


839
SEQ ID NO: 207
SEQ ID NO: 142


840
SEQ ID NO: 208
SEQ ID NO: 142


841
SEQ ID NO: 209
SEQ ID NO: 142


842
SEQ ID NO: 210
SEQ ID NO: 142


843
SEQ ID NO: 211
SEQ ID NO: 142


844
SEQ ID NO: 212
SEQ ID NO: 142


845
SEQ ID NO: 213
SEQ ID NO: 142


846
SEQ ID NO: 214
SEQ ID NO: 142


847
SEQ ID NO: 215
SEQ ID NO: 142


848
SEQ ID NO: 216
SEQ ID NO: 142


849
SEQ ID NO: 217
SEQ ID NO: 142


850
SEQ ID NO: 218
SEQ ID NO: 142


851
SEQ ID NO: 219
SEQ ID NO: 142


852
SEQ ID NO: 220
SEQ ID NO: 142


853
SEQ ID NO: 221
SEQ ID NO: 142


854
SEQ ID NO: 222
SEQ ID NO: 142


855
SEQ ID NO: 223
SEQ ID NO: 142


856
SEQ ID NO: 224
SEQ ID NO: 142


857
SEQ ID NO: 225
SEQ ID NO: 142


858
SEQ ID NO: 226
SEQ ID NO: 142


859
SEQ ID NO: 227
SEQ ID NO: 142


860
SEQ ID NO: 228
SEQ ID NO: 142


861
SEQ ID NO: 229
SEQ ID NO: 142


862
SEQ ID NO: 230
SEQ ID NO: 142


863
SEQ ID NO: 231
SEQ ID NO: 142


864
SEQ ID NO: 232
SEQ ID NO: 142


865
SEQ ID NO: 233
SEQ ID NO: 142


866
SEQ ID NO: 234
SEQ ID NO: 142


867
SEQ ID NO: 235
SEQ ID NO: 142


868
SEQ ID NO: 236
SEQ ID NO: 142


869
SEQ ID NO: 237
SEQ ID NO: 142


870
SEQ ID NO: 238
SEQ ID NO: 142


871
SEQ ID NO: 239
SEQ ID NO: 142


872
SEQ ID NO: 240
SEQ ID NO: 142


873
SEQ ID NO: 241
SEQ ID NO: 142


874
SEQ ID NO: 242
SEQ ID NO: 142


875
SEQ ID NO: 243
SEQ ID NO: 142


876
SEQ ID NO: 244
SEQ ID NO: 142


877
SEQ ID NO: 245
SEQ ID NO: 142


878
SEQ ID NO: 246
SEQ ID NO: 142


879
SEQ ID NO: 247
SEQ ID NO: 142


880
SEQ ID NO: 248
SEQ ID NO: 142


881
SEQ ID NO: 249
SEQ ID NO: 142


882
SEQ ID NO: 250
SEQ ID NO: 142


883
SEQ ID NO: 251
SEQ ID NO: 142


884
SEQ ID NO: 252
SEQ ID NO: 142


885
SEQ ID NO: 253
SEQ ID NO: 142


886
SEQ ID NO: 254
SEQ ID NO: 142


887
SEQ ID NO: 255
SEQ ID NO: 142


888
SEQ ID NO: 256
SEQ ID NO: 142


889
SEQ ID NO: 257
SEQ ID NO: 142


890
SEQ ID NO: 258
SEQ ID NO: 142


891
SEQ ID NO: 259
SEQ ID NO: 142


892
SEQ ID NO: 260
SEQ ID NO: 142


893
SEQ ID NO: 261
SEQ ID NO: 142


894
SEQ ID NO: 262
SEQ ID NO: 142


895
SEQ ID NO: 263
SEQ ID NO: 142


896
SEQ ID NO: 264
SEQ ID NO: 142


897
SEQ ID NO: 265
SEQ ID NO: 142


898
SEQ ID NO: 266
SEQ ID NO: 142


899
SEQ ID NO: 267
SEQ ID NO: 142


900
SEQ ID NO: 268
SEQ ID NO: 142


901
SEQ ID NO: 269
SEQ ID NO: 142


902
SEQ ID NO: 270
SEQ ID NO: 142


903
SEQ ID NO: 271
SEQ ID NO: 142


904
SEQ ID NO: 272
SEQ ID NO: 142


905
SEQ ID NO: 273
SEQ ID NO: 142


906
SEQ ID NO: 274
SEQ ID NO: 142


907
SEQ ID NO: 275
SEQ ID NO: 142


908
SEQ ID NO: 276
SEQ ID NO: 142


909
SEQ ID NO: 277
SEQ ID NO: 142


910
SEQ ID NO: 278
SEQ ID NO: 142


911
SEQ ID NO: 279
SEQ ID NO: 142


912
SEQ ID NO: 280
SEQ ID NO: 142


913
SEQ ID NO: 281
SEQ ID NO: 142


914
SEQ ID NO: 282
SEQ ID NO: 142


915
SEQ ID NO: 283
SEQ ID NO: 142


916
SEQ ID NO: 284
SEQ ID NO: 142


917
SEQ ID NO: 285
SEQ ID NO: 142


918
SEQ ID NO: 286
SEQ ID NO: 142


919
SEQ ID NO: 287
SEQ ID NO: 142


920
SEQ ID NO: 288
SEQ ID NO: 142


921
SEQ ID NO: 289
SEQ ID NO: 142


922
SEQ ID NO: 141
SEQ ID NO: 144


923
SEQ ID NO: 143
SEQ ID NO: 144


924
SEQ ID NO: 145
SEQ ID NO: 144


925
SEQ ID NO: 147
SEQ ID NO: 144


926
SEQ ID NO: 149
SEQ ID NO: 144


927
SEQ ID NO: 151
SEQ ID NO: 144


928
SEQ ID NO: 153
SEQ ID NO: 144


929
SEQ ID NO: 155
SEQ ID NO: 144


930
SEQ ID NO: 156
SEQ ID NO: 144


931
SEQ ID NO: 164
SEQ ID NO: 144


932
SEQ ID NO: 165
SEQ ID NO: 144


933
SEQ ID NO: 166
SEQ ID NO: 144


934
SEQ ID NO: 167
SEQ ID NO: 144


935
SEQ ID NO: 168
SEQ ID NO: 144


936
SEQ ID NO: 169
SEQ ID NO: 144


937
SEQ ID NO: 170
SEQ ID NO: 144


938
SEQ ID NO: 171
SEQ ID NO: 144


939
SEQ ID NO: 172
SEQ ID NO: 144


940
SEQ ID NO: 173
SEQ ID NO: 144


941
SEQ ID NO: 174
SEQ ID NO: 144


942
SEQ ID NO: 175
SEQ ID NO: 144


943
SEQ ID NO: 176
SEQ ID NO: 144


944
SEQ ID NO: 177
SEQ ID NO: 144


945
SEQ ID NO: 178
SEQ ID NO: 144


946
SEQ ID NO: 179
SEQ ID NO: 144


947
SEQ ID NO: 180
SEQ ID NO: 144


948
SEQ ID NO: 181
SEQ ID NO: 144


949
SEQ ID NO: 182
SEQ ID NO: 144


950
SEQ ID NO: 183
SEQ ID NO: 144


951
SEQ ID NO: 184
SEQ ID NO: 144


952
SEQ ID NO: 185
SEQ ID NO: 144


953
SEQ ID NO: 186
SEQ ID NO: 144


954
SEQ ID NO: 187
SEQ ID NO: 144


955
SEQ ID NO: 188
SEQ ID NO: 144


956
SEQ ID NO: 189
SEQ ID NO: 144


957
SEQ ID NO: 190
SEQ ID NO: 144


958
SEQ ID NO: 191
SEQ ID NO: 144


959
SEQ ID NO: 192
SEQ ID NO: 144


960
SEQ ID NO: 193
SEQ ID NO: 144


961
SEQ ID NO: 194
SEQ ID NO: 144


962
SEQ ID NO: 195
SEQ ID NO: 144


963
SEQ ID NO: 196
SEQ ID NO: 144


964
SEQ ID NO: 197
SEQ ID NO: 144


965
SEQ ID NO: 198
SEQ ID NO: 144


966
SEQ ID NO: 199
SEQ ID NO: 144


967
SEQ ID NO: 200
SEQ ID NO: 144


968
SEQ ID NO: 201
SEQ ID NO: 144


969
SEQ ID NO: 202
SEQ ID NO: 144


970
SEQ ID NO: 203
SEQ ID NO: 144


971
SEQ ID NO: 204
SEQ ID NO: 144


972
SEQ ID NO: 205
SEQ ID NO: 144


973
SEQ ID NO: 206
SEQ ID NO: 144


974
SEQ ID NO: 207
SEQ ID NO: 144


975
SEQ ID NO: 208
SEQ ID NO: 144


976
SEQ ID NO: 209
SEQ ID NO: 144


977
SEQ ID NO: 210
SEQ ID NO: 144


978
SEQ ID NO: 211
SEQ ID NO: 144


979
SEQ ID NO: 212
SEQ ID NO: 144


980
SEQ ID NO: 213
SEQ ID NO: 144


981
SEQ ID NO: 214
SEQ ID NO: 144


982
SEQ ID NO: 215
SEQ ID NO: 144


983
SEQ ID NO: 216
SEQ ID NO: 144


984
SEQ ID NO: 217
SEQ ID NO: 144


985
SEQ ID NO: 218
SEQ ID NO: 144


986
SEQ ID NO: 219
SEQ ID NO: 144


987
SEQ ID NO: 220
SEQ ID NO: 144


988
SEQ ID NO: 221
SEQ ID NO: 144


989
SEQ ID NO: 222
SEQ ID NO: 144


990
SEQ ID NO: 223
SEQ ID NO: 144


991
SEQ ID NO: 224
SEQ ID NO: 144


992
SEQ ID NO: 225
SEQ ID NO: 144


993
SEQ ID NO: 226
SEQ ID NO: 144


994
SEQ ID NO: 227
SEQ ID NO: 144


995
SEQ ID NO: 228
SEQ ID NO: 144


996
SEQ ID NO: 229
SEQ ID NO: 144


997
SEQ ID NO: 230
SEQ ID NO: 144


998
SEQ ID NO: 231
SEQ ID NO: 144


999
SEQ ID NO: 232
SEQ ID NO: 144


1000
SEQ ID NO: 233
SEQ ID NO: 144


1001
SEQ ID NO: 234
SEQ ID NO: 144


1002
SEQ ID NO: 235
SEQ ID NO: 144


1003
SEQ ID NO: 236
SEQ ID NO: 144


1004
SEQ ID NO: 237
SEQ ID NO: 144


1005
SEQ ID NO: 238
SEQ ID NO: 144


1006
SEQ ID NO: 239
SEQ ID NO: 144


1007
SEQ ID NO: 240
SEQ ID NO: 144


1008
SEQ ID NO: 241
SEQ ID NO: 144


1009
SEQ ID NO: 242
SEQ ID NO: 144


1010
SEQ ID NO: 243
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1011
SEQ ID NO: 244
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1012
SEQ ID NO: 245
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1013
SEQ ID NO: 246
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1014
SEQ ID NO: 247
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1015
SEQ ID NO: 248
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1016
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1017
SEQ ID NO: 250
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1018
SEQ ID NO: 251
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1019
SEQ ID NO: 252
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1020
SEQ ID NO: 253
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1021
SEQ ID NO: 254
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1022
SEQ ID NO: 255
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1023
SEQ ID NO: 256
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1024
SEQ ID NO: 257
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1025
SEQ ID NO: 258
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1026
SEQ ID NO: 259
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1027
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1028
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1029
SEQ ID NO: 262
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1030
SEQ ID NO: 263
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1031
SEQ ID NO: 264
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1032
SEQ ID NO: 265
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1033
SEQ ID NO: 266
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1034
SEQ ID NO: 267
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1035
SEQ ID NO: 268
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1036
SEQ ID NO: 269
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1037
SEQ ID NO: 270
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1038
SEQ ID NO: 271
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1039
SEQ ID NO: 272
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1040
SEQ ID NO: 273
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1041
SEQ ID NO: 274
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1042
SEQ ID NO: 275
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1043
SEQ ID NO: 276
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1044
SEQ ID NO: 277
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1045
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1046
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1047
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1048
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1049
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1050
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1051
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1052
SEQ ID NO: 285
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1053
SEQ ID NO: 286
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1054
SEQ ID NO: 287
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1055
SEQ ID NO: 288
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1056
SEQ ID NO: 289
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1057
SEQ ID NO: 141
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1058
SEQ ID NO: 143
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1059
SEQ ID NO: 145
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1060
SEQ ID NO: 147
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1061
SEQ ID NO: 149
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1062
SEQ ID NO: 151
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1063
SEQ ID NO: 153
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1064
SEQ ID NO: 155
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1065
SEQ ID NO: 156
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1066
SEQ ID NO: 164
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1067
SEQ ID NO: 165
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1068
SEQ ID NO: 166
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1069
SEQ ID NO: 167
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1070
SEQ ID NO: 168
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1071
SEQ ID NO: 169
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1072
SEQ ID NO: 170
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1073
SEQ ID NO: 171
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1074
SEQ ID NO: 172
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1075
SEQ ID NO: 173
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1076
SEQ ID NO: 174
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1077
SEQ ID NO: 175
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1078
SEQ ID NO: 176
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1079
SEQ ID NO: 177
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1080
SEQ ID NO: 178
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1081
SEQ ID NO: 179
SEQ ID NO: 146


1082
SEQ ID NO: 180
SEQ ID NO: 146


1083
SEQ ID NO: 181
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1084
SEQ ID NO: 182
SEQ ID NO: 146


1085
SEQ ID NO: 183
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1086
SEQ ID NO: 184
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1087
SEQ ID NO: 185
SEQ ID NO: 146


1088
SEQ ID NO: 186
SEQ ID NO: 146


1089
SEQ ID NO: 187
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1090
SEQ ID NO: 188
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1091
SEQ ID NO: 189
SEQ ID NO: 146


1092
SEQ ID NO: 190
SEQ ID NO: 146


1093
SEQ ID NO: 191
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1094
SEQ ID NO: 192
SEQ ID NO: 146


1095
SEQ ID NO: 193
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1096
SEQ ID NO: 194
SEQ ID NO: 146


1097
SEQ ID NO: 195
SEQ ID NO: 146


1098
SEQ ID NO: 196
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1099
SEQ ID NO: 197
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1100
SEQ ID NO: 198
SEQ ID NO: 146


1101
SEQ ID NO: 199
SEQ ID NO: 146


1102
SEQ ID NO: 200
SEQ ID NO: 146


1103
SEQ ID NO: 201
SEQ ID NO: 146


1104
SEQ ID NO: 202
SEQ ID NO: 146


1105
SEQ ID NO: 203
SEQ ID NO: 146


1106
SEQ ID NO: 204
SEQ ID NO: 146


1107
SEQ ID NO: 205
SEQ ID NO: 146


1108
SEQ ID NO: 206
SEQ ID NO: 146


1109
SEQ ID NO: 207
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1110
SEQ ID NO: 208
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1111
SEQ ID NO: 209
SEQ ID NO: 146


1112
SEQ ID NO: 210
SEQ ID NO: 146


1113
SEQ ID NO: 211
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1114
SEQ ID NO: 212
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1115
SEQ ID NO: 213
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1116
SEQ ID NO: 214
SEQ ID NO: 146


1117
SEQ ID NO: 215
SEQ ID NO: 146


1118
SEQ ID NO: 216
SEQ ID NO: 146


1119
SEQ ID NO: 217
SEQ ID NO: 146


1120
SEQ ID NO: 218
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1121
SEQ ID NO: 219
SEQ ID NO: 146


1122
SEQ ID NO: 220
SEQ ID NO: 146


1123
SEQ ID NO: 221
SEQ ID NO: 146


1124
SEQ ID NO: 222
SEQ ID NO: 146


1125
SEQ ID NO: 223
SEQ ID NO: 146


1126
SEQ ID NO: 224
SEQ ID NO: 146


1127
SEQ ID NO: 225
SEQ ID NO: 146


1128
SEQ ID NO: 226
SEQ ID NO: 146


1129
SEQ ID NO: 227
SEQ ID NO: 146


1130
SEQ ID NO: 228
SEQ ID NO: 146


1131
SEQ ID NO: 229
SEQ ID NO: 146


1132
SEQ ID NO: 230
SEQ ID NO: 146


1133
SEQ ID NO: 231
SEQ ID NO: 146


1134
SEQ ID NO: 232
SEQ ID NO: 146


1135
SEQ ID NO: 233
SEQ ID NO: 146


1136
SEQ ID NO: 234
SEQ ID NO: 146


1137
SEQ ID NO: 235
SEQ ID NO: 146


1138
SEQ ID NO: 236
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1139
SEQ ID NO: 237
SEQ ID NO: 146


1140
SEQ ID NO: 238
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1141
SEQ ID NO: 239
SEQ ID NO: 146


1142
SEQ ID NO: 240
SEQ ID NO: 146


1143
SEQ ID NO: 241
SEQ ID NO: 146


1144
SEQ ID NO: 242
SEQ ID NO: 146


1145
SEQ ID NO: 243
SEQ ID NO: 146


1146
SEQ ID NO: 244
SEQ ID NO: 146


1147
SEQ ID NO: 245
SEQ ID NO: 146


1148
SEQ ID NO: 246
SEQ ID NO: 146


1149
SEQ ID NO: 247
SEQ ID NO: 146


1150
SEQ ID NO: 248
SEQ ID NO: 146


1151
SEQ ID NO: 249
SEQ ID NO: 146


1152
SEQ ID NO: 250
SEQ ID NO: 146


1153
SEQ ID NO: 251
SEQ ID NO: 146


1154
SEQ ID NO: 252
SEQ ID NO: 146


1155
SEQ ID NO: 253
SEQ ID NO: 146


1156
SEQ ID NO: 254
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1157
SEQ ID NO: 255
SEQ ID NO: 146


1158
SEQ ID NO: 256
SEQ ID NO: 146


1159
SEQ ID NO: 257
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1160
SEQ ID NO: 258
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1161
SEQ ID NO: 259
SEQ ID NO: 146


