U.S. patent application number 13/572557 was filed with the patent office on 2012-12-20 for crystal structures and methods using same.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Christian Wiesmann.
Application Number | 20120321606 13/572557 |
Document ID | / |
Family ID | 42340706 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120321606 |
Kind Code |
A1 |
Wiesmann; Christian |
December 20, 2012 |
CRYSTAL STRUCTURES AND METHODS USING SAME
Abstract
The present invention relates generally to the fields of
molecular biology and growth factor regulation. More specifically,
the invention concerns modulators of FGFR3 function, and the
identification and uses of said modulators.
Inventors: |
Wiesmann; Christian;
(Bottmingen, CH) |
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
42340706 |
Appl. No.: |
13/572557 |
Filed: |
August 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12661852 |
Mar 24, 2010 |
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13572557 |
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61163222 |
Mar 25, 2009 |
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Current U.S.
Class: |
424/94.3 ;
435/188; 435/194; 703/11 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 2039/505 20130101; C07K 2317/92 20130101; A61P 43/00 20180101;
C07K 2317/732 20130101; C07K 2317/567 20130101; A61K 39/395
20130101; A61K 39/3955 20130101; C07K 2317/76 20130101; A61P 19/00
20180101; C07K 2317/73 20130101; A61P 35/00 20180101; C07K 2317/34
20130101; C07K 2317/56 20130101; C07K 16/2863 20130101; C07K
2317/52 20130101; C07K 16/3038 20130101; C07K 2317/55 20130101;
C07K 16/3061 20130101; C07K 2317/77 20130101; C07K 2319/30
20130101; C07K 2317/565 20130101; C07K 2317/24 20130101; A61K
39/39558 20130101; C07K 2317/75 20130101 |
Class at
Publication: |
424/94.3 ;
435/188; 435/194; 703/11 |
International
Class: |
A61K 38/54 20060101
A61K038/54; C12N 9/12 20060101 C12N009/12; G06F 19/16 20110101
G06F019/16; C12N 9/96 20060101 C12N009/96 |
Claims
1. A crystal of FGFR3 complexed with an anti-FGFR3 antibody
comprising a human FGFR3 comprising sequence of SEQ ID NO:272 or
conservative substitutions thereof complexed with an anti-FGFR3
antibody comprising (a) a light chain variable domain comprising
the amino acid sequence of SEQ ID NO:274 or conservative
substitutions thereof, and (b) a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO:275 or conservative
substitutions thereof.
2. Crystalline form of a complex of FGFR3 and an anti-FGFR3
antibody. space group P2.sub.12.sub.12.sub.1 with cell parameters
of a=58.5 .ANG., b=99.3 .ANG. and c=143.7 .ANG..
3. A crystal of a 1:1 complex of FGFR3 and an anti-FGFR3 antibody
having a space group having a space group symmetry of
P2.sub.12.sub.12.sub.1 and comprising a unit cell having the
dimensions of a, b, and c, wherein a=58.5 .ANG., b=99.3 .ANG. and
c=143.7 .ANG..
4. A cocrystal of FGFR3 with an anti-FGFR3 antibody having the
three-dimensional coordinates of Table 6.
5. The crystal of claim 4, wherein the crystal diffracts X-rays for
the determination of atomic coordinates to a resolution of 5 .ANG.
or better.
6. A composition comprising a crystal of any of claims 1-5, and a
carrier.
7. A molecule or molecular complex comprising at least a portion of
the binding site of FGFR3 or conservative substitution thereof,
wherein the binding site comprises at least one amino acid residue
corresponding to residues 158, 170, 171, 173, 175, and/or 315 or
mixtures thereof, the binding site defined by a set of points
having a root mean square deviation of less than about 0.70 .ANG.
from points representing the backbone atoms of the amino acids as
represented by the structure coordinates listed in Table 6.
8. The molecule or molecular complex of claim 7, wherein the
binding site comprises at least one amino acid residue
corresponding to residues 158, 159, 169, 170, 171, 173, 175, 205,
207, and/or 315 or mixtures thereof.
9. The molecule or molecular complex of claim 7, wherein the
binding site comprises at least one amino acid residue
corresponding to residues 154, 155, 158, 159, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202,
205, 207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279,
280, 281, 282, 283, 314, 315, 316, 317 and/or 318 or mixtures
thereof.
10. A computer-implemented method for causing a display of a
graphical three-dimensional representation of the structure of a
portion of a crystal of FGFR3 complexed with an anti-FGFR3
antibody, or structural homologs thereof, wherein the method
comprises: causing said display of said graphical three-dimensional
representation by a computer system programmed with instructions
for transforming structure coordinates into said graphical
three-dimensional representation of said structure and for
displaying said graphical three-dimensional representation, wherein
said graphical three-dimensional representation is generated by
transforming said structure coordinates into said graphical
three-dimensional representation of said structure, wherein said
structure coordinates comprise structure coordinates of the
backbone atoms of the portion of the crystal, wherein the portion
of the crystal comprises an FGFR3 binding site, and wherein the
crystal has the space group symmetry P2.sub.12.sub.12.sub.1.
11. The computer-implemented method of claim 10, wherein the
FGFR3:anti-FGFR3 antibody crystal comprises a polypeptide
comprising an amino acid sequence shown in Table 6 or conservative
substitution thereof, and further comprises an antibody comprising
(a) a light chain variable domain comprising the amino acid
sequence of SEQ ID NO:274 or conservative substitutions thereof,
and (b) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:275 or conservative substitutions
thereof.
12. The computer-implemented method of claim 10, wherein the
structure coordinates are defined in Table 6.
13. The computer-implemented method of claim 10, wherein the
structure coordinates comprise the structure coordinates of the
backbone atoms of the amino acid residues corresponding to residues
158, 170, 171, 173, 175, and/or 315 of FGFR3.
14. The computer-implemented method of claim 10, wherein the
structure coordinates comprise the structure coordinates of the
backbone atoms of the amino acid residues corresponding to residues
154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169,
170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214,
216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282, 283, 314,
315, 316, 317, 318 of FGFR3.
15. The computer-implemented method of claim 10, wherein the
structure coordinates are determined by homology modeling.
16. A machine-readable data storage medium comprising a data
storage material encoded with machine-readable instructions for:
(a) transforming data into a graphical three-dimensional
representation for the structure of a portion of a crystal of FGFR3
complexed with an anti-FGFR3 antibody, or structural homologs
thereof; and (b) causing the display of said graphical
three-dimensional representation; wherein said data comprise
structure coordinates of the backbone atoms of the amino acids
defining an FGFR3 binding site; and wherein the crystal or
structural homolog has the space group symmetry
P2.sub.12.sub.12.sub.1.
17. A computer system for displaying a three-dimensional graphical
representation for the structure of a portion of a crystal of FGFR3
complexed with an anti-FGFR3 antibody, or structural homologs
thereof, comprising: (a) a machine-readable data storage medium
comprising a data storage material encoded with machine-readable
data, wherein said data comprise structure coordinates of the
backbone atoms of the amino acids defining an FGFR3 binding site,
wherein the crystal or structural homolog has the space group
symmetry P2.sub.12.sub.12.sub.1; (b) a working memory; (c) a
central processing unit coupled to said working memory and to said
machine-readable data storage medium for processing said
machine-readable data into sad three-dimensional graphical
representation; and (d) a display coupled to said central
processing unit for displaying said three-dimensional graphical
representation.
18. A method for obtaining structural information about a molecule
or molecular complex comprising applying at least a portion of the
structure coordinates of a FGFR3 complexed with an anti-FGFR3
antibody to an X-ray diffraction pattern of the molecule or
molecular complex's crystal structure to cause the generation of a
three-dimensional electron density map of at least a portion of the
molecule or molecular complex; wherein the FGFR3:anti-FGFR3
antibody crystal comprises a polypeptide comprising an amino acid
sequence of SEQ ID NO:272 or conservative substitution thereof, and
further comprises an antibody comprising (a) a light chain variable
domain comprising the amino acid sequence of SEQ ID NO:274 or
conservative substitutions thereof, and (b) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:275 or
conservative substitutions thereof; wherein the FGFR3:anti-FGFR3
antibody crystal diffracts x-rays for the determination of atomic
coordinates to a resolution of 5 .ANG. or better.
19. A method of screening for molecules that may be antagonists or
agonists of FGFR3 comprising: (a) computationally screening agents
against a three-dimensional model to identify potential antagonists
or agonists of FGFR3; wherein the three-dimensional model comprises
a three-dimensional model of at least a portion of a crystal of a
FGFR3 complexed with an anti-FGFR3 antibody; wherein the three
dimensional model is generated from at least a portion of the
structure coordinates of the crystal by a computer algorithm for
generating a three-dimensional model of the crystal useful for
identifying agents that are potential antagonists or agonists of
FGFR3; wherein the FGFR3: anti-FGFR3 antibody crystal comprises a
polypeptide comprising an amino acid sequence SEQ ID NO:272 or
conservative substitution thereof, and further comprises an
antibody comprising (a) a light chain variable domain comprising
the amino acid sequence of SEQ ID NO:274 or conservative
substitutions thereof, and (b) a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO:275 or conservative
substitutions thereof; and wherein the FGFR3:anti-FGFR3 antibody
crystal diffracts x-rays for the determination of atomic
coordinates to a resolution of 5 .ANG. or better.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 12/661,852, filed Mar. 24, 2010 which claims priority to U.S.
patent application No. 61/163,222, filed on Mar. 25, 2009, the
contents of which are incorporated herein by reference.
REFERENCE TO TABLE SUBMITTED
[0002] This application is accompanied by a Table submitted in .txt
format which contains the file titled "P4294R1-1C1 Table 6.txt",
the contents of which are incorporated in their entirety herein by
reference.
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120321606A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
molecular biology. More specifically, the invention concerns
anti-FGFR3 antibodies, and uses of same.
BACKGROUND OF THE INVENTION
[0004] Fibroblast growth factors (FGFs) and their receptors (FGFRs)
play critical roles during embryonic development, tissue
homeostasis and metabolism (1-3). In humans, there are 22 FGFs
(FGF1-14, FGF16-23) and four FGF receptors with tyrosine kinase
domain (FGFR1-4). FGFRs consist of an extracellular ligand binding
region, with two or three immunoglobulin-like domains (IgD1-3), a
single-pass transmembrane region, and a cytoplasmic, split tyrosine
kinase domain. FGFR1, 2 and 3 each have two major alternatively
spliced isoforms, designated IIIb and Mc. These isoforms differ by
about 50 amino acids in the second half of IgD3, and have distinct
tissue distribution and ligand specificity. In general, the Mb
isoform is found in epithelial cells, whereas IIIc is expressed in
mesenchymal cells. Upon binding FGF in concert with heparan sulfate
proteoglycans, FGFRs dimerize and become phosphorylated at specific
tyrosine residues. This facilitates the recruitment of critical
adaptor proteins, such as FGFR substrate 2 .alpha. (FRS2.alpha.),
leading to activation of multiple signaling cascades, including the
mitogen-activated protein kinase (MAPK) and PI3K-AKT pathways (1,
3, 4). Consequently, FGFs and their cognate receptors regulate a
broad array of cellular processes, including proliferation,
differentiation, migration and survival, in a context-dependent
manner.
[0005] Aberrantly activated FGFRs have been implicated in specific
human malignancies (1, 5). In particular, the t(4; 14) (p16.3; q32)
chromosomal translocation occurs in about 15-20% of multiple
myeloma patients, leading to overexpression of FGFR3 and correlates
with shorter overall survival (6-9). FGFR3 is implicated also in
conferring chemoresistance to myeloma cell lines in culture (10),
consistent with the poor clinical response of t(4; 14)+ patients to
conventional chemotherapy (8). Overexpression of mutationally
activated FGFR3 is sufficient to induce oncogenic transformation in
hematopoietic cells and fibroblasts (11-14, 15), transgenic mouse
models (16), and murine bone marrow transplantation models (16,
17). Accordingly, FGFR3 has been proposed as a potential
therapeutic target in multiple myeloma. Indeed, several
small-molecule inhibitors targeting FGFRs, although not selective
for FGFR3 and having cross-inhibitory activity toward certain other
kinases, have demonstrated cytotoxicity against FGFR3-positive
myeloma cells in culture and in mouse models (18-22).
[0006] FGFR3 overexpression has been documented also in a high
fraction of bladder cancers (23, 24). Furthermore, somatic
activating mutations in FGFR3 have been identified in 60-70% of
papillary and 16-20% of muscle-invasive bladder carcinomas (24,
25). In cell culture experiments, RNA interference (11, 26) or an
FGFR3 single-chain Fv antibody fragment inhibited bladder cancer
cell proliferation (27). A recent study demonstrated that an FGFR3
antibody-toxin conjugate attenuates xenograft growth of a bladder
cancer cell line through FGFR3-mediated toxin delivery into tumors
(28). However, it remains unclear whether FGFR3 signaling is indeed
an oncogenic driver of in vivo growth of bladder tumors. Moreover,
the therapeutic potential for targeting FGFR3 in bladder cancer has
not been defined on the basis of in vivo models. Publications
relating to FGFR3 and anti-FGFR3 antibodies include co-pending,
co-owned U.S. patent application Ser. No. ______ (attorney docket
P4294R1) filed Mar. 24, 2010, U.S. Patent Publication no.
2005/0147612; Rauchenberger et al, J Biol Chem 278 (40):38194-38205
(2003); WO2006/048877; Martinez-Torrecuadrada et al, (2008) Mol
Cancer Ther 7(4): 862-873; WO2007/144893; Trudel et al. (2006)
107(10): 4039-4046; Martinez-Torrecuadrada et al (2005) Clin Cancer
Res 11 (17): 6280-6290; Gomez-Roman et al (2005) Clin Cancer Res
11:459-465; Direnzo, R et al (2007) Proceedings of AACR Annual
Meeting, Abstract No. 2080; WO2010/002862.
[0007] It is clear that there continues to be a need for agents
that have clinical attributes that are optimal for development as
therapeutic agents. The invention described herein meets this need
and provides other benefits.
[0008] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides a crystal comprising
an anti-FGFR3 antibody and an FGFR3, the crystal having, for
example, the structural coordinates of Table 6. In one aspect, the
invention provides a heavy atom derivative of a crystal of the
invention. In one aspect, the invention provides a composition
comprising a crystal of the invention.
[0010] In one aspect, the invention provides a computer-implemented
method, a computer system and machine-readable data storage medium
comprising a data storage material encoded with machine-readable
instructions for causing a display of a graphical three-dimensional
representation of a structure of a portion of a crystal of
anti-FGFR3 antibody (or structural homolog thereof) in complex with
FGFR3 (or a structural homolog and/or portion thereof). In some
embodiments, the computer is programmed with instructions for
transforming the structure coordinates into the graphical
three-dimensional representation of the structure and/or displaying
the graphical three-dimensional representation. In some
embodiments, the structure coordinates include the coordinates of
the backbone atoms of the portion of the crystal and/or one or more
of the contact residues between the anti-FGFR3 antibody and the
FGFR3 in the complex (e.g., some or all of the coordinates shown in
Table 6).
[0011] In one aspect, the amino acid residues that form a binding
site for an inhibitor binding site on FGFR3 are identified and are
useful, for example, in methods to model the structure of an FGFR3
binding site and to identify agents that can bind or fit into the
binding site. This use includes the rational design of modulators
of FGFR3 activity. For example, these modulators include ligands
that interact with FGFR3 and modulate FGFR/FGF activities.
[0012] In some embodiments, the crystals are formed from an FGFR3
sequence comprising sequence
[0013] ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG
REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPH
RPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVL
KSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA DEAGSV
(SEQ ID NO:272) and an anti-FGFR3 antibody.
[0014] In some embodiments, the crystals are formed from an FGFR3
sequence comprising sequence
[0015] ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG
REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPH
RPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVL
KSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA
DEAGSVHHHHHH (SEQ ID NO:273) and an anti-FGFR3 antibody.
[0016] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising HVR-L1, HVR-L2, HVR-L3,
wherein each, in order, comprises SEQ ID NO:4, 5, 6, and/or a heavy
chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, where
each, in order, contains SEQ ID NO: 1, 2, 3. In some embodiments,
the anti-FGFR3 antibody comprises a light chain variable region
comprising sequence
TABLE-US-00001 (SEQ ID NO: 274)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKR
and a heavy chain variable region comprising sequence
TABLE-US-00002 (SEQ ID NO: 275)
EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGR IY PTN
GSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
ARTYGIYDLYVDYTEYVMDYWGQGTLV.
[0017] In some embodiments, the anti-FGFR3 antibody comprises:
[0018] (a) at least one, two, three, four, or five hypervariable
region (HVR) sequences selected from:
[0019] (i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is
RASQDVDTSLA (SEQ ID NO:87),
[0020] (ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is
SASFLYS (SEQ ID NO:88),
[0021] (iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is
QQSTGHPQT (SEQ ID NO:89),
[0022] (iv)) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is
GFTFTSTGIS (SEQ ID NO:84),
[0023] (v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is
GRIYPTSGSTNYADSVKG (SEQ ID NO:85), and
[0024] (vi) HVR-H3 comprising sequence F1-F20, wherein F1-F20 is
ARTYGIYDLYVDYTEYVMDY (SEQ ID NO:86); and
[0025] (b) at least one variant HVR, where the variant HVR sequence
comprises modification of at least one residue (at least two
residues, at least three or more residues) of the sequence depicted
in SEQ ID NOS:1-18, 48-131 and 140-145. The modification desirably
is a substitution, insertion, or deletion.
[0026] In some embodiments, a HVR-L1 variant comprises 1-6 (1, 2,
3, 4, 5, or 6) substitutions in any combination of the following
positions: A5 (V or D), A6 (V or I), A7 (D, E or S), A8 (T or I),
A9 (A or S) and A10 (V or L). In some embodiments, a HVR-L2 variant
comprises 1-2 (1 or 2) substitutions in any combination of the
following positions: B1 (S or G), B4 (F or S or T) and B6 (A or Y).
In some embodiments, a HVR-L3 variant comprises 1-6 (1, 2, 3, 4, 5,
or 6) substitutions in any combination of the following positions:
C3 (G or S or T), C4 (T or Y or A), C5 (G or S or T or A), C6 (A or
H or D or T or N), C7 (Q or P or S), and C8 (S or Y or L or P or
Q). In some embodiment, a HVR-H1 variant comprises 1-3 (1, 2, or 3)
substitutions in any combination of the following positions: D3 (S
or T), D5 (W or Y or S or T), D6 (S or G or T). In some embodiment,
a HVR-H2 variant comprises 1-6 (1, 2, 3, 4, 5, or 6) substitutions
in any combination of the following positions: E2 (R or S), E6 (Y
or A or L or S or T), E7 (A or Q or D or G or Y or S or N or F), E8
(A or D or G), E9 (T or S), E10 (K or F or T or S), E11 (Y or H or
N or I).
[0027] In some embodiments, the anti-FGFR3 antibody comprises:
[0028] (a) at least one, two, three, four, or five hypervariable
region (HVR) sequences selected from:
[0029] (i) HVR-L1 comprising sequence
RASQX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6A, wherein X.sub.1 is
V or D, X.sub.2 is V or I, X.sub.3 is D, E or S, X.sub.4 is T or I,
X.sub.5 is A or S, and X.sub.6 is V or L (SEQ ID NO:146),
[0030] (ii) HVR-L2 comprising sequence X.sub.1ASFLX.sub.2S wherein
X.sub.1 is S or G and X.sub.2 is A or Y (SEQ ID NO:147),
[0031] (iii) HVR-L3 comprising sequence
QQX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6T, wherein X.sub.1 is
G, S or T, X.sub.2 is T, Y or A, X.sub.3 is G, S, T, or A, X.sub.4
is A, H, D, T, or N, X.sub.5 is Q, P or S, X.sub.6 is S, Y, L, P or
Q (SEQ ID NO:148),
[0032] (iv)) HVR-H1 comprising sequence
GFX.sub.1FX.sub.2X.sub.3TGIS, wherein X.sub.1 is S or T, X.sub.2 is
W, Y, S or T, X.sub.3 is S, G, or T (SEQ ID NO:149),
[0033] (v) HVR-H2 comprising sequence
GRIYPX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6YADSVKG, wherein
X.sub.1 is Y, A, L, 5, or T, X.sub.2 is A, Q, D, G, Y, 5, N or F,
X.sub.3 is A, D, or G, X.sub.4 is T or 5, X.sub.5 is K, F, T, or S,
X.sub.6 is Y, H, N or I (SEQ ID NO:150), and
[0034] (vi) HVR-H3 comprising sequence ARTYGIYDLYVDYTEYVMDY (SEQ ID
NO:151).
[0035] In some embodiments, HVR-L1 comprises sequence
RASQX.sub.1VX.sub.2X.sub.3X.sub.4VA, wherein X.sub.1 is V or D,
X.sub.2 is D, E or S, X.sub.3 is T or I, X.sub.4 is A or S (SEQ ID
NO:152). In some embodiments, HVR-L3 comprises sequence
QQX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6T, wherein X.sub.1 is
S, G, or T, X.sub.2 is Y, T, or A, X.sub.3 is T or G, X.sub.4 is T,
H or N, X.sub.5 is P or S, X.sub.6 is P, Q, Y, or L (SEQ ID
NO:153). In some embodiments, HVR-H2 comprises sequence
GRIYPX.sub.1X.sub.2GSTX.sub.3YADSVKG, wherein X.sub.1 is T or L,
X.sub.2 is N, Y, S, G, A, or Q; X.sub.3 is N or H (SEQ ID
NO:154).
[0036] In one aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, wherein
each, in order, comprises SEQ ID NO:1, 2, 3, and/or a light chain
variable region comprising HVR-L1, HVR-L2, and HVR-L3, where each,
in order, contains SEQ ID NO: 4, 5, 6.
[0037] In another aspect, an-anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, wherein
each, in order, comprises SEQ ID NO:7, 8, 9, and/or a light chain
variable region comprising HVR-L1, HVR-L2, and HVR-L3, where each,
in order, comprises SEQ ID NO: 10, 11, 12.
[0038] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:13, 14, 15, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO:16, 17, 18.
[0039] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO: 48, 49, 50, and/or a light
chain variable region HVR-L1, HVR-L2, and HVR-L3, where each, in
order, comprises SEQ ID NO: 51, 52, 53.
[0040] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO: 54, 55, 56, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 57, 58, 59.
[0041] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:60, 61, 62, 63, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 63, 64, 65.
[0042] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:66, 67, 68, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 69, 70, 71.
[0043] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:72, 73, 74, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 75, 76, 77.
[0044] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:78, 79 80, and/or a light chain
variable region comprising HVR-L1, HVR-L2, and HVR-L3, where each,
in order, comprises SEQ ID NO:81, 82, 83.
[0045] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO: 84, 85, 86, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO:87, 88, 89.
[0046] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO: 90, 91, 92, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO:93, 94, 95.
[0047] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO: 96, 97, 98, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 99, 100, 101.
[0048] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO: 102, 103, 104, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 105, 106, 107.
[0049] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:108, 109, 110, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 111, 112, 113.
[0050] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:114, 115, 116, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO:117, 118, 119.
[0051] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:120, 121, 122, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO: 123, 124, 125.
[0052] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:126, 127, 128, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO:129, 130, 131.
[0053] In another aspect, an anti-FGFR3 antibody comprises a heavy
chain variable region comprising HVR-H1, HVR-H2, HVR-H3, where
each, in order, comprises SEQ ID NO:143, 144, 145, and/or a light
chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, where
each, in order, comprises SEQ ID NO:140, 141, 142.
[0054] The amino acid sequences of SEQ ID NOs:1-18, 48-131 and
140-145 are numbered with respect to individual HVR (i.e., H1, H2
or H3) as indicated in FIG. 1, the numbering being consistent with
the Kabat numbering system as described below.
[0055] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:132 and a light
chain variable region.
[0056] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising SEQ ID NO: 133, and a heavy
chain variable region.
[0057] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:132 and a light
chain variable region comprising SEQ ID NO:133.
[0058] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:134 and a light
chain variable region.
[0059] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising SEQ ID NO: 135, and a heavy
chain variable region.
[0060] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising SEQ ID NO: 139, and a heavy
chain variable region.
[0061] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:134 and a light
chain variable region comprising SEQ ID NO:135.
[0062] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:136 and a light
chain variable region.
[0063] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising SEQ ID NO: 137, and a heavy
chain variable region.
[0064] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:136 and a light
chain variable region comprising SEQ ID NO:137.
[0065] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:138 and a light
chain variable region.
[0066] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising SEQ ID NO: 139, and a heavy
chain variable region.
[0067] In some embodiments, the anti-FGFR3 antibody comprises a
heavy chain variable region comprising SEQ ID NO:138 and a light
chain variable region comprising SEQ ID NO:139.