1162
SEQ ID NO: 260
SEQ ID NO: 146


1163
SEQ ID NO: 261
SEQ ID NO: 146


1164
SEQ ID NO: 262
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1165
SEQ ID NO: 263
SEQ ID NO: 146


1166
SEQ ID NO: 264
SEQ ID NO: 146


1167
SEQ ID NO: 265
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1168
SEQ ID NO: 266
SEQ ID NO: 146


1169
SEQ ID NO: 267
SEQ ID NO: 146


1170
SEQ ID NO: 268
SEQ ID NO: 146


1171
SEQ ID NO: 269
SEQ ID NO: 146


1172
SEQ ID NO: 270
SEQ ID NO: 146


1173
SEQ ID NO: 271
SEQ ID NO: 146


1174
SEQ ID NO: 272
SEQ ID NO: 146


1175
SEQ ID NO: 273
SEQ ID NO: 146


1176
SEQ ID NO: 274
SEQ ID NO: 146


1177
SEQ ID NO: 275
SEQ ID NO: 146


1178
SEQ ID NO: 276
SEQ ID NO: 146


1179
SEQ ID NO: 277
SEQ ID NO: 146


1180
SEQ ID NO: 278
SEQ ID NO: 146


1181
SEQ ID NO: 279
SEQ ID NO: 146


1182
SEQ ID NO: 280
SEQ ID NO: 146


1183
SEQ ID NO: 281
SEQ ID NO: 146


1184
SEQ ID NO: 282
SEQ ID NO: 146


1185
SEQ ID NO: 283
SEQ ID NO: 146


1186
SEQ ID NO: 284
SEQ ID NO: 146


1187
SEQ ID NO: 285
SEQ ID NO: 146


1188
SEQ ID NO: 286
SEQ ID NO: 146


1189
SEQ ID NO: 287
SEQ ID NO: 146


1190
SEQ ID NO: 288
SEQ ID NO: 146


1191
SEQ ID NO: 289
SEQ ID NO: 146


1192
SEQ ID NO: 141
SEQ ID NO: 148


1193
SEQ ID NO: 143
SEQ ID NO: 148


1194
SEQ ID NO: 145
SEQ ID NO: 148


1195
SEQ ID NO: 147
SEQ ID NO: 148


1196
SEQ ID NO: 149
SEQ ID NO: 148


1197
SEQ ID NO: 151
SEQ ID NO: 148


1198
SEQ ID NO: 153
SEQ ID NO: 148


1199
SEQ ID NO: 155
SEQ ID NO: 148


1200
SEQ ID NO: 156
SEQ ID NO: 148


1201
SEQ ID NO: 164
SEQ ID NO: 148


1202
SEQ ID NO: 165
SEQ ID NO: 148


1203
SEQ ID NO: 166
SEQ ID NO: 148


1204
SEQ ID NO: 167
SEQ ID NO: 148


1205
SEQ ID NO: 168
SEQ ID NO: 148


1206
SEQ ID NO: 169
SEQ ID NO: 148


1207
SEQ ID NO: 170
SEQ ID NO: 148


1208
SEQ ID NO: 171
SEQ ID NO: 148


1209
SEQ ID NO: 172
SEQ ID NO: 148


1210
SEQ ID NO: 173
SEQ ID NO: 148


1211
SEQ ID NO: 174
SEQ ID NO: 148


1212
SEQ ID NO: 175
SEQ ID NO: 148


1213
SEQ ID NO: 176
SEQ ID NO: 148


1214
SEQ ID NO: 177
SEQ ID NO: 148


1215
SEQ ID NO: 178
SEQ ID NO: 148


1216
SEQ ID NO: 179
SEQ ID NO: 148


1217
SEQ ID NO: 180
SEQ ID NO: 148


1218
SEQ ID NO: 181
SEQ ID NO: 148


1219
SEQ ID NO: 182
SEQ ID NO: 148


1220
SEQ ID NO: 183
SEQ ID NO: 148


1221
SEQ ID NO: 184
SEQ ID NO: 148


1222
SEQ ID NO: 185
SEQ ID NO: 148


1223
SEQ ID NO: 186
SEQ ID NO: 148


1224
SEQ ID NO: 187
SEQ ID NO: 148


1225
SEQ ID NO: 188
SEQ ID NO: 148


1226
SEQ ID NO: 189
SEQ ID NO: 148


1227
SEQ ID NO: 190
SEQ ID NO: 148


1228
SEQ ID NO: 191
SEQ ID NO: 148


1229
SEQ ID NO: 192
SEQ ID NO: 148


1230
SEQ ID NO: 193
SEQ ID NO: 148


1231
SEQ ID NO: 194
SEQ ID NO: 148


1232
SEQ ID NO: 195
SEQ ID NO: 148


1233
SEQ ID NO: 196
SEQ ID NO: 148


1234
SEQ ID NO: 197
SEQ ID NO: 148


1235
SEQ ID NO: 198
SEQ ID NO: 148


1236
SEQ ID NO: 199
SEQ ID NO: 148


1237
SEQ ID NO: 200
SEQ ID NO: 148


1238
SEQ ID NO: 201
SEQ ID NO: 148


1239
SEQ ID NO: 202
SEQ ID NO: 148


1240
SEQ ID NO: 203
SEQ ID NO: 148


1241
SEQ ID NO: 204
SEQ ID NO: 148


1242
SEQ ID NO: 205
SEQ ID NO: 148


1243
SEQ ID NO: 206
SEQ ID NO: 148


1244
SEQ ID NO: 207
SEQ ID NO: 148


1245
SEQ ID NO: 208
SEQ ID NO: 148


1246
SEQ ID NO: 209
SEQ ID NO: 148


1247
SEQ ID NO: 210
SEQ ID NO: 148


1248
SEQ ID NO: 211
SEQ ID NO: 148


1249
SEQ ID NO: 212
SEQ ID NO: 148


1250
SEQ ID NO: 213
SEQ ID NO: 148


1251
SEQ ID NO: 214
SEQ ID NO: 148


1252
SEQ ID NO: 215
SEQ ID NO: 148


1253
SEQ ID NO: 216
SEQ ID NO: 148


1254
SEQ ID NO: 217
SEQ ID NO: 148


1255
SEQ ID NO: 218
SEQ ID NO: 148


1256
SEQ ID NO: 219
SEQ ID NO: 148


1257
SEQ ID NO: 220
SEQ ID NO: 148


1258
SEQ ID NO: 221
SEQ ID NO: 148


1259
SEQ ID NO: 222
SEQ ID NO: 148


1260
SEQ ID NO: 223
SEQ ID NO: 148


1261
SEQ ID NO: 224
SEQ ID NO: 148


1262
SEQ ID NO: 225
SEQ ID NO: 148


1263
SEQ ID NO: 226
SEQ ID NO: 148


1264
SEQ ID NO: 227
SEQ ID NO: 148


1265
SEQ ID NO: 228
SEQ ID NO: 148


1266
SEQ ID NO: 229
SEQ ID NO: 148


1267
SEQ ID NO: 230
SEQ ID NO: 148


1268
SEQ ID NO: 231
SEQ ID NO: 148


1269
SEQ ID NO: 232
SEQ ID NO: 148


1270
SEQ ID NO: 233
SEQ ID NO: 148


1271
SEQ ID NO: 234
SEQ ID NO: 148


1272
SEQ ID NO: 235
SEQ ID NO: 148


1273
SEQ ID NO: 236
SEQ ID NO: 148


1274
SEQ ID NO: 237
SEQ ID NO: 148


1275
SEQ ID NO: 238
SEQ ID NO: 148


1276
SEQ ID NO: 239
SEQ ID NO: 148


1277
SEQ ID NO: 240
SEQ ID NO: 148


1278
SEQ ID NO: 241
SEQ ID NO: 148


1279
SEQ ID NO: 242
SEQ ID NO: 148


1280
SEQ ID NO: 243
SEQ ID NO: 148


1281
SEQ ID NO: 244
SEQ ID NO: 148


1282
SEQ ID NO: 245
SEQ ID NO: 148


1283
SEQ ID NO: 246
SEQ ID NO: 148


1284
SEQ ID NO: 247
SEQ ID NO: 148


1285
SEQ ID NO: 248
SEQ ID NO: 148


1286
SEQ ID NO: 249
SEQ ID NO: 148


1287
SEQ ID NO: 250
SEQ ID NO: 148


1288
SEQ ID NO: 251
SEQ ID NO: 148


1289
SEQ ID NO: 252
SEQ ID NO: 148


1290
SEQ ID NO: 253
SEQ ID NO: 148


1291
SEQ ID NO: 254
SEQ ID NO: 148


1292
SEQ ID NO: 255
SEQ ID NO: 148


1293
SEQ ID NO: 256
SEQ ID NO: 148


1294
SEQ ID NO: 257
SEQ ID NO: 148


1295
SEQ ID NO: 258
SEQ ID NO: 148


1296
SEQ ID NO: 259
SEQ ID NO: 148


1297
SEQ ID NO: 260
SEQ ID NO: 148


1298
SEQ ID NO: 261
SEQ ID NO: 148


1299
SEQ ID NO: 262
SEQ ID NO: 148


1300
SEQ ID NO: 263
SEQ ID NO: 148


1301
SEQ ID NO: 264
SEQ ID NO: 148


1302
SEQ ID NO: 265
SEQ ID NO: 148


1303
SEQ ID NO: 266
SEQ ID NO: 148


1304
SEQ ID NO: 267
SEQ ID NO: 148


1305
SEQ ID NO: 268
SEQ ID NO: 148


1306
SEQ ID NO: 269
SEQ ID NO: 148


1307
SEQ ID NO: 270
SEQ ID NO: 148


1308
SEQ ID NO: 271
SEQ ID NO: 148


1309
SEQ ID NO: 272
SEQ ID NO: 148


1310
SEQ ID NO: 273
SEQ ID NO: 148


1311
SEQ ID NO: 274
SEQ ID NO: 148


1312
SEQ ID NO: 275
SEQ ID NO: 148


1313
SEQ ID NO: 276
SEQ ID NO: 148


1314
SEQ ID NO: 277
SEQ ID NO: 148


1315
SEQ ID NO: 278
SEQ ID NO: 148


1316
SEQ ID NO: 279
SEQ ID NO: 148


1317
SEQ ID NO: 280
SEQ ID NO: 148


1318
SEQ ID NO: 281
SEQ ID NO: 148


1319
SEQ ID NO: 282
SEQ ID NO: 148


1320
SEQ ID NO: 283
SEQ ID NO: 148


1321
SEQ ID NO: 284
SEQ ID NO: 148


1322
SEQ ID NO: 285
SEQ ID NO: 148


1323
SEQ ID NO: 286
SEQ ID NO: 148


1324
SEQ ID NO: 287
SEQ ID NO: 148


1325
SEQ ID NO: 288
SEQ ID NO: 148


1326
SEQ ID NO: 289
SEQ ID NO: 148


1327
SEQ ID NO: 141
SEQ ID NO: 150


1328
SEQ ID NO: 143
SEQ ID NO: 150


1329
SEQ ID NO: 145
SEQ ID NO: 150


1330
SEQ ID NO: 147
SEQ ID NO: 150


1331
SEQ ID NO: 149
SEQ ID NO: 150


1332
SEQ ID NO: 151
SEQ ID NO: 150


1333
SEQ ID NO: 153
SEQ ID NO: 150


1334
SEQ ID NO: 155
SEQ ID NO: 150


1335
SEQ ID NO: 156
SEQ ID NO: 150


1336
SEQ ID NO: 164
SEQ ID NO: 150


1337
SEQ ID NO: 165
SEQ ID NO: 150


1338
SEQ ID NO: 166
SEQ ID NO: 150


1339
SEQ ID NO: 167
SEQ ID NO: 150


1340
SEQ ID NO: 168
SEQ ID NO: 150


1341
SEQ ID NO: 169
SEQ ID NO: 150


1342
SEQ ID NO: 170
SEQ ID NO: 150


1343
SEQ ID NO: 171
SEQ ID NO: 150


1344
SEQ ID NO: 172
SEQ ID NO: 150


1345
SEQ ID NO: 173
SEQ ID NO: 150


1346
SEQ ID NO: 174
SEQ ID NO: 150


1347
SEQ ID NO: 175
SEQ ID NO: 150


1348
SEQ ID NO: 176
SEQ ID NO: 150


1349
SEQ ID NO: 177
SEQ ID NO: 150


1350
SEQ ID NO: 178
SEQ ID NO: 150


1351
SEQ ID NO: 179
SEQ ID NO: 150


1352
SEQ ID NO: 180
SEQ ID NO: 150


1353
SEQ ID NO: 181
SEQ ID NO: 150


1354
SEQ ID NO: 182
SEQ ID NO: 150


1355
SEQ ID NO: 183
SEQ ID NO: 150


1356
SEQ ID NO: 184
SEQ ID NO: 150


1357
SEQ ID NO: 185
SEQ ID NO: 150


1358
SEQ ID NO: 186
SEQ ID NO: 150


1359
SEQ ID NO: 187
SEQ ID NO: 150


1360
SEQ ID NO: 188
SEQ ID NO: 150


1361
SEQ ID NO: 189
SEQ ID NO: 150


1362
SEQ ID NO: 190
SEQ ID NO: 150


1363
SEQ ID NO: 191
SEQ ID NO: 150


1364
SEQ ID NO: 192
SEQ ID NO: 150


1365
SEQ ID NO: 193
SEQ ID NO: 150


1366
SEQ ID NO: 194
SEQ ID NO: 150


1367
SEQ ID NO: 195
SEQ ID NO: 150


1368
SEQ ID NO: 196
SEQ ID NO: 150


1369
SEQ ID NO: 197
SEQ ID NO: 150


1370
SEQ ID NO: 198
SEQ ID NO: 150


1371
SEQ ID NO: 199
SEQ ID NO: 150


1372
SEQ ID NO: 200
SEQ ID NO: 150


1373
SEQ ID NO: 201
SEQ ID NO: 150


1374
SEQ ID NO: 202
SEQ ID NO: 150


1375
SEQ ID NO: 203
SEQ ID NO: 150


1376
SEQ ID NO: 204
SEQ ID NO: 150


1377
SEQ ID NO: 205
SEQ ID NO: 150


1378
SEQ ID NO: 206
SEQ ID NO: 150


1379
SEQ ID NO: 207
SEQ ID NO: 150


1380
SEQ ID NO: 208
SEQ ID NO: 150


1381
SEQ ID NO: 209
SEQ ID NO: 150


1382
SEQ ID NO: 210
SEQ ID NO: 150


1383
SEQ ID NO: 211
SEQ ID NO: 150


1384
SEQ ID NO: 212
SEQ ID NO: 150


1385
SEQ ID NO: 213
SEQ ID NO: 150


1386
SEQ ID NO: 214
SEQ ID NO: 150


1387
SEQ ID NO: 215
SEQ ID NO: 150


1388
SEQ ID NO: 216
SEQ ID NO: 150


1389
SEQ ID NO: 217
SEQ ID NO: 150


1390
SEQ ID NO: 218
SEQ ID NO: 150


1391
SEQ ID NO: 219
SEQ ID NO: 150


1392
SEQ ID NO: 220
SEQ ID NO: 150


1393
SEQ ID NO: 221
SEQ ID NO: 150


1394
SEQ ID NO: 222
SEQ ID NO: 150


1395
SEQ ID NO: 223
SEQ ID NO: 150


1396
SEQ ID NO: 224
SEQ ID NO: 150


1397
SEQ ID NO: 225
SEQ ID NO: 150


1398
SEQ ID NO: 226
SEQ ID NO: 150


1399
SEQ ID NO: 227
SEQ ID NO: 150


1400
SEQ ID NO: 228
SEQ ID NO: 150


1401
SEQ ID NO: 229
SEQ ID NO: 150


1402
SEQ ID NO: 230
SEQ ID NO: 150


1403
SEQ ID NO: 231
SEQ ID NO: 150


1404
SEQ ID NO: 232
SEQ ID NO: 150


1405
SEQ ID NO: 233
SEQ ID NO: 150


1406
SEQ ID NO: 234
SEQ ID NO: 150


1407
SEQ ID NO: 235
SEQ ID NO: 150


1408
SEQ ID NO: 236
SEQ ID NO: 150


1409
SEQ ID NO: 237
SEQ ID NO: 150


1410
SEQ ID NO: 238
SEQ ID NO: 150


1411
SEQ ID NO: 239
SEQ ID NO: 150


1412
SEQ ID NO: 240
SEQ ID NO: 150


1413
SEQ ID NO: 241
SEQ ID NO: 150


1414
SEQ ID NO: 242
SEQ ID NO: 150


1415
SEQ ID NO: 243
SEQ ID NO: 150


1416
SEQ ID NO: 244
SEQ ID NO: 150


1417
SEQ ID NO: 245
SEQ ID NO: 150


1418
SEQ ID NO: 246
SEQ ID NO: 150


1419
SEQ ID NO: 247
SEQ ID NO: 150


1420
SEQ ID NO: 248
SEQ ID NO: 150


1421
SEQ ID NO: 249
SEQ ID NO: 150


1422
SEQ ID NO: 250
SEQ ID NO: 150


1423
SEQ ID NO: 251
SEQ ID NO: 150


1424
SEQ ID NO: 252
SEQ ID NO: 150


1425
SEQ ID NO: 253
SEQ ID NO: 150


1426
SEQ ID NO: 254
SEQ ID NO: 150


1427
SEQ ID NO: 255
SEQ ID NO: 150


1428
SEQ ID NO: 256
SEQ ID NO: 150


1429
SEQ ID NO: 257
SEQ ID NO: 150


1430
SEQ ID NO: 258
SEQ ID NO: 150


1431
SEQ ID NO: 259
SEQ ID NO: 150


1432
SEQ ID NO: 260
SEQ ID NO: 150


1433
SEQ ID NO: 261
SEQ ID NO: 150


1434
SEQ ID NO: 262
SEQ ID NO: 150


1435
SEQ ID NO: 263
SEQ ID NO: 150


1436
SEQ ID NO: 264
SEQ ID NO: 150


1437
SEQ ID NO: 265
SEQ ID NO: 150


1438
SEQ ID NO: 266
SEQ ID NO: 150


1439
SEQ ID NO: 267
SEQ ID NO: 150


1440
SEQ ID NO: 268
SEQ ID NO: 150


1441
SEQ ID NO: 269
SEQ ID NO: 150


1442
SEQ ID NO: 270
SEQ ID NO: 150


1443
SEQ ID NO: 271
SEQ ID NO: 150


1444
SEQ ID NO: 272
SEQ ID NO: 150


1445
SEQ ID NO: 273
SEQ ID NO: 150


1446
SEQ ID NO: 274
SEQ ID NO: 150


1447
SEQ ID NO: 275
SEQ ID NO: 150


1448
SEQ ID NO: 276
SEQ ID NO: 150


1449
SEQ ID NO: 277
SEQ ID NO: 150


1450
SEQ ID NO: 278
SEQ ID NO: 150


1451
SEQ ID NO: 279
SEQ ID NO: 150


1452
SEQ ID NO: 280
SEQ ID NO: 150


1453
SEQ ID NO: 281
SEQ ID NO: 150


1454
SEQ ID NO: 282
SEQ ID NO: 150


1455
SEQ ID NO: 283
SEQ ID NO: 150


1456
SEQ ID NO: 284
SEQ ID NO: 150


1457
SEQ ID NO: 285
SEQ ID NO: 150


1458
SEQ ID NO: 286
SEQ ID NO: 150


1459
SEQ ID NO: 287
SEQ ID NO: 150


1460
SEQ ID NO: 288
SEQ ID NO: 150


1461
SEQ ID NO: 289
SEQ ID NO: 150


1462
SEQ ID NO: 141
SEQ ID NO: 152


1463
SEQ ID NO: 143
SEQ ID NO: 152


1464
SEQ ID NO: 145
SEQ ID NO: 152


1465
SEQ ID NO: 147
SEQ ID NO: 152


1466
SEQ ID NO: 149
SEQ ID NO: 152


1467
SEQ ID NO: 151
SEQ ID NO: 152


1468
SEQ ID NO: 153
SEQ ID NO: 152


1469
SEQ ID NO: 155
SEQ ID NO: 152


1470
SEQ ID NO: 156
SEQ ID NO: 152


1471
SEQ ID NO: 164
SEQ ID NO: 152


1472
SEQ ID NO: 165
SEQ ID NO: 152


1473
SEQ ID NO: 166
SEQ ID NO: 152


1474
SEQ ID NO: 167
SEQ ID NO: 152


1475
SEQ ID NO: 168
SEQ ID NO: 152


1476
SEQ ID NO: 169
SEQ ID NO: 152


1477
SEQ ID NO: 170
SEQ ID NO: 152


1478
SEQ ID NO: 171
SEQ ID NO: 152


1479
SEQ ID NO: 172
SEQ ID NO: 152


1480
SEQ ID NO: 173
SEQ ID NO: 152


1481
SEQ ID NO: 174
SEQ ID NO: 152


1482
SEQ ID NO: 175
SEQ ID NO: 152


1483
SEQ ID NO: 176
SEQ ID NO: 152


1484
SEQ ID NO: 177
SEQ ID NO: 152


1485
SEQ ID NO: 178
SEQ ID NO: 152


1486
SEQ ID NO: 179
SEQ ID NO: 152


1487
SEQ ID NO: 180
SEQ ID NO: 152


1488
SEQ ID NO: 181
SEQ ID NO: 152


1489
SEQ ID NO: 182
SEQ ID NO: 152


1490
SEQ ID NO: 183
SEQ ID NO: 152


1491
SEQ ID NO: 184
SEQ ID NO: 152


1492
SEQ ID NO: 185
SEQ ID NO: 152


1493
SEQ ID NO: 186
SEQ ID NO: 152


1494
SEQ ID NO: 187
SEQ ID NO: 152


1495
SEQ ID NO: 188
SEQ ID NO: 152


1496
SEQ ID NO: 189
SEQ ID NO: 152


1497
SEQ ID NO: 190
SEQ ID NO: 152


1498
SEQ ID NO: 191
SEQ ID NO: 152


1499
SEQ ID NO: 192
SEQ ID NO: 152


1500
SEQ ID NO: 193
SEQ ID NO: 152


1501
SEQ ID NO: 194
SEQ ID NO: 152


1502
SEQ ID NO: 195
SEQ ID NO: 152


1503
SEQ ID NO: 196
SEQ ID NO: 152


1504
SEQ ID NO: 197
SEQ ID NO: 152


1505
SEQ ID NO: 198
SEQ ID NO: 152


1506
SEQ ID NO: 199
SEQ ID NO: 152


1507
SEQ ID NO: 200
SEQ ID NO: 152


1508
SEQ ID NO: 201
SEQ ID NO: 152


1509
SEQ ID NO: 202
SEQ ID NO: 152


1510
SEQ ID NO: 203
SEQ ID NO: 152


1511
SEQ ID NO: 204
SEQ ID NO: 152


1512
SEQ ID NO: 205
SEQ ID NO: 152


1513
SEQ ID NO: 206
SEQ ID NO: 152


1514
SEQ ID NO: 207
SEQ ID NO: 152


1515
SEQ ID NO: 208
SEQ ID NO: 152


1516
SEQ ID NO: 209
SEQ ID NO: 152


1517
SEQ ID NO: 210
SEQ ID NO: 152


1518
SEQ ID NO: 211
SEQ ID NO: 152


1519
SEQ ID NO: 212
SEQ ID NO: 152


1520
SEQ ID NO: 213
SEQ ID NO: 152


1521
SEQ ID NO: 214
SEQ ID NO: 152


1522
SEQ ID NO: 215
SEQ ID NO: 152


1523
SEQ ID NO: 216
SEQ ID NO: 152


1524
SEQ ID NO: 217
SEQ ID NO: 152


1525
SEQ ID NO: 218
SEQ ID NO: 152


1526
SEQ ID NO: 219
SEQ ID NO: 152


1527
SEQ ID NO: 220
SEQ ID NO: 152


1528
SEQ ID NO: 221
SEQ ID NO: 152


1529
SEQ ID NO: 222
SEQ ID NO: 152


1530
SEQ ID NO: 223
SEQ ID NO: 152


1531
SEQ ID NO: 224
SEQ ID NO: 152


1532
SEQ ID NO: 225
SEQ ID NO: 152


1533
SEQ ID NO: 226
SEQ ID NO: 152


1534
SEQ ID NO: 227
SEQ ID NO: 152


1535
SEQ ID NO: 228
SEQ ID NO: 152


1536
SEQ ID NO: 229
SEQ ID NO: 152


1537
SEQ ID NO: 230
SEQ ID NO: 152


1538
SEQ ID NO: 231
SEQ ID NO: 152


1539
SEQ ID NO: 232
SEQ ID NO: 152


1540
SEQ ID NO: 233
SEQ ID NO: 152


1541
SEQ ID NO: 234
SEQ ID NO: 152


1542
SEQ ID NO: 235
SEQ ID NO: 152


1543
SEQ ID NO: 236
SEQ ID NO: 152


1544
SEQ ID NO: 237
SEQ ID NO: 152


1545
SEQ ID NO: 238
SEQ ID NO: 152


1546
SEQ ID NO: 239
SEQ ID NO: 152


1547
SEQ ID NO: 240
SEQ ID NO: 152


1548
SEQ ID NO: 241
SEQ ID NO: 152


1549
SEQ ID NO: 242
SEQ ID NO: 152


1550
SEQ ID NO: 243
SEQ ID NO: 152


1551
SEQ ID NO: 244
SEQ ID NO: 152


1552
SEQ ID NO: 245
SEQ ID NO: 152


1553
SEQ ID NO: 246
SEQ ID NO: 152


1554
SEQ ID NO: 247
SEQ ID NO: 152


1555
SEQ ID NO: 248
SEQ ID NO: 152


1556
SEQ ID NO: 249
SEQ ID NO: 152


1557
SEQ ID NO: 250
SEQ ID NO: 152


1558
SEQ ID NO: 251
SEQ ID NO: 152


1559
SEQ ID NO: 252
SEQ ID NO: 152


1560
SEQ ID NO: 253
SEQ ID NO: 152


1561
SEQ ID NO: 254
SEQ ID NO: 152


1562
SEQ ID NO: 255
SEQ ID NO: 152


1563
SEQ ID NO: 256
SEQ ID NO: 152


1564
SEQ ID NO: 257
SEQ ID NO: 152


1565
SEQ ID NO: 258
SEQ ID NO: 152


1566
SEQ ID NO: 259
SEQ ID NO: 152


1567
SEQ ID NO: 260
SEQ ID NO: 152


1568
SEQ ID NO: 261
SEQ ID NO: 152


1569
SEQ ID NO: 262
SEQ ID NO: 152


1570
SEQ ID NO: 263
SEQ ID NO: 152


1571
SEQ ID NO: 264
SEQ ID NO: 152


1572
SEQ ID NO: 265
SEQ ID NO: 152


1573
SEQ ID NO: 266
SEQ ID NO: 152


1574
SEQ ID NO: 267
SEQ ID NO: 152


1575
SEQ ID NO: 268
SEQ ID NO: 152


1576
SEQ ID NO: 269
SEQ ID NO: 152


1577
SEQ ID NO: 270
SEQ ID NO: 152


1578
SEQ ID NO: 271
SEQ ID NO: 152


1579
SEQ ID NO: 272
SEQ ID NO: 152


1580
SEQ ID NO: 273
SEQ ID NO: 152


1581
SEQ ID NO: 274
SEQ ID NO: 152


1582
SEQ ID NO: 275
SEQ ID NO: 152


1583
SEQ ID NO: 276
SEQ ID NO: 152


1584
SEQ ID NO: 277
SEQ ID NO: 152


1585
SEQ ID NO: 278
SEQ ID NO: 152


1586
SEQ ID NO: 279
SEQ ID NO: 152


1587
SEQ ID NO: 280
SEQ ID NO: 152


1588
SEQ ID NO: 281
SEQ ID NO: 152


1589
SEQ ID NO: 282
SEQ ID NO: 152


1590
SEQ ID NO: 283
SEQ ID NO: 152


1591
SEQ ID NO: 284
SEQ ID NO: 152


1592
SEQ ID NO: 285
SEQ ID NO: 152


1593
SEQ ID NO: 286
SEQ ID NO: 152


1594
SEQ ID NO: 287
SEQ ID NO: 152


1595
SEQ ID NO: 288
SEQ ID NO: 152


1596
SEQ ID NO: 289
SEQ ID NO: 152


1597
SEQ ID NO: 141
SEQ ID NO: 154


1598
SEQ ID NO: 143
SEQ ID NO: 154


1599
SEQ ID NO: 145
SEQ ID NO: 154


1600
SEQ ID NO: 147
SEQ ID NO: 154


1601
SEQ ID NO: 149
SEQ ID NO: 154


1602
SEQ ID NO: 151
SEQ ID NO: 154


1603
SEQ ID NO: 153
SEQ ID NO: 154


1604
SEQ ID NO: 155
SEQ ID NO: 154


1605
SEQ ID NO: 156
SEQ ID NO: 154


1606
SEQ ID NO: 164
SEQ ID NO: 154


1607
SEQ ID NO: 165
SEQ ID NO: 154


1608
SEQ ID NO: 166
SEQ ID NO: 154


1609
SEQ ID NO: 167
SEQ ID NO: 154


1610
SEQ ID NO: 168
SEQ ID NO: 154


1611
SEQ ID NO: 169
SEQ ID NO: 154


1612
SEQ ID NO: 170
SEQ ID NO: 154


1613
SEQ ID NO: 171
SEQ ID NO: 154


1614
SEQ ID NO: 172
SEQ ID NO: 154


1615
SEQ ID NO: 173
SEQ ID NO: 154


1616
SEQ ID NO: 174
SEQ ID NO: 154


1617
SEQ ID NO: 175
SEQ ID NO: 154


1618
SEQ ID NO: 176
SEQ ID NO: 154


1619
SEQ ID NO: 177
SEQ ID NO: 154


1620
SEQ ID NO: 178
SEQ ID NO: 154


1621
SEQ ID NO: 179
SEQ ID NO: 154


1622
SEQ ID NO: 180
SEQ ID NO: 154


1623
SEQ ID NO: 181
SEQ ID NO: 154


1624
SEQ ID NO: 182
SEQ ID NO: 154


1625
SEQ ID NO: 183
SEQ ID NO: 154


1626
SEQ ID NO: 184
SEQ ID NO: 154


1627
SEQ ID NO: 185
SEQ ID NO: 154


1628
SEQ ID NO: 186
SEQ ID NO: 154


1629
SEQ ID NO: 187
SEQ ID NO: 154


1630
SEQ ID NO: 188
SEQ ID NO: 154


1631
SEQ ID NO: 189
SEQ ID NO: 154


1632
SEQ ID NO: 190
SEQ ID NO: 154


1633
SEQ ID NO: 191
SEQ ID NO: 154


1634
SEQ ID NO: 192
SEQ ID NO: 154


1635
SEQ ID NO: 193
SEQ ID NO: 154


1636
SEQ ID NO: 194
SEQ ID NO: 154


1637
SEQ ID NO: 195
SEQ ID NO: 154


1638
SEQ ID NO: 196
SEQ ID NO: 154


1639
SEQ ID NO: 197
SEQ ID NO: 154


1640
SEQ ID NO: 198
SEQ ID NO: 154


1641
SEQ ID NO: 199
SEQ ID NO: 154


1642
SEQ ID NO: 200
SEQ ID NO: 154


1643
SEQ ID NO: 201
SEQ ID NO: 154


1644
SEQ ID NO: 202
SEQ ID NO: 154


1645
SEQ ID NO: 203
SEQ ID NO: 154


1646
SEQ ID NO: 204
SEQ ID NO: 154


1647
SEQ ID NO: 205
SEQ ID NO: 154


1648
SEQ ID NO: 206
SEQ ID NO: 154


1649
SEQ ID NO: 207
SEQ ID NO: 154


1650
SEQ ID NO: 208
SEQ ID NO: 154


1651
SEQ ID NO: 209
SEQ ID NO: 154


1652
SEQ ID NO: 210
SEQ ID NO: 154


1653
SEQ ID NO: 211
SEQ ID NO: 154


1654
SEQ ID NO: 212
SEQ ID NO: 154


1655
SEQ ID NO: 213
SEQ ID NO: 154


1656
SEQ ID NO: 214
SEQ ID NO: 154


1657
SEQ ID NO: 215
SEQ ID NO: 154


1658
SEQ ID NO: 216
SEQ ID NO: 154


1659
SEQ ID NO: 217
SEQ ID NO: 154


1660
SEQ ID NO: 218
SEQ ID NO: 154


1661
SEQ ID NO: 219
SEQ ID NO: 154


1662
SEQ ID NO: 220
SEQ ID NO: 154


1663
SEQ ID NO: 221
SEQ ID NO: 154


1664
SEQ ID NO: 222
SEQ ID NO: 154


1665
SEQ ID NO: 223
SEQ ID NO: 154


1666
SEQ ID NO: 224
SEQ ID NO: 154


1667
SEQ ID NO: 225
SEQ ID NO: 154


1668
SEQ ID NO: 226
SEQ ID NO: 154


1669
SEQ ID NO: 227
SEQ ID NO: 154


1670
SEQ ID NO: 228
SEQ ID NO: 154


1671
SEQ ID NO: 229
SEQ ID NO: 154


1672
SEQ ID NO: 230
SEQ ID NO: 154


1673
SEQ ID NO: 231
SEQ ID NO: 154


1674
SEQ ID NO: 232
SEQ ID NO: 154


1675
SEQ ID NO: 233
SEQ ID NO: 154


1676
SEQ ID NO: 234
SEQ ID NO: 154


1677
SEQ ID NO: 235
SEQ ID NO: 154


1678
SEQ ID NO: 236
SEQ ID NO: 154


1679
SEQ ID NO: 237
SEQ ID NO: 154


1680
SEQ ID NO: 238
SEQ ID NO: 154


1681
SEQ ID NO: 239
SEQ ID NO: 154


1682
SEQ ID NO: 240
SEQ ID NO: 154


1683
SEQ ID NO: 241
SEQ ID NO: 154


1684
SEQ ID NO: 242
SEQ ID NO: 154


1685
SEQ ID NO: 243
SEQ ID NO: 154


1686
SEQ ID NO: 244
SEQ ID NO: 154


1687
SEQ ID NO: 245
SEQ ID NO: 154


1688
SEQ ID NO: 246
SEQ ID NO: 154


1689
SEQ ID NO: 247
SEQ ID NO: 154


1690
SEQ ID NO: 248
SEQ ID NO: 154


1691
SEQ ID NO: 249
SEQ ID NO: 154


1692
SEQ ID NO: 250
SEQ ID NO: 154


1693
SEQ ID NO: 251
SEQ ID NO: 154


1694
SEQ ID NO: 252
SEQ ID NO: 154


1695
SEQ ID NO: 253
SEQ ID NO: 154


1696
SEQ ID NO: 254
SEQ ID NO: 154


1697
SEQ ID NO: 255
SEQ ID NO: 154


1698
SEQ ID NO: 256
SEQ ID NO: 154


1699
SEQ ID NO: 257
SEQ ID NO: 154


1700
SEQ ID NO: 258
SEQ ID NO: 154


1701
SEQ ID NO: 259
SEQ ID NO: 154


1702
SEQ ID NO: 260
SEQ ID NO: 154


1703
SEQ ID NO: 261
SEQ ID NO: 154


1704
SEQ ID NO: 262
SEQ ID NO: 154


1705
SEQ ID NO: 263
SEQ ID NO: 154


1706
SEQ ID NO: 264
SEQ ID NO: 154


1707
SEQ ID NO: 265
SEQ ID NO: 154


1708
SEQ ID NO: 266
SEQ ID NO: 154


1709
SEQ ID NO: 267
SEQ ID NO: 154


1710
SEQ ID NO: 268
SEQ ID NO: 154


1711
SEQ ID NO: 269
SEQ ID NO: 154


1712
SEQ ID NO: 270
SEQ ID NO: 154


1713
SEQ ID NO: 271
SEQ ID NO: 154


1714
SEQ ID NO: 272
SEQ ID NO: 154


1715
SEQ ID NO: 273
SEQ ID NO: 154


1716
SEQ ID NO: 274
SEQ ID NO: 154


1717
SEQ ID NO: 275
SEQ ID NO: 154


1718
SEQ ID NO: 276
SEQ ID NO: 154


1719
SEQ ID NO: 277
SEQ ID NO: 154


1720
SEQ ID NO: 278
SEQ ID NO: 154


1721
SEQ ID NO: 279
SEQ ID NO: 154


1722
SEQ ID NO: 280
SEQ ID NO: 154


1723
SEQ ID NO: 281
SEQ ID NO: 154


1724
SEQ ID NO: 282
SEQ ID NO: 154


1725
SEQ ID NO: 283
SEQ ID NO: 154


1726
SEQ ID NO: 284
SEQ ID NO: 154


1727
SEQ ID NO: 285
SEQ ID NO: 154


1728
SEQ ID NO: 286
SEQ ID NO: 154


1729
SEQ ID NO: 287
SEQ ID NO: 154


1730
SEQ ID NO: 288
SEQ ID NO: 154


1731
SEQ ID NO: 289
SEQ ID NO: 154









In certain embodiments, the anti-FGFR3 antigen binding protein or fragment thereof comprises a pair of heavy chain and light chain of Table 1.1 above.


In certain embodiments, the anti-FGFR3 antigen binding protein or fragment thereof comprises a VH domain and a VL domain, wherein:


(a) the VH domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122; and the VL domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 60, SEQ ID NO: 61SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132;


(b) the VH domain comprises the amino acid sequence of SEQ ID NO: 8; and the VL domain comprises the amino acid sequence of SEQ ID NO: 9;


(c) the VH domain comprises the amino acid sequence of SEQ ID NO: 10; and the VL domain comprises the amino acid sequence of SEQ ID NO: 11;


(d) the VH domain comprises the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises the amino acid sequence of SEQ ID NO: 13;


(e) the VH domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; and the VL domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;


(f) the VH domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; the VL domain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.


In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 63 or 65, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67 or 69.


Variants of the anti-FGFR3 antigen binding protein or fragment thereof as described herein are also provided. In certain embodiments, the VH domain of a variant is at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122, and the VL domain of the variant is at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, or SEQ ID NO: 132. The amino acid sequence alignment for obtaining the identity can be any conventional amino acid sequence alignment tool, and the sequence alignment algorithms includes Needle-Wunsch algorithm, Smith-Waterman algorithm, or Karling & Altschul algorithm, but is not limited thereto; the amino acid sequence alignment tool includes BLAST (Basic Local Alignment Search Tool), BLAT (BLAST-like Alignment Tool), Grapped BLAST or FASTA, but is not limited thereto. In certain embodiments, the variant has all of the identical heavy chain CDRs and light chain CDRs of the anti-FGFR3 antigen binding protein or fragment thereof as described herein, with modifications in the constant region on the heavy chain and/or the light chain.


In certain embodiments, the anti-FGFR3 antigen binding protein or fragment thereof comprises an antibody heavy chain at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 63 or 65, and an antibody light chain at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 67 or 69.


In certain embodiments, the anti-FGFR3 antigen binding protein or fragment thereof comprises a VH domain and a VL domain, wherein: (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), a CDR-H2 sequence comprising the amino acid sequence of VDPETGGT (SEQ ID NO: 297), and a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAFDY (SEQ ID NO: 301); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSNNNKNY (SEQ ID NO: 302), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NOs: 74, 92, 98, and 104), and a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 57, and the VL domain comprises the amino acid sequence of SEQ ID NO: 19 or 59.


In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 63, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67. In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 65, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the anti-FGFR3 antigen binding protein or fragment thereof comprises a VH domain and a VL domain, wherein: (a) the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), a CDR-H2 sequence comprising the amino acid sequence of VDPETGGT (SEQ ID NO: 297), and a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAFDY (SEQ ID NO: 301); and (b) the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSDNQKNY (SEQ ID NO: 306), a CDR-L2 sequence comprising the amino acid sequence of FAS (SEQ ID NO: 304), and a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75).


In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 57, and the VL domain comprises the amino acid sequence of SEQ ID NO: 61.


In certain embodiments, the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 65, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure comprise one or more sequences with at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any of the sequences of Table 1, Table 3, Table 4, Table 8, Table 13, or Table 14, or protein sequences encoded by the nucleic acid sequences in Table 9.


Also provided herein are human framework regions for anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof. Non-limiting examples of human framework regions are provided below.









TABLE 1.2







Human Framework regions of anti-FGFR3 antigen binding


proteins and antigen-binding fragments thereof









Framework




type
Sequence
Note





Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP
X is any naturally occurring amino acid, and



GQGLEWIGA(X)n2AYNQKFQGRVTITADKSTSTAYMEL
n1, n2, and n3 are numbers at least 3, and less



SSLRSEDTAVYYC(X)n3WGQGTLVTVSS
than 50, each X can be the same or different



SEQ ID NO: 318
amino acid as the amino acid next to it





Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGA(X)n2AYNQKFQGRVTITADKSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 319






Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGA(X)n2AYNQKFQGRVTITADKSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 320






Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGA(X)n2AYNQKFQGRVTITADKSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 321






Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGG(X)n2AYNQKFQGRVTITADRSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 322






Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGG(X)n2AYNQKFQGRVTITADRSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 323






Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGG(X)n2AYNQKFQGRVTITADRSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 324






Heavy chain
QVQLVQSGAEVKKPGASVKVSCKAS(X)n1IHWVRQAP




GQGLEWIGG(X)n2AYNQKFQGRVTITADRSTSTAYMEL




SSLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 325






Heavy chain
EVQLVQSGAEVKKPGATVKLSCKAS(X)n1IHWVQQAPG




KGLEWIGD(X)n2AYAEKFQGRATLTADRSTDTAYMELS




SLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 326






Heavy chain
EVQLVQSGAEVKKPGATVKLSCKAS(X)n1IHWVQQAPG




KGLEWIGD(X)n2AYAEKFQGRATLTADRSTDTAYLELS




SLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 327






Heavy chain
EVQLVQSGAEVKKPGATVKLSCKAS(X)n1IHWVQQAPG




KGLEWIGD(X)n2AYAEKFQGRATLTADRSTDTAYLELS




SLRSEDTAVYYC(X)n3WGQGTLVTVSS




SEQ ID NO: 328






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1ILAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 329






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 330






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 331






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 332






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 333






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 334






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 335






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSLQAE




DVAVYYC(X)n3FGQGTKLEIK




SEQ ID NO: 336






Light chain
DIVMTQSPDSLAVSLGERATINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSVQA




EDVAVYYC(X)n3FGGGTKVEIK




SEQ ID NO: 337






Light chain
DIVMTQSPDSLAVSLGERATINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSVQA




EDVAVYYC(X)n3FGGGTKVEIK




SEQ ID NO: 338






Light chain
DIVMTQSPDSLAVSLGERVTINCKSS(X)n1LAWYQQKPG




QSPKLLIY(X)n2TRESGVPDRFSGSGSGTDFTLTISSVQA




EDVAVYYC(X)n3FGGGTKLEIK




SEQ ID NO: 339









In some embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments comprise three heavy chain CDRs that bind to FGFR3, such as the three heavy chain CDRs of mouse antibody KC18, KE35, KE42, KE58, KE63, or KE94, and a human heavy chain variable region framework. In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments further comprise three light chain CDRs that bind to FGFR3, such as the three light chain CDRs of mouse antibody KC18, KE35, KE42, KE58, KE63, or KE94, and a human light chain variable region framework. In certain embodiments, the human heavy chain variable region framework comprises any one of SEQ ID NOs: 318 to 328. In certain embodiments, the human light chain variable region framework comprises any one of SEQ ID NOs: 329 to 339. In certain embodiments, the human heavy chain variable region framework comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to any one of SEQ ID NOs: 318 to 328. In certain embodiments, the human light chain variable region framework comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to any one of SEQ ID NOs: 329 to 339. In certain embodiments, such anti-FGFR3 antigen binding proteins and antigen-binding fragments bind human FGFR3 with an equilibrium dissociation constant (KD) of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2.89 nM or less, about 2 nM or less, about 1.5 or less, about 1.4 or less, about 1.23 or less, about 1.2 or less, or about 1 nM or less, about 0.8 or less, or about 0.6 or less. “X nM or less” therein includes the embodiment “less than X nM”. “Less” therein can mean e.g. to about 2.8 nM, to about 1.3 nM, to about 1.1 nM, to about 0.7 nM or to about 0.5 nM. These lower limits can be used to form ranges with any of the aforementioned upper limits, such as about 3 nM or <3 nM to about 2.8 nM, about 1.5 nM or <1.5 nM to about 1.3 nM, about 1.3 nM or <1.3 nM to about 1.1 nM, about 0.9 nM or <0.9 nM to about 0.7 nM, or about 0.7 nM or <0.7 nM to about 0.5 nM. “About X” therein can mean e.g. “X±5%”, “X±4%”, “X±4%”, “X±3%”, “X±2%”, “X±1%” or “X±0.5%”. In certain embodiments, the nM values are as obtained by a Surface Plasmon Resonance assay, such as the Biacore assay.


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure are chimeric or humanized antibodies. In certain embodiments, the antigen binding protein is a humanized antibody.


In certain embodiments, the antigen binding protein is a monoclonal antibody.


In certain embodiments, the antigen binding protein comprises one or more full-length antibody heavy chains comprising an Fc region. In certain embodiments, the Fc region is a human IgG1 Fc region. In certain embodiments, the Fc region is a human IgG4 Fc region.


In certain embodiments, the antibody Fc region comprises one or more mutations that reduces Fc effector function. In certain embodiments, the one or more mutations reduces one or more of antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or complement dependent cytotoxicity (CDC). In certain embodiments, the human IgG1 Fc region comprises a L234A and L235A mutation. In certain embodiments, the human IgG4 Fc region comprises a F234A and L235A mutation. These IgG1 and IgG4 mutations are also known as the “LALA” and “FALA” mutations, respectively, and are described in further detail in Xu et al. (Cell Immunol. 2000; 200:16-26). In certain embodiments, the human IgG4 Fc region comprises one or more stabilizing mutations, including, but limited to, mutations in the IgG4 hinge that reduce or prevent the formation of disulfide bonds and in vivo fab arm exchange (FAE). In certain embodiments, the human IgG4 Fc region comprises a S228P mutation. The IgG4 hinge mutation is described in further detail in Angal et al. (Mol. Immunol. 1993; 30:105-108). In certain embodiments, the human IgG4 Fc region comprises a S228P mutation and a L235A mutation. In certain embodiments, the human IgG1 Fc region comprises one or more mutations that alters antibody glycosylation. In certain embodiments, the human IgG1 Fc region comprises one or more of a S298N mutation, a T299A mutation, and a Y300S mutation. In certain embodiments, the human IgG1 Fc region comprises a S298N mutation, a T299A mutation, and a Y300S mutation. The Fc region amino acid positions referred to herein are based on EU antibody numbering.


In certain embodiments, the anti-FGFR3 antigen binding protein fragments of the disclosure comprise or consists of an antibody F(ab), F(ab′)2, Fab′-SH, Fv, or scFv fragment. In certain embodiments, the antigen binding protein fragment comprises an antibody F(ab) fragment.


The antibody F(ab) fragment can be modified with one or more serum half-life extending moieties. In certain embodiments, the antibody F(ab) fragment is conjugated to an antigen binding protein with binding specificity to serum albumin. In certain embodiments, the antigen binding protein with binding specificity to serum albumin is a nanobody. In certain embodiments, the serum albumin is human serum albumin or mouse serum albumin.


In certain embodiments, the antibody F(ab) fragment comprises a heavy chain and a light chain. In certain embodiments, the heavy chain of the F(ab) fragment comprises a heavy chain variable region disclosed herewith, and a light chain variable region disclosed herewith. In certain embodiments, the heavy chain variable region comprises or consists of any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 28, 29, 30, 31, 56, 57, 58, 106, 107, 108, 109, 110, 111, 115, 116, 117, 118, 119, 120, 121, or 112, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to any of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 28, 29, 30, 31, 56, 57, 58, 106, 107, 108, 109, 110, 111, 115, 116, 117, 118, 119, 120, 121, or 112. In certain embodiments, the light chain variable region comprises or consists of any one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 24, 25, 26, 27, 32, 33, 34, 35, 59, 60, 61, 112, 113, 114, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132, or a or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to any of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 24, 25, 26, 27, 32, 33, 34, 35, 59, 60, 61, 112, 113, 114, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132.


In certain embodiments, the heavy chain of the F(ab) fragment comprises or consists of a heavy chain variable region disclosed herewith and a heavy chain constant region. In certain embodiments, the heavy chain constant region is derived from a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, or a combination thereof. In certain embodiments, the heavy chain constant region is a fragment of a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, a human IgG4 Fc region, or a combination thereof. In certain embodiments, the heavy chain constant region comprises or consists of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 5, 36, 37, 38, 39, 40, 41, 44, 45, 46, 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 amino acids of a human IgG Fc region, such as the human IgG1 Fc region (SEQ ID NO: 54).


In certain embodiments, the light chain of the F(ab) fragment comprises or consists of a light chain variable region disclosed herewith, and a light chain constant region. In certain embodiments, the light chain constant region is derived from a human kappa (x) chain, a human lambda (k) chain, or a combination thereof. In certain embodiments, the light chain constant region comprises or consists of a part of IgG1 light constant region. In certain embodiments, the part of the IgG1 light constant region comprises or consists of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 5, 36, 37, 38, 39, 40, 41, 44, 45, 46, 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107 amino acids of SEQ ID NO: 55.


In certain embodiments, the F(ab) fragment comprises one or more point mutations in the C-terminus to reduce anti-Fab antibody binding.


In certain embodiments, the antigen binding protein comprises cross-reactivity to one or both of mouse and cynomolgus FGFR3. In certain embodiments, the cynomolgus FGFR3 is encoded by SEQ ID NO: 136:









(SEQ ID NO: 136)


ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCAGTGGCCATCGT





GGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGC





GAGTGGCAGAAGTGTCCGGCCCGGAGCCCAGCCAGCAGGAGCAGTTGGTC





TTCGGCAGCGGGGACGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGG





TCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCGCAGGGCTGGTGCCCT





CGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCC





CACGAGGACTCTGGGGCCTACAGCTGCCGGCAGCGGCTCACACAGCTCGT





ACTGTGCCACTTCAGTGTGCGGGTGACAGATGCTCCATCCTCGGGAGATG





ACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCC





CCTTACTGGACTCGGCCCGAGCGGATGGACAAGAAGCTGCTGGCTGTGCC





GGCCGCCAACACCGTCCGCTTCCGCTGCCCGGCTGCCGGCAACCCCACTC





CCTCCATCTCCTGGCTGAAGAATGGCAAGGAGTTCCGCGGCGAGCACCGC





ATTGGCGGCATCAAGCTTCGGCACCAGCAGTGGAGCCTGGTCATGGAAAG





CGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTGGTGGAGAACAAGT





TTGGCAGCATCCGGCAGACATACACGCTGGACGTGCTGGAGCGCTCCCCG





CACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCT





GGGCAGCGATGTGGAGTTTCACTGCAAGGTGTACAGTGATGCGCAGCCCC





ACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCC





GACGGCACACCCTACGTCACCGTGCTCAAGACGGCGGGCGCTAATACCAC





CGACAAGGAGCTAGAGGTTCTGTCCTTGCACAACGTCACCTTTGAGGACG





CCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCCCATCAC





TCTGCGTGGCTCGTGGTGCTGCCAGCTGAGGAGGAGCTGGTGGAGGCTGA





CGAGGCGGGCAGTGTGTACGCAGGCATCCTCAGCTACGGGGTGGGCTTCT





TCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGC





ACCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTT





CCCACTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCA





ACACACCGCTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGTCCCACA





CTGGCCAATGTCTCCGAGCTTGAGCTGCCTGCTGACCCCAAATGGGAGCT





GTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTTG





GCCAGGTGGTCATGGCGGAGGCTATCGGCATTGACAAGGACCGGGCCGCC





AAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGATGATGCCACTGACAA





GGACCTGTCAGACCTGGTGTCTGAGATGGAGATGATGAAGATGATTGGGA





AACACAAGAACATTATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCC





CTGTACGTGCTGGTGGAGTACGCGGCCAAGGGCAACCTGAGGGAGTTTCT





GCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGC





CGCCTGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAG





GTGGCCCGAGGCATGGAGTACCTCGCCTCCCAGAAGTGCATCCACAGGGA





CCTGGCTGCTCGAAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCG





CAGACTTCGGGCTGGCCCGCGACGTGCACAACCTTGACTACTACAAGAAG





ACAACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCCTGTT





TGACCGAGTCTACACCCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGC





TCTGGGAGATCTTCACGCTGGGGGGCTCTCCGTACCCCGGCATCCCTGTG





GAGGAGCTCTTCAAGCTGCTGAAGGAGGGTCACCGGATGGACAAGCCGGC





CAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCTG





CGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGT





GTCCTCACTGTGACGTCCACCGACGAGTACCTGGACCTGTCAGCGCCCTT





CGAGCAGTACTCCCCCGGCGGCCAGGACACCCCGAGCTCCAGCTCCTCAG





GGGATGACTCCGTGTTTGCCCACGACCTGCTGCCCCCGGCCCCACCCAGC





AGTGGGGGCTCGCGGACGTGA






In certain embodiments, the cynomolgus FGFR3 is the IIIc isoform, comprising SEQ ID NO: 138, or its mutated version comprising the G380R mutation (SEQ ID NO: 139):









(SEQ ID NO: 138)


MGAPACALALCVAVAIVAGASSESLGTEQRVVGRVAEVSGPEPSQQEQLV





FGSGDAVELSCPPPGGGPMGPTVWVKDGAGLVPSERVLVGPQRLQVLNAS





HEDSGAYSCRQRLTQLVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA





PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGKEFRGEHR





IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP





HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP





DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH





SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS





TPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT





LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA





KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP





LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ





VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK





TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV





EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR





VLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPS





SGGSRT





(SEQ ID NO: 139)


MGAPACALALCVAVAIVAGASSESLGTEQRVVGRVAEVSGPEPSQQEQLV





FGSGDAVELSCPPPGGGPMGPTVWVKDGAGLVPSERVLVGPQRLQVLNAS





HEDSGAYSCRQRLTQLVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA





PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGKEFRGEHR





IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP





HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP





DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH





SAWLVVLPAEEELVEADEAGSVYAGILSYRVGFFLFILVVAAVTLCRLRS





TPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT





LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA





KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP





LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ





VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK





TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV





EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR





VLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPS





SGGSRT






In certain embodiments, the antigen binding protein comprises binding specificity for FGFR3 isoform IIIb and/or isoform IIIc.