[0068] In some embodiments, the anti-FGFR3 antibody comprises: at
least one, two, three, four, five, and/or six hypervariable region
(HVR) sequences selected from the group consisting of:
[0069] (a) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID NO:155),
SASSSVSYMH (SEQ ID NO:156) or LASQTIGTWLA (SEQ ID NO:157),
[0070] (b) HVR-L2 comprising sequence TWIYDTSILAS (SEQ ID NO:158),
RWIYDTSKLAS (SEQ ID NO:159), or LLIYAATSLAD (SEQ ID NO:160),
[0071] (c) HVR-L3 comprising sequence QQWTSNPLT (SEQ ID NO:161),
QQWSSYPPT (SEQ ID NO:162), or QQLYSPPWT (SEQ ID NO:163),
[0072] (d) HVR-H1 comprising sequence GYSFTDYNMY (SEQ ID NO:164),
GYVFTHYNMY (SEQ ID NO:165), or GYAFTSYNMY (SEQ ID NO:166),
[0073] (e) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID
NO:167), WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:168), or
WIGYIDPYIGGTSYNQKFKG (SEQ ID NO:169), and
[0074] (f) HVR-H3 comprising sequence ASPNYYDSSPFAY (SEQ ID
NO:170), ARGQGPDFDV (SEQ ID NO:171), or ARWGDYDVGAMDY (SEQ ID
NO:172).
[0075] In some embodiments, the anti-FGFR3 antibody comprises: at
least one, two, three, four, five, and/or six hypervariable region
(HVR) sequences selected from the group consisting of:
[0076] (a) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID
NO:155),
[0077] (b) HVR-L2 comprising sequence TWIYDTSILAS (SEQ ID
NO:158),
[0078] (c) HVR-L3 comprising sequence QQWTSNPLT (SEQ ID
NO:161),
[0079] (d) HVR-H1 comprising sequence GYSFTDYNMY (SEQ ID
NO:164),
[0080] (e) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID
NO:167), and
[0081] (f) HVR-H3 comprising sequence ASPNYYDSSPFAY (SEQ ID
NO:170).
[0082] In some embodiments, the anti-FGFR3 antibody comprises: at
least one, two, three, four, five, and/or six hypervariable region
(HVR) sequences selected from the group consisting of:
[0083] (a) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID
NO:156),
[0084] (b) HVR-L2 comprising sequence RWIYDTSKLAS (SEQ ID
NO:159),
[0085] (c) HVR-L3 comprising sequence QQWSSYPPT (SEQ ID
NO:162),
[0086] (d) HVR-H1 comprising sequence GYVFTHYNMY (SEQ ID
NO:165),
[0087] (e) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID
NO:168), and
[0088] (f) HVR-H3 comprising sequence ARGQGPDFDV (SEQ ID
NO:171).
[0089] In some embodiments, the anti-FGFR3 antibody comprises: at
least one, two, three, four, five, and/or six hypervariable region
(HVR) sequences selected from the group consisting of:
[0090] (a) HVR-L1 comprising sequence LASQTIGTWLA (SEQ ID
NO:157),
[0091] (b) HVR-L2 comprising sequence LLIYAATSLAD (SEQ ID
NO:160),
[0092] (c) HVR-L3 comprising sequence QQLYSPPWT (SEQ ID
NO:163),
[0093] (d) HVR-H1 comprising sequence GYAFTSYNMY (SEQ ID
NO:166),
[0094] (e) HVR-H2 comprising sequence WIGYIDPYIGGTSYNQKFKG (SEQ ID
NO:169), and
[0095] (f) HVR-H3 comprising sequence ARWGDYDVGAMDY (SEQ ID
NO:172).
[0096] In some embodiments, the anti-FGFR3 antibody comprises (a) a
light chain comprising (i) HVR-L1 comprising sequence SASSSVSYMH
(SEQ ID NO:155); (ii) HVR-L2 comprising sequence TWIYDTSILAS (SEQ
ID NO:158); and (iii) HVR-L3 comprising sequence QQWTSNPLT (SEQ ID
NO:161); and/or (b) a heavy chain comprising (i) HVR-H1 comprising
sequence GYSFTDYNMY (SEQ ID NO:164); (ii) HVR-H2 comprising
sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:167); and (iii) HVR-H3
comprising sequence ASPNYYDSSPFAY (SEQ ID NO:170).
[0097] In some embodiments, the anti-FGFR3 antibody comprises (a) a
light chain comprising (i) HVR-L1 comprising sequence SASSSVSYMH
(SEQ ID NO:156); (ii) HVR-L2 comprising sequence RWIYDTSKLAS (SEQ
ID NO:159); and (iii) HVR-L3 comprising sequence QQWSSYPPT (SEQ ID
NO:162); and/or (b) a heavy chain comprising (i) HVR-H1 comprising
sequence GYVFTHYNMY (SEQ ID NO:165); (ii) HVR-H2 comprising
sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:168); and (iii) HVR-H3
comprising sequence ARGQGPDFDV (SEQ ID NO:171).
[0098] In some embodiments, the anti-FGFR3 antibody comprises (a) a
light chain comprising (i) HVR-L1 comprising sequence LASQTIGTWLA
(SEQ ID NO:157); (ii) HVR-L2 comprising sequence LLIYAATSLAD (SEQ
ID NO:160); and (iii) HVR-L3 comprising sequence QQLYSPPWT (SEQ ID
NO:163); and/or (b) a heavy chain comprising (i) HVR-H1 comprising
sequence GYAFTSYNMY (SEQ ID NO:166); (ii) HVR-H2 comprising
sequence WIGYIDPYIGGTSYNQKFKG (SEQ ID NO:169); and (iii) HVR-H3
comprising sequence ARWGDYDVGAMDY (SEQ ID NO:172).
[0099] Some embodiments of antibodies of the invention comprise a
light chain variable domain of humanized 4D5 antibody (huMAb4D5-8)
(HERCEPTIN.RTM., Genentech, Inc., South San Francisco, Calif., USA)
(also referred to in U.S. Pat. No. 6,407,213 and Lee et al., J.
Mol. Biol. (2004), 340(5):1073-1093) as depicted in SEQ ID NO:173
below.
TABLE-US-00003 (SEQ ID NO: 173) 1 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (HVR residues are
underlined)
In one embodiment, the huMAb4D5-8 light chain variable domain
sequence is modified at one or more of positions 30, 66, and 91
(Asn, Arg, and His as indicated in bold/italics above,
respectively). In a particular embodiment, the modified huMAb4D5-8
sequence comprises Ser in position 30, Gly in position 66, and/or
Ser in position 91. Accordingly, in one embodiment, an antibody of
the invention comprises a light chain variable domain comprising
the sequence depicted in SEQ ID NO:174 below:
TABLE-US-00004 (SEQ ID NO: 174) 1 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (HVR residues are
underlined)
Substituted residues with respect to huMAb4D5-8 are indicated in
bold/italics.
[0100] Antibodies of the invention can comprise any suitable
framework variable domain sequence, provided binding activity to
FGFR3 is substantially retained. For example, in some embodiments,
antibodies of the invention comprise a human subgroup III heavy
chain framework consensus sequence. In one embodiment of these
antibodies, the framework consensus sequence comprises a
substitution at position 71, 73, and/or 78. In some embodiments of
these antibodies, position 71 is A, 73 is T and/or 78 is A. In one
embodiment, these antibodies comprise heavy chain variable domain
framework sequences of huMAb4D5-8 (HERCEPTIN.RTM., Genentech, Inc.,
South San Francisco, Calif., USA) (also referred to in U.S. Pat.
Nos. 6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol.
(2004), 340(5):1073-1093). In one embodiment, these antibodies
further comprise a human .kappa.I light chain framework consensus
sequence. In a particular embodiment, these antibodies comprise
light chain HVR sequences of huMAb4D5-8 as described in U.S. Pat.
Nos. 6,407,213 & 5,821,337.) In one embodiment, these
antibodies comprise light chain variable domain sequences of
huMAb4D5-8 (HERCEPTIN.RTM., Genentech, Inc., South San Francisco,
Calif., USA) (also referred to in U.S. Pat. Nos. 6,407,213 &
5,821,337, and Lee et al., J. Mol. Biol. (2004),
340(5):1073-1093).
[0101] In one embodiment, an antibody of the invention comprises a
heavy chain variable domain, wherein the framework sequence
comprises the sequence of SEQ ID NOS:19 and 203-205, 20 and
206-208, 21 and 209-211, 22 and 212-214, 23 and 215-217, 24 and
218-220, 25 and 221-223, 26 and 224-226, 27 and 227-229, 28 and
230-232, 29 and 233-235, 30 and 236-238, 31 and 239-241, 32 and
242-244, 33 and 245-247, 34 and 248-250, 35 and 251-253, 36 and
254-256, and/or 37 and 257-259, and HVR H1, H2, and H3 sequences
are SEQ ID NOS:13, 14 and/or 15, respectively. In another
embodiment, the framework sequence comprises the sequence of SEQ ID
NOS: 19 and 203-205, 20 and 206-208, 21 and 209-211, 22 and
212-214, 23 and 215-217, 24 and 218-220, 25 and 221-223, 26 and
224-226, 27 and 227-229, 28 and 230-232, 29 and 233-235, 30 and
236-238, 31 and 239-241, 32 and 242-244, 33 and 245-247, 34 and
248-250, 35 and 251-253, 36 and 254-256, and/or 37 and 257-259, and
HVR H1, H2, and H3 sequences are SEQ ID NOS:48, 49 and/or 50,
respectively. In yet another embodiment, the framework sequence
comprises the sequence of SEQ ID NOS: 19 and 203-205, 20 and
206-208, 21 and 209-211, 22 and 212-214, 23 and 215-217, 24 and
218-220, 25 and 221-223, 26 and 224-226, 27 and 227-229, 28 and
230-232, 29 and 233-235, 30 and 236-238, 31 and 239-241, 32 and
242-244, 33 and 245-247, 34 and 248-250, 35 and 251-253, 36 and
254-256, and/or 37 and 257-259, and HVR H1, H2, and H3 sequences
are SEQ ID NOS:84, 85, and/or 86, respectively. In a further
embodiment, the framework sequence comprises the sequence of SEQ ID
NOS: 19 and 203-205, 20 and 206-208, 21 and 209-211, 22 and
212-214, 23 and 215-217, 24 and 218-220, 25 and 221-223, 26 and
224-226, 27 and 227-229, 28 and 230-232, 29 and 233-235, 30 and
236-238, 31 and 239-241, 32 and 242-244, 33 and 245-247, 34 and
248-250, 35 and 251-253, 36 and 254-256, and/or 37 and 257-259, and
HVR H1, H2, and H3 sequences are SEQ ID NOS:108, 109, and/or 110,
respectively.
[0102] In a particular embodiment, an antibody of the invention
comprises a light chain variable domain, wherein the framework
sequence comprises the sequence of SEQ ID NOS:38 and 260-262, 39
and 263-265, 40 and 266-268, and/or 41 and 269-271, and HVR L1, L2,
and L3 sequences are SEQ ID NOS:16, 17, and/or 18, respectively. In
another embodiment, an antibody of the invention comprises a light
chain variable domain, wherein the framework sequence comprises the
sequence of SEQ ID NOS: 38 and 260-262, 39 and 263-265, 40 and
266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences
are SEQ ID NOS:51, 52 and/or 53, respectively. In an additional
embodiment, an antibody of the invention comprises a light chain
variable domain, wherein the framework sequence comprises the
sequence of SEQ ID NOS: 38 and 260-262, 39 and 263-265, 40 and
266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences
are SEQ ID NOS:87, 88 and/or 89, respectively. In yet another
embodiment, an antibody of the invention comprises a light chain
variable domain, wherein the framework sequence comprises the
sequence of SEQ ID NOS: 38 and 260-262, 39 and 263-265, 40 and
266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences
are SEQ ID NOS:111, 112, and/or 113, respectively.
[0103] In another aspect, an antibody of the invention comprises a
heavy chain variable domain comprising the sequence of SEQ ID
NO:132 and/or a light chain variable domain comprising the sequence
of SEQ ID NO:133. In another aspect, an antibody of the invention
comprises a heavy chain variable domain comprising the sequence of
SEQ ID NO:134 and/or a light chain variable domain comprising the
sequence of SEQ ID NO:135. In another aspect, an antibody of the
invention comprises a heavy chain variable domain comprising the
sequence of SEQ ID NO:136 and/or a light chain variable domain
comprising the sequence of SEQ ID NO:137. In another aspect, an
antibody of the invention comprises a heavy chain variable domain
comprising the sequence of SEQ ID NO:138 and/or a light chain
variable domain comprising the sequence of SEQ ID NO:139.
[0104] In one aspect, the invention provides an anti-FGFR3 antibody
that binds a polypeptide comprising, consisting essentially of or
consisting of the following amino acid sequence: LAVPAANTVRFRCPA
(SEQ ID NO:179) and/or SDVEFHCKVYSDAQP (SEQ ID NO:180).
[0105] In some embodiments, the antibody binds a polypeptide
comprising, consisting essentially of or consisting of amino acid
numbers 164-178 and/or 269-283 of human FGFR3.
[0106] In one embodiment, an anti-FGFR3 antibody of the invention
specifically binds an amino acid sequence having at least 50%, 60%,
70%, 80%, 90%, 95%, 98% sequence identity or similarity with the
sequence LAVPAANTVRFRCPA (SEQ ID NO:179) and/or SDVEFHCKVYSDAQP
(SEQ ID NO:180).
[0107] In one aspect, the anti-FGFR3 antibody of the present
invention binds to at least one, two, three, four, or any number up
to all of residues 154, 155, 158, 159, 161, 162, 163, 164, 165,
166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205,
207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280,
281, 282, 283, 314, 315, 316, 317 and/or 318 of FGFR3-IIIb
polypeptide, or equivalent residues of FGFR3-IIIc polypeptide. One
of ordinary skill in the art understands how to align FGFR3
sequences in order identify corresponding residues between
respective FGFR3 sequences. Combinations of two or more residues
can include any of residues 154, 155, 158, 159, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202,
205, 207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279,
280, 281, 282, 283, 314, 315, 316, 317 and/or 318, or mixtures
thereof, of FGFR3-IIIb polypeptide, or equivalent residues of
FGFR3-IIIc polypeptide. In some embodiments, the anti-FGFR3
antibody binds to at least one, two, three, four, or any number up
to all of residues 158, 159, 169, 170, 171, 173, 175, 205, 207,
and/or 315, or mixtures thereof, of FGFR3-IIIb polypeptide, or
equivalent residues of FGFR3-IIIc polypeptide. In some embodiments,
the anti-FGFR3 antibody binds to at least one, two three, four, or
any number up to all of residues 158, 170, 171, 173, 175, and/or
315, or mixtures thereof, of FGFR3-IIIb polypeptide, or equivalent
residues of FGFR3-IIIc polypeptide.
[0108] In one aspect, the invention provides methods of screening
for a candidate inhibitor substance that inhibits FGFR3 activity,
said method comprising: detecting binding, if any, of the candidate
substance to a FGFR3 binding site, wherein the candidate substance
is identified by a method comprising comparing amount of FGFR3
activity in a sample with amount of FGFR3 activity in a reference
sample comprising similar amounts of FGFR3 as the first sample but
that has not been contacted with said candidate substance, whereby
a decrease in amount of FGFR3 activity in the first sample compared
to the reference sample indicates that the candidate substance is
capable of inhibiting FGFR3 activity.
[0109] In another aspect, the invention provides methods of
screening for a candidate inhibitory substance that inhibits FGFR3
activation, the method comprising screening for a candidate
inhibitory substance that binds FGFR3 binding site and inhibits
FGFR3 activity.
[0110] In some embodiments, the methods comprising selecting for a
substance that binds to at least one, two, three, four, or any
number up to all of residues 158, 170, 171, 173, 175, and/or 315 of
the sequence of FGFR3-IIIb polypeptide, or equivalent residues of
FGFR3-IIIc polypeptide. In some embodiments, the methods comprising
selecting for a substance that binds to at least one, two, three,
four, or any number up to all of residues 154, 155, 158, 159, 161,
162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,
175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241, 246, 247,
248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317, 318 of the
sequence of FGFR3-IIIb polypeptide, or equivalent residues of
FGFR3-IIIc polypeptide.
[0111] In some embodiments, the sample comprises FGFR3 and FGFR3
ligand (such as FGF1 or FGF9). In some embodiments, the sample
comprises a mammalian cell expressing FGFR3. In some embodiments,
the FGFR3 is transgenically expressed. In some embodiments, the
sample comprises a Ba/FC cell expressing FGFR3.
[0112] In some embodiments, FGFR3 activity comprises FGF (such as
FGF1 and/or FGF9) binding, FGFR3 downstream molecular signaling,
FGFR3 phosphorylation, FGFR3 binding to a ligand (e.g., FGF1,
FGF9), FGFR3 dimerization, promotion of formation of monomeric
FGFR3, and/or treatment and/or prevention of a tumor, cell
proliferative disorder or a cancer; and/or treatment or prevention
of a disorder associated with FGFR3 expression and/or activity
(such as increased FGFR3 expression and/or activity). In some
embodiments, decrease in amount of FGFR3 activity is reduction or
blocking of FGF (such as FGF1 and/or FGF9) binding to FGFR3,
reduction or blocking of FGFR3 activation, reduction or blocking of
FGFR3 downstream molecular signaling), reduction or blocking of
FGFR3 dimerization and/or treatment and/or prevention of a tumor,
cell proliferative disorder or a cancer; and/or treatment or
prevention of a disorder associated with FGFR3 expression and/or
activity (such as increased FGFR3 expression and/or activity).
[0113] The invention also provides an antagonist molecule that
inhibits FGFR3, wherein the molecule binds to at least one, two,
three, four, or any number up to all of residues 158, 170, 171,
173, 175, and/or 315 of the sequence of FGFR3-IIIb polypeptide, or
equivalent residues of FGFR3-IIIc polypeptide. In some embodiments,
the antagonist molecule binds to at least one, two, three, four, or
any number up to all of residues 154, 155, 158, 159, 161, 162, 163,
164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177,
202, 205, 207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278,
279, 280, 281, 282, 283, 314, 315, 316, 317, 318 of the sequence of
FGFR3-IIIb polypeptide, or equivalent residues of FGFR3-IIIc
polypeptide. In some embodiments, the antagonist molecule comprises
an antibody.
[0114] The invention also provides methods using the antagonist
molecules, including treatment and diagnostic methods.
[0115] In another aspect, the disclosure includes FGFR3
polypeptides and polynucleotides encoding the polypeptides. The
disclosure includes a polynucleotide encoding a polypeptide and/or
a polypeptide having at least 90% sequence identity to the
polypeptide comprising sequence of amino acids 154-318 of human
FGFR3, not including the polypeptide having the amino acid sequence
of human FGFR3. In some embodiments, the disclosure includes a
polynucleotide encoding a polypeptide and/or a polypeptide having
at least 90% sequence identity to the polypeptide comprising any of
amino acid residue 154 to amino acid residue 177, amino acid
residue 202 to amino acid reside 217, amino acid residue 241 to
amino acid residue 248, amino acid residue 278 to amino acid
residue 283 and/or amino acid residue 314 to amino acid residue 318
FGFR3, not including the polypeptide comprising the amino acid
sequence of FGFR3. In some embodiments, the polypeptide binds a
human FGFR3 ligand (such as FGF1 or FGF9).
BRIEF DESCRIPTION OF THE DRAWINGS
[0116] FIGS. 1A, 1B and 1C: Heavy chain and light chain HVR loop
sequences of anti-FGFR3 antibodies. The figures show the heavy
chain HVR sequences, H1, H2, and H3, and light chain HVR sequences,
L1, L2, and L3. Sequence numbering is as follows:
[0117] Clone 184.6 (HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ ID NO:2;
HVR-H3 is SEQ ID NO:3; HVR-L1 is SEQ ID NO:4; HVR-L2 is SEQ ID
NO:5; HVR-L3 is SEQ ID NO:6);
[0118] Clone 184.6.1 (HVR-H1 is SEQ ID NO:7; HVR-H2 is SEQ ID NO:8;
HVR-H3 is SEQ ID NO:9; HVR-L1 is SEQ ID NO:10; HVR-L2 is SEQ ID
NO:11; HVR-L3 is SEQ ID NO:12)
[0119] Clone 184.6.58 (HVR-H1 is SEQ ID NO:13; HVR-H2 is SEQ ID
NO:14; HVR-H3 is SEQ ID NO:15; HVR-L1 is SEQ ID NO:16; HVR-L2 is
SEQ ID NO:17; HVR-L3 is SEQ ID NO:18)
[0120] Clone 184.6.62 (HVR-H1 is SEQ ID NO:48; HVR-H2 is SEQ ID
NO:49; HVR-H3 is SEQ ID NO:50; HVR-L1 is SEQ ID NO:51; HVR-L2 is
SEQ ID NO:52; HVR-L3 is SEQ ID NO:53)
[0121] Clone 184.6.21 (HVR-H1 is SEQ ID NO:54; HVR-H2 is SEQ ID
NO:55; HVR-H3 is SEQ ID NO:56; HVR-L1 is SEQ ID NO:57; HVR-L2 is
SEQ ID NO:58; HVR-L3 is SEQ ID NO:59)
[0122] Clone 184.6.49 (HVR-H1 is SEQ ID NO:60; HVR-H2 is SEQ ID
NO:61; HVR-H3 is SEQ ID NO:62; HVR-L1 is SEQ ID NO:63; HVR-L2 is
SEQ ID NO:64; HVR-L3 is SEQ ID NO:65)
[0123] Clone 184.6.51 (HVR-H1 is SEQ ID NO:66; HVR-H2 is SEQ ID
NO:67; HVR-H3 is SEQ ID NO:68; HVR-L1 is SEQ ID NO:69; HVR-L2 is
SEQ ID NO:70; HVR-L3 is SEQ ID NO:71)
[0124] Clone 184.6.52 (HVR-H1 is SEQ ID NO:72; HVR-H2 is SEQ ID
NO:73; HVR-H3 is SEQ ID NO:74; HVR-L1 is SEQ ID NO:75; HVR-L2 is
SEQ ID NO:76; HVR-L3 is SEQ ID NO:77)
[0125] Clone 184.6.92 (HVR-H1 is SEQ ID NO:78; HVR-H2 is SEQ ID
NO:79; HVR-H3 is SEQ ID NO:80; HVR-L1 is SEQ ID NO:81; HVR-L2 is
SEQ ID NO:82; HVR-L3 is SEQ ID NO:83)
[0126] Clone 184.6.1.N54S (HVR-H1 is SEQ ID NO:84; HVR-H2 is SEQ ID
NO:85; HVR-H3 is SEQ ID NO:86; HVR-L1 is SEQ ID NO:87; HVR-L2 is
SEQ ID NO:88; HVR-L3 is SEQ ID NO:89)
[0127] Clone 184.6.1.N54G (HVR-H1 is SEQ ID NO:90; HVR-H2 is SEQ ID
NO:91; HVR-H3 is SEQ ID NO:92; HVR-L1 is SEQ ID NO:93; HVR-L2 is
SEQ ID NO:94; HVR-L3 is SEQ ID NO:95)
[0128] Clone 184.6.1.N54A (HVR-H1 is SEQ ID NO:96; HVR-H2 is SEQ ID
NO:97; HVR-H3 is SEQ ID NO:98; HVR-L1 is SEQ ID NO:99; HVR-L2 is
SEQ ID NO:100; HVR-L3 is SEQ ID NO:101)
[0129] Clone 184.6.1.N54Q (HVR-H1 is SEQ ID NO:102; HVR-H2 is SEQ
ID NO:103; HVR-H3 is SEQ ID NO:104; HVR-L1 is SEQ ID NO:105; HVR-L2
is SEQ ID NO:106; HVR-L3 is SEQ ID NO:107)
[0130] Clone 184.6.58.N54S (HVR-H1 is SEQ ID NO:108; HVR-H2 is SEQ
ID NO:109; HVR-H3 is SEQ ID NO:110; HVR-L1 is SEQ ID NO:111; HVR-L2
is SEQ ID NO:112; HVR-L3 is SEQ ID NO:113)
[0131] Clone 184.6.58.N54G (HVR-H1 is SEQ ID NO:114; HVR-H2 is SEQ
ID NO:115; HVR-H3 is SEQ ID NO:116; HVR-L1 is SEQ ID NO:117; HVR-L2
is SEQ ID NO:118; HVR-L3 is SEQ ID NO:119)
[0132] Clone 184.6.58.N54A (HVR-H1 is SEQ ID NO:120; HVR-H2 is SEQ
ID NO:121; HVR-H3 is SEQ ID NO:122; HVR-L1 is SEQ ID NO:123; HVR-L2
is SEQ ID NO:124; HVR-L3 is SEQ ID NO:125)
[0133] Clone 184.6.58.N54Q (HVR-H1 is SEQ ID NO:126; HVR-H2 is SEQ
ID NO:127; HVR-H3 is SEQ ID NO:128; HVR-L1 is SEQ ID NO:129; HVR-L2
is SEQ ID NO:130; HVR-L3 is SEQ ID NO:131).