In certain embodiments, the antigen binding protein specifically binds to FGFR3, and does not bind to one or more of FGFR1, FGFR2, and FGFR4, or does not have detectable binding to one or more of FGFR1, FGFR2, and FGFR4. In certain embodiments, the antigen binding protein does not bind to each of FGFR1, FGFR2, and FGFR4, or does not have detectable binding to each of FGFR1, FGFR2, and FGFR4. In certain embodiments, the antigen binding protein binds to each of FGFR1, FGFR2, and FGFR4 with an affinity of about 100 μM, 500 μM, 1000 μM, or greater. In certain embodiments, the antigen binding protein does not bind to one or more of FGFR1, FGFR2, and FGFR4 over a background measurement, as determined by a Biacore affinity analysis.


In certain embodiments, the antigen binding protein binds human FGFR3 with an equilibrium dissociation constant (KD) of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2.89 nM or less, about 2 nM or less, about 1.5 or less, about 1.4 or less, about 1.23 or less, about 1.2 or less, or about 1 nM or less, about 0.8 or less, or about 0.6 or less. “X nM or less” therein includes the embodiment “less than X nM”. “Less” therein can mean e.g. to about 2.8 nM, to about 1.3 nM, to about 1.1 nM, to about 0.7 nM or to about 0.5 nM. These lower limits can be used to form ranges with any of the aforementioned upper limits, such as about 3 nM or <3 nM to about 2.8 nM, about 1.5 nM or <1.5 nM to about 1.3 nM, about 1.3 nM or <1.3 nM to about 1.1 nM, about 0.9 nM or <0.9 nM to about 0.7 nM, or about 0.7 nM or <0.7 nM to about 0.5 nM. “About X” therein can mean e.g. “X±5%”, “X±4%”, “X±4%”, “X±3%”, “X±2%”, “X±1%” or “X±0.5%”. In certain embodiments, the nM values are as obtained by a Surface Plasmon Resonance assay, such as the Biacore assay. In certain embodiments, the Biacore assay is carried out at about 4° C., 10° C., 15° C., 20° C., 25° C., 30° C., or 37° C.


In certain embodiments, the antigen binding protein binds human FGFR3 with an off rate (Kd) of about 10−2 s−1 or less, about 5×10−3 s−1 or less, about 2×10−3 s−1 or less, about 10−3 s−1 or less, about 9×10−4 s−1 or less, about 8×10−4 s−1 or less, about 7×10−4 s−1 or less, about 6×10−4 s−1 or less, about 5×10−4 s−1 or less, about 4×10−4 s−1 or less, or about 3.5×10−4 s−1 or less. “Less” therein can mean e.g. to about 3×10−4 s−1. “About X” therein can mean e.g. “X±10%”, “X±5%”, “X±4%”, “X±4%”, “X±3%”, “X±2%”, “X±1%” or “X±0.5%”.


In certain embodiments, the antigen binding protein inhibits ligand-induced FGFR3 dimerization with IC50 of about 5 μg/ml or less, about 4 μg/ml or less, about 3 μg/ml or less, about 2 μg/ml or less, about 1 μg/ml or less, about 0.9 μg/ml or less, about 0.8 μg/ml or less, about 0.7 μg/ml or less, about 0.6 μg/ml or less, about 0.5 μg/ml or less, about 0.4 μg/ml or less, or about 0.3 μg/ml or less. “Less” therein can mean e.g. to about 0.25 μg/ml (or less, e.g. to about 0.2 μg/ml or about 0.1 μg/ml). “About X” therein can mean e.g. “X±10%”, “X±5%”, “X±4%”, “X±4%”, “X±3%”, “X±2%”, “X±1%” or “X±0.5%”. In certain embodiments, the nM values are as obtained by a Homogenous Time-Resolved Fluorescence (HTRF) assay at about 4° C., 10° C., 15° C., 20° C., 25° C., 30° C., or 37° C.


In certain embodiments, the antigen binding protein inhibits FGFR3 receptor activation and downstream signaling with IC50 of about 5 μg/ml or less, about 4 μg/ml or less, about 3 μg/ml or less, about 2 μg/ml or less, about 1 μg/ml or less, about 0.9 μg/ml or less, about 0.8 μg/ml or less, about 0.7 μg/ml or less, about 0.6 μg/ml or less, about 0.5 μg/ml or less, about 0.4 μg/ml or less, or about 0.3 μg/ml or less. “Less” therein can mean e.g. to about 0.25 μg/ml (or less, e.g. to about 0.2 μg/ml or about 0.1 μg/ml). About X” therein can mean e.g. “X±10%”, “X±5%”, “X±4%”, “X±4%”, “X±3%”, “X±2%”, “X±1%” or “X±0.5%”.%”. In certain embodiments, the nM values are as obtained by a homogenous time-resolved fluorescence (HTRF) assay.


Inhibition of FGFR3 receptor activation and downstream signaling can be determined by any means known in the art. In certain embodiments, inhibition of FGFR3 receptor activation and downstream signaling is measured by determining Erk phosphorylation. A decrease in Erk phosphorylation indicates inhibition of FGFR3 activation. Erk phosphorylation can be determined using a homogenous time-resolved fluorescence (HTRF) assay. In certain embodiments, the assay is performed in chondrocytes. In certain embodiments, the assay is performed in mouse primary rib chondrocytes.


In certain embodiments, the antigen binding protein inhibits the activity of an FGFR3G380R mutant. In certain embodiments, the antigen binding protein inhibits the activity of a human FGFR3G380R mutant, a mouse FGFR3G380R mutant, and/or a cynomolgus FGFR3G380R mutant. In certain embodiments, the human FGFR3G380R mutant is represented by the amino acid sequence set forth in SEQ ID NO: 133.


In certain embodiments, the antigen binding protein or fragment thereof is capable of penetrating a bone growth plate.


In certain embodiments, the antigen binding protein or fragment thereof is capable of decreasing the binding of FGFR3 with its ligand in a bone growth plate.


Anti-FGFR3 Antigen-Binding Protein Epitopes


In one aspect, the disclosure provides an antigen-binding protein or fragment thereof with binding specificity to a fibroblast growth factor receptor 3 (FGFR3) epitope, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the antigen binding protein binds a human FGFR3 polypeptide comprising the amino acid sequence of SEQ ID NO: 134, recited below.









(SEQ ID NO: 134)


DTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG





REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYT





LDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVE





VNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAG





NSIGFSHHSAWLVVLPAEEELVE






In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure bind a human FGFR3 polypeptide comprising the amino acid sequence of SEQ ID NO: 134, recited above. The amino acid sequence recited above corresponds to the D2D3 region of FGFR3 isoform IIIc, specifically to amino acid D143 to E365 of FGFR3 isoform IIc.


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure bind an epitope of human FGFR3 polypeptide comprising the N-terminus of the D2 region (amino acids D143 to L163) of SEQ ID NO: 133, shown above.


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure bind an epitope of human FGFR3 polypeptide comprising the N-terminus of the D2 region (amino acids D143 to N170) of SEQ ID NO: 133, shown above.


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure bind an epitope of human FGFR3 polypeptide comprising the N-terminus and middle of the D2 region (amino acids D143 to D160 and G197 to L213) of SEQ ID NO: 133, shown above.


In certain embodiments, the one or more epitopes of the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure are determined by hydrogen deuterium exchange (HDX) mass spectrometry. HDX is performed by measuring the amide hydrogen deuterium exchange on FGFR3 over time. HDX mass spectrometry is described in further detail in Pradzińska et al. (Amino Acids. 48: 2809-2820. 2016).


In certain embodiments, the anti-FGFR3 antigen binding proteins and antigen-binding fragments thereof of the disclosure compete with a reference binding protein for binding to the human FGFR3 polypeptide D2 region.


In one aspect, the disclosure provides an antigen-binding protein or fragment thereof with binding specificity to a fibroblast growth factor receptor 3 (FGFR3) epitope, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the antigen binding protein competes for binding to FGFR3 with an antibody comprising VH/VL domain amino acid sequence pairs selected from the group consisting of: SEQ ID NO: 6/SEQ ID NO: 7, SEQ ID NO: 8/SEQ ID NO: 9, SEQ ID NO: 10/SEQ ID NO: 11, SEQ ID NO: 12/SEQ ID NO: 13, SEQ ID NO: 14/SEQ ID NO: 15, and SEQ ID NO: 16/SEQ ID NO: 17.


Expression of Antigen-Binding Proteins


In one aspect, polynucleotides encoding the binding proteins (e.g., antigen-binding proteins and antigen-binding fragments thereof) disclosed herein are provided. Methods of making binding proteins comprising expressing these polynucleotides are also provided.


Polynucleotides encoding the binding proteins disclosed herein are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of the binding proteins. Accordingly, in certain aspects, the disclosure provides expression vectors comprising polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.


The term “vector” or “expression vector” is used herein to mean vectors used in accordance with the present disclosure as a vehicle for introducing into and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may readily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the disclosure will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.


Numerous expression vector systems may be employed for the purposes of this disclosure. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.


In other embodiments, the binding proteins may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980, which is incorporated by reference herein in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.


More generally, once a vector or DNA sequence encoding a binding protein, e.g. an antibody or fragment thereof, has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host can be by electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.


As used herein, the term “transformation” shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype.


Along those same lines, “host cells” refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of polypeptides from recombinant hosts, the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the “cells” may mean either from spun down whole cells, from supernatant of lysed cells culture, or from the cell culture containing both the medium and the suspended cells.


In one embodiment, a host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HEK (human kidney line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte), 293 (human kidney). In one embodiment, the cell line provides for altered glycosylation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-out CHO cell lines (POTELLIGENT® cells) (Biowa, Princeton, N.J.)). In one embodiment, NSO cells may be used. CHO cells are particularly useful. Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from authors of published literature.


In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography.


Genes encoding the binding proteins featured in the disclosure can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard, it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed, i.e., those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the binding proteins can become part of inclusion bodies. In some embodiments, the binding proteins are then isolated, purified and assembled into functional molecules. In some embodiments, the binding proteins of the disclosure are expressed in a bacterial host cell. In some embodiments, the bacterial host cell is transformed with an expression vector comprising a nucleic acid molecule encoding a binding protein of the disclosure.


In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microbes, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)), is commonly used. This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.


Methods of Preparing/Administering Binding Proteins


Methods of preparing and administering binding proteins (e.g., antigen-binding proteins and antigen-binding fragments thereof disclosed herein) to a subject are also provided. The route of administration of the antigen binding proteins and antigen-binding fragments thereof of the current disclosure may be oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the current disclosure, a form for administration would be a solution for injection, e.g. for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer, a surfactant, optionally a stabilizer agent, etc. However, in other methods compatible with the teachings herein, the modified antibodies can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.


Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that syringability for injection exists. It should be stable under the conditions of manufacture and storage, and should also be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium. The proper fluidity can be maintained, for example, by the use of a coating, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.


Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents. Isotonic agents, for example, sugars, polyalcohols, may also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.


In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a modified binding protein by itself or in combination with other active agents) in a required amount in an appropriate solvent with one or a combination of ingredients enumerated, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and any required other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation typically include vacuum drying and freeze-drying, which yield a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit.


Effective doses of the compositions of the present disclosure, for the treatment of a disease or disorder vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated to optimize safety and efficacy.


Binding proteins described herein can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring levels of binding protein or antigen in the subject. Alternatively, binding proteins can be administered as a sustained release formulation, in which case less frequent administration is required. For antibodies, dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show the longest half-life, followed by chimeric antibodies and non-human antibodies.


The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present binding protein are administered to a subject not already in the disease state to enhance the subject's resistance. In terms of achondroplasia, a prophylactic treatment is understood as a method of preventing or alleviating one or more symptoms of the disorder. In terms of cancer, a prophylactic treatment is understood as a method of preventing the happening of cancer, or alleviating one or more symptoms of the cancer. Such an amount is defined to be a “prophylactic effective dose.” In this use, the precise amounts again depend upon the subject's state of health and general immunity. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the patient shows partial or complete amelioration of disease symptoms. Thereafter, the patient can be administered a prophylactic regime.


Binding proteins described herein can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).


While the binding proteins may be administered as described immediately above, it must be emphasized that in other embodiments binding proteins may be administered to otherwise healthy subjects as a first line therapy. In such embodiments the binding proteins may be administered to subjects that have not, and are not, undergoing one or more other therapies, or subjects that have, or are undergoing one or more other therapies. As used herein, the administration of binding proteins, e.g. antibodies or fragments thereof, in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant, or contemporaneous administration or application of the therapy and the disclosed binding proteins. The administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment.


As previously discussed, the binding proteins of the present disclosure may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders.


Pharmaceutical compositions in accordance with the present disclosure typically include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of the binding protein that, shall be held to mean an amount sufficient to achieve effective binding to an antigen and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the antigen-binding protein or antigen-binding fragment thereof.


In keeping with the scope of the present disclosure, the antigen-binding protein or antigen-binding fragment thereof may be administered to a human or other animal in accordance with the methods of treatment herein in an amount sufficient to produce a therapeutic or prophylactic effect. The binding proteins of the disclosure can be administered to such human or other animal in a conventional dosage form prepared by combining the binding protein with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. In some embodiments, a cocktail comprising one or more species of binding proteins described in the current disclosure may prove to be particularly effective.


Methods of Treatment and Inhibition of FGFR3 Activity


The anti-FGFR3 antigen binding proteins or fragments thereof are useful for the treatment of FGFR3-mediated diseases or disorders. As used herein, the term “FGFR3-mediated disease or disorder” refers to any disease or disorder that is the result of aberrant FGFR3 activity. In certain embodiments, the disease or disorder is caused by FGFR3 over activity or over expression. Non-limiting examples of FGFR3-mediated diseases or disorders include achondroplasia, hypochondroplasia, and cancer.


The anti-FGFR3 antigen binding proteins or fragments thereof are useful for the reduction of FGFR3 activity. As used herein, an “FGFR3 activity” refers to any signaling event associated with the dimerization of FGFR3 on the surface of a cell. The FGFR3 activity can be one or both of an extracellular activity and an intracellular activity. FGFR3 activities include, but are not limited to, FGFR3 dimerization, extracellular signal-regulated kinase (Erk) phosphorylation, mitogen-activated protein kinase kinase (MKK, MEK, or MAP2K) phosphorylation, FGFR3 cytoplasmic tyrosine kinase domain activity, and FGF ligand binding activity (e.g., FGF18 binding to FGFR3).


As used herein, the term “achondroplasia” refers to a genetic disorder caused by mutations in the FGFR3 gene that make the resulting protein overactive. The anti-FGFR3 antigen binding proteins or fragments thereof are useful for the reduction of one or more symptoms of achondroplasia. Achondroplasia symptoms include, but are not limited to, shortening of the proximal limbs, brachydactyly (i.e., short fingers and toes with trident hands), large head with prominent forehead frontal bossing, small midface with a flattened nasal bridge, spinal kyphosis (convex curvature) or lordosis (concave curvature), varus (i.e., bowleg) or valgus (i.e., knock knee, ear infections (due to Eustachian tube blockages)), sleep apnea (central or obstructive), and hydrocephalus. Achondroplasia may be diagnosed through the measurement of one or more of proximal limb length (e.g., femur and tibia length), finger and toe length, head circumference (e.g., skull length), and lumbar vertebrae length, although other anatomical measurements can be employed. Achondroplasia may be diagnosed through genetic testing to detect one or more mutations in the FGFR3 gene.


In one aspect, the disclosure provides a method for treating a FGFR3-mediated disease or disorder in a subject, comprising administering to a subject in need thereof the antigen binding protein or fragment thereof described herein.


As used herein, the term “subject,” “patient,” or “individual” refers to a human or non-human animal. The term “non-human animal” includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, avian species (e.g., chickens), amphibians, and reptiles. In certain embodiments, the subject is a mammal such as a non-human primate, sheep, dog, cat, rabbit, ferret or rodent. In certain embodiments, the subject is a cynomolgus monkey. In certain embodiments, the subject is a human. In certain embodiments, the subject is a child. In certain embodiments, the subject is an adolescent.


In certain embodiments, the FGFR3-mediated disease or disorder is achondroplasia. In certain embodiments, the achondroplasia is FGFR3G380R+ achondroplasia, meaning the subject to be treated contains the G380R mutation in its FGFR3 gene, either homozygous or heterozygous.


In certain embodiments, a subject diagnosed as having achondroplasia or risk of achondroplasia is treated with a binding protein disclosed herewith. In certain embodiments, the treatment leads to one or more effects selected from the group consisting of increased bone length (e.g., femur length and/or tibia length), increased bone diameter (e.g., femur diameter), increased growth plate volume (e.g., femur growth plate volume), increased vertebrae length, increased skull length, increased bone volume, increased skull volume, corrected vertebral abnormalities (e.g., increased Kyphosis Index), and improved bone age (e.g., more developed secondary ossification center), as compared to a control subject not receiving the treatment that is of the same development stage.


Accordingly, a method of improving one or more bone features in a subject is also provided. In certain embodiments, the method comprises administrating a binding protein or an antigen-binding fragment thereof disclosed herewith to a subject in need. In certain embodiments, the improved bone feature is selected from the group consisting of bone length (e.g., femur length or tibia length), bone diameter (e.g., femur diameter), growth plate volume, vertebrae length, skull length, bone volume, skull volume, Kyphosis Index, and improved bone age.


In certain embodiments, the FGFR3-mediated disease or disorder is cancer. In certain embodiments, the cancer is selected from the group consisting of bladder cancer, melanoma, urothelial cancer, and endometrial cancer.


In another aspect, the disclosure provides a method for treating achondroplasia in a subject, comprising administering to a subject in need thereof an antigen binding protein or an antigen-binding fragment thereof with binding specificity to an FGFR3 epitope, wherein the antigen binding protein or the antigen binding fragment thereof does not bind to one or more of FGFR1, FGFR2, and FGFR4.


In another aspect, the disclosure provides a method for inhibiting one or both of FGFR3 activity and expression in a bone growth plate of a subject, comprising administering to a subject an antigen binding protein or an antigen binding fragment thereof with binding specificity to an FGFR3 epitope, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4. In certain embodiments, an FGFR3 antibody as described herewith is used.


Therapeutic and Prophylactic Uses


The disclosure provides therapeutic uses of its binding proteins or an antigen binding protein fragment thereof with binding specificity to an FGFR3 epitope, wherein the antigen binding protein fragment does not bind to one or more of FGFR1, FGFR2, and FGFR4) corresponding to the methods of treatment disclosed above. For instance, the disclosure provides a binding protein or the antigen binding protein fragment thereof as described herein for use in medicine. In some embodiments, it provides a binding protein, or an antigen binding protein fragment thereof as described herein for use as an FGFR3-activity inhibiting medicament. In some embodiments, it provides a binding protein, or an antigen binding protein fragment thereof as described herein for use in treating an FGFR3-mediated disorder. Examples of such disorders are given herein. In some embodiments, it provides a binding protein, or antigen binding protein fragment thereof as described herein for use in preventing one or more symptoms of achondroplasia, as exemplified above. In some embodiments, it provides a binding protein, or antigen binding protein fragment thereof as described herein for use in preventing cancer, as exemplified above. Embodiments described herein regarding methods of treatment, administration, subjects and all other aspects relevant to therapy and prevention also apply to these uses of binding proteins.


It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following example, which is included for purposes of illustration only and is not intended to be limiting.


EXAMPLE
Example 1—Generation of Anti-FGFR3 Antibodies

Generation of Immunogen


Anti-FGFR3 monoclonal antibodies were developed using 300.19 pre-B lymphoblast cells (ATCC, Manassas, Va.) transformed with DNA encoding full-length human FGFR3 (see FIG. 1, GenBank ID NP_000133.1, FGFR3 isoform IIIc) with the mutation G380R (SEQ ID NO: 133) which was expressed on the cell surface (FGFR3G380R-300.19 cell). The FGFR3G380R open reading frame was subcloned into the pXL-MCS vector and transfected into 300.19 cells (Immunogen 1). Separately, DNA encoding the human FGFR3 extra cellular domain (ECD) protein (Immunogen 2) was subcloned into the pTT5 vector (Invitrogen), and was prepared using Expi293 cells (Invitrogen). FGFR3 extra cellular domain (ECD) protein (Immunogen 2):









(SEQ ID NO: 140)


GGLNDIFEAQKIEWHEHHHHHHEDQVDPRLIDGKIQPEPESLGTEQRVVG





RAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVP





SERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGD





DEDGEDEAEDTGVDTGAPYVVTRPERMDKKLLAVPAANTVRFRCPAAGNP





TPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVEN





KFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQ





PHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFE





DAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSAS






FGFR3 expression on FGFR3G380R-300.19 cells was monitored and quantified by FACS analysis using the comparator antibody, GT184.6.1, described further in Yin et al. (Mol. Cancer Ther. 14(10): 2270-8. 2015). Anti-FGFR1 (MAB765), anti-FGFR2 (MAB6843), anti-FGFR3 (MAB766), and anti-FGFR4 (MAB6852) antibodies were used as negative controls (R&D Systems, Minneapolis, Minn.). The FGFR3 ECD protein was quantified using an enzyme-linked immunosorbent assay (ELISA) with the antibodies described further below.


The cells expressing FGFR3 were maintained at 37° C. under 5% CO2 in RPMI (Gibco) supplemented with heat inactivated fetal bovine serum (FBS) (Hyclone). Cells were prepared for injection by substituting the above culture medium with phosphate buffered (Ca—Mg free) saline (CMF-PBS) supplemented with 5 mM EDTA and harvesting the cells in that buffer. The harvested cells were pelleted by centrifugation at 500×g for 5 minutes, washed once by resuspending the pellet in CMF-PBS and centrifuging as before, counted and adjusted to the appropriate volume (such as 5×106 cells in 0.2 ml) for injection by resuspending the cell pellet in CMF-PBS. The FGFR3 ECD protein was used to boost antibody titers in animals before sacrificing the mouse.