[0134] Clone 184.6.1.NS D30E (HVR-H1 is SEQ ID NO:143; HVR-H2 is
SEQ ID NO:144; HVR-H3 is SEQ ID NO:145; HVR-L1 is SEQ ID NO:140;
HVR-L2 is SEQ ID NO:141; HVR-L3 is SEQ ID NO:142).
[0135] Amino acid positions are numbered according to the Kabat
numbering system as described below.
[0136] FIGS. 2A and 2B: depict (A) the amino acid sequences of the
heavy chain variable regions and light chain variable regions of
anti-FGFR3 antibodies 184.6.1.N54S, 184.6.58, and 184.6.62; and (B)
the hypervariable regions of anti-FGFR3 antibodies 1G6, 6G1, and
15B2.
[0137] FIGS. 3A, 3B, and 4: depict exemplary acceptor human
consensus framework sequences for use in practicing the instant
invention with sequence identifiers as follows:
Variable Heavy (VH) Consensus Frameworks (FIG. 3A, 3B)
[0138] human VH subgroup I consensus framework minus Kabat CDRs
(SEQ ID NOS:19 and 203-205) human VH subgroup I consensus framework
minus extended hypervariable regions (SEQ ID NOS:20 and 206-208, 21
and 209-211, 22 and 212-214) human VH subgroup II consensus
framework minus Kabat CDRs (SEQ ID NOS:23 and 215-217) human VH
subgroup II consensus framework minus extended hypervariable
regions (SEQ ID NOS:24 and 218-220, 25 and 221-223, 26 and 224-226)
human VH subgroup II consensus framework minus extended human VH
subgroup III consensus framework minus Kabat CDRs (SEQ ID NOS:27
and 227-229) human VH subgroup III consensus framework minus
extended hypervariable regions (SEQ ID NOS:28 and 230-232, 29 and
233-235, 30 and 236-238) human VH acceptor framework minus Kabat
CDRs (SEQ ID NOS:31 and 239-241) human VH acceptor framework minus
extended hypervariable regions (SEQ ID NOS:32 and 242-244, 33 and
2245-247) human VH acceptor 2 framework minus Kabat CDRs (SEQ ID
NOS:34 and 248-250) human VH acceptor 2 framework minus extended
hypervariable regions (SEQ ID NOS:35 and 251-253, 36 and 254-256,
37 and 257-259)
Variable Light (VL) Consensus Frameworks (FIG. 4)
[0139] human VL kappa subgroup I consensus framework (SEQ ID NO:38
and 260-262) human VL kappa subgroup II consensus framework (SEQ ID
NO:39 and 263-265) human VL kappa subgroup III consensus framework
(SEQ ID NO:40 and 266-268) human VL kappa subgroup IV consensus
framework (SEQ ID NO:41 and 269-271)
[0140] FIG. 5: depicts framework region sequences of huMAb4D5-8
light (SEQ ID NOS:42-45) and heavy chains (SEQ ID NOS:46, 47, 175,
176). Numbers in superscript/bold indicate amino acid positions
according to Kabat.
[0141] FIG. 6: depicts modified/variant framework region sequences
of huMAb4D5-8 light (SEQ ID NOS:42, 43, 177, 45) and heavy chains
(SEQ ID NOS:46, 47, 178, and 176). Numbers in superscript/bold
indicate amino acid positions according to Kabat.
[0142] FIG. 7: FGFR3 knockdown in bladder cancer cell RT112
inhibits proliferation and induces G1 cell cycle arrest in vitro,
and suppresses tumor growth in vivo. Three different FGFR3 shRNAs
were cloned into a Tet-inducible expression vector. RT112 cells
stably expressing FGFR3 shRNAs or a control shRNA were established
with puromycin selection. (A) Representative blots showing FGFR3
expression in selected clones treated with or without doxycycline
(Dox, 0, 0.1 and 1 .mu.g/ml, left to right). (B)
[.sup.3H]-thymidine incorporation by RT112 stable cells. RT112
stable clones were cultured with or without 1 .mu.g/ml doxycycline
for 3 days prior to 16 hour-incubation with [.sup.3H]-thymidine (1
.mu.Ci per well). Counts of incorporated [.sup.3H]-thymidine were
normalized to that from cells without doxycycline induction. Error
bars represent SEM. (C) DNA fluorescence flow cytometry histograms
of RT112 stable cells. RT112 clones expressing control shRNA or
FGFR3 shRNA4 were cultured with or without 1 .mu.g/ml doxycycline
for 72 hours, and the nuclei were stained with propidium iodide
(PI). Similar results were obtained for FGFR3 shRNA2 and 6 (FIG.
16). (D) The growth of RT112 cells expressing control shRNA (n=9
per treatment group) or FGFR3 shRNA4 (n=11 per treatment group) in
mice. Mice were given 5% sucrose alone or supplemented with 1 mg/ml
doxycycline, and tumor size was measured twice a week. Error bars
represent SEM. Similar results were obtained for FGFR3 shRNA2 and 6
(FIG. 16). Lower panel: Expression of FGFR3 protein in tumor
lysates extracted from control shRNA or FGFR3 shRNA4 stable cell
xenograft tissues.
[0143] FIG. 8: R3Mab blocks FGF/FGFR3 interaction. (A) Selective
binding of human FGFR3 by R3Mab. Human FGFR1-4 Fc chimeric proteins
were immobilized and incubated with increasing amount of R3Mab.
Specific binding was detected using an anti-human Fab antibody.
(B-C) Blocking of FGF1 binding to human FGFR3-IIIb (B) or IIIc (C)
by R3Mab. Specific binding was detected by using a biotinylated
FGF1-specific polyclonal antibody. (D-E) Blocking of FGF9 binding
to human FGFR3-IIIb (D) or Mc (E) by R3Mab. Specific binding was
detected by using a biotinylated FGF9-specific polyclonal antibody.
Error bars represent standard error of the mean (SEM) and are
sometimes smaller than symbols.
[0144] FIG. 9: R3Mab inhibits Ba/F3 cell proliferation driven by
wild type and mutated FGFR3. (A) Inhibitory effect of R3Mab on the
viability of Ba/F3 cells expressing wild type human FGFR3-IIIb.
Cells were cultured in medium without FGF1 (no FGF1), or in the
presence of 10 ng/ml FGF1 plus 10 .mu.g/ml heparin alone (FGF1), or
in combination with a control antibody (Control) or R3Mab. Cell
viability was assessed with CellTiter-Glo (Promega) after 72 hr
incubation with antibodies. (B) Inhibition of FGFR3 and MAPK
phosphorylation by R3Mab in Ba/F3-FGFR3-IIIb.sup.WT stable cells.
Cells were treated with 15 ng/ml FGF1 and 10 .mu.g/ml heparin (+)
or heparin alone (-) for 10 minutes, following pre-incubation with
a Control Ab (Ctrl), decreasing amount of R3Mab (1, 0.2, 0.04
.mu.g/ml respectively) in PBS, or PBS alone (Mock) for 3 hours.
Lysates were immunoblotted to assess phosphorylation of FGFR3 and
p44/42 MAPK with antibodies to pFGFR.sup.Y653/654 and
pMAPK.sup.Thr202/Tyr204 respectively. (C) Schematic representation
of FGFR3 mutation hot spots and frequency in bladder cancer
(sequence numbering depicted is based on the FGFR3-IIIb isoform
amino acid sequence) based on published data (32). TM,
transmembrane domain; TK1 and TK2, tyrosine kinase domain 1 and 2.
(D-H) Inhibitory effect of R3Mab on the viability of Ba/F3 cells
expressing cancer-associated FGFR3 mutants. G372C is derived from
Mc isoform, and the rest are derived from Mb isoform. Sequence
numbering for all mutants is based on the FGFR3-IIIb isoform amino
acid sequence (including the G372C mutant, which would be numbered
G370C based on the FGFR3-IIIc isoform amino acid sequence). Cell
viability was assessed after 72 hour incubation with antibodies as
described in (A). Error bars represent SEM.
[0145] FIG. 10: Epitope mapping for R3Mab and crystal structure of
the complex between R3Mab Fab fragment and IgD2-D3 of human
FGFR3-IIIb. (A) Epitope determined by the binding of 13 peptides
spanning IgD2-D3 of human FGFR3 to R3Mab. Each biotinylated peptide
was captured onto streptavidin-coated microtiter well and incubated
with R3Mab. Specifically bound R3Mab was detected using a goat
anti-human IgG antibody. (B) Sequence alignment of human FGFR3
peptides 3 (LAVPAANTVRFRCPA (SEQ ID NO:179) and 11 (SDVEFHCKVYSDAQP
(SEQ ID NO:180) with extracellular segments of human FGFR1 (peptide
3: HAVPAAKTVKFKCPS (SEQ ID NO:181); peptide 11: SNVEFMCKVYSDPQP
(SEQ ID NO:182)). FGFR1 residues engaged in the primary FGF2-FGFR1
interaction, heparin binding, and receptor-receptor association are
shown in bold, italics, and underlined font, respectively.
Functional assignment of FGFR1 residues is based on Plotnikov et al
(34). (C) Structure of R3Mab Fab (shown in ribbon-helix, light
chain grey, heavy chain black) in complex with human FGFR IgD2-D3
(shown in molecular surface, white). Receptor residues involved in
ligand binding and dimerization are colored in grey/crosshatched
and dark grey respectively based on Plotnikov et al (34). (D) The
close-up of the crystal structure shows that CDR-H3 and -H2 from
the Fab constitute the major interaction sites with IgD2 and IgD3
of FGFR3. (E) Superposition of FGFR3-IIIc-FGF1 complex (PDB code
1RY7) with FGFR3-IIIb-Fab complex. FGFR3-IIIc and FGF1 are colored
in grey and dark grey respectively. FGFR3-IIIb is shown in white
and the Fab is shown in light grey for light chain, dark grey for
heavy chain. IgD2 was used as the anchor for superposition. Note
the well-superposed IgD2 from both structures and the new
conformation adopted by IgD3 of FGFR3-IIIb when bound by R3Mab. (F)
Another representation of the superposition of FGFR3-IIIc-FGF1
complex (PDB code 1RY7) with FGFR3-IIIb-Fab complex. FGFR3-IIIc and
FGF1 are shown as molecular surfaces that are grey/mesh texture and
dark grey/dotted texture, respectively. FGFR3-IIIb is shown in
white and the Fab is shown in grey for light chain, black for heavy
chain. IgD2 was used as the anchor for superposition. Note the
well-superposed IgD2 from both structures and the new conformation
adopted by IgD3 of FGFR3-IIIb when bound by R3Mab.
[0146] FIG. 11: R3Mab inhibits proliferation, clonal growth and
FGFR3 signaling in bladder cancer cells expressing wild type or
mutated FGFR3.sup.S249C. (A) Inhibition of [.sup.3H]-thymidine
incorporation by R3Mab in bladder cancer cell line RT112. Error
bars represent SEM. (B) Blocking of FGF1-activated FGFR3 signaling
by R3Mab (15 .mu.g/ml) in bladder cancer cell line RT112 as
compared to treatment medium alone (Mock) or a control antibody
(Ctrl). Cell lysates were immunoprecipitated with anti-FGFR3
antibody and assessed for FGFR3 phosphorylation with an
anti-phospho-tyrosine antibody (4G10). Lysates were immunoblotted
to detect phosphorylation of AKT (pAKT.sup.S473) and p44/42 MAPK
(pMAPK.sup.Thr202/Tyr204). (C) Inhibition of clonal growth by R3Mab
(10 .mu.g/ml) in bladder cancer cell line UMUC-14 (harboring
FGFR3.sup.S249C) as compared to treatment medium alone (Mock) or a
control antibody (Ctrl). (D) Quantitation of the study in (C)
reporting the number of colonies larger than 120 .mu.m in diameter
per well from a replicate of 12 wells. Error bars represent SEM.
P<3.4.times.10.sup.-9 versus Mock or Ctrl. (E) Inhibition of
FGFR3 phosphorylation in UMUC-14 cells by R3Mab (15 .mu.g/ml).
FGFR3 phosphorylation was analyzed as in (B). Note constitutive
phosphorylation of FGFR3 in this cell line.
[0147] FIG. 12: R3Mab decreases steady-state level of
disulfide-linked FGFR3.sup.S249C dimer by driving the dimer-monomer
equilibrium toward monomeric state. (A) Effect of R3Mab on
FGFR3.sup.S249C dimer in UMUC-14 cells. Cells were incubated with
R3Mab (15 .mu.g/ml) or a control antibody (Ctrl) for 3 hours, and
whole cell lysates were analyzed by immunoblot under non-reducing
and reducing conditions. (B) Effect of free-sulfhydryl blocker DTNB
on FGFR3.sup.S249C dimer-monomer equilibrium in UMUC-14 cells.
UMUC-14 cells were treated with increasing concentration of DTNB
for 3 hours, and cell lysates were analyzed as in (A). (C) Effect
of R3Mab on purified recombinant FGFR3.sup.S249C dimer in vitro.
FGFR3.sup.S249C dimer composed of IgD2-D3 was purified through
size-exclusion column, and incubated with PBS (Mock), a control
antibody (Ctrl), or R3Mab at 37.degree. C. Samples were collected
at indicated time for immunoblot analysis under non-reducing
conditions. FGFR3 dimer-monomer was detected using anti-FGFR3
hybridoma antibody 6G1 (A-C).
[0148] FIG. 13: R3Mab inhibits xenograft growth of bladder cancer
cells and allograft growth of Ba/F3-FGFR3.sup.S249C (A) Effect of
R3Mab on the growth of pre-established RT112 bladder cancer
xenografts compared with vehicle control. n=10 per group. (B)
Inhibition of FGFR3 signaling in RT112 tumor tissues by R3Mab. In a
separate experiment, RT112 xenograft tumors that were treated with
15 mg/kg of a control antibody (Ctrl) or R3Mab for 48 hours or 72
hours were collected (n=3 per group), homogenized and analyzed for
FRS2.alpha. and MAPK activation by immunoblot. (C) Effect of R3Mab
on the growth of pre-established Ba/F3-FGFR3.sup.S249C allografts.
n=10 per group. (D) Effect of R3Mab on the growth of
pre-established UMUC-14 bladder cancer xenografts, n=10 per group.
(E) Effect of R3Mab on FGFR3.sup.S249C dimer and signaling in
UMUC-14 tumor tissues. UMUC-14 xenograft tumors that were treated
with 30 mg/kg of a control antibody (Ctrl) or R3Mab for 24 hours or
72 hours were collected (n=3 per group), homogenized, and analyzed
for FGFR3.sup.S249C dimer-monomer as well as MAPK activation by
immunoblot. FGFR3 dimer-monomer was detected using an anti-FGFR3
rabbit polyclonal antibody sc9007 to avoid interference from mouse
IgG in tumor lysates. Error bars represent SEM.
[0149] FIG. 14: ADCC contributes to the anti-tumor efficacy of
R3Mab in t(4; 14) positive multiple myeloma models. (A-B) Effect of
R3Mab on the growth of pre-established OPM2 (A) and KMS11 (B)
myeloma xenografts. n=10 per group. (C-F) Cytolysis of myeloma cell
lines OPM2 (C) and KMS11 (D), or bladder cancer cell lines RT112
(E) and UMUC-14 (F) induced by R3Mab in cell culture. Myeloma or
bladder cancer cells were incubated with freshly isolated human
PBMC in the presence of R3Mab or a control antibody. Cytotoxicity
was determined by measuring LDH released in the supernatant. (G-H)
Effect of R3Mab or its DANA mutant on the growth of pre-established
OPM2 (G) and KMS11 (H) myeloma xenografts. n=10 per group. Error
bars represent SEM and are sometimes smaller than symbols.
[0150] FIG. 15: Knockdown of FGFR3 with siRNA inhibits cell
proliferation of bladder cancer cell lines. Six to seven different
FGFR3 siRNAs and three non-specific control siRNAs were designed
and synthesized in Genentech. Bladder cancer cell lines RT112 (A),
SW780 (B), RT4 (C) and UMUC-14 (D) were plated into 96-well plate
(3000 cells per well) and allowed to attach overnight, and
transiently transfected with 25 nM siRNA in complex with RNAiMax
(Invitrogen). 72 hr post-transfection, [.sup.3H]-thymidine (1
.mu.Ci per well) was added to the culture (A, C, and D) for another
16 hour incubation. Incorporated [.sup.3H]-thymidine was
quantitated with TopCount. Data were normalized to that from cells
transfected with RNAiMax alone (Mock). Error bars represent SEM.
Lower panel: Representative blots showing FGFR3 expression in siRNA
transfected cells. (B) Cell viability was measured with
CellTiter-Glo (Promega) 96 hours after transfection. Error bars
represent SEM.
[0151] FIG. 16: FGFR3 knockdown in bladder cancer cell line RT112
induces G1 cell cycle arrest in vitro, and suppresses tumor growth
in vivo. Three different FGFR3 RNAs were designed and cloned into a
Tet-inducible shRNA expression retroviral vector. RT112 stable
clones expressing FGFR3 shRNAs or control shRNA were established
with puromycin selection. (A) DNA fluorescence flow cytometry
histograms of propidium iodide (PI)-stained nuclei obtained from
RT112 stable cells expressing FGFR3 shRNA2 or shRNA6 following
treatment with or without 1 .mu.g/ml doxycycline for 72 hours. (B)
The growth of RT112 stable cells expressing FGFR3 shRNA2-4 (n=11
per treatment group) or FGFR3shRNA6-16 (n=10 per treatment group)
in nu/nu mice. Tumor bearing mice received 5% sucrose only (solid
circle) or 5% sucrose plus 1 mg/ml doxycycline (solid square), and
tumors were measured with calipers twice a week. Error bars
represent SEM.
[0152] FIG. 17: Effect of anti-FGFR3 hybridoma antibodies 16G, 6G1
and 15B2 on Ba/F3 cell proliferation driven by wild type and
mutated FGFR3. Anti-FGFR3 hybridoma antibodies were generated by
immunizing BALB/c mice with human FGFR3-IIIb/Fc or human
FGFR3-IIIc/Fc chimera. Fused hybridoma cells were selected using
hypoxanthin-aminopterin-thymidine selection in Medium D from the
ClonaCell.RTM. hybridoma selection kit (StemCell Technologies,
Inc., Vancouver, BC, Canada). Hybridoma antibodies were
sequentially screened for their ability to bind to FGFR3-IIIb and
FGFR3-IIIc by ELISA and to recognize cell surface FGFR3 by FACS.
Selected hybridomas were then cloned by limiting dilution. 16G, 6G1
and 15B2 are clones used to assess the effect on the proliferation
of Ba/F3 cells expressing wild type or mutated FGFR3 similarly as
described in FIG. 9A. Error bars represent SEM.
[0153] FIG. 18: Comparison of R3Mab epitopes determined by peptide
mapping and crystal structure analysis. (A) Epitope revealed by the
structure of the R3Mab Fab fragment in complex with the
extracellular IgD2-D3 segment of human FGFR3. FGFR3 residues
contacted by Fab heavy chain and light chain are colored in black
and grey, respectively. (B) Location of peptides 3 and 11 on
FGFR3.
[0154] FIG. 19: R3Mab inhibits proliferation and FGFR3 signaling in
bladder cancer cells containing wild type or mutated
FGFR3.sup.S249C. (A) Inhibition of cell viability by R3Mab in
bladder cancer cell line RT4. Cell viability was assessed with
CellTiter-Glo (Promega) after 96 hr incubation with the antibody.
Error bars represent SEM. (B) Blocking of FGF1-activated FGFR3
signaling by R3Mab (15 ug/ml) in bladder cancer cell line RT4. (C)
Inhibition of [.sup.3H]-thymidine incorporation by R3Mab in bladder
cancer cell line RCC-97-7 (containing FGFR3.sup.S249C). Error bars
represent SEM. (D) Inhibition of FGFR3 phosphorylation in TCC-97-7
cells by R3Mab (15 ug/ml). (E) Decrease of FGFR3.sup.S249C dimer in
TCC-97-7 cells after 3 hours incubation with R3Mab (15 ug/ml)
compared with a control antibody (Ctrl).
[0155] FIG. 20: Effect of endocytosis inhibitors on the
internalization of R3Mab and FGFR3.sup.S249C dimer in UMUC-14
cells. (A) Effect of endocytosis inhibitors on the internalization
of R3Mab. UMUC-14 cells, pre-treated with various endocytosis
inhibitor or DMSO for 1 hour at 37.degree. C., were incubated with
R3Mab (15 ug/ml) for 3 hours at 37.degree. C. to allow
internalization. A low pH wash was used to remove cell surface
R3Mab to visualize internalized antibody. Cells were fixed and
stained with Alexa 488-labeled anti-human IgG. Image was taken
using confocal microscopy. (B) Effect of endocytosis inhibitors on
FGFR3.sup.S249C dimer in UMUC-14 cells treated with R3Mab. UMUC-14
cells, pre-treated with various endocytosis inhibitor or DMSO for 1
hour at 37.degree. C., were incubated with mock (Lane 1), a control
antibody (Lane 2), or R3Mab (15 ug/ml, Lane 3) for 3 hours at
37.degree. C. Cell lysates were analyzed for FGFR3 protein under
non-reducing or reducing conditions by immunoblot. Note that
chlorpromazine (inhibitor of clathrin-mediated endocytosis) and
genistein (pan-inhibitor of endocytosis) blocked R3Mab
internalization, but had no effect on R3Mab-induced decrease of
FGFR3.sup.S249C dimer.
[0156] FIG. 21: Detection sensitivity of different anti-FGFR3
antibodies toward monomeric and dimeric FGFR3.sup.S249C under
non-reducing conditions. UMUC-14 cells were lysed after treatment
with R3Mab (Lane 1), a control IgG1 (Lane 2), or PBS (Lane 3) for 3
hours, and cell lysates were subject to immunoblot analyses under
reducing or non-reducing conditions. Note that 6G1 (murine
hybridoma antibody generated at Genentech) detected both
FGFR3.sup.S249C dimer and monomer, whereas sc9007 (rabbit
polyclonal antibody, Santa Cruz Biotechnology) or sc13121 (murine
hybridoma antibody, Santa Cruz Biotechnology) preferentially
detected the dimeric FGFR3.sup.S249C.
[0157] FIG. 22: Effect of R3Mab on the proliferation of t(4; 14)+
multiple myeloma cells. (A) Inhibitory effect of R3Mab on
[.sup.3H]-thymidine incorporation by UTMC-2 cells. UTMC-2 cells
were grown in medium containing R3Mab or a control antibody in the
presence of 25 ng/ml FGF9 and 5 ug/ml heparin or heparin alone (No
FGF9). After 6 days incubation, [.sup.3H]-thymidine was added for
16 hr incubation. Data were normalized to that from cells grown in
the absence of FGF9 and antibody. (B-C) Effect of R3Mab on
[.sup.3H]-thymidine incorporation by OPM2 (B) and KMS11 (C) cells.
Cells grown in 1.5% FBS medium were treated with R3Mab or a control
antibody for 6 days. Data were normalized to that from untreated
cells. Error bars represent SEM.
[0158] FIG. 23: Cell surface expression levels of FGFR3 in myeloma
and bladder cancer cells. (A) Cell surface FGFR3 expression in
myeloma cells and bladder cancer cells assessed by FACS analysis.
Cells were stained with phycoerythin-conjugated mouse mAb against
human FGFR3 (FAB766P, R&D Systems) or phycoerythin-conjugated
isotype control mouse IgG1 (BD Pharmingen). (B) Scatchard analysis
of FGFR3 density in myeloma cells and bladder cancer cells. R3Mab
was radioiodinated, and incubated with cells in suspension with
excess unlabeled antibody. After incubation at RT for 2 hours,
cells were pelleted by centrifugation and washed twice.
Specifically bound .sup.125I was determined. Receptor density and
binding affinity (Kd) represent the mean from two binding
experiments.
[0159] FIG. 24: Effect of R3Mab or its DANA mutant on xenograft
growth of bladder carcinoma cells. (A) Effect of R3Mab and its DANA
mutant (50 mg/kg each) on the growth of pre-established RT112
tumors. (B) Effect of R3Mab and its DANA mutant (50 mg/kg each) on
the growth of pre-established UMUC-14 tumors. Error bars represent
SEM.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques
[0160] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds.,
(2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A
LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed.
(1987)).
DEFINITIONS
[0161] The term "binding site," as used herein, refers to a region
of a molecule or molecular complex that, as a result of its shape,
distribution of electrostatic charge, presentation of hydrogen-bond
acceptors or hydrogen-bond donors, and/or distribution of nonpolar
regions, favorably associates with a ligand. Thus, a binding site
may include or consist of features such as cavities, surfaces, or
interfaces between domains. Ligands that may associate with a
binding site include, but are not limited to, cofactors,
substrates, receptors, agonists, and antagonists. The term binding
site includes a functional binding site and/or a structural binding
site. A structural binding site can include "in contact" amino acid
residues as determined from examination of a three-dimensional
structure. "Contact" can be determined using Van der Waals radii of
atoms or by proximity sufficient to exclude solvent, typically
water, from the space between the ligand and the molecule or
molecular complex. In some embodiments, a FGFR3 residue in contact
with an anti-FGFR3 antibody (e.g., YW184.6) or other substrate or
inhibitor is a residue that has one atom within about 5 .ANG. of an
anti-FGFR3 antibody residue. Alternatively, "in contact" residue
may be those that have a loss of solvent accessible surface area of
at least about 10 .ANG. and, more preferably at least about 50
.ANG. to about 300 .ANG.. Loss of solvent accessible surface can be
determined by the method of Lee & Richards (J Mol Biol. 1971
Feb. 14; 55(3):379-400) and similar algorithms known to those
skilled in the art, for instance as found in the SOLV module from
C. Broger of F. Hoffman-La Roche in Basel Switzerland.
[0162] Some of the "in contact" amino acid residues, if substituted
with another amino acid type, may not cause any change in a
biochemical assay, a cell-based assay, or an in vivo assay used to
define a functional binding site but may contribute to the
formation of a three dimensional structure. A functional binding
site includes amino acid residues that are identified as binding
site residues based upon loss or gain of function, for example,
loss of binding to ligand upon mutation of the residue. In some
embodiments, the amino acid residues of a functional binding site
are a subset of the amino acid residues of the structural binding
site.
[0163] The term "FGFR3 binding site" refers to a region of FGFR3
that can favorably associate with a ligand. In some embodiments,
the FGFR3 binding site may comprise, consist essentially of, or
consist of one or more of the amino acid residues 154, 155, 158,
159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,
173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241,
246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317
and/or 318 of FGFR3-IIIb polypeptide (e.g., human FGFR3-IIIb
disclosed in UniProKB/Swiss-Prot accession number P22607.sub.--2),
and mixtures thereof, or equivalent residues of FGFR3-IIIc
polypeptide (e.g., human FGFR3-IIIc disclosed in
UniProKB/Swiss-Prot accession number P22607). In some embodiments,
the FGFR3 binding site may comprise, consist essentially of, or
consist of one or more of the amino acid residues 158, 159, 169,
170, 171, 173, 175, 205, 207, and/or 315 of FGFR3-IIIb polypeptide,
and mixtures thereof, or equivalent residues of FGFR3-IIIc
polypeptide. In some embodiments, the FGFR3 binding site may
comprise, consist essentially of, or consist of one or more of the
amino acid residues 158, 170, 171, 173, 175, and/or 315, or
mixtures thereof, of FGFR3-IIIb polypeptide, or equivalent residues
of FGFR3-IIIc polypeptide. A structurally equivalent ligand binding
site is defined by a root mean square deviation from the structure
coordinates of the backbone atoms of the amino acids that make up
binding sites in FGFR3 for anti-FGFR3 antibody YW184.6 of at most
about 0.70 .ANG., preferably about 5 .ANG..
[0164] "Crystal" as used herein, refers to one form of a solid
state of matter in which atoms are arranged in a pattern that
repeats periodically in three-dimensions, typically forming a
lattice.
[0165] "Complementary or complement" as used herein, means the fit
or relationship between two molecules that permits interaction,
including for example, space, charge, three-dimensional
configuration, and the like.
[0166] The term "corresponding" or "corresponds" refers to an amino
acid residue or amino acid sequence that is found at the same
position or positions in a sequence when the amino acid position or
sequences are aligned with a reference sequence. In some
embodiments, the reference sequence is a human FGFR3-IIIb disclosed
in UniProKB/Swiss-Prot accession number P22607.sub.--2) or a human
FGFR3-IIIc disclosed in UniProKB/Swiss-Prot accession number
P22607. It will be appreciated that when the amino acid position or
sequence is aligned with the reference sequence the numbering of
the amino acids may differ from that of the reference sequence.
[0167] "Heavy atom derivative", as used herein, means a derivative
produced by chemically modifying a crystal with a heavy atom such
as Hg, Au, or a halogen.
[0168] "Structural homolog" of FGFR3 as used herein refers to a
protein that contains one or more amino acid substitutions,
deletions, additions, or rearrangements with respect to the amino
acid sequence of FGFR3, but that, when folded into its native
conformation, exhibits or is reasonably expected to exhibit at
least a portion of the tertiary (three-dimensional) structure of
the FGFR3. In some embodiments, a portion of the three dimensional
structure refers to structural domains of the FGFR3, including the
an extracellular ligand binding region, with two or three
immunoglobulin-like domains (IgD1-3), a single-pass transmembrane
region, and a cytoplasmic, split tyrosine kinase domain. Homolog
tertiary structure can be probed, measured, or confirmed by known
analytic or diagnostic methods, for example, X-ray, NMR, circular
dichroism, a panel of monoclonal antibodies that recognize native
FGFR3, and like techniques. For example, structurally homologous
molecules can have substitutions, deletions or additions of one or
more contiguous or noncontiguous amino acids, such as a loop or a
domain. Structurally homologous molecules also include "modified"
FGFR3 molecules that have been chemically or enzymatically
derivatized at one or more constituent amino acid, including side
chain modifications, backbone modifications, and N- and C-terminal
modifications including acetylation, hydroxylation, methylation,
amidation, and the attachment of carbohydrate or lipid moieties,
cofactors, and like modifications.
[0169] "Molecular complex", as used herein, refers to a combination
of bound substrate or ligand with polypeptide, such as an antibody
bound to FGFR3, or a ligand bound to FGFR3.
[0170] "Machine-readable data storage medium", as used herein,
means a data storage material encoded with machine-readable data,
wherein a machine programmed with instructions for using such data
and is capable of displaying data in the desired format, for
example, a graphical three-dimensional representation of molecules
or molecular complexes.
[0171] "Scalable," as used herein, means the increasing or
decreasing of distances between coordinates (configuration of
points) by a scalar factor while keeping the angles essentially the
same.
[0172] "Space group symmetry", as used herein, means the whole
symmetry of the crystal that combines the translational symmetry of
a crystalline lattice with the point group symmetry. A space group
is designated by a capital letter identifying the lattice type (P,
A, F, etc.) followed by the point group symbol in which the
rotation and reflection elements are extended to include screw axes
and glide planes. Note that the point group symmetry for a given
space group can be determined by removing the cell centering symbol
of the space group and replacing all screw axes by similar rotation
axes and replacing all glide planes with mirror planes. The point
group symmetry for a space group describes the true symmetry of its
reciprocal lattice.
[0173] "Unit cell", as used herein, means the atoms in a crystal
that are arranged in a regular repeating pattern, in which the
smallest repeating unit is called the unit cell. The entire
structure can be reconstructed from knowledge of the unit cell,
which is characterized by three lengths (a, b and c) and three
angles (.alpha., .beta. and .gamma.). The quantities a and b are
the lengths of the sides of the base of the cell and .gamma. is the
angle between these two sides. The quantity c is the height of the
unit cell. The angles .alpha. and .beta. describe the angles
between the base and the vertical sides of the unit cell.
[0174] "X-ray diffraction pattern" means the pattern obtained from
X-ray scattering of the periodic assembly of molecules or atoms in
a crystal. X-ray crystallography is a technique that exploits the
fact that X-rays are diffracted by crystals. X-rays have the proper
wavelength (in the .ANG.ngstrom (.ANG.) range, approximately 10-8
cm) to be scattered by the electron cloud of an atom of comparable
size. Based on the diffraction pattern obtained from X-ray
scattering of the periodic assembly of molecules or atoms in the
crystal, the electron density can be reconstructed. Additional
phase information can be extracted either from the diffraction data
or from supplementing diffraction experiments to complete the
reconstruction (the phase problem in crystallography). A model is
then progressively built into the experimental electron density,
refined against the data to produce an accurate molecular
structure.
[0175] X-ray structure coordinates define a unique configuration of
points in space. Those of skill in the art understand that a set of
structure coordinates for a protein or a protein/ligand complex, or
a portion thereof, define a relative set of points that, in turn,
define a configuration in three dimensions. A similar or identical
configuration can be defined by an entirely different set of
coordinates, provided the distances and angles between coordinates
remain essentially the same. In addition, a configuration of points
can be defined by increasing or decreasing the distances between
coordinates by a scalar factor, while keeping the angles
essentially the same.
[0176] "Crystal structure" generally refers to the
three-dimensional or lattice spacing arrangement of repeating
atomic or molecular units in a crystalline material. The crystal
structure of a crystalline material can be determined by X-ray
crystallographic methods, see for example, "Principles of Protein
X-Ray Crystallography," by Jan Drenth, Springer Advanced Texts in
Chemistry, Springer Verlag; 2nd ed., February 1999, ISBN:
0387985875, and "Introduction to Macromolecular Crystallography,"
by Alexander McPherson, Wiley-Liss, Oct. 18, 2002, ISBN:
0471251224.
[0177] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0178] The phrase "substantially similar," or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is
preferably less than about 50%, preferably less than about 40%,
preferably less than about 30%, preferably less than about 20%,
preferably less than about 10% as a function of the value for the
reference/comparator antibody.
[0179] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Desirably the Kd is 1.times.10.sup.-7, 1.times.10.sup.-8,
5.times.10.sup.-8, 1.times.10.sup.-9, 3.times.10.sup.-9,
5.times.10.sup.-9, or even 1.times.10.sup.-10 or stronger. Affinity
can be measured by common methods known in the art, including those
described herein. Low-affinity antibodies generally bind antigen
slowly and tend to dissociate readily, whereas high-affinity
antibodies generally bind antigen faster and tend to remain bound
longer. A variety of methods of measuring binding affinity are
known in the art, any of which can be used for purposes of the
present invention. Specific illustrative embodiments are described
in the following.
[0180] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay that measures solution
binding affinity of Fabs for antigen by equilibrating Fab with a
minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol. Biol. 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5
.mu.g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM
sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)
bovine serum albumin in PBS for two to five hours at room
temperature (approximately 23.degree. C.). In a non-adsorbant plate
(Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed with
serial dilutions of a Fab of interest (e.g., consistent with
assessment of an anti-VEGF antibody, Fab-12, in Presta et al.,
(1997) Cancer Res. 57:4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., 65 hours) to insure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
Tween-20 in PBS. When the plates have dried, 150 .mu.l/well of
scintillant (MicroScint-20; Packard) is added, and the plates are
counted on a Topcount gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
According to another embodiment the Kd or Kd value is measured by
using surface plasmon resonance assays using a BIAcore.TM.-2000 or
a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIAcore IIIc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate
of 5 .mu.l/minute to achieve approximately 10 response units (RU)
of coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 .mu.l/min. In some embodiments, the
following modifications are used for the surface Plasmon resonance
assay method: antibody is immobilized to CM5 biosensor chips to
achieve approximately 400 RU, and for kinetic measurements,
two-fold serial dilutions of target protein (e.g., FGFR3-IIIb or
-IIIc) (starting from 67 nM) are injected in PBST buffer at
25.degree. C. with a flow rate of about 30 ul/minute. Association
rates (k.sub.on) and dissociation rates (k.sub.off) are calculated
using a simple one-to-one Langmuir binding model (BIAcore
Evaluation Software version 3.2) by simultaneous fitting the
association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen, Y., et al., (1999) J. Mol.
Biol. 293:865-881. If the on-rate exceeds 10.sup.6 M.sup.-1
S.sup.-1 by the surface plasmon resonance assay above, then the
on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red
cuvette.
[0181] An "on-rate" or "rate of association" or "association rate"
or "k.sub.on" according to this invention can also be determined
with the same surface plasmon resonance technique described above
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CM5
chips at .about.10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CM5, BIAcore IIIc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 .mu.g/ml (.about.0.2 uM) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 .mu.l/min. In some embodiments, the
following modifications are used for the surface Plasmon resonance
assay method: antibody is immobilized to CM5 biosensor chips to
achieve approximately 400 RU, and for kinetic measurements,
two-fold serial dilutions of target protein (e.g., FGFR3-IIIb or
-IIIc) (starting from 67 nM) are injected in PBST buffer at
25.degree. C. with a flow rate of about 30 ul/minute. Association
rates (k.sub.on) and dissociation rates (k.sub.off) are calculated
using a simple one-to-one Langmuir binding model (BIAcore
Evaluation Software version 3.2) by simultaneous fitting the
association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) was calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen, Y., et al., (1999) J. Mol.
Biol. 293:865-881. However, if the on-rate exceeds 10.sup.6
M.sup.-1 S.sup.-1 by the surface plasmon resonance assay above,
then the on-rate is preferably determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stir red cuvette.
[0182] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label. Other types of modifications include, for example, "caps,"
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
ply-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and a basic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"), P(O)R,
P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or R' is
independently H or substituted or unsubstituted alkyl (1-20 C)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0183] "Percent (%) amino acid sequence identity" with respect to a
peptide or polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the specific peptide or polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2, wherein the complete
source code for the ALIGN-2 program is provided in Table A below.
The ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc. and the source code has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through
Genentech, Inc., South San Francisco, Calif. or may be compiled
from the source code provided in, e.g., WO2007/001851. The ALIGN-2
program should be compiled for use on a UNIX operating system,
preferably digital UNIX V4.0D. All sequence comparison parameters
are set by the ALIGN-2 program and do not vary.
[0184] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A.
[0185] In some embodiments, two or more amino acid sequences are at
least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments,
two or more amino acid sequences are at least 95%, 97%, 98%, 99%,
or even 100% identical. Unless specifically stated otherwise, all %
amino acid sequence identity values used herein are obtained as
described in the immediately preceding paragraph using the ALIGN-2
computer program.
[0186] The term "FGFR3," as used herein, refers, unless
specifically or contextually indicated otherwise, to any native or
variant (whether native or synthetic) FGFR3 polypeptide (e.g.,
FGFR3-IIIb isoform or FGFR3-IIIc isoform). The term "native
sequence" specifically encompasses naturally occurring truncated
forms (e.g., an extracellular domain sequence or a transmembrane
subunit sequence), naturally occurring variant forms (e.g.,
alternatively spliced forms) and naturally-occurring allelic
variants. The term "wild-type FGFR3" generally refers to a
polypeptide comprising an amino acid sequence of a naturally
occurring FGFR3 protein. The term "wild type FGFR3 sequence"
generally refers to an amino acid sequence found in a naturally
occurring FGFR3.
[0187] "Ligand", as used herein, refers to an agent or compound
that associates with a binding site on a molecule, for example,
FGFR3 binding sites, and may be an antagonist or agonist of FGFR3
activity. Ligands include molecules that mimic anti-FGFR3 antibody
(e.g., R3Mab) binding to FGFR3. A ligand may be any native or
variant (whether native or synthetic) FGFR3 ligand (for example,
FGF1, FGF2, FGF4, FGF8, FGF9, FGF17, FGF18, FGF23) polypeptide. The
term "native sequence" specifically encompasses naturally occurring
truncated forms (e.g., an extracellular domain sequence or a
transmembrane subunit sequence), naturally occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants. The term "wild-type FGFR3 ligand" generally refers to a
polypeptide comprising an amino acid sequence of a naturally
occurring FGFR3 ligand protein. The term "wild type FGFR3 ligand
sequence" generally refers to an amino acid sequence found in a
naturally occurring FGFR3 ligand.
[0188] "Compound" refers to molecule that associates with FGFR3 or
a pharmaceutically acceptable salt, ester, amide, prodrug, isomer,
or metabolite, thereof. "Pharmaceutically acceptable salt" refers
to a formulation of a compound that does not compromise the
biological activity and properties of the compound. Pharmaceutical
salts can be obtained by reacting a binding-active compound of the
disclosure with inorganic or organic acids such as hydrochloric
acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric
acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic
acid, salicylic acid and the like.
[0189] "FGFR3 activation" refers to activation, or phosphorylation,
of the FGFR3 receptor. Generally, FGFR3 activation results in
signal transduction (e.g. that caused by an intracellular kinase
domain of a FGFR3 receptor phosphorylating tyrosine residues in
FGFR3 or a substrate polypeptide). FGFR3 activation may be mediated
by FGFR ligand binding to a FGFR3 receptor of interest. FGFR3
ligand (e.g., such as FGF1 or FGF9) binding to FGFR3 may activate a
kinase domain of FGFR3 and thereby result in phosphorylation of
tyrosine residues in the FGFR3 and/or phosphorylation of tyrosine
residues in additional substrate polypeptides(s).
[0190] The term "constitutive" as used herein, as for example
applied to receptor kinase activity, refers to continuous signaling
activity of a receptor that is not dependent on the presence of a
ligand or other activating molecules. Depending on the nature of
the receptor, all of the activity may be constitutive or the
activity of the receptor may be further activated by the binding of
other molecules (e.g. ligands). Cellular events that lead to
activation of receptors are well known among those of ordinary
skill in the art. For example, activation may include
oligomerization, e.g., dimerization, trimerization, etc., into
higher order receptor complexes. Complexes may comprise a single
species of protein, i.e., a homomeric complex. Alternatively,
complexes may comprise at least two different protein species,
i.e., a heteromeric complex. Complex formation may be caused by,
for example, overexpression of normal or mutant forms of receptor
on the surface of a cell. Complex formation may also be caused by a
specific mutation or mutations in a receptor.
[0191] The term "ligand-independent" as used herein, as for example
applied to receptor signaling activity, refers to signaling
activity that is not dependent on the presence of a ligand. A
receptor having ligand-independent kinase activity will not
necessarily preclude the binding of ligand to that receptor to
produce additional activation of the kinase activity.
[0192] The term "ligand-dependent" as used herein, as for example
applied to receptor signaling activity, refers to signaling
activity that is dependent on the presence of a ligand.
[0193] The term "mutation", as used herein, means a difference in
the amino acid or nucleic acid sequence of a particular protein or
nucleic acid (gene, RNA) relative to the wild-type protein or
nucleic acid, respectively. A mutated protein or nucleic acid can
be expressed from or found on one allele (heterozygous) or both
alleles (homozygous) of a gene, and may be somatic or germ line. In
the instant invention, mutations are generally somatic. Mutations
include sequence rearrangements such as insertions, deletions, and
point mutations (including single nucleotide/amino acid
polymorphisms).
[0194] To "inhibit" is to decrease or reduce an activity, function,
and/or amount as compared to a reference.
[0195] An agent possesses "agonist activity or function" when an
agent mimics at least one of the functional activities of a
polypeptide of interest (e.g., FGFR ligand, such as FGF1 or
FGF9).
[0196] An "agonist antibody", as used herein, is an antibody which
mimics at least one of the functional activities of a polypeptide
of interest (e.g., FGFR ligand, such as FGF1 or FGF9).
[0197] The term "Fc region", as used herein, generally refers to a
dimer complex comprising the C-terminal polypeptide sequences of an
immunoglobulin heavy chain, wherein a C-terminal polypeptide
sequence is that which is obtainable by papain digestion of an
intact antibody. The Fc region may comprise native or variant Fc
sequences. Although the boundaries of the Fc sequence of an
immunoglobulin heavy chain might vary, the human IgG heavy chain Fc
sequence is usually defined to stretch from an amino acid residue
at about position Cys226, or from about position Pro230, to the
carboxyl terminus of the Fc sequence. The Fc sequence of an
immunoglobulin generally comprises two constant domains, a CH2
domain and a CH3 domain, and optionally comprises a CH4 domain. The
C-terminal lysine (residue 447 according to the EU numbering
system) of the Fc region may be removed, for example, during
purification of the antibody or by recombinant engineering of the
nucleic acid encoding the antibody. Accordingly, a composition
comprising an antibody having an Fc region according to this
invention can comprise an antibody with K447, with all K447
removed, or a mixture of antibodies with and without the K447
residue.
[0198] By "Fc polypeptide" herein is meant one of the polypeptides
that make up an Fc region. An Fc polypeptide may be obtained from
any suitable immunoglobulin, such as IgG.sub.1, IgG.sub.2,
IgG.sub.3, or IgG.sub.4 subtypes, IgA, IgE, IgD or IgM. In some
embodiments, an Fc polypeptide comprises part or all of a wild type
hinge sequence (generally at its N terminus). In some embodiments,
an Fc polypeptide does not comprise a functional or wild type hinge
sequence.
[0199] A "blocking" antibody or an antibody "antagonist" is one
which inhibits or reduces biological activity of the antigen it
binds. Preferred blocking antibodies or antagonist antibodies
completely inhibit the biological activity of the antigen.
[0200] A "naked antibody" is an antibody that is not conjugated to
a heterologous molecule, such as a cytotoxic moiety or
radiolabel.
[0201] An antibody having a "biological characteristic" of a
designated antibody is one which possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies that bind to the same antigen.
[0202] In order to screen for antibodies which bind to an epitope
on an antigen bound by an antibody of interest, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed.
[0203] To increase the half-life of the antibodies or polypeptide
containing the amino acid sequences of this invention, one can
attach a salvage receptor binding epitope to the antibody
(especially an antibody fragment), as described, e.g., in U.S. Pat.
No. 5,739,277. For example, a nucleic acid molecule encoding the
salvage receptor binding epitope can be linked in frame to a
nucleic acid encoding a polypeptide sequence of this invention so
that the fusion protein expressed by the engineered nucleic acid
molecule comprises the salvage receptor binding epitope and a
polypeptide sequence of this invention. As used herein, the term
"salvage receptor binding epitope" refers to an epitope of the Fc
region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
or IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule (e.g., Ghetie et al., Ann. Rev.
Immunol. 18:739-766 (2000), Table 1). Antibodies with substitutions
in an Fc region thereof and increased serum half-lives are also
described in WO00/42072, WO 02/060919; Shields et al., J. Biol.
Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem. 279:6213-6216
(2004)). In another embodiment, the serum half-life can also be
increased, for example, by attaching other polypeptide sequences.
For example, antibodies or other polypeptides useful in the methods
of the invention can be attached to serum albumin or a portion of
serum albumin that binds to the FcRn receptor or a serum albumin
binding peptide so that serum albumin binds to the antibody or
polypeptide, e.g., such polypeptide sequences are disclosed in
WO01/45746. In one preferred embodiment, the serum albumin peptide
to be attached comprises an amino acid sequence of DICLPRWGCLW (SEQ
ID NO:183). In another embodiment, the half-life of a Fab is
increased by these methods. See also, Dennis et al. J. Biol. Chem.
277:35035-35043 (2002) for serum albumin binding peptide
sequences.
[0204] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule that contains, preferably, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of
the reference nucleic acid molecule or polypeptide. A fragment may
contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400,
500, 600, or more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 120, 140, 160, 180, 190, 200 amino acids or more.
[0205] The phrase "little to no agonist function" with respect to
an antibody of the invention, as used herein, means the antibody
does not elicit a biologically meaningful amount of agonist
activity, e.g., upon administration to a subject. As would be
understood in the art, amount of an activity may be determined
quantitatively or qualitatively, so long as a comparison between an
antibody of the invention and a reference counterpart can be done.
The activity can be measured or detected according to any assay or
technique known in the art, including, e.g., those described
herein. The amount of activity for an antibody of the invention and
its reference counterpart can be determined in parallel or in
separate runs. In some embodiments, a bivalent antibody of the
invention does not possess substantial agonist function.
[0206] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (e.g., full length or intact monoclonal antibodies),
polyclonal antibodies, multivalent antibodies, multispecific
antibodies (e.g., bispecific antibodies so long as they exhibit the
desired biological activity) and may also include certain antibody
fragments (as described in greater detail herein). An antibody can
be human, humanized, and/or affinity matured.
[0207] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The
[0208] CDRs in each chain are held together in close proximity by
the FR regions and, with the CDRs from the other chain, contribute
to the formation of the antigen-binding site of antibodies (see
Kabat et al., Sequences of Proteins of Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in binding an
antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity.
[0209] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0210] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by a flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0211] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0212] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0213] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known. "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion preferably retains at least one,
preferably most or all, of the functions normally associated with
that portion when present in an intact antibody. Examples of
antibody fragments include Fab, Fab', F(ab')2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. In one
embodiment, an antibody fragment comprises an antigen binding site
of the intact antibody and thus retains the ability to bind
antigen. In another embodiment, an antibody fragment, for example
one that comprises the Fc region, retains at least one of the
biological functions normally associated with the Fc region when
present in an intact antibody, such as FcRn binding, antibody half
life modulation, ADCC function and complement binding. In one
embodiment, an antibody fragment is a monovalent antibody that has
an in vivo half life substantially similar to an intact antibody.