Cells were maintained in RPMI medium by seeding about 3-5×105 cells per ml in a T75 flask and grown for approximately 24-48 hours with selection antibiotic to eliminate the cells not carrying FGFR3 plasmid. Cell surface expression of FGFR3 on cells was verified by FACS analysis prior to use as an immunogen.


Generation of Anti-FGFR3 Antibodies—Hybridoma Strategy


Six eight-week old female FGFR3 KO (C57BL/6mFGFR3−) mice from the Jackson Laboratory (Bar Harbor, Me.) were immunized with the FGFR3-transfected cells (Immunogen 1). Separately, wild-type Balb/c mice were immunized with the FGFR3 ECD protein described above (Immunogen 2). A group of mice were primed intraperitoneally on day 0 with FGFR3-expressing cells, FGFR3G380R-300.19 cells, in PBS without adjuvants, administrated on days 14, 28, 42 and 56, followed by an intraperitoneal boost on day 77 with the FGFR3 ECD protein in PBS without adjuvants. Each injection was administrated with approximately 5×106 cells in a volume of 200 μl and 50 μg protein in a volume of 100 μl PBS. Immunizations were performed in two-week intervals and the IgG titer was evaluated by ELISA with FGFR3 ECD protein to assess immune response.


The mice were sacrificed for harvesting the spleen and placed in approximately 10 ml of pre-warmed serum-free DMEM (Hyclone) at 37° C. in a petri dish. The splenocytes were teased out of the capsule using forceps and transferred to a 15-ml conical tube. The cells were then washed three times with pre-warmed serum-free IMDM (Hyclone) and cells from multiple mice were pooled.


The fusion partner cell, FO B lymphoblasts (ATCC, Manassas, Va.), for the immunized spleen cells was established with a hypoxanthine/aminopterin/thymidine (HAT)-sensitive and IgG non-secreting myeloma cell line. Prior to the fusion, FO B lymphoblasts were maintained in IMDM medium supplemented with 10% FBS (37° C., 7% CO2) ensuring that the cells were in logarithmic growth phase on the day of the fusion.


The fusion protocol was derived from Lerner (Yale J Biol Med, 1981, 54 (5) 387-402) and Gefter et al. (Somatic Cell Genet, 1977, 3 (2) 231-236). Before the fusion, the spleen cells and the logarithmic phase myeloma cells were washed three times with serum-free IMDM and counted. For each fusion, 1-1.5×108 spleen cells were mixed with 2-3×107 myeloma cells in a 50-ml conical polypropylene tube and cells were washed once with serum-free IMDM. The ratio of spleen cells to myeloma cells was 5:1. The tubes were centrifuged at 500×g for 10 minutes to pellet the cells. After aspiration of the supernatant, the pellets were gently resuspended by tapping the bottom of the tubes. The tubes were then placed in a beaker of 37° C. water. All subsequent fusion steps were carried out in the beaker of 37° C. water.


Next, 1 ml of polyethylene glycol 1500 (PEG) (Roche Applied Science, Indianapolis, Ind.) preheated to 37 C was slowly added to the cell pellet over the course of about 1 minute, while gently rocking the tube. The cells were incubated in the PEG for one minute followed by addition of 1 ml serum-free IMDM added dropwise to the pellet over the course of 30 seconds, and then 9 ml of serum-free IMDM were added to the pellet for one minute. The tube was then centrifuged at 500×g for 10 minutes at room temperature, and the supernatant was aspirated. The pellet was resuspended in 200 ml of filtered complete hybridoma production media, IMDM (Hyclone) supplemented with 10% FBS (Hyclone), 1× non-essential amino acid (Gibco), 1 mM sodium pyruvate (Gibco), 1× pen-strep (Gibco) and 1×HAT (Sigma). The resuspended cells were then seeded in ten 96-well flat-bottom microtiter plates, in a volume of about 200 μl/well. The plates were kept in an incubator at 37° C., 7% CO2.


A primary hybridoma screen was designed to select hybridoma clones producing antibodies which recognized native FGFR3 epitopes. Supernatants from wells growing clones, typically on days 10−14 post-fusion, were incubated with Chinese hamster ovary (CHO) cells stably expressing FGFR3 (FGFR3-CHO). Antibody binding was detected by FACS using a fluorescently labeled goat anti-mouse secondary antibody. Clones were considered positive if labeled supernatant samples were greater than 10-fold over background in the FACS analysis. Selected positive clones were transferred to 24-well flat bottom plates and expanded for a second screen to confirm selection. Supernatants from 24-well plates were incubated with hFGFR3G380R-300.19 cells and detected by a fluorescently labeled goat anti-mouse secondary antibody. More than 4,000 hybridomas were generated and screened by FACS assay described above. Only 25 clones were confirmed specific to FGFR3. Positive clones were expanded for purified antibody production, VH and VL gene sequencing and cryo-preservation.


Generation of Anti-FGFR3 Antibodies—Phage-Display Strategy


Three scFv phage libraries and two fab fragment phage libraries were screened, with 5 separate screening campaigns performed. A first campaign screened the scFv phage libraries against the hFGFR3 isoform IIIc dimer. 2,700 scFv antibodies were screened with only 13 antibodies identified as potential candidates. A second campaign screened fab and scFv libraries against the hFGFR3 isoform IIIc dimer. 1,940 antibodies were screened with only 13 antibodies identified as potential candidates. A third and fourth campaign screened fab and scFv libraries against the hFGFR3 isoform IIIc dimer, the hFGFR3 isoform IIIc his tagged monomer, and hFGFR3 isoform IIIc-expressing cells. 9,770 antibodies were screened with only 36 antibodies identified as potential candidates. A fifth campaign screened fab libraries against the hFGFR3 isoform IIIc monomer and mouse FGFR3 (mFGFR3) isoform IIIc monomer, and hFGFR3 isoform IIIc- and mFGFR3 isoform IIIc-expressing cells. 2,990 antibodies were screened with only 57 antibodies identified as potential candidates. Between the five screening campaigns, 17,400 antibodies were screened. Among those screened antibodies, only 48 were found to be cross-reactive between human, mouse, and cyno, and only 15 were found to block FGFR3 ligand binding.


FGFR family member proteins (human FGFR1α IIIb, FGFR1α IIIc, FGFR1β IIIb, FGFR1β IIIc, FGFR2α IIIc, FGFR2β IIIb, FGFR3 IIIb, FGFR3 IIIc, FGFR4; mouse FGFR3; and cynomolgus FGFR3 recombinant proteins) were purchased from R&D Systems (Minneapolis, Minn.) for identifying FGFR3 specific clones by ELISA. cDNA encoding the following proteins were all subcloned into the pTT5 vector (Invitrogen): partial ECD and chimeric ECD proteins of FGFR3; human D2D3 (hD2D3) and D3 (hD3) of human FGFR3; mouse D2D3 (mD2D3) and D3 (mD3) of mouse FGFR3; cynomolgus D2D3 (cyD2D3) and D3 (cyD3) of cynomolgus FGFR3; and hD1-cyD2D3 and hD1-mD2D3 recombinant proteins. Proteins were prepared by transient transfection using Lipofectamine 2000 (Invitrogen) in Expi293 cells (Invitrogen). Supernatants from the selected clones were incubated in 96-well plates coated with the recombinant FGFR proteins and detected with goat anti-mouse IgG horseradish peroxidase (Jackson Immunoresearch, West Grove, Pa.) followed by chemiluminescent detection.


Cell Based-Binding Assays for Anti-FGFR3 Antibodies


A cell-based binding assay was used to characterize the anti-FGFR3 antibodies generated above. Full-length human, mouse, and cynomolgus FGFR3 cDNAs were subcloned into different pcDNA 3.1 (Invitrogen) vectors and transfected into 300.19 cells (ATCC). The cDNA of FGFR3 Ig domains D2D3 was subcloned into pcDNA 3.1 and transfected into CHO (D2D3-CHO) (ATCC) cells. Full-length human FGFR3G380R, mouse FGFR3, and cynomolgus FGFR3G380R cDNAs were also subcloned into different pcDNA 3.1 vectors. Stable cell lines expressing FGFR3 were generated by transfection of human FGFR3G380R, mouse FGFR3, and cynomolgus FGFR3G380R cDNA plasmid constructs into CHO (hFGFR3G380R-CHO and cyFGFR3G380R-CHO) and human embryonic kidney (HEK, mFGFR3-HEK) (ATCC) cells using the Lipofectamine 2000 kit (Invitrogen). Cell lines were maintained in F-12K medium supplemented with 10% FBS for CHO cells and in DMEM supplemented with 10% FBS for HEK cells overnight, then cultured in the presence of geneticin (0.5 ml/ml) for 10-14 days. Isolated single colonies were picked and grown in separate wells until sufficient clonal cells were expanded. Stable clones resistant to geneticin and expressing high copies of FGFR3 protein were identified by FACS assay using GT184.6.1.


Cell-based antibody binding to wild-type FGFR3, mutant FGFR3, and D2D3 FGFR3 expressed on CHO and HEK cells was accessed by FACS analysis. Cells were incubated with anti-FGFR3 antibodies in 1% bovine serum albumin in PBS (BPBS). After three washes, the cells were incubated with a fluorescent-conjugated secondary antibody (Invitrogen).


The results indicated that several antibodies bind specifically to FGFR3 expressed on the cells. For example, clones KC18, KE35, KE42, KE58, KE63, and KE94, with mouse and human isotype controls as negative controls and GT184.6.1 as a positive control, were tested for binding to CHO, HEK and 300.19 cells transfected to express wild-type FGFR3, mutant FGFR3, and the ECD domain of FGFR3. All antibodies bound to the cells as described above, with similar binding profiles and titration kinetics. The negative control antibodies exhibited only background reactivities. The binding of anti-FGFR3 antibodies to mouse primary rib chondrocytes was also observed by FACS analysis.


The binding specificities of antibodies KC18, KE35, KE42, KE58, KE63, and KE94 were also tested using GT184.6.1 as positive control. Unlike GT184.6.1, which bound to both hFGFR2 and hFGFR3, KC18, KE35, KE42, KE58, KE63, and KE94 only bound to hFGFR3, indicating high FGFR3 specificities.


Biacore Affinity Analysis


The N-terminal ECD proteins of FGFR3 from human and mouse were produced with a terminal avidin tag and used in a forward format Biacore assay where proteins were immobilized on a Biacore chip and then the kinetics of antibody interaction with the proteins on the chip were determined. The proteins were immobilized on a Biacore chip for approximately 10,000 response units (RU). Then the antibodies were exposed to the chip for kinetic measurements, following the manufacturer's recommendations (GE Healthcare). Sensorgrams were fit to a 1:1 binding model and analyzed using double-reference subtraction by T200 Evaluation software (GE Healthcare). Affinities of the tested antibodies are shown in Table 2.









TABLE 2





anti-FGFR3 antibodies binding specificities and affinities

















FGFR3
Selectivity
Reactivity















antibodies
FGFR1
FGRF2
FGFR3
FGFR4
hFGFR3IIIc
hFGFR3IIIb
mFGFR3
cynoFGFR3





KC18


+

+++
+++
+++
+++


KE35


+

+++
+++
+++
+++


KE42


+

+++
+++
+++
+++


KE58


+

+++
+++
+++
+++


KE63


+

+++
+++
+++
+++


KE94


+

+++
+++
+++
+++


Control mAb:

+
+

++
++
+++
+++


GT184













Affinity (hFGFR3)
Affinity (mFGFR3)













FGFR3
Ka
Kd
KD


KD


antibodies
(M−1s−1)
(s−1)
(nM)
Ka
Kd
(nM)





KC18
7.92E+05
3.47E−04
1.4
1.74E+05
1.10E−03
6.30


KE35
4.14E+05
1.20E−03
2.89
5.71E+5 
1.08E−03
1.89


KE42
4.60E+05
5.67E−04
1.23
7.39E+5 
6.00E−04
0.81


KE58
1.15E+06
1.43E−03
1.2
1.67E+05
9.18E−04
5.50


KE63
8.87E+05
7.45E−04
0.8
4.36E+05
2.91E−03
6.70


KE94
9.40E+05
5.95E−04
0.6
7.05E+05
1.48E−03
2.10


Control mAb:
4.23E+05
1.20E−03
2.8
4.15E+05
1.52E−03
3.7


GT184









Sequence Analysis of Anti-FGFR3 Monoclonal Antibodies


Prior to sequence analyses, the isotype of each clone was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Applied Science, Indianapolis, Ind.). The heavy (VH) and light (VL) chain variable regions of clones were sequenced using 5′RACE (Rapid Amplification of cDNA Ends) Kit (Clontech, Mountain View, Calif.) as per the manufacturer's instruction. Total RNA was extracted from each hybridoma clone using the RNeasy Miniprep kit according to the manufacturer's instructions (Qiagen, Germantown, Md.). Full-length first strand cDNAs containing 5′ends were generated by polymerase chain reaction (PCR) using 5′RACE kits (Clontech). The VH and VL genes were amplified by TA cloning kit (Invitrogen) with primers, 5′primer (5′RACE kit, Clontech), and 3′ primer of IgG isotype specific heavy chain and 3′primer of kappa light chain.











IgG1 3′ Primer:



(SEQ ID NO: 1)



5′-TATGCAAGGCTTACAACCACA-3′







IgG2b 3′ Primer:



(SEQ ID NO: 2)



5′-GTTAGGAGCTGGGCATTTGTGACACTCC-3′







IgG2c 3′ Primer:



(SEQ ID NO: 3)



5′-GTTAGGTGCTGGGCATTTGCATGGAGGACAGGG-3′







IgG3 3′ Primer:



(SEQ ID NO: 4)



5′-GTTTGGTGGGCATGAAGAACCCGGGG-3′







Kappa light chain 3′Primer:



(SEQ ID NO: 5)



5′-CTCATTCCTGTTGAAGCTCTTGAC-3′






The amplified cDNAs were cloned into pCR2.1 vectors (Invitrogen) and transformed using a TOPO-TA cloning kit (Invitrogen) as per the manufacturer's instructions. Plasmids were isolated from 2 ml of an LB culture, inoculated from a single colony and grown overnight, using QIAprep spin miniprep kit (Qiagen) and sequenced using M13 Forward and M13 Reverse primers included in the TOPO-TA cloning kit (Invitrogen). The VH and VL gene sequences were analyzed using the IMGT V-Quest web server to identify and confirm variable region sequences. VH and VL sequences of each antibody are provided below in Table 3:









TABLE 3







Antibody VH and VL amino acid sequences








Antibody ID
Sequence





KC18 VH
QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGDI



DPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTYDG



YPYAMDYWGQGTSVTVSS (SEQ ID NO: 6)





KC18 VL
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNNQKNYLAWYQQKPGQSPK



LLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYRTF



GGGTKLEIK (SEQ ID NO: 7)





KE35 VH
EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWIGY



INPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCARERD



YDGAMDYWGQGTSVTVSS (SEQ ID NO: 8)





KE35 VL
DIQMTQSPSSLSASLGGKVTITCKASQDINKFIAWYQHKPGKGPRLLIHYTST



LQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLWTFGGGTKLEIK



(SEQ ID NO: 9)





KE42 VH
EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWIG



YINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCARER



DYDGSMDFWGQGTSVTVSS (SEQ ID NO: 10)





KE42 VL
DIQMTQSPSSLSASLGGKVTITCKASQDINKFIAWYQHKPGKGPRLLIHYTST



LQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYFCLQYDNLLWTFGGGTKLEIK



(SEQ ID NO: 11)





KE58 VH
EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIGD



INPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREED



FDGFDYWGQGTTLTVSS (SEQ ID NO: 12)





KE58 VL
DIVMTQSHKFMSTSVGDRVSITCKASQDVSTGVAWYQQKPGQSPQLLIYWA



STRHTGVPDRFTGSGSGTDYILTIRSVQAEDLALYYCQQHYSTPLTFGAGTK



LELK (SEQ ID NO: 13)





KE63 VH
QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGGID



PETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNYDGY



SQTMDYWGQGTSVTVSS (SEQ ID NO: 14)





KE63 VL
NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSP



KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSYT



FGGGTKLEMK (SEQ ID NO: 15)





KE94 VH
QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGAID



PETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNYDGY



SRTMDYWGQGTSVTVSS (SEQ ID NO: 16)





KE94 VL
NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSP



KLLIYWASTRESGVPDRFTGSGSGTDFSLSISSVQTEDLAVYYCHQYLSSYTF



GGGTRLEMK (SEQ ID NO: 17)









The CDR regions for the VH and VL sequences recited above are provided below in Table 4:









TABLE 4





Antibody Heavy chain and Light chain CDR regions







HEAVY Chain










VH Chain ID
HCDR1-IMGT
HCDR2-IMGT
HCDR3-IMGT





KC18_VH
GDTFTDFE
IDPETGGT
TRTYDGYPYAMDY



(SEQ ID NO: 70)
(SEQ ID NO: 71)
(SEQ ID NO: 72)





KE35_VH
GYTFTDYN
INPNNGGT
ARERDYDGAMDY



(SEQ ID NO: 76)
(SEQ ID NO: 77)
(SEQ ID NO: 78)





KE42_VH
GYTFTDYN
INPNNGGT
ARERDYDGSMDF



(SEQ ID NO: 82)
(SEQ ID NO: 83)
(SEQ ID NO: 84)





KE58_VH
GYTVTDYY
INPNNGVT
AREEDFDGFDY



(SEQ ID NO: 88)
(SEQ ID NO: 89)
(SEQ ID NO: 90)





KE63_VH
GSTFSDEE
IDPETGGT
TRNYDGYSQTMDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)





KE94_VH
GSTFTDFE
IDPETGGT
TRNYDGYSRTMDY



(SEQ ID NO: 100)
(SEQ ID NO: 101)
(SEQ ID NO: 102)










LIGHT Chain










VL Chain ID
LCDR1-IMGT
LCDR2-IMGT
LCDR3-IMGT





KC18_VL
QSLLYSNNQKNY
WAS
QQYYSYRT



(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)





KE35_VL
QDINKF
YTS
LQYDNLLWT



(SEQ ID NO: 79)
(SEQ ID NO: 80)
(SEQ ID NO: 81)





KE42_VL
QDINKF
YTS
LQYDNLLWT



(SEQ ID NO: 85)
(SEQ ID NO: 86)
(SEQ ID NO: 87)





KE58_VL
QDVSTG
WAS
QQHYSTPLT



(SEQ ID NO: 91)
(SEQ ID NO: 92)
(SEQ ID NO: 93)





KE63_VL
QSVLYSSNQKNY
WAS
HQYLSSYT



(SEQ ID NO: 97)
(SEQ ID NO: 98)
(SEQ ID NO: 99)





KE94_VL
QSVLYSSNQKNY
WAS
HQYLSSYT



(SEQ ID NO: 103)
(SEQ ID NO: 104)
(SEQ ID NO: 105)









Inhibition of FGF1 Ligand Binding by Anti-FGFR3 Antibodies


To test if the isolated antibodies could inhibit the binding between FGF1 ligand and FGFR3, an FGF1 ligand blocking assay was conducted. For the FGFR1 ligand blocking assay, human FGFR3 cells, FGFR3-300.19 and FGFR3G380R-300.19, were pre-incubated with various concentration of anti-FGFR3 antibodies and isotype controls, prior to addition of human FGF1 ligand (R&D Systems). Binding of hFGF1 to FGFR3 expressed on the cells was determined by FACS assay using biotinylated anti-FGF1 antibody followed by incubation with a streptavidin-fluorescent secondary antibody. The anti-FGFR3 antibodies which bound to the EC domain, including KC18, KE35, KE42, KE58, KE63, and KE94, all blocked binding of FGF1 to FGFR3 on these cells.


An ELISA-based blocking assay using human and mouse FGF1 ligand was conducted. Each well of 96-well plates was coated with either human or mouse FGFR3 proteins to capture both/either anti-hFGFR3 antibody and/or FGF1 ligand, then incubated with various concentrations of anti-FGFR3 antibody and isotype controls, prior to addition of human FGF1 ligand. Binding of FGF1 ligand was determined by addition of a substrate to induce a quantifiable chemiluminescence reaction. Inhibition of FGF1 binding to human FGFR3 and mouse FGFR3 proteins by the anti-FGFR3 antibodies was detected by ELISA, respectively.


Epitope Binning of Anti-FGFR3 Antibodies


Using a Biacore T100 (GE Healthcare, Piscataway, N.J.), anti-FGFR3 antibodies were immobilized on a Biacore CM5 chip and ECD proteins of FGFR3 were injected. Competition of second anti-FGFR3 antibodies (Ab2) bound to FGFR3 protein captured by immobilized anti-FGFR3 antibodies (Ab1) were determined. The anti-FGFR3 antibodies were immobilized on a Biacore chip for approximately 100 response units (RU). Flow cell 1 remained blank for reference subtraction on each chip. The ECD FGFR3 protein and mouse anti-FGFR3 antibodies (Ab2) prepared in HBS-EP+ running buffer were injected for 3 minutes and 5 minutes at a flow rate of 50 μl/min, respectively. The Ab1 surface was regenerated between cycles using 10 mM glycine-HCl (pH 2.0) at 50 μl/min for 1 minutes. Sensorgrams were fit to a 1:1 binding model and analyzed using double-reference subtraction by BiaEvaluation software (GE Healthcare). It was determined that anti-FGFR3 antibodies KC18, KE35, KE42, KE58, KE63, and KE94 competed for the same region on FGFR3 around the D2 domain.


Hydrogen deuterium exchange (HDX) mass spectrometry was also used to determine the epitopes on FGFR3 for these antibodies by measuring the amide hydrogen deuterium exchange on FGFR3. HDX mass spectrometry measured amide hydrogen deuterium exchange over time and quenched at 0° C. and pH 2.5 along with protease digestion. Briefly, the antibody to be tested and antigen were mixed such that about 90% of the antibody is bound to the antigen at room temperature. Deuterium exchange was performed with a 10-fold dilution into D2O at neutral pH. Quenching was then performed by holding the reaction mixture for 1 minute at about 0 to 1° C. to reduce disulfide bonds. Protease digestion was performed with protease XIII and pepsin. HDX mass spectrometry is described in further detail in Pradzińska et al. (Amino Acids. 48: 2809-2820. 2016). The FGFR3 sequence used in the HDX assay is recited below, and corresponds to the D2D3 region, corresponding to D143 to E365 of FGFR3 Isoform IIIc:









(SEQ ID NO: 134)


DTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG





REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYT





LDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVE





VNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAG





NSIGFSHHSAWLVVLPAEEELVE






The results (FIG. 2) indicated that KE35 and KE58 bind to the N-terminus of the D2 region (D143 to L163); KE42 binds to N-terminus of the D2 region (D143 to N170); while KC18, KE94, and KE63 bind to N-terminus and middle of the D2 region (D143 to D160 and G197 to L213).


Internalization Assay


Internalization of anti-FGFR3 antibodies was evaluated in KMS-11 cells (JCRB cell bank, Japan). The KMS-11 cells were incubated with each antibody at a final concentration of 2 μg/ml for 30 minutes, 1, 2, 5, and 24 hours, then placed at 4° C. before washing the cells with PBS (Ca2+ and Mg2+). A subset of samples was washed with glycine-HCl buffer (pH 4) and fixed with 4% paraformaldehyde (Sigma). Another subset of samples was permeabilized with Triton X-100 (Sigma). The cells with immunofluorescence staining buffer, containing dye and fluorescent conjugated secondary antibody, were imaged by a PerkinElmer Opera high-throughput automated microscope and analyzed using the PerkinElmer Columbus image management system (PerkinElmer, Hopkinton, Mass.). Internalization of anti-FGFR3 antibodies was observed in KMS-11 cells treated with glycine-HCl and Triton X-100. The cell surface staining of each antibody was detected in a subset without treatment of glycine-HCl and Triton X-100 (FIG. 3). The ratio of the fluorescence signal for internalized antibody (+pH3/+TX100) to total antibody (−pH3/+TX100) then allows comparison of the degree of internalization for different antibodies (FIG. 4). The results indicate that the anti-FGFR3 antibodies were effectively internalized into the KMS-11 cells.