For e.g., such an antibody fragment may comprise on antigen binding
arm linked to an Fc sequence capable of conferring in vivo
stability to the fragment.
[0214] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six hypervariable regions;
three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A
number of hypervariable region delineations are in use and are
encompassed herein. The Kabat Complementarity Determining Regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). Chothia refers instead to the
location of the structural loops (Chothia and Lesk, J. Mol. Biol.
196:901-917 (1987)). The AbM hypervariable regions represent a
compromise between the Kabat CDRs and Chothia structural loops, and
are used by Oxford Molecular's AbM antibody modeling software. The
"contact" hypervariable regions are based on an analysis of the
available complex crystal structures. The residues from each of
these hypervariable regions are noted below.
TABLE-US-00005 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
Hypervariable regions may comprise "extended hypervariable regions"
as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3)
in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or
95-102 (H3) in the VH. The variable domain residues are numbered
according to Kabat et al., supra for each of these definitions.
[0215] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0216] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the
following review articles and references cited therein: Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994).
[0217] "Chimeric" antibodies (immunoglobulins) have a portion of
the heavy and/or light chain identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; and Morrison
et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Humanized
antibody as used herein is a subset of chimeric antibodies.
[0218] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0219] An "antigen" is a predetermined antigen to which an antibody
can selectively bind. The target antigen may be polypeptide,
carbohydrate, nucleic acid, lipid, hapten or other naturally
occurring or synthetic compound. Preferably, the target antigen is
a polypeptide.
[0220] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0221] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
[0222] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al., Proc Nat. Acad.
Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155
(1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol.
Biol. 226:889-896 (1992).
[0223] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0224] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 or Presta
U.S. Pat. No. 6,737,056 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al., PNAS
(USA) 95:652-656 (1998).
[0225] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0226] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)) and regulates homeostasis of immunoglobulins. WO
00/42072 (Presta) describes antibody variants with improved or
diminished binding to FcRs. The content of that patent publication
is specifically incorporated herein by reference. See, also,
Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).
[0227] Methods of measuring binding to FcRn are known (see, e.g.,
Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and serum
half life of human FcRn high affinity binding polypeptides can be
assayed, e.g., in transgenic mice or transfected human cell lines
expressing human FcRn, or in primates administered with the Fc
variant polypeptides.
[0228] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0229] Polypeptide variants with altered Fc region amino acid
sequences and increased or decreased C1q binding capability are
described in U.S. Pat. No. 6,194,551B1 and WO 99/51642. The
contents of those patent publications are specifically incorporated
herein by reference. See, also, Idusogie et al., J. Immunol.
164:4178-4184 (2000).
[0230] The term "Fc region-comprising polypeptide" refers to a
polypeptide, such as an antibody or immunoadhesin, which comprises
an Fc region. The C-terminal lysine (residue 447 according to the
EU numbering system) of the Fc region may be removed, for example,
during purification of the polypeptide or by recombinant
engineering the nucleic acid encoding the polypeptide. Accordingly,
a composition comprising a polypeptide having an Fc region
according to this invention can comprise polypeptides with K447,
with all K447 removed, or a mixture of polypeptides with and
without the K447 residue.
[0231] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a VL or VH
framework derived from a human immunoglobulin framework, or from a
human consensus framework. An acceptor human framework "derived
from" a human immunoglobulin framework or human consensus framework
may comprise the same amino acid sequence thereof, or may contain
pre-existing amino acid sequence changes. Where pre-existing amino
acid changes are present, preferably no more than 5 and preferably
4 or less, or 3 or less, pre-existing amino acid changes are
present. Where pre-existing amino acid changes are present in a VH,
preferably those changes are only at three, two, or one of
positions 71H, 73H, and 78H; for instance, the amino acid residues
at those positions may be 71A, 73T, and/or 78A. In one embodiment,
the VL acceptor human framework is identical in sequence to the VL
human immunoglobulin framework sequence or human consensus
framework sequence.
[0232] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residue in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al.
[0233] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al. In one embodiment,
[0234] the VH subgroup III consensus framework amino acid sequence
comprises at least a portion or all of each of the following
sequences
TABLE-US-00006 (SEQ ID NO: 184) EVQLVESGGGLVQPGGSLRLSCAAS-H1- (SEQ
ID NO: 185) WVRQAPGKGLEWV-H2- (SEQ ID NO: 186)
RFTISRDNSKNTLYLQMNSLRAEDTAVYYC-H3- (SEQ ID NO: 187)
WGQGTLVTVSS.
[0235] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al. In one embodiment,
the VH subgroup I consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00007 (SEQ ID NO: 188) DIQMTQSPSSLSASVGDRVTITC-L1- (SEQ ID
NO: 189) WYQQKPGKAPKLLIY-L2- (SEQ ID NO: 190)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-L3- (SEQ ID NO: 191)
FGQGTKVEIK.
[0236] As used herein, "antibody mutant" or "antibody variant"
refers to an amino acid sequence variant of an antibody wherein one
or more of the amino acid residues of the species-dependent
antibody have been modified. Such mutants necessarily have less
than 100% sequence identity or similarity with the
species-dependent antibody. In one embodiment, the antibody mutant
will have an amino acid sequence having at least 75% amino acid
sequence identity or similarity with the amino acid sequence of
either the heavy or light chain variable domain of the
species-dependent antibody, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, and most
preferably at least 95%. Identity or similarity with respect to
this sequence is defined herein as the percentage of amino acid
residues in the candidate sequence that are identical (i.e. same
residue) or similar (i.e. amino acid residue from the same group
based on common side-chain properties, see below) with the
species-dependent antibody residues, after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent
sequence identity. None of N-terminal, C-terminal, or internal
extensions, deletions, or insertions into the antibody sequence
outside of the variable domain shall be construed as affecting
sequence identity or similarity
Compositions and Methods of the Invention
[0237] The present disclosure includes a crystalline form and a
crystal structure of FGFR3 complexed with an anti-FGFR3 antibody,
and methods of using the FGFR3:anti-FGFR3 antibody crystal
structure and structural coordinates to identify homologous
proteins and to design or identify agents that can modulate the
function of FGFR3 or the FGFR3-anti-FGFR3 antibody complex. The
present disclosure also includes the three-dimensional
configuration of points derived from the structure coordinates of
at least a portion of an FGFR3 molecule or molecular complex, as
well as structurally equivalent configurations, as described below.
The three-dimensional configuration includes points derived from
structure coordinates of, e.g., the FGFR3:anti-FGFR3 antibody
complex, representing the locations of a plurality of the amino
acids defining the FGFR3-anti-FGFR3 antibody complex binding
site.
[0238] In some embodiments, the three-dimensional configuration
includes points derived from structure coordinates representing the
locations of the backbone atoms of a plurality of amino acids
defining the FGFR3-anti-FGFR3 antibody complex or the FGFR3 binding
site of, e.g., the FGFR3:anti-FGFR3 antibody complex.
Alternatively, the three-dimensional configuration includes points
derived from structure coordinates representing the locations of
the side chain and the backbone atoms (other than hydrogens) of a
plurality of the amino acids defining the FGFR3-anti-FGFR3 antibody
complex.
[0239] The disclosure also includes the scalable three-dimensional
configuration of points derived from structure coordinates of
molecules or molecular complexes that are structurally homologous
to FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody
complex, as well as structurally equivalent configurations.
Structurally homologous molecules or molecular complexes are
defined below. Advantageously, structurally homologous molecules
can be identified using the structure coordinates of the FGFR3,
anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex or
extracellular fragment(s) of FGFR3 according to a method of the
disclosure.
[0240] The configurations of points in space derived from structure
coordinates according to the disclosure can be visualized as, for
example, a holographic image, a stereodiagram, a model, or a
computer-displayed image, and the disclosure thus includes such
images, diagrams or models.
[0241] The crystal structure and structural coordinates can be used
in methods, for example, for obtaining structural information of a
related molecule, and for identifying and designing agents that
modulate FGFR3, or the FGFR3:anti-FGFR3 antibody complex.
[0242] In some embodiments, the FGFR3 binding site may comprise,
consist essentially of, or consist of one or more of the amino acid
residues 154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210,
212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282,
283, 314, 315, 316, 317 and/or 318, or mixtures thereof, of
FGFR3-IIIb polypeptide (e.g., human FGFR3-IIIb disclosed in
(UniProKB/Swiss-Prot accession number P22607.sub.--2), or
equivalent residues of FGFR3-IIIc polypeptide (e.g., human
FGFR3-IIIc disclosed in UniProKB/Swiss-Prot accession number
P22607). In some embodiments, the FGFR3 binding site may comprise,
consist essentially of, or consist of one or more of the amino acid
residues 158, 159, 169, 170, 171, 173, 175, 205, 207, and/or 315,
or mixtures thereof, of FGFR3-IIIb polypeptide, or equivalent
residues of FGFR3-IIIc polypeptide. In some embodiments, the FGFR3
binding site may comprise, consist essentially of, or consist of
one or more of the amino acid residues 158, 170, 171, 173, 175,
and/or 315, or mixtures thereof, of FGFR3-IIIb polypeptide, or
equivalent residues of FGFR3-IIIc polypeptide.
FGFR3 Polypeptides, Polynucleotides and Variants Thereof
[0243] FGFR3 nucleic acid and amino acid sequences are known in the
art. Nucleic acid sequence encoding the FGFR3 can be designed using
the amino acid sequence of the desired region of FGFR3. As is
well-known in the art, there are two major splice isoforms of
FGFR3, FGFR3-IIIb and FGFR3-IIIc. FGFR3 sequences are well-known in
the art and may include the sequence of UniProKB/Swiss-Prot
accession number P22607 (FGFR3-IIIc) or P22607.sub.--2
(FGFR3-IIIb). FGFR3 mutations have been identified and are
well-known in the art and include the following mutations (with
reference to the sequences shown in UniProKB/Swiss-Prot accession
number P22607 (FGFR3-IIIc) or P22607.sub.--2 (FGFR3-IIIb):
TABLE-US-00008 FGFR3-IIIb FGFR3-IIIc R248C R248C S249C S249C G372C
G370C Y375C Y373C G382R G380R K652E K650E
[0244] The present disclosure also includes a polypeptides
comprising, consisting essentially of, or consisting of a portion
or fragment of the FGFR3, and polynucleotides encoding the
polypeptides.
[0245] An embodiment of a polypeptide fragment comprises, consists
essentially of, or consists of any of amino acid residue starting
from amino acid residue 154 to amino acid residue 164 and ending at
amino acid residue 178 to amino acid 283 of human FGFR3 (e.g.,
human UniProKB/Swiss-Prot accession number P22607.sub.--2 (human
FGFR3-IIIb)). An embodiment of a polypeptide fragment comprises,
consists essentially of, or consists of any of amino acid residue
154 to amino acid residue 177, amino acid residue 202 to amino acid
reside 217, amino acid residue 241 to amino acid residu3 248, amino
acid residue 278 to amino acid residue 283 and/or amino acid
residue 314 to amino acid residue 318. An embodiment of a
polypeptide fragment comprises, consists essentially of, or
consists of any of amino acid residue 164 to amino acid residue 164
to residue 178, residue 269 to residue 283 and/or residue 154 to
residue 318. In some embodiments, the polypeptide portion has the
ability to bind to FGFR3 ligand.
[0246] The present disclosure also includes variants of FGFR3.
Variants include those polypeptides that have amino acid
substitutions, deletions, and additions (such as at least 1, 2, 3,
4, 5, 6, 7, 8, 9 10, or more amino acid substitutions, deletions
and additions). In some embodiments, the variant comprises 1, 2, 3,
4, 5, 6, 7, 8, 9 10 or more conservative substitutions (relative to
a reference sequence, such as a human FGFR3 reference sequence).
Amino acid substitutions can be made for example to replace
cysteines and eliminate formation of disulfide bonds. Amino acid
substitutions can also be made to change proteolytic cleavage
sites. Other variants can be made at the FGFR3 inhibitor binding
site. In other embodiments, the variants of the FGFR3 bind FGFR3
ligand with the same or higher affinity than the wild type FGFR3.
In some embodiments, the variants of the FGFR3 bind an FGFR3
inhibitor (e.g. anti-FGFR3 antibody) with the same or higher
affinity that the wild type FGFR3.
Fusion Proteins
[0247] FGFR3 polypeptides, variants, or structural homolog or
portions thereof, may be fused to a heterologous polypeptide or
compound. The heterologous polypeptide is a polypeptide that has a
different function than that of the FGFR3. Examples of heterologous
polypeptide include polypeptides that may act as carriers, may
extend half life, may act as epitope tags, may provide ways to
detect or purify the fusion protein. Heterologous polypeptides
include KLH, albumin, salvage receptor binding epitopes,
immunoglobulin constant regions, and peptide tags. Peptide tags
useful for detection or purification include FLAG, gD protein,
polyhistidine tags, hemagluthinin from influenza virus, T7 tag, S
tag, Strep tag, chloramiphenicol acetyl transferase, biotin,
glutathione-S transferase, green fluorescent protein and maltose
binding protein. Compounds that can be combined with the FGFR3,
variants or structural homolog or portions thereof, include
radioactive labels, protecting groups, and carbohydrate or lipid
moieties.
Polynucleotides, Vectors and Host Cells
[0248] FGFR3, variants or fragments thereof can be prepared by
introducing appropriate nucleotide changes into DNA encoding FGFR3,
or by synthesis of the desired polypeptide variants.
[0249] Polynucleotide sequences encoding the polypeptides described
herein can be obtained using standard recombinant techniques.
Desired polynucleotide sequences may be isolated and sequenced from
appropriate source cells. Alternatively, polynucleotides can be
synthesized using nucleotide synthesizer or PCR techniques. Once
obtained, sequences encoding the polypeptides or variant
polypeptides are inserted into a recombinant vector capable of
replicating and expressing heterologous polynucleotides in a host
cell. Many vectors that are available and known in the art can be
used for the purpose of the present invention. Selection of an
appropriate vector will depend mainly on the size of the nucleic
acids to be inserted into the vector and the particular host cell
to be transformed with the vector. Each vector contains various
components, depending on its function (amplification or expression
of heterologous polynucleotide, or both) and its compatibility with
the particular host cell in which it resides. The vector components
generally include, but are not limited to: an origin of replication
(in particular when the vector is inserted into a prokaryotic
cell), a selection marker gene, a promoter, a ribosome binding site
(RBS), a signal sequence, the heterologous nucleic acid insert and
a transcription termination sequence.
[0250] In general, plasmid vectors containing replicon and control
sequences, which are derived from a species compatible with the
host cell are used in connection with these hosts. The vector
ordinarily carries a replication site, as well as marking
sequences, which are capable of providing phenotypic selection in
transformed cells. For example, E. coli is typically transformed
using pBR322, a plasmid derived from an E. coli species. pBR322
contains genes encoding ampicillin (Amp) and tetracycline (Tet)
resistance and thus provides easy means for identifying transformed
cells. pBR322, its derivatives, or other microbial plasmids or
bacteriophage may also contain, or be modified to contain,
promoters which can be used by the microbial organism for
expression of endogenous proteins.
[0251] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0252] Either constitutive or inducible promoters can be used in
the present invention, in accordance with the needs of a particular
situation, which can be ascertained by one skilled in the art. A
large number of promoters recognized by a variety of potential host
cells are well known. The selected promoter can be operably linked
to cistron DNA encoding a polypeptide described herein by removing
the promoter from the source DNA via restriction enzyme digestion
and inserting the isolated promoter sequence into the vector of
choice. Both the native promoter sequence and many heterologous
promoters may be used to direct amplification and/or expression of
the target genes. However, heterologous promoters are preferred, as
they generally permit greater transcription and higher yields of
expressed target gene as compared to the native target polypeptide
promoter.
[0253] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .beta.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the polypeptides or variant
polypeptides (Siebenlist et al. (1980) Cell 20: 269) using linkers
or adaptors to supply any required restriction sites.
[0254] In embodiments, each cistron within a recombinant vector
comprises a secretion signal sequence component that directs
translocation of the expressed polypeptides across a membrane. In
general, the signal sequence may be a component of the vector, or
it may be a part of the polypeptide encoding DNA that is inserted
into the vector. The signal sequence selected for the purpose of
this invention should be one that is recognized and processed (i.e.
cleaved by a signal peptidase) by the host cell. For prokaryotic
host cells that do not recognize and process the signal sequences
native to the heterologous polypeptides, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group consisting of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders,
LamB, PhoE, PelB, OmpA and MBP.
[0255] Prokaryotic host cells suitable for expressing polypeptides
include Archaebacteria and Eubacteria, such as Gram-negative or
Gram-positive organisms. Examples of useful bacteria include
Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),
Enterobacteria, Pseudomonas species (e.g., P. aeruginosa),
Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus,
Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Preferably,
gram-negative cells are used. Preferably the host cell should
secrete minimal amounts of proteolytic enzymes, and additional
protease inhibitors may desirably be incorporated in the cell
culture.
[0256] Besides prokaryotic host cells, eukaryotic host cell systems
are also well established in the art. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plants and plant cells. Examples of useful
mammalian host cell lines include Chinese hamster ovary (CHO) and
COS cells. More specific examples include monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); and mouse mammary tumor (MMT 060562, ATCC
CCL51).
Polypeptide Production
[0257] Host cells are transformed or transfected with the
above-described expression vectors and cultured in conventional
nutrient media modified as appropriate for inducing promoters,
selecting transformants, or amplifying the genes encoding the
desired sequences.
[0258] Transfection refers to the taking up of an expression vector
by a host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO4 precipitation and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
[0259] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0260] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
preferred embodiments, the media also contains a selection agent,
chosen based on the construction of the expression vector, to
selectively permit growth of prokaryotic cells containing the
expression vector. For example, ampicillin is added to media for
growth of cells expressing ampicillin resistant gene.
[0261] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0262] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the preferred
temperature ranges from about 20.degree. C. to about 39.degree. C.,
more preferably from about 25.degree. C. to about 37.degree. C.,
even more preferably at about 30.degree. C. The pH of the medium
may be any pH ranging from about 5 to about 9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8
to about 7.4, and more preferably about 7.0.
[0263] If an inducible promoter is used in the expression vector,
protein expression is induced under conditions suitable for the
activation of the promoter. For example, if a PhoA promoter is used
for controlling transcription, the transformed host cells may be
cultured in a phosphate-limiting medium for induction. A variety of
other inducers may be used, according to the vector construct
employed, as is known in the art.
[0264] Eukaryotic host cells are cultured under conditions suitable
for expression of the FGFR3 and/or KD polypeptides. The host cells
used to produce the polypeptides may be cultured in a variety of
media. Commercially available media such as Ham's F10 (Sigma),
Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for
culturing the host cells. In addition, any of the media described
in one or more of Ham et al., 1979, Meth. Enz. 58:44, Barnes et
al., 1980, Anal. Biochem. 102: 255, U.S. Pat. No. 4,767,704, U.S.
Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No.
4,560,655, or U.S. Pat. No. 5,122,469, WO 90/103430, WO 87/00195,
and U.S. Pat. No. Re. 30,985 may be used as culture media for the
host cells. Any of these media may be supplemented as necessary
with hormones and/or other growth factors (such as insulin,
transferrin, or epidermal growth factor), salts (such as sodium
chloride, calcium, magnesium, and phosphate), buffers (such as
HEPES.TM.), nucleotides (such as adenosine and thymidine),
antibiotics (such as GENTAMYCIN.TM.), trace elements (defined as
inorganic compounds usually present at final concentrations in the
micromolar range), and glucose or an equivalent energy source.
Other supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0265] Polypeptides described herein expressed in a host cell may
be secreted into and/or recovered from the periplasm of the host
cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated there from. Cells may be removed from the culture and
the culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as fractionation on immunoaffinity or ion-exchange columns;
ethanol precipitation; reverse phase HPLC; chromatography on silica
or on a cation exchange resin such as DEAE; chromatofocusing;
SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for
example, Sephadex G-75; hydrophobic affinity resins, ligand
affinity using a suitable antigen immobilized on a matrix and
Western blot assay.
[0266] Polypeptides that are produced may be purified to obtain
preparations that are substantially homogeneous for further assays
and uses. Standard protein purification methods known in the art
can be employed. The following procedures are exemplary of suitable
purification procedures: fractionation on immunoaffinity or
ion-exchange columns, ethanol precipitation, reverse phase HPLC,
chromatography on silica or on a cation-exchange resin such as
DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation,
and gel filtration using, for example, Sephadex G-75.
[0267] Antibody Production
[0268] For recombinant production of an antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for
further cloning (amplification of the DNA) or for expression. DNA
encoding the antibody is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of the antibody). Many vectors are available. The
choice of vector depends in part on the host cell to be used.
Generally, preferred host cells are of either prokaryotic or
eukaryotic (generally mammalian) origin.
[0269] Generating Antibodies Using Prokaryotic Host Cells:
[0270] Vector Construction
[0271] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0272] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0273] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .pi.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0274] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g. the presence
or absence of a nutrient or a change in temperature.
[0275] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0276] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .beta.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the target light and heavy chains
(Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors
to supply any required restriction sites.
[0277] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0278] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB-strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).
[0279] The present invention provides an expression system in which
the quantitative ratio of expressed polypeptide components can be
modulated in order to maximize the yield of secreted and properly
assembled antibodies of the invention. Such modulation is
accomplished at least in part by simultaneously modulating
translational strengths for the polypeptide components.
[0280] One technique for modulating translational strength is
disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes
variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a series of amino acid or nucleic acid
sequence variants can be created with a range of translational
strengths, thereby providing a convenient means by which to adjust
this factor for the desired expression level of the specific chain.
TIR variants can be generated by conventional mutagenesis
techniques that result in codon changes which can alter the amino
acid sequence, although silent changes in the nucleotide sequence
are preferred. Alterations in the TIR can include, for example,
alterations in the number or spacing of Shine-Dalgarno sequences,
along with alterations in the signal sequence. One method for
generating mutant signal sequences is the generation of a "codon
bank" at the beginning of a coding sequence that does not change
the amino acid sequence of the signal sequence (i.e., the changes
are silent). This can be accomplished by changing the third
nucleotide position of each codon; additionally, some amino acids,
such as leucine, serine, and arginine, have multiple first and
second positions that can add complexity in making the bank. This
method of mutagenesis is described in detail in Yansura et al.
(1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.
[0281] Preferably, a set of vectors is generated with a range of
TIR strengths for each cistron therein. This limited set provides a
comparison of expression levels of each chain as well as the yield
of the desired antibody products under various TIR strength
combinations. TIR strengths can be determined by quantifying the
expression level of a reporter gene as described in detail in
Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational
strength comparison, the desired individual TIRs are selected to be
combined in the expression vector constructs of the invention.
[0282] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhuA (.DELTA.tonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP41 kanR (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli, 1776 (ATCC 31,537) and E.
coli RV308 (ATCC 31,608) are also suitable. These examples are
illustrative rather than limiting. Methods for constructing
derivatives of any of the above-mentioned bacteria having defined
genotypes are known in the art and described in, for example, Bass
et al., Proteins, 8:309-314 (1990). It is generally necessary to
select the appropriate bacteria taking into consideration
replicability of the replicon in the cells of a bacterium. For
example, E. coli, Serratia, or Salmonella species can be suitably
used as the host when well known plasmids such as pBR322, pBR325,
pACYC177, or pKN410 are used to supply the replicon. Typically the
host cell should secrete minimal amounts of proteolytic enzymes,
and additional protease inhibitors may desirably be incorporated in
the cell culture.
[0283] Antibody Production
[0284] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0285] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0286] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0287] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0288] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the preferred
temperature ranges from about 20.degree. C. to about 39.degree. C.,
more preferably from about 25.degree. C. to about 37.degree. C.,
even more preferably at about 30.degree. C. The pH of the medium
may be any pH ranging from about 5 to about 9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8
to about 7.4, and more preferably about 7.0.
[0289] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. Preferably,
the phosphate-limiting medium is the C.R.A.P medium (see, for e.g.,
Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety
of other inducers may be used, according to the vector construct
employed, as is known in the art.
[0290] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0291] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, preferably about 1,000 to 100,000
liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0292] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0293] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
[0294] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0295] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
[0296] Antibody Purification
[0297] In one embodiment, the antibody protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed. The
following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
[0298] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the full length antibody
products of the invention. Protein A is a 41kD cell wall protein
from Staphylococcus aureas which binds with a high affinity to the
Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
[0299] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
Finally the antibody of interest is recovered from the solid phase
by elution.
[0300] Generating antibodies using eukaryotic host cells:
[0301] The vector components generally include, but are not limited
to, one or more of the following:
[0302] a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence.
[0303] (i) Signal Sequence Component
[0304] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected preferably is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0305] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0306] (ii) Origin of Replication
[0307] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used only because it contains the early promoter.
[0308] (iii) Selection Gene Component
[0309] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0310] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0311] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0312] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity (e.g., ATCC CRL-9096).
[0313] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0314] (iv) Promoter Component
[0315] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody polypeptide nucleic acid. Promoter sequences are known
for eukaryotes. Virtually alleukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors.
[0316] Antibody polypeptide transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0317] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0318] (v) Enhancer Element Component
[0319] Transcription of DNA encoding the antibody polypeptide of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0320] (vi) Transcription Termination Component
[0321] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0322] (vii) Selection and Transformation of Host Cells
[0323] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0324] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0325] (viii) Culturing the Host Cells
[0326] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0327] (ix) Purification of Antibody
[0328] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0329] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0330] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0331] Activity Assays
[0332] The antibodies can be characterized for their
physical/chemical properties and biological functions by various
assays known in the art.
[0333] The purified immunoglobulins can be further characterized by
a series of assays including, but not limited to, N-terminal
sequencing, amino acid analysis, non-denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange chromatography and papain digestion.
[0334] In certain embodiments of the invention, the immunoglobulins
produced herein are analyzed for their biological activity. In some
embodiments, the immunoglobulins of the present invention are
tested for their antigen binding activity. The antigen binding
assays that are known in the art and can be used herein include
without limitation any direct or competitive binding assays using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays.
[0335] 2. Crystals and Crystal Structures
[0336] The present disclosure provides crystals of an
FGFR3:anti-FGFR3 antibody complex as well as the crystal structure
of FGFR3:anti-FGFR3 antibody as determined therefrom. In some
embodiments, the crystals are formed from an FGFR3 sequence
comprising sequence
[0337] ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG
REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPH
RPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVL
KSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA DEAGSV
(SEQ ID NO:272) and an anti-FGFR3 antibody.
[0338] In some embodiments, the crystals are formed from an FGFR3
sequence comprising sequence
[0339] ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNG
REFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPH
RPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVL
KSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA
DEAGSVHHHHHH (SEQ ID NO:273) and an anti-FGFR3 antibody.
[0340] In some embodiments, the anti-FGFR3 antibody comprises a
light chain variable region comprising HVR-L1, HVR-L2, HVR-L3,
wherein each, in order, comprises SEQ ID NO:4, 5, 6, and/or a heavy
chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, where
each, in order, contains SEQ ID NO: 1, 2, 3. In some embodiments,
the anti-FGFR3 antibody comprises a light chain variable region
comprising sequence
TABLE-US-00009 (SEQ ID NO: 274)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKR
and a heavy chain variable region comprising sequence
TABLE-US-00010 (SEQ ID NO: 275)
EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGR IY PTN
GSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
ARTYGIYDLYVDYTEYVMDYWGQGTLV.
[0341] In some embodiments, the anti-FGFR3 antibody comprises any
antibody disclosed herein or disclosed in co-pending co-owned U.S.
Ser. No. ______, filed Mar. 24, 2010 (attorney docket P4294R1).
[0342] In a specific embodiment, the structure of FGFR3 complexed
with an anti-FGFR3 antibody was solved by molecular replacement
with the program PHASER. The crystals belonged to space group
P2.sub.12.sub.12.sub.1 with cell parameters of a=58.5 .ANG., b=99.3
.ANG. and c=143.7 .ANG..
[0343] The crystals are useful to provide the crystal structure and
to provide a stable form of the molecule for storage.
[0344] Each of the constituent amino acids of FGFR3:anti-FGFR3
antibody is defined by a set of structure coordinates as set forth
in Table 6. The term "structure coordinates" refers to Cartesian
coordinates derived from mathematical equations related to the
patterns obtained on diffraction of a monochromatic beam of X-rays
by the atoms (scattering centers) of a FGFR3 and FGFR3:anti-FGFR3
antibody, in crystal form. The diffraction data are used to
calculate an electron density map of the repeating unit of the
crystal. The electron density maps are then used to establish the
positions of the individual atoms of the FGFR3, anti-FGFR3 antibody
and FGFR3:anti-FGFR3 antibody complex.
[0345] Slight variations in structure coordinates can be generated
by mathematically manipulating the FGFR3 and FGFR3:anti-FGFR3
antibody complex structure coordinates. For example, the structure
coordinates as set forth in Table 6 could be manipulated by
crystallographic permutations of the structure coordinates,
fractionalization of the structure coordinates, integer additions
or subtractions to sets of the structure coordinates, inversion of
the structure coordinates, or any combination of the above.
Alternatively, modifications in the crystal structure due to
mutations, additions, substitutions, deletions, and combinations
thereof, of amino acids, or other changes in any of the components
that make up the crystal, could also yield variations in structure
coordinates. Such slight variations in the individual coordinates
will have little effect on overall shape. If such variations are
within an acceptable standard error as compared to the original
coordinates, the resulting three-dimensional shape is considered to
be structurally equivalent. Structural equivalence is described in
more detail below.
[0346] It should be noted that slight variations in individual
structure coordinates of the FGFR3, anti-FGFR3 antibody and
FGFR3:anti-FGFR3 antibody complex would not be expected to
significantly alter the nature of chemical entities such as ligands
that could associate with a binding site or other structural
features of FGFR3. In this context, the phrase "associating with"
refers to a condition of proximity between a ligand, or portions
thereof, and a FGFR3 molecule or portions thereof. The association
may be non-covalent, wherein the juxtaposition is energetically
favored by hydrogen bonding, van der Waals forces, and/or
electrostatic interactions, or it may be covalent.
[0347] FGFR3 residues that form a binding site for a modulator
(e.g., an antagonist or agonist) of FGFR3 are described in the
present application. The identification of a binding site for a
modulator on FGFR3 can be used to design new classes of FGFR3
modulators, such as antagonists, agonists, and like agents, having
therapeutic applications, such as, for treating cancer.
[0348] 3. Structurally Equivalent Crystal Structures
[0349] Various computational analyses can be used to determine
whether a molecule or portions of the molecule defining structure
features are "structurally equivalent," defined in terms of its
three-dimensional structure, to all or part of an activated unbound
FGFR3 or FGFR3 bound to an inhibitor, such as an anti-FGFR3
antibody, or FGFR3 present in FGFR3:anti-FGFR3 antibody complex.
Such analyses may be carried out in current software applications,
such as the Molecular Similarity application of QUANTA (Molecular
Simulations IIIc., San Diego, Calif.), Version 4.1, and as
described in the accompanying User's Guide.
[0350] The Molecular Similarity application permits comparisons
between different structures, different conformations of the same
structure, and different parts of the same structure. A procedure
used in Molecular Similarity to compare structures comprises: 1)
loading the structures to be compared; 2) defining the atom
equivalences in these structures; 3) performing a fitting
operation; and 4) analyzing the results.
[0351] One structure is identified as the target (i.e., the fixed
structure); all remaining structures are working structures (i.e.,
moving structures). Since atom equivalency within QUANTA is defined
by user input, for the purpose of this disclosure equivalent atoms
are defined as protein backbone atoms (N, C.alpha., C, and O) for
all conserved residues between the two structures being compared. A
conserved residue is defined as a residue that is structurally or
functionally equivalent. Only rigid fitting operations are
considered.
[0352] When a rigid fitting method is used, the working structure
is translated and rotated to obtain an optimum fit with the target
structure. The fitting operation uses an algorithm that computes
the optimum translation and rotation to be applied to the moving
structure, such that the root mean square difference of the fit
over the specified pairs of equivalent atom is an absolute minimum.
This number, given in Angstroms, is reported by QUANTA.
[0353] Structurally equivalent crystal structures have portions of
the two molecules that are substantially identical, within an
acceptable margin of error. The margin of error can be calculated
by methods known to those of skill in the art. In some embodiments,
any molecule or molecular complex or any portion thereof, that has
a root mean square deviation of conserved residue backbone atoms
(N, C.alpha., C, O) of less than about 0.70 .ANG., preferably 0.5
.ANG.. For example, structurally equivalent molecules or molecular
complexes are those that are defined by the entire set of structure
coordinates listed in Table 7 or 8.+-.a root mean square deviation
from the conserved backbone atoms of those amino acids of not more
than 0.70 .ANG., preferably 0.5 .ANG.. The term "root mean square
deviation" means the square root of the arithmetic mean of the
squares of the deviations. It is a way to express the deviation or
variation from a trend or object. For purposes of this disclosure,
the "root mean square deviation" defines the variation in the
backbone of a protein from the backbone of FGFR3 complex (as
defined by the structure coordinates of the complex as described
herein) or a defining structural feature thereof
[0354] 4. Structurally Homologous Molecules, Molecular Complexes,
and Crystal Structures
[0355] Structure coordinates can be used to aid in obtaining
structural information about another crystallized molecule or
molecular complex. The method of the disclosure allows
determination of at least a portion of the three-dimensional
structure of molecules or molecular complexes that contain one or
more structural features that are similar to structural features of
at least a portion of the FGFR3, anti-FGFR3 antibody, or
FGFR3:anti-FGFR3 antibody complex. These molecules are referred to
herein as "structurally homologous" to FGFR3, anti-FGFR3 antibody,
or FGFR3:anti-FGFR3 antibody complex. Similar structural features
can include, for example, regions of amino acid identity, conserved
active site or binding site motifs, and similarly arranged
secondary structural elements (for example, binding sites for FGFR3
ligand on FGFR3).
[0356] Optionally, structural homology is determined by aligning
the residues of the two amino acid sequences to optimize the number
of identical amino acids along the lengths of their sequences; gaps
in either or both sequences are permitted in making the alignment
in order to optimize the number of identical amino acids, although
the amino acids in each sequence must nonetheless remain in their
proper order. Two amino acid sequences are compared using the BLAST
program, version 2.0.9, of the BLAST 2 search algorithm, as
described by Tatusova et al., and available at
http:www.ncbi.nlm.nih.gov/BLAST/. Preferably, the default values
for all BLAST 2 search parameters are used, including
matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap
x_dropoff=50, expect=10, wordsize=3, and filter on. In the
comparison of two amino acid sequences using the BLAST search
algorithm, structural similarity is referred to as "identity." In
some embodiments, a structurally homologous molecule is a protein
that has an amino acid sequence having at least 80% identity with a
wild type or recombinant amino acid sequence of FGFR3, in some
embodiments human FGFR3-IIIb or human FGFR3-IIIc. More preferably,
a protein that is structurally homologous to FGFR3 includes at
least one contiguous stretch of at least 50 amino acids that has at
least 80% amino acid sequence identity with the analogous portion
of the wild type or recombinant FGFR3. Methods for generating
structural information about the structurally homologous molecule
or molecular complex are well known and include, for example,
molecular replacement techniques.
[0357] Therefore, in another embodiment this disclosure provides a
method of utilizing molecular replacement to obtain structural
information about a molecule or molecular complex whose structure
is unknown comprising:
[0358] (a) generating an X-ray diffraction pattern from a
crystallized molecule or molecular complex of unknown or
incompletely known structure; and
[0359] (b) applying at least a portion of the structural
coordinates of FGFR3 complex to the X-ray diffraction pattern to
generate a three-dimensional electron density map of the molecule
or molecular complex whose structure is unknown or incompletely
known.
[0360] By using molecular replacement, all or part of the structure
coordinates of FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3
antibody complex as provided by this disclosure can be used to
determine the unsolved structure of a crystallized molecule or
molecular complex more quickly and efficiently than attempting to
determine such information ab initio. Coordinates of structural
features of FGFR3 can be utilized including that of trypsin-like
serine protease domain.
[0361] Molecular replacement can provide an accurate estimation of
the phases for an unknown or incompletely known structure. Phases
are one factor in equations that are used to solve crystal
structures, and this factor cannot be determined directly.
Obtaining accurate values for the phases, by methods other than
molecular replacement, can be a time-consuming process that
involves iterative cycles of approximations and refinements and
greatly hinders the solution of crystal structures. However, when
the crystal structure of a protein containing at least a
structurally homologous portion has been solved, molecular
replacement using the known structure provide a useful estimate of
the phases for the unknown or incompletely known structure.
[0362] Thus, this method involves generating a preliminary model of
a molecule or molecular complex whose structure coordinates are
unknown, by orienting and positioning the relevant portion of the
FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex
within the unit cell of the crystal of the unknown molecule or
molecular complex. This orientation or positioning is conducted so
as best to account for the observed X-ray diffraction pattern of
the crystal of the molecule or molecular complex whose structure is
unknown. Phases can then be calculated from this model and combined
with the observed X-ray diffraction pattern amplitudes to generate
an electron density map of the structure. This map, in turn, can be
subjected to established and well-known model building and
structure refinement techniques to provide a final, accurate
structure of the unknown crystallized molecule or molecular complex
(see for example, Lattman, 1985. Methods in Enzymology
115:55-77).
[0363] Structural information about a portion of any crystallized
molecule or molecular complex that is sufficiently structurally
homologous to a portion of FGFR3, anti-FGFR3 antibody, or
FGFR3:anti-FGFR3 antibody complex can be solved by this method. In
addition to a molecule that shares one or more structural features
with the FGFR3, such as the extracellular ligand binding region,
with two or three immunoglobulin-like domains (IgD1-3) and an acid
box, and/or FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3
antibody complex as described above, a molecule that has similar
bioactivity, such as the same ligand binding activity as FGFR3
and/or anti-FGFR3 antibody, may also be sufficiently structurally
homologous to a portion of the FGFR3 and/or antiFGFR3 antibody to
permit use of the structure coordinates of FGFR3:anti-FGFR3
antibody to solve its crystal structure or identify structural
features that are similar to those identified in the FGFR3
described herein. It will be appreciated that amino acid residues
in the structurally homologous molecule identified as corresponding
to the FGFR3 structural feature may have different amino acid
numbering.
[0364] In one embodiment of the disclosure, the method of molecular
replacement is utilized to obtain structural information about a
molecule or molecular complex, wherein the molecule or molecular
complex includes at least one FGFR3, anti-FGFR3 antibody, or
FGFR3:anti-FGFR3 antibody complex subunit or homolog. In the
context of the present disclosure, a "structural homolog" of the
FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex is
a protein that contains one or more amino acid substitutions,
deletions, additions, or rearrangements with respect to the amino
acid sequence of FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3
antibody complex but that, when folded into its native
conformation, exhibits or is reasonably expected to exhibit at
least a portion of the tertiary (three-dimensional) structure of at
least a portion of FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3
antibody complex. A portion of the FGFR3 includes the binding site
for an FGFR3 inhibitor.
[0365] A heavy atom derivative of FGFR3 is also included as a FGFR3
homolog. The term "heavy atom derivative" refers to derivatives of
FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex
produced by chemically modifying a crystal of FGFR3 or both. In
practice, a crystal is soaked in a solution containing heavy metal
atom salts, or organometallic compounds, e.g., lead chloride, gold
thiomalate, thiomersal or uranyl acetate, which can diffuse through
the crystal and bind to the surface of the protein. The location(s)
of the bound heavy metal atom(s) can be determined by X-ray
diffraction analysis of the soaked crystal. This information, in
turn, is used to generate the phase information used to construct
three-dimensional structure of the protein (Blundell, et al., 1976,
Protein Crystallography, Academic Press, San Diego, Calif.).
[0366] Variants may be prepared, for example, by expression of
FGFR3 cDNA previously altered in its coding sequence by
oligonucleotide-directed mutagenesis as described herein. Variants
may also be generated by site-specific incorporation of unnatural
amino acids into FGFR3 proteins using known biosynthetic methods
(Noren, et al., 1989, Science 244:182-88). In this method, the
codon encoding the amino acid of interest in wild-type FGFR3 is
replaced by a "blank" nonsense codon, TAG, using
oligonucleotide-directed mutagenesis. A suppressor tRNA directed
against this codon is then chemically aminoacylated in vitro with
the desired unnatural amino acid. The aminoacylated tRNA is then
added to an in vitro translation system to yield a mutant FGFR3
with the site-specific incorporated unnatural amino acid.
[0367] For example, structurally homologous molecules can contain
deletions or additions of one or more contiguous or noncontiguous
amino acids, such as a loop or a domain. Structurally homologous
molecules also include "modified" FGFR3, anti-FGFR3 antibody, or
FGFR3:anti-FGFR3 antibody complex that have been chemically or
enzymatically derivatized at one or more constituent amino acid,
including side chain modifications, backbone modifications, and N-
and C-terminal modifications including acetylation, hydroxylation,
methylation, amidation, and the attachment of carbohydrate or lipid
moieties, cofactors, and like modifications. It will be appreciated
that amino acid residues in the structurally homologous molecule
identified as corresponding to activated FGFR3 or other structural
feature of the FGFR3 may have different amino acid numbering.
[0368] The structure coordinates of FGFR3 are also particularly
useful to solve or model the structure of crystals of FGFR3,
anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex homologs,
which are co-complexed with a variety of ligands (e.g., a ligand
binding the antagonist binding site). This approach enables the
determination of the optimal sites for interaction between ligand,
including candidate FGFR3 ligands. Potential sites for modification
within the various binding sites (such as an FGFR3 binding site) of
the molecule can also be identified. This information provides an
additional tool for determining more efficient binding
interactions, for example, increased hydrophobic or polar
interactions, between FGFR3 and a ligand. For example,
high-resolution X-ray diffraction data collected from crystals
exposed to different types of solvent allows the determination of
where each type of solvent molecule resides. Small molecules that
bind tightly to those sites can then be designed and synthesized
and tested for their FGFR3 affinity, and/or inhibition
activity.
[0369] All of the complexes referred to above may be studied using
well-known X-ray diffraction techniques and may be refined versus
1.5-3.5 .ANG. resolution X-ray data to an R-factor of about 0.30 or
less using computer software, such as X-PLOR (Yale University,
distributed by Molecular Simulations, Inc.) (see for example,
Blundell, et al. 1976. Protein Crystallography, Academic Press, San
Diego, Calif., and Methods in Enzymology, Vol. 114 & 115, H. W.
Wyckoff et al., eds., Academic Press (1985)). This information may
thus be used to optimize known FGFR3 modulators, and more
importantly, to design new FGFR3 modulators.
[0370] The disclosure also includes the unique three-dimensional
configuration defined by a set of points defined by the structure
coordinates for a molecule or molecular complex structurally
homologous to FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3
antibody complex as determined using the method of the present
disclosure, structurally equivalent configurations, and magnetic
storage media including such set of structure coordinates.
[0371] 5. Homology Modeling
[0372] Using homology modeling, a computer model of a homolog,
e.g., an FGFR3 homolog, can be built or refined without
crystallizing the homolog. First, a preliminary model of the
homolog is created by sequence alignment with FGFR3, anti-FGFR3
antibody, or FGFR3:anti-FGFR3 antibody complex secondary structure
prediction, the screening of structural libraries, or any
combination of those techniques. Computational software may be used
to carry out the sequence alignments and the secondary structure
predictions. Structural incoherences, e.g., structural fragments
around insertions and deletions, can be modeled by screening a
structural library for peptides of the desired length and with a
suitable conformation. For prediction of the side chain
conformation, a side chain rotamer library may be employed. If the
homolog has been crystallized, the final homology model can be used
to solve the crystal structure of the homolog by molecular
replacement, as described above. Next, the preliminary model is
subjected to energy minimization to yield an energy-minimized
model. The energy-minimized model may contain regions where
stereochemistry restraints are violated, in which case such regions
are remodeled to obtain a final homology model. The homology model
is positioned according to the results of molecular replacement,
and subjected to further refinement including molecular dynamics
calculations.
[0373] 6. Methods for Identification of Modulators of FGFR3
[0374] Potent and selective ligands that modulate activity
(antagonists and agonists) can be identified using the
three-dimensional model of the FGFR3 using the coordinates of Table
6. In some embodiments, the three-dimensional model of the binding
site on FGFR3 and/or other structural features are produced using
the coordinates of Table 6. Using this model, candidate ligands
that interact with the FGFR3, e.g., the FGFR3 binding site, are
assessed for the desired characteristics (e.g., interaction with
FGFR3) by fitting against the model, and the result of the
interactions is predicted. Agents predicted to be molecules capable
of modulating the activity of FGFR3 can then be further screened or
confirmed against known bioassays. For example, the ability of an
agent to inhibit the effects of FGFR3 can be measured using assays
known in the art. Using the modeling information and the assays
described, one can identify agents that possess FGFR3-modulating
properties. Modulators of FGFR3 of the present disclosure can
include compounds or agents having, for example, inhibitory
activity.
[0375] Ligands which can interact with FGFR3 can also be identified
using commercially available modeling software, such as docking
programs, in which the solved crystal structure coordinates of
Table 6 can be computationally represented and screened against a
large virtual library of small molecules or virtual fragment
molecules for interaction with a site of interest, such as the
FGFR3 binding site. Preferred small molecules or fragments
identified in this way can be synthesized and contacted with the
FGFR3. The resulting molecular complex or association can be
further analyzed by, for example, NMR or X-ray co-crystallography,
to optimize the FGFR3 ligand interaction and/or desired biological
activity. Fragment-based drug discovery methods are known and
computational tools for their use are commercially available, for
example "SAR by NMR" (Shukers, S. B., et al., Science, 1996, 274,
1531-1534), "Fragments of Active Structures" (www.stromix.com;
Nienaber, V. L., et al., Nat. Biotechnol., 2000, 18, 1105-1108),
and "Dynamic Combinatorial X-ray Crystallography" (e.g., permitting
self-selection by the protein molecule of self-assembling
fragments; Congreve, M. S., et al., Angew. Chem., Int. Ed., 2003,
42, 4479-4482). Still other molecular modeling, and like methods
are discussed below and in the Examples.
[0376] In another embodiment, a candidate modulator can be
identified using a biological assay such as binding to FGFR3,
modulation (e.g., inhibition) of FGFR3 ligand activation of FGFR3,
modulation (e.g., inhibition) of FGFR3 biological activity. The
candidate modulator can then serve as a model to design similar
agents and/or to modify the candidate modulator for example, to
improve characteristics such as binding to FGFR3. Design or
modification of candidate modulators can be accomplished using the
crystal structure coordinates and available software.
[0377] Binding Site and Other Structural Features
[0378] The present disclosure provides information inter alia about
the shape and structure of a binding site of FGFR3 in the presence
of an inhibitor (anti-FGFR3 antibody). Binding sites are of
significant utility in fields such as drug discovery. The
association of natural ligands or substrates with the binding sites
of their corresponding receptors or enzymes is the basis of many
biological mechanisms of action. Similarly, many drugs exert their
biological effects through association with the binding sites of
receptors and enzymes. Such associations may occur with all or any
part of the binding site. An understanding of such associations
helps lead to the design of drugs having more favorable
associations with their target, and thus improved biological
effects. Therefore, this information is valuable in designing
potential modulators of FGFR3 binding sites, as discussed in more
detail below.
[0379] The amino acid constituents of a FGFR3 binding site as
defined herein are positioned in three dimensions. The structural
coordinates of FGFR3 with a bound inhibitor are in Table 6. In one
aspect, the structure coordinates defining a binding site of FGFR3
include structure coordinates of all atoms in the constituent amino
acids; in another aspect, the structure coordinates of a binding
site include structure coordinates of just the backbone atoms of
the constituent atoms. FGFR3 that is bound to an inhibitor has a
different conformation than when inhibitor is not bound. In the
bound state, a number of amino acid residues form a pocket.
[0380] The FGFR3 binding site may be defined by those amino acids
whose backbone atoms are situated within about 5 .ANG. of one or
more constituent atoms of a bound ligand.
[0381] Rational Drug Design
[0382] Computational techniques can be used to screen, identify,
select, design ligands, and combinations thereof, capable of
associating with FGFR3 or structurally homologous molecules.
Candidate modulators of FGFR3 may be identified using functional
assays, such as binding to FGFR3, and novel modulators designed
based on the structure of the candidate molecules so identified.
Knowledge of the structure coordinates for FGFR3 permits, for
example, the design, the identification of synthetic compounds, and
like processes, and the design, the identification of other
molecules and like processes, that have a shape complementary to
the conformation of the FGFR3 binding sites. In particular,
computational techniques can be used to identify or design ligands,
such as agonists and/or antagonists, that associate with a FGFR3
binding site. Antagonists may bind to or interfere with all or a
portion of an active site of FGFR3, and can be competitive,
non-competitive, or uncompetitive inhibitors. Once identified and
screened for biological activity, these agonists, antagonists, and
combinations thereof, may be used therapeutically and/or
prophylactically, for example, to block FGFR3 activity and thus
prevent the onset and/or further progression of diseases associated
with FGFR3 activity. Structure-activity data for analogues of
ligands that bind to or interfere with FGFR3 binding sites can also
be obtained computationally.
[0383] In some embodiments, agonists or antagonists can be designed
to include components that preserve and/or strengthen the
interactions. Such antagonists or agonists would include components
that are able to interact, for example, hydrogen bond with the
charged amino acids found in, e.g., either an antagonist binding
site of FGFR3 (activated or unactivated, bound to substrate or
unbound to substrate) or FGFR3 bound to an inhibitor or both.