Dimerization of FGFR3 Assay


Inhibition of FGFR3 dimerization by anti-FGFR3 antibodies was evaluated by a chemiluminescent assay. U20S cells co-expressing a fusion protein of β-galactosidase-prolink (PK) and FGFR3 (FGFR3-PK) and β-galactosidase-enzyme acceptor (EA) and FGFR3 (FGFR3-EA) were developed by DiscoveRx (Fremont, Calif.). β-galactosidase is in an inactive form and when dimerization occurs the prolink and enzyme acceptor come together and are cleaved by the now activated β-galactosidase. The cells were pre-incubated with anti-FGFR3 antibodies at various concentrations before addition of FGF18 ligand to induce dimerization of FGFR3 proteins, then incubated at 37° C. overnight. The substrate was added to the cells and chemiluminescent signal was measured by EnVision (PerkinElmer). Blocking of FGFR3 dimerization by anti-FGFR3 antibodies was observed (FIG. 5A-FIG. 5C).


Humanization


Humanized variants of KC18, KE63 and KE94 clones were generated as follows: 1) Structural models of the Fv regions of these antibodies in complex with FGFR3 were generated by the Molecular Operating Environment (MOE) from the Chemical Computing Group using KC18-FGFR3 complex structure as template given their high sequence similarity with KC18 (FIG. 6); 2) The VH and VL sequences were searched against the human germline sequence, and the most similar human germline Fv sequence and J region were identified. CDRs from KC18, KE63 and KE94 were then grafted onto the framework of the closest human germline genes; 3) Analysis was performed to identify amino acids that were likely to be important for the FGFR3 binding properties of the antibodies. Selected Vernier zone and key contact residues were mutated back to mouse residues after examination within the structural models; and 4) Potential liability sites were removed.


Heavy chain of KC18_Hrw1-3 and _HV1.69rw2-4, and light chain of KC18_Lrw1-3 were designed by the method described above. Further analysis to introduce back-mutations, stabilizing mutations and to eliminate other unwanted motifs, additional heavy and light chains of KC18 variants were identified KC18_VH1, VH1b-c, VH2-3, VH3b-c and VH4, and KC18_VL1, VL1b-d, VL2, VL3, VL3b, VL4, VL5 and VL6, respectively. Heavy chains KE63 VH1-6 and light chains KE63_VL1-4 were designed. Particularly, initial KE63 (KE63_VH1 and KE63_VL1) humanized constructs were designed, then further modifications to the heavy and light chain were designed (KE63_VH2-6 and KE63_VL2-4) to introduce back-mutations, stabilizing mutations and to eliminate other unwanted motifs. Similar methods were also used to design KE94 humanized constructs KE94VH-6. Notably, KE63 and KE94 share the same set of VL designs, namely KE63_VL1-4 here. Tables 5, 6, and 7 indicate the strategy used to pair the heavy and light chain variants for KC18, KE63 and KE94, respectively, including the human and mouse germline identity percentage.









TABLE 5





Human and mouse germline identity percentage of the designed KC18 variants























KC18


Lrw1
Lrw2
Lrw3
VL1
VL1d
VL1c


















Human
93.07%
96.04%
95.05%
95.05%
93.07%
94.06%



Mouse
88.12%
87.13%
86.14%
88.12%
88.12%
89.11%















Hrw1
83.67%
74.49%
KC18_Hu1
KC18_Hu7 
KC18_Hu13





Hrw2
84.73%
75.51%
KC18_Hu2
KC18_Hu8 
KC18_Hu14





Hrw3
84.69%
75.51%
KC18_Hu3
KC18_Hu9 
KC18_Hu15





HV1.69rw2
83.67%
72.45%
KC18_Hu4
KC18_Hu10
KC18_Hu16





HV1.69rw3
83.67%
72.45%
KC18_Hu5
KC18 Hu11
KC18_Hu17





HV1.69rw4
83.67%
72.45%
KC18_Hu6
KC18_Hu12
KC18_Hu18





VH1
80.61%
75.51%



KC18_Hu19

KC18_Hu23


VH1b
84.69%
73.47%








VH1c
82.65%
73.47%




KC18_Hu38



VH2
77.55%
78.57%








VH3
84.69%
73.47%



KC18_Hu20

KC18_Hu24


VH3b
85.71%
74.49%








VH3c
84.69%
74.49%




KC18_Hu39



VH4
78.57%
79.59%



















KC18


VL1b
VL2
VL3
VL3b
VL4
VL5
VL6



















Human
94.06%
90.10%
86.14%
85.15%
81.19%
81.19%
81.19%



Mouse
87.13%
91.09%
79.21%
80.20%
86.14%
81.19%
85.15%
















Hrw1
83.67%
74.49%









Hrw2
84.73%
75.51%









Hrw3
84.69%
75.51%









HV1.69rw2
83.67%
72.45%









HV1.69rw3
83.67%
72.45%









HV1.69rw4
83.67%
72.45%









VH1
80.61%
75.51%


KC18_Hu29






VH1b
84.69%
73.47%
KC18_Hu40


KC18_Hu31

KC18_Hu35



VH1c
82.65%
73.47%









VH2
77.55%
78.57%

KC18_Hu27


KC18_Hu35

KC18_Hu37


VH3
84.69%
73.47%


KC18_Hu30






VH3b
85.71%
74.49%
KC18_Hu41


KC18_Hu32





VH3c
84.69%
74.49%









VH4
78.57%
79.59%

KC18_Hu28


KC18_Hu34
















TABLE 6







Human and mouse germline identity percentage of the designed KE63 variants













KE63


KE63_VL1
KE63_VL2
KE63_VL3
KE63_VL4
















Human
93.07%
93.07%
91.09%
91.09%



Mouse
90.00%
89.00%
87.00%
87.00%













KE63_VH1
83.67%
74.49%
KE63_Hu1
KE63_Hu2
KE63_Hu3
KE63_Hu4


KE63_VH2
86.73%
73.47%
KE63_Hu5
KE63_Hu6
KE63_Hu7
KE63_Hu8


KE63_VH3
85.71%
74.49%
KE63_Hu9
KE63_Hu10
KE63_Hu11
KE63_Hu12


KE63_VH4
86.73%
73.47%
KE63_Hu13
KE63_Hu14
KE63_Hu15
KE63_Hu16


KE63_VH5
82.65%
73.47%
KE63_Hu17
KE63_Hu18
KE63_Hu19
KE63_Hu20


KE63_VH6
81.63%
72.45%
KE63_Hu21
KE63_Hu22
KE63_Hu23
KE63_Hu24
















TABLE 7







Human and mouse germline identity percentage of the designed KE94 variants













KE94


KE94_VL1
KE94_VL2
KE94_VL3
KE94_VL4
















Human
93.07%
93.07%
91.09%
91.09%



Mouse
90.00%
89.00%
87.00%
87.00%













KE94_VH1
82.65%
77.55%
KE94_Hu1
KE94_Hu2
KE94_Hu3
KE94_Hu4


KE94_VH2
85.71%
76.53%
KE94_Hu5
KE94_Hu6
KE94_Hu7
KE94_Hu8


KE94_VH3
84.69%
77.55%
KE94_Hu9
KE94_Hu10
KE94_Hu11
KE94_Hu12


KE94_VH4
83.67%
78.57%
KE94_Hu13
KE94_Hu14
KE94_Hu15
KE94_Hu16


KE94_VH5
82.65%
77.55%
KE94_Hu17
KE94_Hu18
KE94_Hu19
KE94_Hu20


KE94_VH6
81.63%
76.53%
KE94_Hu21
KE94_Hu22
KE94_Hu23
KE94_Hu24









Amino acid sequences for the humanized variants of KC18, KE64 and KE94, without the constant regions, are depicted below in Table 8. Nucleic acid sequences for the humanized variants of KC18, KE64 and KE94, without the constant regions, are depicted below in Table 9. The heavy and light constant regions are indicated in SEQ ID NO: 54 and SEQ ID NO: 55, respectively.









TABLE 8







Amino acid sequences of humanized variants of KC18, KE64


and KE94








Antibody ID
Sequence





KC18 Hu18 VH
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGL



EWIGDVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDT



AVYYCTRTYDGYPYAFDYWGQGTLVTVSS (SEQ ID NO: 18)





KC18 Hu18 VL
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSNNNKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKVEIK (SEQ ID NO: 19)





KE63 Hu01 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFSDFEIHWVRQAPGQGL



EWIGGIDPETGGTAYNQKFQGRVTITADRSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSQTFDYWGQGTLVTVSS (SEQ ID NO: 20)





KE63 Hu02 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFSDFEIHWVRQAPGQGL



EWIGGIDPETGGTAYNQKFQGRVTITADRSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSQTFDYWGQGTLVTVSS (SEQ ID NO: 21)





KE63 Hu03 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFSDFEIHWVRQAPGQGL



EWIGGIDPETGGTAYNQKFQGRVTITADRSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSQTFDYWGQGTLVTVSS (SEQ ID NO: 22)





KE63 Hu04 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFSDFEIHWVRQAPGQGL



EWIGGIDPETGGTAYNQKFQGRVTITADRSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSQTFDYWGQGTLVTVSS (SEQ ID NO: 23)





KE63 Hu01 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY



CHQYLSSYTFGQGTKLEIK (SEQ ID NO: 24)





KE63 Hu02 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY



CHQYLSPYTFGQGTKLEIK (SEQ ID NO: 25)





KE63 Hu03 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKP



GQSPKLLIYYASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY



CHQYLSPYTFGQGTKLEIK (SEQ ID NO: 26)





KE63 Hu04 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKP



GQSPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC



HQYLSPYTFGQGTKLEIK (SEQ ID NO: 27)





KE94 Hu01 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFTDFEIHWVRQAPGQGL



EWIGAIDPETGGTAYNQKFQGRVTITADKSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSRTFDYWGQGTLVTVSS (SEQ ID NO: 28)





KE94 Hu02 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFTDFEIHWVRQAPGQGL



EWIGAIDPETGGTAYNQKFQGRVTITADKSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSRTFDYWGQGTLVTVSS (SEQ ID NO: 29)





KE94 Hu03 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFTDFEIHWVRQAPGQGL



EWIGAIDPETGGTAYNQKFQGRVTITADKSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSRTFDYWGQGTLVTVSS (SEQ ID NO: 30)





KE94 Hu04 VH
QVQLVQSGAEVKKPGASVKVSCKASGSTFTDFEIHWVRQAPGQGL



EWIGAIDPETGGTAYNQKFQGRVTITADKSTSTAYMELSSLRSEDTA



VYYCTRNYDGYSR1FDYWGQGTLVTVSS (SEQ ID NO: 31)





KE94 Hu01 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY



CHQYLSSYTFGQGTKLEIK (SEQ ID NO: 32)





KE94 Hu02 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY



CHQYLSPYTFGQGTKLEIK (SEQ ID NO: 33)





KE94 Hu03 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKP



GQSPKLLIYYASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY



CHQYLSPYTFGQGTKLEIK (SEQ ID NO: 34)





KE94 Hu04 VL
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKP



GQSPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC



HQYLSPYTFGQGTKLEIK (SEQ ID NO: 35)





IgG1 Constant Heavy
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL



TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK



VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP



EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR



VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ



VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT



TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ



KSLSLSPG (SEQ ID NO: 54)





IgG1 Constant Light
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA



LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ



GLSSPVTKSFNRGEC (SEQ ID NO: 55)





KC18Hrw1
QVQLVQSGAEVKKPGASVKVSCKASGDTFTDFEIHWVRQAPGQGL



EWIGDIDPETGSTSYAQKFQGRATLTADRSTSTAYMELSSLRSEDTA



VYYCTRTYDGYPYAMDYWGQGTLVTVSS (SEQ ID NO: 106)





KC18Hrw2
QVQLVQSGAEVKKPGASVKVSCKASGDTFTDYEIHWVRQAPGQGL



EWIGDIDPETGSTSYAQKFQGRATLTADRSTSTAYMELSSLRSEDTA



VYYCTRTYDGYPYAMDYWGQGTLVTVSS (SEQ ID NO: 107)





KC18Hrw3
QVQLVQSGAEVKKPGASVKVSCKASGDTFTDYEIHWVRQAPGQGL



EWIGDIDPETGSTSYAQKFQGRATLTADRSTSTAYMELSSLRSEDTA



VYYCTRTYDGYPYAMDYWGQGTSVTVSS (SEQ ID NO: 108)





KC18HV1-69rw2
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGL



EWIGDVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDT



AVYYCTRTYDGYPYAMDYWGQGTLVTVSS (SEQ ID NO: 109)





KC18HV1-69rw3
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGL



EWIGDVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDT



AVYYCTRTYEGYPYAMDYWGQGTLVTVSS (SEQ ID NO: 110)





KC18HV1-69rw4
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGL



EWIGDVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDT



AVYYCTRTYDGYPYAFDYWGQGTLVTVSS (SEQ ID NO: 111)





KC18Lrw1
DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKVEIK (SEQ ID NO: 112)





KC18Lrw2
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKVEIK (SEQ ID NO: 113)





KC18Lrw3
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSNNNKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKVEIK (SEQ ID NO: 114)





KC18_CL_VH1
QVQLVQSGAEVVKPGATVKISCKASGDTFTDFEIHWVQQAPGKGL



EWIGDIDPETGGTAYNEKFQGRATLTADRSTSTAYMELSSLRSEDT



AVYYCARTYDGYPYAMDYWGQGTTVTVSS (SEQ ID NO: 115)





KC18_CL_VH1b
QVQLVQSGAEVVKPGATVKISCKASGYTFTDFEIHWVQQAPGKGL



EWIGDVDPETGGTAYAEKFQGRATITADRSTSTAYMELSSLRSEDT



AVYYCARTYDGYPYAMDYWGQGTTVTVSS (SEQ ID NO: 116)





KC18_CL_VH1c
QVQLVQSGAEVVKPGATVKISCKASGYTFTDFEIHWVQQAPGKGL



EWIGDVEPETGGTAYAEKFQGRATITADRSTSTAYMELSSLRSEDA



AVYYCARTYDGYPYAMDVWGQGTTVTVSS (SEQ ID NO: 117)





KC18_CL_VH2
QVQLVQSGAEVVKPGATVKLSCKASGDTFTDFEIHWVKQAPGKGL



EWIGDIDPETGGTAYNEKFQGRATLTADRSTSTAYMELSSLRSEDT



AVYYCTRTYDGYPYAMDYWGQGTTVTVSS (SEQ ID NO: 118)





KC18_CL_VH3
QVQLVQSGAEVVKPGASVKVSCKASGDTFTDFEIHWVKQAPGQGL



EWIGDIDPESGGTAYNQKFQGRVTMTADRSISTAYMELSRLRSDDT



AVYYCARTYDGYPYAMDYWGQGTTVTVSS (SEQ ID NO: 119)





KC18_CL_VH3b
QVQLVQSGAEVVKPGASVKVSCKASGYTFTDFEIHWVKQAPGQGL



EWIGDIDPESGGTAYNQKFQGRVTMTADRSISTAYMELSRLRSDDT



AVYYCARTYDGYPYAMDYWGQGTTVTVSS (SEQ ID NO: 120)





KC18_CL_VH3c
QVQLVQSGAEVVKPGASVKVSCKASGYTFTDFEIHWVKQAPGQGL



EWIGDIEPESGGTAYNQKFQGRVTMTADRSISTAYMELSRLRSDDA



AVYYCARTYDGYPYAMDVWGQGTTVTVSS (SEQ ID NO: 121)





KC18_CL_VH4
QVQLVQSGAEVVKPGASVKLSCKASGDTFTDFEIHWVKQAPGQGL



EWIGDIDPETGGTAYNQKFQGRATLTADRSSSTAYMELSRLRSDDT



AVYYCTRTYDGYPYAMDYWGQGTTVTVSS (SEQ ID NO: 122)





KC18_CL_VL1
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKLEIK (SEQ ID NO: 123)





KC18_CL_VL1b
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSNNQKNYLAWYQQKP



GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKLEIK (SEQ ID NO: 124)





KC18_CL_VL1c
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKLEIK (SEQ ID NO: 125)





KC18_CL_VL1d
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSSNQKNYLAWYQQKP



GQSPKLLIYYASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYY



CQQYYSYRTFGGGTKLEIK (SEQ ID NO: 126)





KC18_CL_VL2
DIVMTQSPSSLAVSLGERVTMNCKSSQSLLYSNNQKNYLAWYQQK



PGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVY



YCQQYYSYRTFGGGTKLEIK (SEQ ID NO: 127)





KC18_CL_VL3
DIVMTQSPLSLPVTVGEPVSISCRSSQSLLHSNNQKNYLAWYLQKPG



QSPQLLIYWGSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC



QQYYSYRTFGGGTKLEIK (SEQ ID NO: 128)





KC18_CL_VL3b
DIVMTQSPLSLPVTVGEPVSISCRSSQSLLYSNNQKNYLAWYLQKPG



QSPQLLIYWGSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC



QQYYSYRTFGGGTKLEIK (SEQ ID NO: 129)





KC18_CL_VL4
DIVMTQSPLSLAVTVGEKVSISCRSSQSLLYSNNQKNYLAWYQQKP



GQSPKLLIYWGSTRESGVPDRFSGSGSGTDFTLTISRVEAEDVGVYY



CQQYYSYRTFGGGTKLEIK (SEQ ID NO: 130)





KC18_CL_VL5
DIQMTQSPSSLSVSVGDRVTMTCRASQGISYSNNQKNYLAWYQQK



PGKSPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSVQAEDVAVY



YCQQYYSYRTFGGGTKLEIK (SEQ ID NO: 131)





KC18_CL_VL6
DIQMTQSPSSLSVSVGDRVTMTCRSSQSLLYSNNQKNYLAWYQQK



PGKSPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSVQAEDVAVY



YCQQYYSYRTFGGGTKLEIK (SEQ ID NO: 132)
















TABLE 9







Nucleic acid sequences of humanized variants of KC18, KE64 and KE94


(HC: heavy chain; LC: light chain)








Antibody ID
Sequence





KC18 Hu18 HC
GAGGTACAACTTGTCCAGTCAGGTGCGGAGGTTAAAAAACCGGG



GGCCACAGTTAAACTGAGCTGCAAGGCTAGCGGTGATACTTTTA



CCGATTTTGAGATACACTGGGTTCAGCAGGCTCCGGGGAAAGGG



CTTGAATGGATTGGTGATGTTGACCCCGAAACGGGCGGAACCGC



GTATGCAGAGAAGTTTCAAGGTAGGGCAACGCTCACTGCGGACA



GAAGCACAGACACGGCATACATGGAGCTTAGTTCTCTCCGCTCT



GAGGATACCGCTGTTTATTATTGTACTAGAACCTATGATGGATAT



CCATACGCATTCGATTATTGGGGGCAAGGGACTCTTGTCACAGT



CAGCTCCGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGCCCC



TTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGTGCC



TTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGGAATT



CAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTTC



AGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGT



CCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATA



AGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCA



TGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCCGAACT



GCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCGAAGGA



CACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTCGTGGT



GGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTGGTACG



TGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCGAGAGA



GGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCTGACCG



TGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGTGCAA



AGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACCATTA



GCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCCTT



CCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCAC



TTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATG



GGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTC



CCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGA



CCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTTCCTGTT



CAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAAAGTCA



CTGAGCCTGTCTCCCGGA (SEQ ID NO: 36)





KC18 Hu18 LC
GACATAGTGATGACTCAATCCCCAGATTCACTCGCCGTATCACTC



GGAGAAAGAGCCACAATTAATTGTAAGAGTAGTCAGTCAGTCCT



TTACTCTAATAACAACAAAAATTACCTGGCCTGGTACCAACAGA



AACCAGGTCAATCTCCTAAACTGCTTATCTACTGGGCTAGTACCC



GAGAATCAGGAGTTCCCGATAGGTTTTCTGGGTCTGGGAGCGGC



ACCGACTTCACACTCACAATCTCTAGCGTACAGGCTGAAGATGT



AGCGGTGTATTACTGCCAACAGTATTATTCATACAGGACCTTCG



GTGGTGGCACCAAAGTAGAAATCAAACGCACTGTGGCAGCCCCT



TCTGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGT



ACCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAG



GCCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTA



ATTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCAC



CTACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGA



AAAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTT



CTAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 37)





KE63 Hu01 HC
CAGGTACAGTTGGTACAGTCAGGAGCGGAGGTTAAAAAACCAG



GGGCGTCTGTGAAAGTCTCATGTAAAGCGAGCGGAAGCACGTTT



AGCGATTTCGAGATTCACTGGGTGAGACAAGCACCCGGTCAGGG



CCTGGAATGGATTGGAGGGATCGACCCGGAAACAGGGGGTACA



GCATATAACCAAAAGTTTCAGGGACGGGTCACTATAACGGCTGA



CAGGAGCACGTCAACTGCGTATATGGAATTGTCCAGTTTGAGGT



CAGAAGATACGGCAGTCTACTACTGCACAAGAAATTATGATGGA



TACTCTCAAACGTTTGATTATTGGGGTCAGGGGACCCTGGTAAC



AGTCAGCTCAGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGC



CCCTTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGT



GCCTTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGG



AATTCAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGT



GCTTCAGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGT



GCCGTCCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAA



TCATAAGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCA



AATCATGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCC



GAACTGCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCG



AAGGACACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTC



GTGGTGGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTG



GTACGTGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCG



AGAGAGGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCT



GACCGTGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGT



GCAAAGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACC



ATTAGCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATAC



CCTTCCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCAC



TCACTTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCG



AATGGGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCAC



CCCTCCCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAA



GCTGACCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTT



CCTGTTCAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAA



AGTCACTGAGCCTGTCTCCCGGA (SEQ ID NO: 38)





KE63 Hu02 HC
CAGGTACAGTTGGTACAGTCAGGAGCGGAGGTTAAAAAACCAG



GGGCGTCTGTGAAAGTCTCATGTAAAGCGAGCGGAAGCACGTTT



AGCGATTTCGAGATTCACTGGGTGAGACAAGCACCCGGTCAGGG



CCTGGAATGGATTGGAGGGATCGACCCGGAAACAGGGGGTACA



GCATATAACCAAAAGTTTCAGGGACGGGTCACTATAACGGCTGA



CAGGAGCACGTCAACTGCGTATATGGAATTGTCCAGTTTGAGGT



CAGAAGATACGGCAGTCTACTACTGCACAAGAAATTATGATGGA



TACTCTCAAACGTTTGATTATTGGGGTCAGGGGACCCTGGTAAC



AGTCAGCTCAGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGC



CCCTTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGT



GCCTTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGG



AATTCAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGT



GCTTCAGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGT



GCCGTCCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAA



TCATAAGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCA



AATCATGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCC



GAACTGCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCG



AAGGACACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTC



GTGGTGGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTG



GTACGTGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCG



AGAGAGGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCT



GACCGTGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGT



GCAAAGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACC



ATTAGCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATAC



CCTTCCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCAC



TCACTTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCG



AATGGGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCAC



CCCTCCCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAA



GCTGACCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTT



CCTGTTCAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAA



AGTCACTGAGCCTGTCTCCCGGA (SEQ ID NO: 39)