[0384] In some embodiments, for FGFR3, antagonist or agonist
molecules are designed or selected that can interact with at least
one or all amino acid residues that comprise, consist essentially
of, or consist of at least one amino acid residue corresponding to
an amino acid residue in one or more of the binding site, or
mixtures thereof.
[0385] Comparison of the binding site on FGFR3 to analogous sites
of related receptors will direct design of inhibitors that favor
FGFR3 over the related receptors. The crystal structures of other
related receptors, if they are available can be utilized to
maximize fit and/or interaction with FGFR3 binding site and
minimize the fit and/or interactions with amino acids in the
corresponding positions in other receptors.
[0386] Data stored in a machine-readable storage medium that is
capable of displaying a graphical three-dimensional representation
of the structure of FGFR3 or a structurally homologous molecule or
molecular complex, as identified herein, or portions thereof may
thus be advantageously used for drug discovery. The structure
coordinates of the ligand are used to generate a three-dimensional
image that can be computationally fit to the three-dimensional
image of FGFR3 and anti-FGFR3 antibody, or a structurally
homologous molecule. The three-dimensional molecular structure
encoded by the data in the data storage medium can then be
computationally evaluated for its ability to associate with
ligands. When the molecular structures encoded by the data is
displayed in a graphical three-dimensional representation on a
computer screen, the protein structure can also be visually
inspected for potential association with ligands.
[0387] One embodiment of the method of drug design involves
evaluating the potential association of a candidate ligand with
FGFR3 or a structurally homologous molecule or homologous complex,
particularly with at least one amino acid residue in a binding site
(e.g., a binding site) of the FGFR3 or a portion of the binding
site. The method of drug design thus includes computationally
evaluating the potential of a selected ligand to associate with any
of the molecules or molecular complexes set forth above. This
method includes the steps of: (a) employing computational means,
for example, such as a programmable computer including the
appropriate software known in the art or as disclosed herein, to
perform a fitting operation between the selected ligand and a
ligand binding site or a subsite of the ligand binding site of the
molecule or molecular complex; and (b) analyzing the results of the
fitting operation to quantify the association between the ligand
and the ligand binding site. Optionally, the method further
comprises analyzing the ability of the selected ligand to interact
with amino acids in the FGFR3 binding site and/or subsite. The
method may also further comprise optimizing the fit of the ligand
for the binding site of FGFR3 as compared to other receptors.
Optionally, the selected ligand can be synthesized, cocrystallized
with FGFR3, and further modifications to selected ligand can be
made to enhance inhibitory activity or fit in the binding pocket.
In addition as described previously, portions of anti-FGFR3
antibody that bind to FGFR3 can be modified and utilized in the
method described herein. Other structural features of the FGFR3
and/or FGFR3:anti-FGFR3 antibody complex can also be analyzed in
the same manner.
[0388] In another embodiment, the method of drug design involves
computer-assisted design of ligand that associates with FGFR3, its
homologs, or portions thereof. Ligands can be designed in a
step-wise fashion, one fragment at a time, or may be designed as a
whole or de novo. Ligands can be designed based on the structure of
molecules that can modulate at least one biological function of
FGFR3, such as anti-FGFR3 antibody and other naturally occurring
inhibitors of FGFR3. In addition, the inhibitors can be modeled on
other known inhibitors of receptors, such as FGFRs.
[0389] In some embodiments, to be a viable drug candidate, the
ligand identified or designed according to the method must be
capable of structurally associating with at least part of a FGFR3
binding site (e.g., a FGFR3 binding site), and must be able,
sterically and energetically, to assume a conformation that allows
it to associate with the FGFR3 binding site. Non-covalent molecular
interactions important in this association include hydrogen
bonding, van der Waals interactions, hydrophobic interactions,
and/or electrostatic interactions. In some embodiments, an agent
may contact at least one amino acid position in the FGFR3 binding
site (e.g., a binding site) for an inhibitor, such as anti-FGFR3
antibody. Conformational considerations include the overall
three-dimensional structure and orientation of the ligand in
relation to the ligand binding site, and the spacing between
various functional groups of a ligand that directly interact with
the FGFR3 binding site or homologs thereof.
[0390] Optionally, the potential binding of a ligand to a FGFR3
binding site is analyzed using computer modeling techniques prior
to the actual synthesis and testing of the ligand. If these
computational experiments suggest insufficient interaction and
association between it and the FGFR3 binding site, testing of the
ligand is obviated. However, if computer modeling indicates a
strong interaction, the molecule may then be synthesized and tested
for its ability to bind to or interfere with a FGFR3 binding site.
Binding assays to determine if a compound actually modulates FGFR3
activity can also be performed and are well known in the art.
[0391] Several methods can be used to screen ligands or fragments
for the ability to associate with a FGFR3 binding site (e.g., an
antagonist binding site). This process may begin by visual
inspection of, for example, a FGFR3 binding site on the computer
screen based on the FGFR3 structure coordinates or other
coordinates which define a similar shape generated from the
machine-readable storage medium. Selected ligands may then be
positioned in a variety of orientations, or docked, within the
binding site. Docking may be accomplished using software such as
QUANTA and SYBYL, followed by energy minimization and molecular
dynamics with standard molecular mechanics force fields, such as
CHARMM and AMBER.
[0392] Specialized computer programs may also assist in the process
of selecting ligands. Examples include GRID (Hubbard, S. 1999.
Nature Struct. Biol. 6:711-4); MCSS (Miranker, et al. 1991.
Proteins 11:29-34) available from Molecular Simulations, San Diego,
Calif.; AUTODOCK (Goodsell, et al. 1990. Proteins 8:195-202)
available from Scripps Research Institute, La Jolla, Calif.; and
DOCK (Kuntz, et al. 1982. J. Mol. Biol. 161:269-88) available from
University of California, San Francisco, Calif.
[0393] FGFR3 binding ligands can be designed to fit a FGFR3 binding
site, optionally as defined by the binding of a known modulator or
one identified as modulating the activity of FGFR3. There are many
ligand design methods including, without limitation, LUDI (Bohm,
1992. J. Comput. Aided Molec. Design 6:61-78) available from
Molecular Simulations IIIc., San Diego, Calif.; LEGEND (Nishibata,
Y., and Itai, A. 1993. J. Med. Chem. 36:2921-8) available from
Molecular Simulations Inc., San Diego, Calif.; LeapFrog, available
from Tripos Associates, St. Louis, Mo.; and SPROUT (Gillet, et al.
1993. J. Comput. Aided Mol. Design 7:127-53) available from the
University of Leeds, UK.
[0394] Once a compound has been designed or selected by the above
methods, the efficiency with which that ligand may bind to or
interfere with a FGFR3 binding site may be tested and optimized by
computational evaluation. FGFR3 binding site ligands may interact
with the binding site in more than one conformation that is similar
in overall binding energy. In those cases, the deformation energy
of binding is taken to be the difference between the free energy of
the ligand and the average energy of the conformations observed
when the ligand binds to the protein.
[0395] A ligand designed or selected as binding to or interfering
with a FGFR3 binding site may be further computationally optimized
so that in its bound state it would preferably lack repulsive
electrostatic interaction with the target enzyme and with the
surrounding water molecules. Such non-complementary electrostatic
interactions include repulsive charge-charge, dipole-dipole, and
charge-dipole interactions.
[0396] Specific computer software is available to evaluate compound
deformation energy and electrostatic interactions. Examples of
programs designed for such uses include: Gaussian 94, revision C
(M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa.); AMBER, version 4.1
(P. A. Kollman, University of California at San Francisco,);
QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif.);
Insight II/Discover (Molecular Simulations, Inc., San Diego,
Calif.); DelPhi (Molecular Simulations, Inc., San Diego, Calif.);
and AMSOL (Quantum Chemistry Program Exchange, Indiana University).
These programs can be implemented, for instance, using a Silicon
Graphics workstation, such as an Indigo2 with IMPACT graphics.
Other hardware systems and software packages will be known to those
skilled in the art.
[0397] Another approach encompassed by this disclosure is the
computational screening of small molecule databases for ligands or
compounds that can bind in whole, or in part, to a FGFR3 binding
site whether in bound or unbound conformation. In this screening,
the quality of fit of such ligands to the binding site may be
judged either by shape complementarity or by estimated interaction
energy (Meng, et al., 1992. J. Comp. Chem., 13:505-24). In
addition, these small molecule databases can be screened for the
ability to interact with the amino acids in the FGFR3 binding site
as identified herein.
[0398] A compound that is identified or designed as a result of any
of these methods can be obtained (or synthesized) and tested for
its biological activity, for example, binding and/or inhibition of
FGFR3 activity.
[0399] Another method involves assessing agents that are
antagonists or agonists of the FGFR3 receptor. A method comprises
applying at least a portion of the crystallography coordinates of
Table 6 to a computer algorithm that generates a three-dimensional
model of a FGFR3:anti-FGFR3 antibody complex or the FGFR3 suitable
for designing molecules that are antagonists or agonists and
searching a molecular structure database to identify potential
antagonists or agonists. In some embodiments, a portion of the
structural coordinates of Table 6 that define a structural feature,
for example, all or a portion of a binding site (e.g., an
antagonist binding site) for an inhibitor on FGFR3. The method may
further comprise synthesizing or obtaining the agonist or
antagonist and contacting the agonist or antagonist with the FGFR3
and selecting the antagonist or agonist that modulates the FGFR3
activity compared to a control without the agonist or antagonists
and/or selecting the antagonist or agonist that binds to the
FGFR3.
[0400] A compound that is identified or designed as a result of any
of these methods can be obtained (or synthesized) and tested for
its biological activity, for example, binding to FGFR3 and/or
modulation of FGFR3 activity.
[0401] 7. Machine-Readable Storage Media
[0402] Transformation of the structure coordinates for all or a
portion of FGFR3, anti-FGFR3 antibody or the FGFR3:anti-FGFR3
antibody complex, or one of its ligand binding sites, or
structurally homologous molecules as defined below, or for the
structural equivalents of any of these molecules or molecular
complexes as defined above, into three-dimensional graphical
representations of the molecule or complex can be conveniently
achieved through the use of commercially-available software.
[0403] The disclosure thus further provides a machine-readable
storage medium including a data storage material encoded with
machine-readable data wherein a machine programmed with
instructions for using said data displays a graphical
three-dimensional representation of any of the molecule or
molecular complexes of this disclosure that have been described
above. In a preferred embodiment, the machine-readable data storage
medium includes a data storage material encoded with
machine-readable data wherein a machine programmed with
instructions for using the abovementioned data displays a graphical
three-dimensional representation of a molecule or molecular complex
including all or any parts of an unbound FGFR3, a FGFR3 ligand
binding site for an inhibitor or pseudo substrate, or FGFR3-like
ligand binding site, anti-FGFR3 antibody, FGFR3:anti-FGFR3 antibody
complex as defined above. In another preferred embodiment, the
machine-readable data storage medium includes a data storage
material encoded with machine readable data wherein a machine
programmed with instructions for using the data displays a
graphical three-dimensional representation of a molecule or
molecular complex.+-.a root mean square deviation from the atoms of
the amino acids of not more than 0.05 .ANG..
[0404] In an alternative embodiment, the machine-readable data
storage medium includes a data storage material encoded with a
first set of machine readable data which includes the Fourier
transform of structure coordinates, and wherein a machine
programmed with instructions for using the data is combined with a
second set of machine readable data including the X-ray diffraction
pattern of a molecule or molecular complex to determine at least a
portion of the structure coordinates corresponding to the second
set of machine readable data.
[0405] For example, a system for reading a data storage medium may
include a computer including a central processing unit ("CPU"), a
working memory which may be, for example, RAM (random access
memory) or "core" memory, mass storage memory (such as one or more
disk drives or CD-ROM drives), one or more display devices (e.g.,
cathode-ray tube ("CRT") displays, light emitting diode ("LED")
displays, liquid crystal displays ("LCDs"), electroluminescent
displays, vacuum fluorescent displays, field emission displays
("FEDs"), plasma displays, projection panels, etc.), one or more
user input devices (e.g., keyboards, microphones, mice, track
balls, touch pads, etc.), one or more input lines, and one or more
output lines, all of which are interconnected by a conventional
bidirectional system bus. The system may be a stand-alone computer,
or may be networked (e.g., through local area networks, wide area
networks, intranets, extranets, or the internet) to other systems
(e.g., computers, hosts, servers, etc.). The system may also
include additional computer controlled devices such as consumer
electronics and appliances.
[0406] Input hardware may be coupled to the computer by input lines
and may be implemented in a variety of ways. Machine-readable data
of this disclosure may be inputted via the use of a modem or modems
connected by a telephone line or dedicated data line. Alternatively
or additionally, the input hardware may include CD-ROM drives or
disk drives. In conjunction with a display terminal, a keyboard may
also be used as an input device.
[0407] Output hardware may be coupled to the computer by output
lines and may similarly be implemented by conventional devices. By
way of example, the output hardware may include a display device
for displaying a graphical representation of a binding site of this
disclosure using a program such as QUANTA as described herein.
Output hardware might also include a printer, so that hard copy
output may be produced, or a disk drive, to store system output for
later use.
[0408] In operation, a CPU coordinates the use of the various input
and output devices, coordinates data accesses from mass storage
devices, accesses to and from working memory, and determines the
sequence of data processing steps. A number of programs may be used
to process the machine-readable data of this disclosure. Such
programs are discussed in reference to the computational methods of
drug discovery as described herein. References to components of the
hardware system are included as appropriate throughout the
following description of the data storage medium.
[0409] Machine-readable storage devices useful in the present
disclosure include, but are not limited to, magnetic devices,
electrical devices, optical devices, and combinations thereof.
Examples of such data storage devices include, but are not limited
to, hard disk devices, CD devices, digital video disk devices,
floppy disk devices, removable hard disk devices, magneto-optic
disk devices, magnetic tape devices, flash memory devices, bubble
memory devices, holographic storage devices, and any other mass
storage peripheral device. It should be understood that these
storage devices include necessary hardware (e.g., drives,
controllers, power supplies, etc.) as well as any necessary media
(e.g., disks, flash cards, etc.) to enable the storage of data.
[0410] 8. Therapeutic Use
[0411] FGFR3 modulator compounds obtained by methods of the
invention are useful in a variety of therapeutic settings. For
example, FGFR3 antagonists designed or identified using the crystal
structure of FGFR3 complex can be used to treat disorders or
conditions where inhibition or prevention of FGFR3 binding or
activity is indicated.
[0412] Likewise, FGFR3 agonists designed or identified using the
binding site and/or crystal structures provided herein can be used
to treat disorders or conditions where induction or stimulation of
FGFR3 is indicated.
[0413] An indication can be, for example, inhibition or stimulation
of FGFR3 activation and the concomitant activation of a complex set
of intracellular pathways that lead to cell growth in a variety of
cell types. Yet another indication can be, for example, in
inhibition or stimulation of the FGFR3 signaling pathway. Still yet
another indication can be, for example, in inhibition or
stimulation of invasive tumor growth and metastasis.
[0414] The following are examples of the methods and compositions
of the invention. It is understood that various other embodiments
may be practiced, given the general description provided above.
EXAMPLES
Materials and Methods
Cell Lines and Cell Culture
[0415] The cell line RT4 was obtained from American Type Cell
Culture Collection. Cell lines RT112, OPM2 and Ba/F3 were purchased
from German Collection of Microorganisms and Cell Cultures (DSMZ,
(Germany)). Multiple myeloma cell line KMS11 was kindly provided by
Dr. Takemi Otsuki at Kawasaki Medical School (Japan). Bladder
cancer cell line TCC-97-7 was a generous gift from Dr. Margaret
Knowles at St James's University Hospital (Leeds, UK). UMUC-14 cell
line was obtained from Dr. H. B. Grossman (currently at University
of Texas M.D. Anderson Cancer Center, TX). The cells were
maintained with RPMI medium supplemented with 10% fetal bovine
serum (FBS) (Sigma), 100 U/ml penicillin, 0.1 mg/ml streptomycin
and L-glutamine under conditions of 5% CO.sub.2 at 37.degree.
C.
FGFR3.sup.S249C Dimerization Studies
[0416] UMUC-14 cells were grown in cysteine-free medium, treated
with R3Mab or DTNB for 3 hr, and cell lysates were subject to
immunoblot analysis under reducing or non-reducing conditions. For
in vitro dimerization studies, FGFR3-IIIb.sup.S249C (residues
143-374) was cloned into pAcGP67A vector and expressed in T.ni Pro
cells. The recombinant protein was purified through Ni-NTA column
followed by Superdex 5200 column. Dimeric FGFR3.sup.S249C was
eluted in 25 mM Tris (pH 7.5) and 300 mM NaCl. R3Mab (1 .mu.M) was
incubated with FGFR3.sup.S249C dimer (0.1 .mu.M) at 37.degree. C.
under the following conditions: 100 mM KH.sub.2PO4 (pH 7.5), 25
.mu.M DTT, 1 mM EDTA and 0.75 mg/ml BSA. Aliquots of the reaction
were taken at indicated time points and the reaction was stopped by
adding sample buffer without .beta.-mercaptoethanol. Dimer-monomer
was analyzed by immunoblot.
Xenograft Studies
[0417] All studies were approved by Genentech's Institutional
Animal Care and Use Committee. Female nu/nu mice or CB17 severe
combined immunodeficiency (SCID) mice, 6-8 weeks of age, were
purchased from Charles River Laboratory (Hollister, Calif.). Female
athymic nude mice were obtained from the National Cancer
Institute-Frederick Cancer Center. Mice were maintained under
specific pathogen-free conditions. RT112 shRNA stable cells
(7.times.10.sup.6), RT112 (7.times.10.sup.6), Ba/F3-FGFR3.sup.S249C
(5.times.10.sup.6), OPM2 (15.times.10.sup.6), or KMS11 cells
(20.times.10.sup.6) were implanted subcutaneously into the flank of
mice in a volume of 0.2 ml in HBSS/matrigel (1:1 v/v, BD
Biosciences). UMUC-14 cells (5.times.10.sup.6) were implanted
without matrigel. Tumors were measured twice weekly using a
caliper, and tumor volume was calculated using the formula: V=0.5
a.times.b.sup.2, where a and b are the length and width of the
tumor, respectively. When the mean tumor volume reached 150-200
mm.sup.3, mice were randomized into groups of 10 and were treated
twice weekly with intraperitoneal (i.p) injection of R3Mab (0.3-50
mg/kg), or a control human IgG1 diluted in HBSS. Control animals
were given vehicle (HBSS) alone.
Statistics
[0418] Pooled data are expressed as mean+/-SEM. Unpaired Student's
t tests (2-tailed) were used for comparison between two groups. A
value of P<0.05 was considered statistically significant in all
experiments.
Generation of FGFR3 shRNA Stable Cells
[0419] Three independent FGFR3 shRNA were cloned into pHUSH vector
as described (50). The sequence for FGFR3 shRNAs used in the
studies is as follows:
TABLE-US-00011 shRNA2: (SEQ ID NO: 192)
5'GATCCCCGCATCAAGCTGCGGCATCATTCAAGAGATGATGCCGCAGCT
TGATGCTTTTTTGGAAA; shRNA4: (SEQ ID NO: 193)
5'-GATCCCCTGCACAACCTCGACTACTATTCAAGAGATAGTAGTCGAGG
TTGTGCATTTTTTGGAAA-3'; shRNA6: (SEQ ID NO: 194)
5'-GATCCCCAACCTCGACTACTACAAGATTCAAGAGATCTTGTAGTAGT
CGAGGTTTTTTTTGGAAA-3'.
All constructs were confirmed by sequencing. EGFP control shRNA was
described in our previous study (50). The shRNA containing
retrovirus was produced by co-transfecting GP2-293 packaging cells
(Clontech Laboratories, Mountain View, Calif.) with VSV-G (Clontech
Laboratories) and pHUSH-FGFR3 shRNA constructs, and viral
supernatants were harvested 72 hr after transfection, and cleared
of cell debris by centrifugation for transduction experiment.
[0420] RT112 cells were maintained in RPMI 1640 medium containing
tetracycline-free FBS (Clontech Laboratories), and transduced with
retroviral supernatant in the presence of 4 .mu.g/ml polybrene. 72
hours after infection, 2 .mu.g/ml puromycin (Clontech Laboratories)
was added to the medium to select stable clones expressing shRNA.
Stable cells were isolated, treated with 0.1 or 1 .mu.g/ml
doxycycline (Clontech Laboratories) for 4 days, and inducible
knockdown of FGFR3 protein expression was assessed by Western
blotting analysis. Cell cycle analyses were performed as described
(51).
Selecting Phage Antibodies Specific for FGFR3
[0421] Human phage antibody libraries with synthetic diversities in
the selected complementary determining regions (H1, H2, H3, L3),
mimicking the natural diversity of human IgG repertoire were used
for panning. The Fab fragments were displayed bivalently on the
surface of M13 bacteriophage particles (52). His-tagged IgD2-D3 of
human FGFR3-IIIb and Mc were used as antigens. 96-well MaxiSorp
immunoplates (Nunc) were coated overnight at 4.degree. C. with
FGFR3-IIIb-His protein or FGFR3-IIIC-His protein (10 .mu.g/ml) and
blocked for 1 hour with PBST buffer (PBS with 0.05% Tween 20)
supplemented with 1% BSA. The antibody phage libraries were added
and incubated overnight at room temperature (RT). The plates were
washed with PBST buffer and bound phage were eluted with 50 mM HCl
and 500 mM NaCl for 30 minutes and neutralized with equal volume of
1M Tris base. Recovered phages were amplified in E. coli XL-1 blue
cells. During subsequent selection rounds, the incubation time of
the phage antibodies was decreased to 2 hours and the stringency of
plate washing was gradually increased (53). Unique and specific
phage antibodies that bind to both Mb and IIIc isoforms of FGFR3
were identified by phage ELISA and DNA sequencing. Out of 400
clones screened, four were selected to reformat to full length IgGs
by cloning VL and VH regions of individual clones into LPG3 and
LPG4 vectors, respectively, transiently expressed in mammalian
cells, and purified with protein A columns (54). Clone 184.6 was
selected for affinity maturation.
[0422] For affinity maturation, phagemid displaying monovalent Fab
on the surface of M13 bacteriophage (52) served as the library
template for grafting light chain (VL) and heavy chain (VH)
variable domains of the phage Ab. Stop codons was incorporated in
CDR-L3. A soft randomization strategy was adopted for affinity
maturation as described (53). Two different combinations of CDR
loops, H1/H2/L3, H3/L3, or L1/L2/L3 were selected for
randomization. For selecting affinity-matured clones, phage
libraries were sorted against FGFR3-IIIb or IIIc-His protein,
subjected to plate sorting for the first round and followed by four
rounds of solution phase sorting as described (52). After five
rounds of panning, a high-throughput single-point competitive phage
ELISA was used to rapidly screen for high-affinity clones as
described (55). Clones with low ratio of the absorbance at 450 nm
in the presence of 10 nM FGFR3-His to that in the absence of
FGFR3-His were chosen for further characterization.
[0423] Clones 184.6.1, 184.6.21, 184.6.49, 184.6.51, 184.6.58,
184.6.62 and 184.6.92 significantly reduced viability of
Ba/F3-FGFR3-IIIb, Ba/F3-FGFR3-IIIc and Ba/F3-FGFR3-S249C cell
lines, and clone 184.6.52 significantly reduced the viability of
the Ba/F3-FGFR3-S249C cell line. The increased inhibitory activity
ranged from about 50-fold (clone 184.6.52) to about 100-fold
(clones 184.6.1, 184.6.21, 184.6.49, 184.6.51, 184.6.58, 184.6.62
and 184.6.92) greater than parent clone 184.6, depending on the
cell line assayed. Binding kinetics of clones 184.6.1, 184.6.58,
and 184.6.62 to FGFR3-IIIb and FGFR3-IIIc were determined using
BIAcore as follows:
TABLE-US-00012 FGFR3-IIIb KD (M) FGFR3-IIIc KD (M) 184.6 3.80E-08
1.10E-07 184.6.1 2.64E-10 1.44E-09 184.6.58 1.90E-10 8.80E-10
184.6.62 1.20E-10 2.24E-09
Clones 184.6.1, 184.6.58, and 184.6.62 also showed improved
inhibition of FGFR3 downstream signaling in Ba/F3-FGFR3 cells,
RT112 cells and OPM2 cells.