KE63 Hu03 HC
CAGGTACAGTTGGTACAGTCAGGAGCGGAGGTTAAAAAACCAG



GGGCGTCTGTGAAAGTCTCATGTAAAGCGAGCGGAAGCACGTTT



AGCGATTTCGAGATTCACTGGGTGAGACAAGCACCCGGTCAGGG



CCTGGAATGGATTGGAGGGATCGACCCGGAAACAGGGGGTACA



GCATATAACCAAAAGTTTCAGGGACGGGTCACTATAACGGCTGA



CAGGAGCACGTCAACTGCGTATATGGAATTGTCCAGTTTGAGGT



CAGAAGATACGGCAGTCTACTACTGCACAAGAAATTATGATGGA



TACTCTCAAACGTTTGATTATTGGGGTCAGGGGACCCTGGTAAC



AGTCAGCTCAGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGC



CCCTTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGT



GCCTTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGG



AATTCAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGT



GCTTCAGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGT



GCCGTCCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAA



TCATAAGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCA



AATCATGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCC



GAACTGCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCG



AAGGACACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTC



GTGGTGGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTG



GTACGTGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCG



AGAGAGGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCT



GACCGTGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGT



GCAAAGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACC



ATTAGCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATAC



CCTTCCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCAC



TCACTTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCG



AATGGGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCAC



CCCTCCCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAA



GCTGACCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTT



CCTGTTCAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAA



AGTCACTGAGCCTGTCTCCCGGA (SEQ ID NO: 40)





KE63 Hu04 HC
CAGGTACAGTTGGTACAGTCAGGAGCGGAGGTTAAAAAACCAG



GGGCGTCTGTGAAAGTCTCATGTAAAGCGAGCGGAAGCACGTTT



AGCGATTTCGAGATTCACTGGGTGAGACAAGCACCCGGTCAGGG



CCTGGAATGGATTGGAGGGATCGACCCGGAAACAGGGGGTACA



GCATATAACCAAAAGTTTCAGGGACGGGTCACTATAACGGCTGA



CAGGAGCACGTCAACTGCGTATATGGAATTGTCCAGTTTGAGGT



CAGAAGATACGGCAGTCTACTACTGCACAAGAAATTATGATGGA



TACTCTCAAACGTTTGATTATTGGGGTCAGGGGACCCTGGTAAC



AGTCAGCTCAGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGC



CCCTTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGT



GCCTTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGG



AATTCAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGT



GCTTCAGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGT



GCCGTCCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAA



TCATAAGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCA



AATCATGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCC



GAACTGCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCG



AAGGACACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTC



GTGGTGGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTG



GTACGTGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCG



AGAGAGGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCT



GACCGTGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGT



GCAAAGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACC



ATTAGCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATAC



CCTTCCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCAC



TCACTTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCG



AATGGGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCAC



CCCTCCCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAA



GCTGACCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTT



CCTGTTCAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAA



AGTCACTGAGCCTGTCTCCCGGA (SEQ ID NO: 41)





KE63 Hu01 LC
GATATAGTTATGACACAGAGCCCTGACTCTCTGGCTGTGAGTTTG



GGCGAGCGAGTAACCATTAATTGTAAGAGTTCTCAATCCGTCCT



CTACTCAAGCAACCAGAAAAATTACCTCGCGTGGTACCAGCAAA



AACCAGGACAGAGCCCCAAACTCTTGATCTATTGGGCGTCCACC



CGAGAGAGTGGCGTGCCAGATCGGTTTTCAGGTTCTGGATCTGG



TACCGACTTCACCCTTACAATCTCAAGCCTGCAAGCAGAGGATG



TCGCAGTTTATTATTGCCATCAGTACCTGAGCAGCTACACATTCG



GACAAGGAACGAAACTGGAAATCAAACGCACTGTGGCAGCCCC



TTCTGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGG



TACCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGA



GGCCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGT



AATTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCA



CCTACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACG



AAAAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTT



TCTAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ



ID NO: 42)





KE63 Hu02 LC
GACATAGTAATGACCCAAAGTCCAGATTCTTTGGCCGTATCTTTG



GGTGAGCGCGTTACCATCAACTGTAAGTCTTCCCAGTCTGTGTTG



TACTCATCTAATCAAAAAAACTACCTCGCTTGGTACCAGCAGAA



GCCAGGTCAAAGCCCGAAACTGCTTATTTATTGGGCGTCTACGC



GAGAGTCTGGGGTCCCCGATCGGTTTTCAGGGTCAGGCTCTGGC



ACTGATTTTACTCTGACTATTTCATCCCTCCAAGCCGAAGACGTG



GCAGTGTATTACTGCCACCAGTATTTGAGCCCTTACACGTTTGGG



CAGGGGACTAAACTTGAAATCAAGCGCACTGTGGCAGCCCCTTC



TGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGTAC



CGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAGGC



CAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTAAT



TCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAA



AAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTC



TAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 43)





KE63 Hu03 LC
GATATTGTGATGACTCAGTCACCTGACAGTCTGGCGGTTTCTTTG



GGCGAAAGAGTGACTATAAATTGCAAAAGCAGCCAGTCAGTTCT



CTATTCCGACAATCAAAAGAACTATCTCGCATGGTATCAGCAGA



AGCCAGGGCAATCCCCAAAATTGCTTATATACTATGCATCAACG



CGCGAAAGCGGTGTACCCGATCGGTTTTCAGGAAGTGGCAGTGG



GACCGACTTTACGCTGACAATCTCTTCCCTTCAAGCGGAGGATGT



CGCGGTTTATTATTGTCATCAGTATCTGAGTCCTTACACCTTTGG



TCAAGGGACGAAGTTGGAGATCAAACGCACTGTGGCAGCCCCTT



CTGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGTA



CCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAGG



CCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTAA



TTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAA



AAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTC



TAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 44)





KE63 Hu04 LC
GACATCGTAATGACCCAGTCCCCCGATAGTCTGGCTGTGTCTTTG



GGCGAGAGGGTAACGATAAACTGTAAATCAAGTCAGTCAGTGCT



TTACTCAGATAACCAGAAGAACTATCTTGCGTGGTATCAGCAAA



AGCCCGGACAGTCTCCAAAACTTCTTATATATTTCGCTTCTACCA



GAGAATCAGGTGTACCAGACCGCTTTTCTGGAAGCGGCTCTGGT



ACTGACTTTACCCTGACAATTAGTAGCTTGCAAGCTGAAGATGTT



GCGGTATATTATTGTCACCAATACTTGAGTCCCTATACTTTTGGC



CAAGGGACAAAACTGGAAATAAAGCGCACTGTGGCAGCCCCTTC



TGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGTAC



CGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAGGC



CAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTAAT



TCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAA



AAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTC



TAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 45)





KE94 Hu01 HC
CAGGTTCAGCTGGTACAATCTGGCGCGGAAGTCAAAAAGCCAGG



CGCAAGTGTTAAAGTGTCTTGCAAGGCTTCAGGATCTACCTTTAC



AGATTTTGAAATCCACTGGGTAAGACAAGCACCTGGCCAGGGGC



TGGAATGGATTGGTGCCATAGACCCTGAGACGGGAGGAACCGC



ATATAACCAGAAATTCCAAGGTCGAGTGACTATTACTGCGGACA



AGTCAACATCAACTGCCTATATGGAGCTGTCTTCTTTGAGGTCAG



AGGATACAGCAGTTTACTACTGCACTAGAAATTACGATGGTTAT



TCACGGACCTTCGATTATTGGGGTCAAGGCACTCTGGTGACCGT



GAGTTCCGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGCCCC



TTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGTGCC



TTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGGAATT



CAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTTC



AGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGT



CCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATA



AGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCA



TGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCCGAACT



GCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCGAAGGA



CACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTCGTGGT



GGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTGGTACG



TGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCGAGAGA



GGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCTGACCG



TGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGTGCAA



AGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACCATTA



GCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCCTT



CCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCAC



TTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATG



GGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTC



CCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGA



CCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTTCCTGTT



CAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAAAGTCA



CTGAGCCTGTCTCCCGGA (SEQ ID NO: 46)





KE94 Hu02 HC
CAGGTTCAGCTGGTACAATCTGGCGCGGAAGTCAAAAAGCCAGG



CGCAAGTGTTAAAGTGTCTTGCAAGGCTTCAGGATCTACCTTTAC



AGATTTTGAAATCCACTGGGTAAGACAAGCACCTGGCCAGGGGC



TGGAATGGATTGGTGCCATAGACCCTGAGACGGGAGGAACCGC



ATATAACCAGAAATTCCAAGGTCGAGTGACTATTACTGCGGACA



AGTCAACATCAACTGCCTATATGGAGCTGTCTTCTTTGAGGTCAG



AGGATACAGCAGTTTACTACTGCACTAGAAATTACGATGGTTAT



TCACGGACCTTCGATTATTGGGGTCAAGGCACTCTGGTGACCGT



GAGTTCCGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGCCCC



TTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGTGCC



TTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGGAATT



CAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTTC



AGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGT



CCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATA



AGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCA



TGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCCGAACT



GCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCGAAGGA



CACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTCGTGGT



GGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTGGTACG



TGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCGAGAGA



GGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCTGACCG



TGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGTGCAA



AGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACCATTA



GCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCCTT



CCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCAC



TTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATG



GGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTC



CCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGA



CCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTTCCTGTT



CAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAAAGTCA



CTGAGCCTGTCTCCCGGA (SEQ ID NO: 47)





KE94 Hu03 HC
CAGGTTCAGCTGGTACAATCTGGCGCGGAAGTCAAAAAGCCAGG



CGCAAGTGTTAAAGTGTCTTGCAAGGCTTCAGGATCTACCTTTAC



AGATTTTGAAATCCACTGGGTAAGACAAGCACCTGGCCAGGGGC



TGGAATGGATTGGTGCCATAGACCCTGAGACGGGAGGAACCGC



ATATAACCAGAAATTCCAAGGTCGAGTGACTATTACTGCGGACA



AGTCAACATCAACTGCCTATATGGAGCTGTCTTCTTTGAGGTCAG



AGGATACAGCAGTTTACTACTGCACTAGAAATTACGATGGTTAT



TCACGGACCTTCGATTATTGGGGTCAAGGCACTCTGGTGACCGT



GAGTTCCGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGCCCC



TTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGTGCC



TTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGGAATT



CAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTTC



AGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGT



CCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATA



AGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCA



TGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCCGAACT



GCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCGAAGGA



CACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTCGTGGT



GGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTGGTACG



TGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCGAGAGA



GGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCTGACCG



TGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGTGCAA



AGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACCATTA



GCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCCTT



CCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCAC



TTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATG



GGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTC



CCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGA



CCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTTCCTGTT



CAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAAAGTCA



CTGAGCCTGTCTCCCGGA (SEQ ID NO: 48)





KE94 Hu04 HC
CAGGTTCAGCTGGTACAATCTGGCGCGGAAGTCAAAAAGCCAGG



CGCAAGTGTTAAAGTGTCTTGCAAGGCTTCAGGATCTACCTTTAC



AGATTTTGAAATCCACTGGGTAAGACAAGCACCTGGCCAGGGGC



TGGAATGGATTGGTGCCATAGACCCTGAGACGGGAGGAACCGC



ATATAACCAGAAATTCCAAGGTCGAGTGACTATTACTGCGGACA



AGTCAACATCAACTGCCTATATGGAGCTGTCTTCTTTGAGGTCAG



AGGATACAGCAGTTTACTACTGCACTAGAAATTACGATGGTTAT



TCACGGACCTTCGATTATTGGGGTCAAGGCACTCTGGTGACCGT



GAGTTCCGCTTCAACCAAGGGACCTTCTGTCTTTCCTCTGGCCCC



TTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCACTCGGGTGCC



TTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTGGAATT



CAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTTC



AGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGT



CCTCTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATA



AGCCTTCTAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCA



TGTGACAAGACCCACACCTGTCCGCCCTGTCCGGCACCCGAACT



GCTGGGTGGCCCTTCCGTGTTCCTTTTCCCTCCAAAGCCGAAGGA



CACTCTTATGATTTCTCGCACTCCCGAAGTGACTTGCGTCGTGGT



GGATGTGTCCCATGAGGATCCAGAGGTCAAGTTCAACTGGTACG



TGGACGGTGTGGAAGTCCACAACGCCAAGACTAAGCCGAGAGA



GGAACAGTACAATTCAACCTATCGGGTGGTGAGCGTCCTGACCG



TGCTGCACCAGGACTGGCTTAACGGAAAGGAGTACAAGTGCAA



AGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGACCATTA



GCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCCTT



CCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCAC



TTGTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATG



GGAGTCCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTC



CCGTGCTGGACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGA



CCGTGGATAAGTCTCGCTGGCAGCAAGGGAATGTGTTTTCCTGTT



CAGTGATGCATGAGGCCCTTCATAATCATTACACCCAAAAGTCA



CTGAGCCTGTCTCCCGGA (SEQ ID NO: 49)





KE94 Hu01 LC
GATATAGTTATGACACAGAGCCCTGACTCTCTGGCTGTGAGTTTG



GGCGAGCGAGTAACCATTAATTGTAAGAGTTCTCAATCCGTCCT



CTACTCAAGCAACCAGAAAAATTACCTCGCGTGGTACCAGCAAA



AACCAGGACAGAGCCCCAAACTCTTGATCTATTGGGCGTCCACC



CGAGAGAGTGGCGTGCCAGATCGGTTTTCAGGTTCTGGATCTGG



TACCGACTTCACCCTTACAATCTCAAGCCTGCAAGCAGAGGATG



TCGCAGTTTATTATTGCCATCAGTACCTGAGCAGCTACACATTCG



GACAAGGAACGAAACTGGAAATCAAACGCACTGTGGCAGCCCC



TTCTGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGG



TACCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGA



GGCCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGT



AATTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCA



CCTACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACG



AAAAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTT



TCTAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ



ID NO: 50)





KE94 Hu02 LC
GACATAGTAATGACCCAAAGTCCAGATTCTTTGGCCGTATCTTTG



GGTGAGCGCGTTACCATCAACTGTAAGTCTTCCCAGTCTGTGTTG



TACTCATCTAATCAAAAAAACTACCTCGCTTGGTACCAGCAGAA



GCCAGGTCAAAGCCCGAAACTGCTTATTTATTGGGCGTCTACGC



GAGAGTCTGGGGTCCCCGATCGGTTTTCAGGGTCAGGCTCTGGC



ACTGATTTTACTCTGACTATTTCATCCCTCCAAGCCGAAGACGTG



GCAGTGTATTACTGCCACCAGTATTTGAGCCCTTACACGTTTGGG



CAGGGGACTAAACTTGAAATCAAGCGCACTGTGGCAGCCCCTTC



TGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGTAC



CGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAGGC



CAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTAAT



TCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAA



AAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTC



TAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 51)





KE94 Hu03 LC
GATATTGTGATGACTCAGTCACCTGACAGTCTGGCGGTTTCTTTG



GGCGAAAGAGTGACTATAAATTGCAAAAGCAGCCAGTCAGTTCT



CTATTCCGACAATCAAAAGAACTATCTCGCATGGTATCAGCAGA



AGCCAGGGCAATCCCCAAAATTGCTTATATACTATGCATCAACG



CGCGAAAGCGGTGTACCCGATCGGTTTTCAGGAAGTGGCAGTGG



GACCGACTTTACGCTGACAATCTCTTCCCTTCAAGCGGAGGATGT



CGCGGTTTATTATTGTCATCAGTATCTGAGTCCTTACACCTTTGG



TCAAGGGACGAAGTTGGAGATCAAACGCACTGTGGCAGCCCCTT



CTGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGTA



CCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAGG



CCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTAA



TTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAA



AAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTC



TAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 52)





KE94 Hu04 LC
GACATCGTAATGACCCAGTCCCCCGATAGTCTGGCTGTGTCTTTG



GGCGAGAGGGTAACGATAAACTGTAAATCAAGTCAGTCAGTGCT



TTACTCAGATAACCAGAAGAACTATCTTGCGTGGTATCAGCAAA



AGCCCGGACAGTCTCCAAAACTTCTTATATATTTCGCTTCTACCA



GAGAATCAGGTGTACCAGACCGCTTTTCTGGAAGCGGCTCTGGT



ACTGACTTTACCCTGACAATTAGTAGCTTGCAAGCTGAAGATGTT



GCGGTATATTATTGTCACCAATACTTGAGTCCCTATACTTTTGGC



CAAGGGACAAAACTGGAAATAAAGCGCACTGTGGCAGCCCCTTC



TGTGTTTATCTTCCCACCCTCCGACGAGCAGCTCAAGTCCGGTAC



CGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCCAAGAGAGGC



CAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCGGTAAT



TCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAA



AAGCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTC



TAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID



NO: 53)









Affinities of the humanized KC18 antibodies to human and mouse FGFR3 were determined and are shown in Table 10 below. The affinities of these humanized antibodies are comparable to the parental KC18 antibody.









TABLE 10







Affinities to human and mouse FGFR3 of the humanized KC18 antibodies









KC18
hFGFR3
mFGFR3













Variants
ka
kd
KD
ka
kd
KD





Hu7
1.96E+05
1.06E−3
5.41E−09
1.98E+05
1.26E−3
6.36E−09


Hu10
2.78E+05
1.32E−3
4.73E−09
2.86E+05
1.47E−3
5.15E−09


Hu12
3.27E+05
5.27E−04
1.61E−09
3.37E+05
5.83E−04
1.73E−09


Hu13
1.89E+05
8.57E−04
4.52E−09
1.93E+05
1.02E−3
5.28E−09


Hu16
2.76E+05
1.04E−3
3.76E−09
2.78E+05
1.18E−3
4.23E−09


Hu18
3.34E+05
4.17E−04
1.25E−09
3.37E+05
4.79E−04
1.42E−09


Hu19
2.25E+05
6.75E−04
2.99E−09
2.35E+05
8.85E−04
3.78E−09


Hu23
2.41E+05
6.48E−04
2.68E−09
2.54E+05
8.50E−04
3.35E−09


Hu27
2.14E+05
8.34E−04
3.90E−09
2.20E+05
1.01E−3
4.60E−09


Hu28
2.13E+05
9.49E−04
4.46E−09
2.10E+05
1.14E−3
5.41E−09


Hu33
1.66E+05
1.14E−3
6.89E−09
1.68E+05
1.54E−3
9.15E−09


Hu34
1.55E+05
1.06E−3
6.85E−09
1.64E+05
1.34E−3
8.14E−09


KC18
2.13E+05
5.80E−04
2.73E−09
2.20E+05
8.32E−04
3.78E−09









Humanized antibodies KC18_Hu42 to KC18_49 were designed based on the sequence of KC_Hu18 to further remove liability sites, such as oxidation sites in FRWH3 and CDRH3, a deamidation site in CDRL1, and an oxidation site in CDRL2 (Table 11). At the end, two additional variants each for VH and VL were designed, which resulted in 8 more variants (KC18_Hu42-49) in addition to KC18_Hu18 (Table 12).









TABLE 11







Liability motifs in KC18_Hu18. The IMGT definition was


used. Liability residues are underlined in the sequences.












Liability
Modi-


Involved
Proposed


Motif
fication
Region
Sequence
in binding
mutations





W
Oxidation
HFW2
IHWVQQAPGKGLEWIGD
No
Y





(SEQ ID NO: 340)







M
Oxidation
HFW3
AYAEKFQGRATLTADRSTD
No
L





TAYMELSSLRSEDTAVYYC







(SEQ ID NO: 341)







W
Oxidation
HFW4

WGQGTLVTVSS

No
Y





(SEQ ID NO: 342)







NNN
Deamidation
LCDR1
QSVLYSNNNKNY
Yes
DNQ





(SEQ ID NO: 302)







W
Oxidation
LCDR2

WAS (SEQ ID NO: 74)

Yes
Y and F









A sequence alignment of Hu18 and the newly designed variants (Hu42-49) is shown in FIG. 7. Mutations of Hu18 residues are underlined.









TABLE 12







Human and mouse germline identity percentage of the desiged KC18 variants.















KC18_VL_3
KC18_VL_14
KC18_VL_15





(SEQ ID NO: 59)
(SEQ ID NO: 60)
(SEQ ID NO: 61)












Human
95.05%
92.08%
92.08%


KC18
Mouse
86.14%
87.13%
87.13%















KC18_VH_6
83.67%
72.45%
KC18_Hu18
KC18_Hu42
KC18_Hu43


(SEQ ID NO: 56)







KC18_VH_15
82.65%
71.43%
KC18_Hu44
KC18_Hu45
KC18_Hu46


(SEQ ID NO: 57)







KC18_VH_16
81.63%
70.41%
KC18_Hu47
KC18_Hu48
KC18_Hu49


(SEQ ID NO: 58)
















TABLE 13







Designed KC18 variant amino acid sequences.








Antibody ID
Sequence





KC18_VH_6
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSS (SEQ ID NO: 56)





KC18_VH_15
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSS (SEQ ID NO: 57)





KC18_VH_16
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSS (SEQ ID NO: 58)





KC18_VL_3
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSNNNKNYLAWYQQKPGQSP



KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSY



RTFGGGTKVEIK (SEQ ID NO: 59)





KC18_VL_14
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKPGQSP



KLLIYYASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYR



TFGGGTKLEIK (SEQ ID NO: 60)





KC18_VL_15
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKPGQSP



KLLIYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYR



TFGGGTKLEIK (SEQ ID NO: 61)
















TABLE 14







Designed KC18 variant sequences.








Antibody ID
Sequence





KC18 Hu44
GAGGTTCAGTTGGTGCAAAGTGGGGCCGAGGTTAAAAAACCAGGTGC


Heavy chain
AACCGTGAAACTGTCCTGCAAGGCGAGTGGTGATACATTTACAGATTT


nucleic acids
TGAAATTCATTGGGTACAGCAGGCACCCGGAAAGGGATTGGAATGGAT


(with constant
AGGAGATGTGGACCCGGAGACTGGCGGAACCGCGTACGCGGAGAAAT


region)
TTCAGGGCAGAGCCACTTTGACGGCGGATAGAAGTACGGATACTGCCT



ACCTTGAACTGAGTTCCTTGCGGTCCGAAGATACGGCAGTTTACTATTG



TACTCGCACGTATGATGGCTACCCATACGCTTTCGATTATTGGGGACAA



GGCACTCTCGTGACCGTATCTTCAGCTTCAACCAAGGGACCTTCTGTCT



TTCCTCTGGCCCCTTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCAC



TCGGGTGCCTTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTG



GAATTCAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTT



CAGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGTCCT



CTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATAAGCCTTC



TAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCATGTGACAAGA



CCCACACCTGTCCGCCCTGTCCGGCACCCGAACTGCTGGGTGGCCCTTC



CGTGTTCCTTTTCCCTCCAAAGCCGAAGGACACTCTTATGATTTCTCGC



ACTCCCGAAGTGACTTGCGTCGTGGTGGATGTGTCCCATGAGGATCCA



GAGGTCAAGTTCAACTGGTACGTGGACGGTGTGGAAGTCCACAACGCC



AAGACTAAGCCGAGAGAGGAACAGTACAATTCAACCTATCGGGTGGT



GAGCGTCCTGACCGTGCTGCACCAGGACTGGCTTAACGGAAAGGAGTA



CAAGTGCAAAGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGA



CCATTAGCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCC



TTCCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCACTT



GTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATGGGAGT



CCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTCCCGTGCTGG



ACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGACCGTGGATAAGTC



TCGCTGGCAGCAAGGGAATGTGTTTTCCTGTTCAGTGATGCATGAGGC



CCTTCATAATCATTACACCCAAAAGTCACTGAGCCTGTCTCCCGGA



(SEQ ID NO: 62)





KC18 Hu44
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG


Heavy Chain
DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT


amino acids
YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV


(with constant
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT


region)
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK



DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN



STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ



VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG



(SEQ ID NO: 63)





KC18 Hu46
GAGGTTCAGTTGGTGCAAAGTGGGGCCGAGGTTAAAAAACCAGGTGC


Heavy Chain
AACCGTGAAACTGTCCTGCAAGGCGAGTGGTGATACATTTACAGATTT


nucleic acids
TGAAATTCATTGGGTACAGCAGGCACCCGGAAAGGGATTGGAATGGAT


(with constant
AGGAGATGTGGACCCGGAGACTGGCGGAACCGCGTACGCGGAGAAAT


region)
TTCAGGGCAGAGCCACTTTGACGGCGGATAGAAGTACGGATACTGCCT



ACCTTGAACTGAGTTCCTTGCGGTCCGAAGATACGGCAGTTTACTATTG



TACTCGCACGTATGATGGCTACCCATACGCTTTCGATTATTGGGGACAA



GGCACTCTCGTGACCGTATCTTCAGCTTCAACCAAGGGACCTTCTGTCT



TTCCTCTGGCCCCTTCAAGCAAGAGCACTTCCGGAGGGACTGCCGCAC



TCGGGTGCCTTGTGAAAGATTACTTCCCAGAGCCTGTCACCGTCAGCTG



GAATTCAGGCGCTCTGACTAGCGGAGTGCACACCTTCCCCGCTGTGCTT



CAGTCCTCCGGACTCTACTCTCTGAGCAGCGTGGTGACCGTGCCGTCCT



CTTCTCTGGGGACCCAGACTTATATCTGCAACGTCAATCATAAGCCTTC



TAATACCAAGGTGGACAAGAAGGTGGAACCCAAATCATGTGACAAGA



CCCACACCTGTCCGCCCTGTCCGGCACCCGAACTGCTGGGTGGCCCTTC



CGTGTTCCTTTTCCCTCCAAAGCCGAAGGACACTCTTATGATTTCTCGC



ACTCCCGAAGTGACTTGCGTCGTGGTGGATGTGTCCCATGAGGATCCA



GAGGTCAAGTTCAACTGGTACGTGGACGGTGTGGAAGTCCACAACGCC



AAGACTAAGCCGAGAGAGGAACAGTACAATTCAACCTATCGGGTGGT



GAGCGTCCTGACCGTGCTGCACCAGGACTGGCTTAACGGAAAGGAGTA



CAAGTGCAAAGTGTCAAACAAGGCACTGCCCGCTCCGATCGAAAAGA



CCATTAGCAAAGCTAAGGGCCAGCCCAGAGAACCCCAAGTCTATACCC



TTCCACCCAGCCGGGACGAGCTGACCAAAAACCAGGTGTCACTCACTT



GTCTCGTGAAGGGTTTCTACCCCTCAGACATCGCCGTCGAATGGGAGT



CCAATGGTCAGCCAGAGAACAACTACAAAACCACCCCTCCCGTGCTGG



ACAGCGACGGGTCTTTCTTTCTCTACTCAAAGCTGACCGTGGATAAGTC



TCGCTGGCAGCAAGGGAATGTGTTTTCCTGTTCAGTGATGCATGAGGC



CCTTCATAATCATTACACCCAAAAGTCACTGAGCCTGTCTCCCGGA



(SEQ ID NO: 64)





KC18 Hu46
EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG


Heavy Chain
DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT


amino acids
YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV


(with constant
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT


region)
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK



DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN



STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ



VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG



(SEQ ID NO: 65)





KC18 Hu44
GACATTGTAATGACACAATCTCCCGACTCTCTTGCAGTCAGCTTGGGTG


Light Chain
AACGAGCAACTATAAATTGTAAAAGCAGCCAGTCTGTACTCTACTCTA


nucleic acids
ATAACAACAAGAACTACCTCGCATGGTATCAGCAGAAACCGGGGCAA


(with constant
AGTCCTAAACTTTTGATCTATTGGGCAAGTACCAGGGAGAGCGGAGTA


region)
CCCGACAGATTCAGCGGGTCTGGATCAGGCACCGATTTTACTCTCACC



ATTTCTTCAGTTCAAGCTGAAGACGTCGCAGTCTACTACTGCCAGCAGT



ATTACAGTTACCGAACTTTTGGCGGTGGAACAAAAGTGGAAATAAAGC



GCACTGTGGCAGCCCCTTCTGTGTTTATCTTCCCACCCTCCGACGAGCA



GCTCAAGTCCGGTACCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTAC



CCAAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAG



CGGTAATTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCA



CCTACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAAAA



GCACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTCTAGCCCT



GTGACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID NO: 66)





KC18 Hu44
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSNNNKNYLAWYQQKPGQSP


Light Chain
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSY


amino acids
RTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV


(with constant
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE


region)
VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67)





KC18 Hu46
GACATAGTTATGACCCAGTCTCCAGACTCCCTCGCAGTTTCTCTCGGCG


Light Chain
AGAGAGTAACAATCAACTGTAAGTCATCACAGTCCGTACTCTACTCTG


nucleic acids
ACAACCAAAAGAATTATTTGGCTTGGTATCAGCAAAAGCCAGGACAAA


(with constant
GCCCCAAACTTCTTATCTATTTTGCCAGCACTAGGGAGTCCGGGGTACC


region)
CGACCGCTTTAGTGGCTCAGGTTCTGGGACAGACTTTACACTGACCATT



TCTAGCGTACAGGCTGAAGACGTTGCAGTCTACTACTGCCAGCAATAC



TATTCTTACAGAACGTTTGGCGGGGGCACAAAGTTGGAGATCAAACGC



ACTGTGGCAGCCCCTTCTGTGTTTATCTTCCCACCCTCCGACGAGCAGC



TCAAGTCCGGTACCGCCTCTGTCGTCTGCCTGCTGAACAATTTCTACCC



AAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCACTGCAAAGCG



GTAATTCACAAGAGTCAGTCACCGAACAAGACTCAAAGGACAGCACCT



ACTCACTGTCATCCACCCTGACTCTCTCAAAGGCTGACTACGAAAAGC



ACAAAGTGTATGCTTGTGAAGTCACTCATCAGGGCCTTTCTAGCCCTGT



GACCAAGAGCTTCAACAGAGGCGAATGC (SEQ ID NO: 68)





KC18 Hu46
DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKPGQSP


Light Chain
KLLIYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYR


amino acids
TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ


(with constant
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV


region)
THQGLSSPVTKSFNRGEC (SEQ ID NO: 69)
















TABLE 15







Humanized Anti-FGFR3 Fab fragment heavy chain amino


acid sequences.








Antibody ID
Sequence





Fab Heavy chain
QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 141)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 143)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 145)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 147)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 149)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDEEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 151)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 153)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 155)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 156)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 164)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 165)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 166)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKS (SEQ ID NO: 167)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDEEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 168)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDNEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 169)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 170)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 171)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 172)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 173)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 174)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 175)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPK (SEQ ID NO: 176)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 177)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDNEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 178)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 179)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 180)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 181)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEP (SEQ ID NO: 182)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEP (SEQ ID NO: 183)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEP (SEQ ID NO: 184)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEP (SEQ ID NO: 185)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEP (SEQ ID NO: 186)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEP (SEQ ID NO: 187)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEP (SEQ ID NO: 188)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEP (SEQ ID NO: 189)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEP (SEQ ID NO: 190)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVE (SEQ ID NO: 191)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVE (SEQ ID NO: 192)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVE (SEQ ID NO: 193)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVE (SEQ ID NO: 194)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVE (SEQ ID NO: 195)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVE (SEQ ID NO: 196)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVE (SEQ ID NO: 197)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVE (SEQ ID NO: 198)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVE (SEQ ID NO: 199)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKV (SEQ ID NO: 200)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKV (SEQ ID NO: 201)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKV (SEQ ID NO: 202)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKV (SEQ ID NO: 203)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKV (SEQ ID NO: 204)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKV (SEQ ID NO: 205)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKV (SEQ ID NO: 206)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKV (SEQ ID NO: 207)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKV (SEQ ID NO: 208)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKK (SEQ ID NO: 209)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKK (SEQ ID NO: 210)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKK (SEQ ID NO: 211)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKK (SEQ ID NO: 212)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKK (SEQ ID NO: 213)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKK (SEQ ID NO: 214)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKK (SEQ ID NO: 215)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKK (SEQ ID NO: 216)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKK (SEQ ID NO: 217)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDK (SEQ ID NO: 218)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDK (SEQ ID NO: 219)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDK (SEQ ID NO: 220)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDK (SEQ ID NO: 221)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDK (SEQ ID NO: 222)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDK (SEQ ID NO: 223)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDK (SEQ ID NO: 224)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDK (SEQ ID NO: 225)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDK (SEQ ID NO: 226)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 227)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 228)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 229)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 230)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 231)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 232)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 233)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 234)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 235)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 236)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 237)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 238)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 239)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 240)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 241)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 242)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 243)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHTC (SEQ ID NO: 244)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 245)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 246)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 247)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 248)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 249)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 250)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 251)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 252)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 253)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 254)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 255)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 256)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 257)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 258)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 259)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 260)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 261)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKTH (SEQ ID NO: 262)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 263)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 264)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 265)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 266)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 267)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 268)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 269)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 270)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDKT (SEQ ID NO: 271)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 272)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 273)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 274)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 275)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 276)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDFEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 277)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 278)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 279)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCDK (SEQ ID NO: 280)



QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWIGD



IDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYCTRTY



DGYPYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 281)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWI



GYINPNNGGTRYNQKFKGKATLTVNKSSSTAYMELRSLTSEDSAVYYCA



RERDYDGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 282)



EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLQWI



GYINPNNGGTNYNQNFKDKATLTVNKSSTTAYMELRSLTSEDSAVYYCA



RERDYDGSMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 283)



EVQLQQSGPDLVKPGASVKISCKASGYTVTDYYMNWVKQSHGKSLEWIG



DINPNNGVTTYNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE



EDFDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD



YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 284)



QVQLQQSGAELVRPGASVTLSCKASGSTFSDFEIHWVKQTPVHGLEWIGG



IDPETGGTAYNQKFKGKAILTADRSSSTAYMELRSLTSEDSAVYYCTRNY



DGYSQTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 285)



QVQLQQSGAELVRPGASVTLSCKASGSTFTDNEIHWVKQTPVHGLEWIGA



IDPETGGTAYNQKFKGKAILTAVKSSSTAYMGLRSLTSEDSAVYYCTRNY



DGYSRTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK



DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY



ICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 286)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYMELSSLRSEDTAVYYCTR



TYDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL



VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ



TYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 287)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEWIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 288)



EVQLVQSGAEVKKPGATVKLSCKASGDTFTDFEIHWVQQAPGKGLEYIG



DVDPETGGTAYAEKFQGRATLTADRSTDTAYLELSSLRSEDTAVYYCTRT



YDGYPYAFDYYGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV



KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT



YICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 289)
















TABLE 16







Humanized Anti-FGFR3 Fab fragment light chain amino acid


sequences.








Antibody ID
Sequence





Fab Light chain
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNNQKNYLAWYQQKPGQS



PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSY



RTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV



QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE



VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 142)



DIQMTQSPSSLSASLGGKVTITCKASQDINKFIAWYQHKPGKGPRLLIHYTS



TLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLWTFGGGTKL



EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL



QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP



VTKSFNRGEC (SEQ ID NO: 144)



DIQMTQSPSSLSASLGGKVTITCKASQDINKFIAWYQHKPGKGPRLLIHYTS



TLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYFCLQYDNLLWTFGGGTKL



EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL



QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP



VTKSFNRGEC (SEQ ID NO: 146)



DIVMTQSHKFMSTSVGDRVSITCKASQDVSTGVAWYQQKPGQSPQLLIY



WASTRHTGVPDRFTGSGSGTDYILTIRSVQAEDLALYYCQQHYSTPLTFG



AGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK



VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ



GLSSPVTKSFNRGEC (SEQ ID NO: 148)



NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQS



PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSS



YTFGGGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK



VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC



EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 150)



NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQS



PKLLIYWASTRESGVPDRFTGSGSGTDFSLSISSVQTEDLAVYYCHQYLSS



YTFGGGTRLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV



QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE



VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 152)



DIVMTQSPDSLAVSLGERATINCKSSQSVLYSNNNKNYLAWYQQKPGQSP



KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSY



RTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV



QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE



VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67)



DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKPGQSP



KLLIYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYR



TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ



WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV



THQGLSSPVTKSFNRGEC (SEQ ID NO: 69)



DIVMTQSPDSLAVSLGERVTINCKSSQSVLYSDNQKNYLAWYQQKPGQSP



KLLIYYASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYR



TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ



WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV



THQGLSSPVTKSFNRGEC (SEQ ID NO: 154)









In certain embodiments, an anti-FGFR3 F(ab) fragment of the present application comprises a heavy chain of Table 15, and a light chain in Table 16.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 141, 164, 173, 182, 191, 200, 209, 218, 227, 236, 245, 254, 263, 272, or 281, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 142.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 143, 165, 174, 183, 192, 201, 210, 219, 228, 237, 246, 255, 264, 273, or 282, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 144.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 145, 166, 175, 184, 193, 202, 211, 220, 229, 238, 247, 256, 265, 274, or 283 and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 146.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 147, 167, 176, 185, 194, 203, 212, 221, 230, 239, 248, 257, 266, 275, or 284, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 148.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 149, 168, 177, 186, 195, 204, 213, 222, 231, 240, 249, 258, 267, 276, or 285, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 150.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 151, 169, 178, 187, 196, 205, 214, 223, 232, 241, 250, 259, 268, 277, or 286, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 152.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, or 287, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, or 287, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 153, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, or 287, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, or 289, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 67.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, or 289, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 154.


In certain embodiments, the antibody F(ab) fragment heavy chain comprises the amino acid sequence of SEQ ID NO: 156, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, or 289, and the antibody F(ab) fragment light chain comprises the amino acid sequence of SEQ ID NO: 69.


In Vitro Analysis


Antibodies were screened for binding and specificity to FGFR3. Activity was assessed using a Homogenous Time-Resolved Fluorescence (HTRF) assay to evaluate the level of inhibition on Erk phosphorylation using mouse primary rib chondrocytes. Briefly, primary mouse rib chondrocyte cells isolated from the achondroplasia mouse model were pretreated for 2 hours with anti-FGFR3 antibodies with a concentration ranging from 0.016 μg/ml to 100 μg/ml. Cells were then stimulated with FGF18 for 5 minutes. The reaction was then stopped, total Erk was measured using the HTRF assay, and Phospho-Erk (Thr202/Tyr204) was measured in a separate HTRF assay. The percent inhibition of Erk phosphorylation was measured by taking the ratio of Phospho-Erk over total Erk and multiplying by 100. Particularly, mouse antibodies KC18, KE63, and KE94 and their corresponding Fab fragments were tested in the HTRF assay, all of which inhibited Erk phosphorylation (FIG. 9A and FIG. 9B).


To determine the effects of different mouse antibody formats on inhibition of Erk phosphorylation, KC18 mouse antibody in various formats, including full-length antibody (IgG), Fab, a one-armed, monovalent antibody (MetMab), and pegylated (PEG) Fab fragment were tested in the HTRF assay and compared to isotype control (Iso). Also, KC18 Fab with half-life extension using a human albumin nanobody (KC18 Fab-HLE) was tested, with and without human serum albumin (HSA) or mouse serum albumin (MSA).


The full-length KC18 mouse antibody has a heavy chain of SEQ ID NO: 291 (with mIgG2a Fc sequence), and a light chain of SEQ ID NO: 292. The KC18 mouse Fab antibody has a heavy chain of SEQ ID NO: 293, and a light chain of SEQ ID NO: 294.









(SEQ ID NO: 291)


QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWI





GDIDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYC





TRTYDGYPYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVT





LGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTS





STWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPPVAGP





SVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT





AQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKGLPSSIER





TISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWT





NNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHE





GLHNHHTTKSFSRTPGK





(SEQ ID NO: 292)


DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNNQKNYLAWYQQKPGQ





SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQ





YYSYRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF





YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE





RHNSYTCEATHKTSTSPIVKSFNRNEC





(SEQ ID NO: 293)


QVQLQQSGAELVRPGASVTLSCKASGDTFTDFEIHWVKQTPVHGLEWI





GDIDPETGGTAYNQKFRGRAMLTADRSSSTAYMELRSLTSEDSAVYYC





TRTYDGYPYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVT





LGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTS





STWPSQSITCNVAHPASSTKVDKKI





(SEQ ID NO: 294)


DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNNQKNYLAWYQQKPGQ





SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQ





YYSYRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF





YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE





RHNSYTCEATHKTSTSPIVKSFNRNEC






All formats inhibited Erk phosphorylation (FIG. 10A to FIG. 10C). The antibody format referred to in FIG. 10A as “MetMab” is described in further detail in Merchant et al. (PNAS. 110(32): E2987-E2996. 2013).


Humanized FGFR3 antibodies, including Hu18, Hu44, and Hu46, were also tested in the HTRF assay, and found to inhibit Erk phosphorylation (FIG. 11). The IC50s of these humanized antibodies were determined, as well (FIG. 8A to FIG. 8C, respectively).


In Vivo Analysis


For in vivo evaluation of the antibodies, an achondroplasia mouse model (Ach) was used. The achondroplasia mouse is a transgenic mouse that overexpresses the mouse FGFR3 protein having the G380R mutation under control of the collagen II promoter (Shazeeb et al., (2018) Sci Rep 8, 469). The mice were genotyped at 1 day of age, and randomized to either a control (i.e., saline) group or an antibody dosed group. Achondroplasia mice (G380R) received a daily dose subcutaneously (SC) from 3 days of age to 20 days of age. The mice were then euthanized, and bones were collected at 21 days of age for microCT analysis. Tibias, femurs, vertebrae and skulls were used for the analysis, and lengths were measured using 3D microCT analysis. AMIRA (V6.0.1, FEI, Hillsboro, Oreg., USA) was used for all numerical analysis of bone lengths. The lengths of leg bones were measured using seed points along the bone, and a 3D length tool in AMIRA. The results indicated that achondroplasia (Ach) mice treated with KC18 Fab had a significantly increased tibia and femur length (FIG. 12A and FIG. 12B), a significantly increased vertebra and skull length (FIG. 13A and FIG. 13B), a significantly increased brain volume (FIG. 14), and corrected vertebral abnormalities as measured by Kyphosis Index (FIG. 15A-FIG. 15B). Ach mice treated with KC18 Fab also had several improved bone parameters, including femur growth plate volume, and femur diameter (FIG. 16A and FIG. 16B). Ach mice treated with KC18 Fab also had improved bone age as shown by more developed secondary ossification center in tibia (FIG. 17) compared to those treated with vehicle.


In summary, achondroplasia is the most common form of dwarfism due to activating mutations in the FGFR3 gene. FGFR3 protein is expressed in the growth plate and its function is to regulate proper growth. However, the activating mutations lead to excessive inhibition of chondrocyte proliferation and differentiation which is the main cause for the short stature and other skeletal deformities. The antibodies generated herein are specific to FGFR3 and inhibit FGFR3 activity. The mechanism of action of the antibody is to inhibit ligand binding and prevent activation of the receptor. The antibodies described herein can block ligand activation and subsequently inhibit downstream signaling measured by the decrease in Erk phosphorylation. This translates into inhibition of receptor activity. In vivo testing of anti-FGFR3 antibodies in the Ach mouse model demonstrated efficacy on the axial and appendicular skeleton. More specifically, a significant increase in femur and tibia length, skull length as well as lumbar length were observed.

Claims
  • 1. An antigen-binding protein or antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 3 (FGFR3), comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GDTFTDFE (SEQ ID NO: 70), a CDR-H2 sequence comprising the amino acid sequence of VDPETGGT (SEQ ID NO: 297), and a CDR-H3 sequence comprising the amino acid sequence of TRTYDGYPYAFDY (SEQ ID NO: 301); andwherein the VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVLYSNNNKNY (SEQ ID NO: 302), a CDR-L2 sequence comprising the amino acid sequence of WAS (SEQ ID NO: 74), and a CDR-L3 sequence comprising the amino acid sequence of QQYYSYRT (SEQ ID NO: 75).
  • 2. The antigen binding protein or antigen-binding fragment thereof of claim 1, wherein the VH domain is at least about 90% identical to the amino acid sequence of SEQ ID NO: 57, and the VL domain is at least about 90% identical to the amino acid sequence of SEQ ID NO: 19.
  • 3. The antigen binding protein or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain is at least 95% identical to the amino acid sequence of SEQ ID NO: 63, and the antibody light chain is at least 95% identical to the amino acid sequence of SEQ ID NO: 67.
  • 4. The antigen binding protein or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 63, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 67.
  • 5. The antigen binding protein or antigen-binding fragment thereof of claim 1, wherein the antigen binding protein or antigen-binding binding fragment thereof comprises an Fc region.
  • 6. The antigen binding protein or antigen-binding fragment thereof of claim 5, wherein the Fc region is a human IgG1 Fc region.
  • 7. The antigen binding protein or antigen-binding fragment thereof of claim 1, wherein the antigen binding protein or antigen-binding binding fragment thereof comprises an antibody F(ab), F(ab′)2, Fab′-SH, Fv, or scFv fragment.
  • 8. The antigen binding protein or antigen-binding fragment thereof of claim 7, wherein the antibody F(ab) fragment comprises the sequence of SEQ ID NO: 56 followed by the first about 100 to about 110 amino acids of SEQ ID NO: 54.
  • 9. The antigen binding protein or antigen-binding fragment thereof of claim 7, wherein the antibody F(ab) fragment comprises a heavy chain comprising the sequence of SEQ ID NO: 57 followed by the first about 100 to about 110 amino acids of SEQ ID NO: 54.
  • 10. The antigen binding protein or antigen-binding fragment thereof of claim 7, wherein the antibody F(ab) fragment comprises a heavy chain comprising the sequence of SEQ ID NO: 58 followed by the first about 100 to about 110 amino acids of SEQ ID NO: 54.
  • 11. A pharmaceutical composition comprising the antigen binding protein or antigen-binding fragment of claim 1, and a pharmaceutically acceptable carrier.
  • 12. The antigen binding protein or antigen-binding fragment thereof of claim 1, wherein the antigen binding protein or antigen-binding binding fragment thereof comprises an antibody F(ab) fragment.
  • 13. An antigen-binding protein or antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 3 (FGFR3), comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein: the VH domain comprises the amino acid sequence of SEQ ID NO: 57, and
  • 14. A pharmaceutical composition comprising the antigen binding protein or antigen-binding fragment of claim 13, and a pharmaceutically acceptable carrier.
  • 15. An antibody F(ab) fragment, wherein the antibody F(ab) fragment comprises an antibody F(ab) fragment heavy chain comprising the amino acid sequence of SEQ ID NO: 155, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, or 288, and an antibody F(ab) fragment light chain comprising the amino acid sequence of SEQ ID NO: 67.
  • 16. A pharmaceutical composition comprising the antigen binding protein or antigen-binding fragment of claim 15, and a pharmaceutically acceptable carrier.
RELATED APPLICATION

This application claims the benefit of priority of U.S. Provisional Application No. 63/068,575, filed Aug. 21, 2020, the content of which is incorporated by reference in its entirety for all purposes.

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Related Publications (1)
Number Date Country
20220056142 A1 Feb 2022 US
Provisional Applications (1)
Number Date Country
63068575 Aug 2020 US