[0424] Clone 184.6.1 was selected. A sequence modification, N54S,
was introduced into HVR H2 at residue 54, to improve
manufacturability, creating clone 184.6.1N54S. Clones 184.6.1 and
184.6.1N54S displayed comparable binding kinetics (measured in
Biacore assays) and comparable activity in the Ba/F3 cell viability
assay. Additional HVR H2 variants were generated: N54S was
introduced in clone 184.6.58, and N54G, N54A, or N54Q were
introduced in clone 184.6.1 and 184.6.58. These clones showed
comparable activity in the Ba/F3 cell viability assay to parent
clones 184.6.1 or 184.6.58.
[0425] Another sequence modification, D30E, was introduced into HVR
L1 of clone 184.6.1N54S, creating clone 184.6.1NSD30E. Clone
184.6.1NSD30E and clone 184.6.1N54S showed comparable binding
kinetics and comparable activity in the BA/F3 cell viability assay
to parent clones 184.6.1 or 184.6.58.
[0426] As used herein, "R3Mab" refers to anti-FGFR3 antibody clones
184.6.1N54S, 184.6.1, or 184.6. Clone 184.6.1N54S was used in
figures and experiments referencing "R3Mab", except in the
experiments leading to the results shown in the following figures
(for which the antibody used is shown in parentheses): FIGS. 9B
(clone 184.6.1), 10 (clone 184.6), 11A and B (clone 184.6), 13
(clone 184.6.1), 14A (clone 184.6.1), 14B, G, and H (clone 184.6),
19 (clone 184.6.1), and 22B and C (clone 184.6.1).
BIAcore/Surface Plasmon Resonance (SRP) Analysis to Determine
Antibody Binding Affinities
[0427] Binding affinities of R3Mab to FGFR3 were measured by
Biacore/SRP using a BIAcore.TM.-3000 instrument as described (52)
with the following modifications. R3Mab was directly coated on CM5
biosensor chips to achieve approximately 400 response units (RU).
For kinetic measurement, two-fold serial dilutions of FGFR3-IIIb or
IIIc-His protein (starting from 67 nM) were injected in PBST buffer
at 25.degree. C. with a flow rate of 30 .mu.l/minute. Association
rates (Kon, per mol/s) and dissociation rates (Koff, per s) were
calculated using a simple one-one Langmuir binding model (BIAcore
Evaluation Software version 3.2). The equilibrium dissociation
constant (Kd, per mol) was calculated as the ratio of Koff/Kon.
[0428] Binding affinities of mouse hybridoma antibodies to FGFR3
were measured by Biacore/SRP as follows. Human FGFR3-IIIb or Mc was
coupled onto three different flow cells (FC), FC2, FC3 and FC4, of
a BIACORE.TM. CM5 sensor chip to achieve the response unit (RU)
about 50 RU. Immobilization was achieved by random coupling through
amino groups using a protocol provided by the manufacturer.
Sensorgrams were recorded for binding of hybridoma-derived
anti-FGFR3 murine IgG or the Fab fragment to these surfaces at
25.degree. C. by injection of a series of solutions ranging from
250 nM to 0.48 nM in 2-fold increments at a flow rate of 300 min.
Between each injection, 10 mM Glycine-HCl pH 1.7 was served as the
buffer to regenerate the sensor chip. The signal from the reference
cell (FC1) was subtracted from the observed sensorgram at FC2, FC3
and FC4. Kinetic constants were calculated by nonlinear regression
fitting of the data according to a 1:1 Langmuir binding model using
BIAcore evaluation software (version 3.2) supplied by the
manufacturer.
ELISA Binding Studies
[0429] cDNAs encoding the extracellular domains (ECD) of human
FGFR1-IIIb, Inc, FGFR2-IIIb and Mc, FGFR3-IIIb and Mc, and FGFR4
were cloned into pRK-based vector to generate human FGFR-human Fc
chimeric proteins. The recombinant proteins were produced by
transiently transfecting Chinese hamster ovary (CHO) cells and
purified via protein A affinity chromatography. To test binding of
antibodies to human FGFRs, Maxisorp 96-well plates (Nunc) were
coated overnight at 4.degree. C. with 50 .mu.l of 2 .mu.g/ml of
FGFR ECD-human Fc chimeric proteins. After blocking with
phosphate-buffered saline (PBS)/3% BSA, FGFR3 antibody was added
and incubated at RT for 2 hours. Specifically bound FGFR3 antibody
was detected using an HRP-conjugated anti-human Fab and the TMB
peroxidase colorigenic substrate (KPL, Gaithersburg, Md.).
[0430] To test the effect of antibodies to FGFR3 on FGF/FGFR3
interaction, FGFR3-Fc chimeric proteins were captured on Maxisorp
plate coated with anti-human immunoglobulin Fc.gamma.
fragment-specific antibody (Jackson Immunoresearch, West Grove,
Pa.). After wash, increasing amount of FGFR3 antibody was added to
the plate and incubated for 30 minutes. Then, FGF1 or FGF9 and
heparin were added for incubation at RT for 2 hours. The plates
were washed and incubated for 1 hour with biotinylated
FGF1-specific polyclonal antibody (BAF232) or biotinylated FGF9
antibody (BAF273, R&D Systems), followed by detection with
streptavidin-HRP and TMB.
Generation of Ba/F3-FGFR3 Stable Cells cDNA encoding full-length
human FGFR3-IIIb or IIIc was cloned into pQCXIP vector (Clontech
Laboratories, Mountain View, Calif.) to generate pQCXIP-FGFR3-IIIb
or Mc. Specific mutations, i.e., R248C, S249C, G372C, Y375C and
K652E, were introduced into the cDNA via QuickChange (Stratagene,
La Jolla, Calif.). To generate Ba/F3 stable cells expressing wild
type or mutant FGFR3, various pQCXIP-FGFR3 constructs were
co-transfected into packaging cells GP2-293 with VSV-G plasmid
(Clontech Laboratories). After selection with 2 .mu.g/ml puromycin
for two weeks, cells expressing wild type or mutant FGFR3 were
stained with Phycoerythrin-conjugated anti-human FGFR3 mAb
(FAB766P, R&D Systems), and selected through
fluorescence-activated cell sorting (FACS) for functional assays.
For cell proliferation assay in 96-well micro-titer plate, the
following cell density was used: For cells expressing wild type
FGFR3-IIIb and FGFR3-K652E: 5,000 cells/well; for the rest: 10,000
cells/well. Cells were seeded in RPMI 1640 medium supplemented with
10% fetal bovine serum, 10 ng/ml FGF1 plus 10 .mu.g/ml heparin
(Sigma-Aldrich, St. Louis, Mo.). R3Mab was added at indicated
concentration and mouse hybridoma FGFR3 antibodies were added at
2000 to 0.49 ng/ml (in four-fold serial dilutions) in the
FGFR3-IIIb experiment and 5000 to 1.2 ng/ml (in four-fold serial
dilutions) in the FGFR3-IIIc experiment. After incubation for 72
hours, cell viability was assessed with CellTiter-Glo (Promega,
Madison, Wis.).
Cell Proliferation Assay
[0431] For proliferation assays for RT112, RT4 and TCC-97-7 cells,
3000 cells/well were seeded into 96-well micro-titer plate and were
allowed to adhere overnight. The medium was then replaced with low
serum medium (0.5% FBS) with control or R3Mab at concentrations
indicated. Following 4 days incubation, 1 .mu.Ci of
[Methyl-.sup.3H] thymidine (PerkinElmer, Waltham, Mass.) was added
to each well, and incubated for additional 16 hours. Cells were
transferred to UniFilters using Packard Filtermate Harvester, and
[.sup.3H]-thymidine incorporated into the genomic DNA of growing
cells was measured using TopCount (PerkinElmer). In some cases,
cell viability was assessed with CellTiter-Glo (Promega) following
incubation with antibodies for 4 days. Values are presented as
means+/-SE of quadruplets.
Clonal Growth Assay
[0432] The effect of R3Mab on cell clonogenicity was assessed
following a previously described protocol (50). In brief, 400
UMUC-14 cells were seeded into 6-well plate in DMEM medium
supplemented with 10% fetal bovine serum to allow adhesion
overnight. Then R3Mab or control antibody diluted in 0.1% BSA
medium was added to a final concentration of 10 .mu.g/ml. Equal
volume of 0.1% BSA medium alone (Mock) was used as another control.
The cells were incubated for about 12 days until cells in control
groups formed sufficiently large colonies. Colonies were stained
with 0.5% crystal violet, and the number and size of colonies were
quantitated using GelCount (Oxford, UK). The number of colonies
larger than 120 .mu.m in diameter was presented as mean+/-SEM
(n=12).
Immunoprecipitation and Immunoblotting Analyses
[0433] To study the effect of antibodies on FGFR3 signaling, cells
were starved in serum-free medium overnight prior to the beginning
of treatment. Cells were incubated with either antibodies diluted
in 0.1% BSA (w/v), RPMI 1640 medium, or with 0.1% BSA medium alone
(Mock). After 3 hours at 37.degree. C., FGF1 (final concentration
of 15 ng/ml) and heparin (final concentration of 5-10 .mu.g/ml)
were added to half of the samples. As controls, a similar volume of
heparin alone was added to the other half of samples. The
incubation was continued for 10 min. Supernatants were removed by
aspiration, and cells were washed with ice-cold PBS, then lysed in
RIPA buffer (Upstate, Charlottesville, Va.) supplemented with 1 mM
sodium orthovanadate and Complete protease inhibitor cocktail
(Roche Applied Science, Indianapolis, Ind.). The lysates were
cleared of insoluble materials by centrifugation.
[0434] FGFR3 was immunoprecipitated using a rabbit polyclonal
antibody (sc-123, Santa Cruz Biotechnology, Santa Cruz, Calif.) and
analyzed by sodium dodecyl-polyacrylamide gel electrophoresis
(SDS-PAGE) and Western blot. Phosphorylated FGFR3 was assessed with
a monoclonal antibody against phospho-tyrosine (4G10, Upstate).
Total FGFR3 was probed with a monoclonal antibody against FGFR3
(sc-13121, Santa Cruz Biotechnology). Phosphorylation and
activation of FGFR3 signaling pathway were probed using the
following antibodies: anti-FGFR.sup.Y653/654,
anti-FRS2.alpha..sup.Y196, anti-phospho-p44/42 MAPK.sup.T202/Y204,
anti-total p44/42 MAPK and anti-AKT.sup.S473 were obtained from
Cell Signaling Technology (Danvers, Mass.); and anti-total
FRS2.alpha. (sc-8318) was purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif.). The blots were visualized using a
chemiluminescent substrate (ECL Plus, Amersham Pharmacia Biotech,
Piscataway, N.J.).
Antibody Epitope Mapping
[0435] To determine the epitope of R3Mab, 13 overlapping peptides,
each of 15 amino acids in length, were synthesized to cover the
extracellular domain of human FGFR3 from residues 138 to 310. The
peptides were biotinylated at the C-terminus, and captured on
streptavidin plates (Pierce, Rockford, Ill.) overnight. After
blocking with PBS/3% BSA, the plates were incubated with R3Mab and
detected using an HRP-conjugated anti-human IgG (Jackson
Immunoresearch) and the TMB peroxidase colorigenic substrate (KPL,
Gaithersburg, Md.).
[0436] Mouse anti-human FGFR3 hybridoma antibodies 1G6, 6G1, and
15B2 were tested in ELISA assay to identify their binding epitopes.
1G6, 6G1 and 15B2 bind to human FGFR IgD2-IgD3 (both IIIb and IIIc
isoforms), whereas 5B8 only binds IgD2-IgD3 of human FGFR3-IIIb. In
a competition assay, 1G6, 6G1 and 15B2 competed with each other to
bind human FGFR3, suggesting that 1G6, 6G1 and 15B2 have
overlapping epitopes. None of the hybridoma antibodies competed
with phage antibody 184.6, suggesting that the hybridoma antibodies
have distinct epitope(s) from 184.6.
Preparation and Molecular Cloning of Mouse Anti-FGFR3 Antibodies
1G6, 6G1, and 15B2
[0437] BALB/c mice were immunized 12 times with 2.0 .mu.g of
FGFR3-IIIb (rhFGFR3 (IIIB)/Fc Chimera, from R&D Systems,
catalog #1264-FR, lot # CYHO25011, or with 2.0 .mu.g of FGFR3-IIIc
(rhFGFR3 (IIIc)/Fc Chimera, from R&D Systems, catalog #766-FR,
lot # CWZ055041, resuspended in monophosphoryl lipid A/trehalose
dicorynomycolate adjuvant (Corixa, Hamilton, Mont.) into each hind
footpad twice a week. Three days after final boost, popliteal lymph
nodes were fused with mouse myeloma cell line P3X63Ag.U.1, via
electrofusion (Hybrimune, Cyto Pulse Sciences, Glen Burnie, Md.).
Fused hybridoma cells were selected from unfused popliteal node or
myeloma cells using hypoxanthin-aminopterin-thymidine (HAT)
selection in Medium D from the ClonaCell.RTM. hybridoma selection
kit (StemCell Technologies, Inc., Vancouver, BC, Canada). Culture
supernatants were initially screened for its ability to bind to
FGFR3-IIIb and FGFR3-IIIc by ELISA, and hybridomas of interest were
subsequently screened for its ability to stain by FACS on
transfected FGFR3-IIIb Ba/F cells and control Ba/F, as well as
antibody blocking activity. Selected hybridomas were then cloned by
limiting dilution.
[0438] Total RNA was extracted from hybridoma cells producing the
mouse anti human FGFRIII monoclonal antibody 1G6 and 15B2, using
RNeasy Mini Kit (Qiagen, Germany). The variable light (VL) and
variable heavy (VH) domains were amplified using RT-PCR with the
following degenerate primers:
TABLE-US-00013 1G6: Light chain (LC) forward: (SEQ ID NO: 195)
5'-GTCAGATATCGTKCTSACMCARTCTCCWGC-3' Heavy chain (HC) forward: (SEQ
ID NO: 196) 5'-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3' 6G1: Light
chain (LC) forward: (SEQ ID NO: 197)
5'-GTCAGATATCGTGCTGACMCARTCTCC-3' Heavy chain (HC) forward: (SEQ ID
NO: 198) 5'-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3' 15B2: Light
chain (LC) forward: (SEQ ID NO: 199)
5'-GTACGATATCCAGATGACMCARTCTCC-3' Heavy chain (HC) forward: (SEQ ID
NO: 200) 5'-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3'
[0439] Light chain and Heavy chain reverse primer for all three
clones are as followed:
TABLE-US-00014 Light chain reverse: (SEQ ID NO: 201)
5'-TTTDAKYTCCAGCTTGGTACC-3' Heavy chain reverse: (SEQ ID NO: 202)
5'-ACAGTGGGCCCTTGGTGGAGGCTGMRGAGACDGTGASHRDRGT-3'.
[0440] The forward primers were specific for the N-terminal amino
acid sequence of the VL and VH region. The LC and HC reverse
primers were designed to anneal to a region in the constant light
(CL) and constant heavy domain 1 (CH1), respectively, which are
highly conserved across species.
[0441] Amplified VL was cloned into a pRK mammalian cell expression
vector (Shields et al, (2000) J. Biol. Chem. 276:659) containing
the human kappa constant domain. Amplified VH was inserted to a pRK
mammalian cell expression vector encoding the full-length human
IgG1 constant domain. The sequence of the heavy and light chains
was determined using conventional methods.
Crystallization, Structure Determination and Refinement
[0442] The human FGFR3-IIIb ECD (residues 143-374) was cloned into
pAcGP67A vector (BD Bioscience, San Jose, Calif.), produced in T.ni
Pro cells and purified using Ni-NTA column followed by size
exclusion chromatography. The R3Mab Fab was expressed in E. coli
and purified sequentially over a protein G affinity column, an SP
sepharose column and a Superdex 75 column. The R3Mab Fab had the
following sequence:
TABLE-US-00015 Light chain: (SEQ ID NO: 276)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC,
and Heavy chain: (SEQ ID NO: 277)
EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGR IY
PTNGSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARA
RTYGIYDLYVDYTEYVMDYWGQGTLVASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH
[0443] Fab-FGFR3 complex was generated by incubating the Fab with
an excess of FGFR3 ECD, and the complex was then deglycosylated and
purified over a Superdex-200 sizing column in 20 mM TrisCl pH 7.5
and 200 mM NaCl buffer. The complex-containing fractions were
pooled and concentrated to 20 mg/ml and used in crystallization
trials. Crystals used in the structure determination were grown at
4.degree. C. from the following condition: 0.1 M sodium cacodylate
pH 6.5, 40% MPD and 5% PEG8000 using vapor diffusion method. Data
was processed using HKL2000 and Scalepack (56). The structure was
solved with molecular replacement using program Phaser (57) and the
coordinates of 1RY3 (FGFR3) and 1N8Z (Fab-fragment). The model was
completed using program Coot (58) and the structure refined to
R/R.sub.free of 20.4%/24.3% with program Refmac (59). Coordinates
and structure factors were deposited in the Protein Data Bank with
accession code 3GRW and are also disclosed in U.S. Ser. No.
61/163,222, filed on Mar. 25, 2009, the contents of which is hereby
incorporated by reference.
ADCC Assay
[0444] Human PBMCs were isolated by Ficoll gradient centrifugation
of heparinized blood, and ADCC was measured using the multiple
myeloma cell lines OPM2 or KMS11 or bladder cancer cell lines RT112
or UMUC-14 as target and PBMCs as effector cells at a 1:100
target:effector ratio. The target cells (10,000 cells/well) were
treated with R3Mab or with control human IgG1 for 4 hours at
37.degree. C. Cytotoxicity was determined by measuring LDH release
using the CytoTox-ONE Homogeneous Membrane Integrity Assay
following manufacturer's instructions (Promega, Madison, Wis.). The
results are expressed as percentage of specific cytolysis using the
formula: Cytotoxicity (%)=[(Experimental lysis-Experimental
spontaneous lysis)/(Target maximum lysis-target spontaneous
lysis)].times.100, where spontaneous lysis is the nonspecific
cytolysis in the absence of antibody, and target maximum lysis is
induced by 1% Triton X-100.
Results
[0445] Inducible shRNA Knockdown of FGFR3 Attenuates Bladder Cancer
Growth In Vivo
[0446] As a prelude to assessing the importance of FGFR3 for
bladder tumor growth in vivo, we examined the effect of FGFR3
knockdown in vitro. Several FGFR3 small interfering (si) RNAs
effectively downregulated FGFR3 in bladder cancer cell lines
expressing either WT (RT112, RT4, SW780) or mutant (UMUC-14, S249C
mutation) FGFR3. FGFR3 knockdown in all four cell lines markedly
suppressed proliferation in culture (FIG. 15). Next, we generated
stable RT112 cell lines expressing doxycycline-inducible FGFR3
shRNA. Induction of three independent FGFR3 shRNAs by doxycycline
diminished FGFR3 expression, whereas induction of a control shRNA
targeting EGFP had no effect (FIG. 7A). In the absence of exogenous
FGF, doxycycline treatment reduced [.sup.3H]-thymidine
incorporation by cells expressing different FGFR3 shRNAs, but not
control shRNA (FIG. 7B), confirming that FGFR3 knockdown inhibits
proliferation. Further analysis of exponentially growing RT112
cells revealed that FGFR3 knockdown over a 72 hr treatment with
doxycycline markedly and specifically reduced the percentage of
cells in the S and G2 phases of the cell cycle, with a concomitant
increase of cells in G1 phase (FIG. 7C). Similar effect was
observed with two other FGFR3 shRNAs (FIG. 16A). No significant
numbers of cells with a sub-diploid DNA content were detected,
suggesting no change in apoptosis levels. Hence, the inhibitory
effect of FGFR3 knockdown on the proliferation of RT112 cells is
mainly due to attenuation of cell cycle progression.
[0447] We next evaluated the effect of FGFR3 knockdown on the
growth of pre-established RT112 tumor xenografts in mice. FGFR3
knockdown substantially and specifically suppressed tumor growth
(FIG. 7D, top panels and FIG. 16B). Analysis of day 45 tumor
samples confirmed effective FGFR3 knockdown upon doxycycline
induction of FGFR3 shRNA as compared to control shRNA (FIG. 7D,
bottom panels). These results demonstrate that FGFR3 is critically
important both in vitro and in vivo for the growth of RT bladder
cancer cells.
Generation of a Blocking Anti-FGFR3 Monoclonal Antibody
[0448] To examine further the importance of FGFR3 in tumor growth
and to explore the potential of this receptor as a therapeutic
target, we developed an antagonistic anti-FGFR3 monoclonal antibody
(dubbed R3Mab) using a phage display approach. We selected this
particular antibody based on its ability to block both ligand
binding and dimerization by FGFR3, and its unique capacity to
inhibit not only WT FGFR3 but also the most prevalent
cancer-associated mutants of this receptor (see below). R3Mab
targets the extracellular IgD2 and IgD3 domains of FGFR3, which are
necessary and sufficient for FGF binding (4). R3Mab bound both the
IIIb and Mc isoforms of human FGFR3, but showed no detectable
binding to FGFR1, FGFR2 or FGFR4 (FIG. 8A). Biacore analysis
indicated that R3Mab had similar apparent affinity to murine,
cynomolgus monkey and human FGFR3-IIIc (data not shown). The
affinity of R3Mab to human FGFR3 is shown in Table 2.
TABLE-US-00016 TABLE 2 Affinity of R3Mab to human FGFR3 determined
by BIAcore analysis. R3 Ab captured on chips Human FGFR3 ECD
kon/(1/Ms) koff(1/s) Kd(M) IIIb 1.80E+06 2.00E-04 1.11E-10 IIIc
9.10E+04 3.20E-04 3.52E-09
[0449] We next tested the ability of R3Mab to block FGFR3 binding
to FGF1 and FGF9. R3Mab strongly inhibited binding of FGF1 to
FGFR3-IIIb and -IIIc, with half-maximal inhibitory concentrations
(IC.sub.50) of 0.3 nM and 1.7 nM, respectively (FIG. 8B,C).
Similarly, R3Mab efficiently blocked FGF9 binding to FGFR3-IIIb and
-IIIc, with an IC.sub.50 of 1.1 nM and 1.3 nM, respectively (FIG.
8D,E).
R3Mab Inhibits WT FGFR3 and its Most Prevalent Cancer-Associated
Mutant Variants
[0450] To examine whether R3Mab inhibits cell proliferation driven
by WT or mutant FGFR3, we took advantage of the observation that
ectopic FGFR3 expression in murine pro-B cell Ba/F3 confers
interleukin (IL)-3-independent, FGF1-dependent proliferation and
survival (29). In the absence of FGF1 and IL-3, Ba/F3 cells stably
expressing WT FGFR3 were not viable, while FGF1 greatly enhanced
their proliferation (FIG. 9A). R3Mab specifically blocked
FGF1-stimulated Ba/F3-FGFR3 cell proliferation in a dose-dependent
manner (FIG. 9A). We next evaluated the impact of R3Mab on FGFR3
signaling in these cells. FGF1 induced phosphorylation and
activation of FGFR3 and concomitant activation of p44/42 MAPK,
while R3Mab effectively suppressed the activation of both molecules
(FIG. 9B).
[0451] In bladder cancer, somatic activating mutations in FGFR3
cluster within the linker region between IgD2 and IgD3, the
extracellular juxtamembrane domain, or the kinase domain (FIG. 9C).
The extracellular missense substitutions most often give rise to an
unpaired cysteine, leading to ligand-independent dimerization of
FGFR3. These mutations cause markedly different levels of
constitutive FGFR3 activation, possibly owing to a differential
impact on the orientation of the cytoplasmic kinase domain (30,
31). The most frequent mutations are S249C, Y375C, R248C, G372C,
and K652E, which together account for 98% of all FGFR3 mutations in
bladder cancer (32). We reasoned that an optimal therapeutic agent
should block not only the WT FGFR3 protein, which is overexpressed
in certain cancers, but also the most prevalent tumor-associated
FGFR3 mutants. To assess R3Mab further, we generated Ba/F3 cell
lines stably expressing each of the five most common FGFR3 mutant
variants. All mutants were expressed at similar levels at the cell
surface, and the cysteine mutants dimerized spontaneously without
ligand (data not shown). Cell lines expressing different cysteine
mutants exhibited a variable growth response to FGF1, consistent
with earlier findings (30, 31). As previously reported (33), cells
expressing FGFR3.sup.R248C displayed constitutive,
ligand-independent proliferation, and were not responsive to FGF1
(FIG. 9D). Similarly, the most frequent mutation, FGFR3.sup.S249C,
conferred ligand-independent proliferation (FIG. 9E). Remarkably,
R3Mab suppressed constitutive proliferation driven by either mutant
(FIG. 9 D,E). Cells expressing the juxtamembrane domain mutations
FGFR3.sup.G372C (FIG. 9F) or FGFR3.sup.Y375C (FIG. 9G) required
FGF1 for proliferation, and their growth was completely blocked by
R3Mab. Cells expressing FGFR3.sup.K652E showed weak
ligand-independent proliferation and significant growth in response
to FGF1 (33). R3Mab did not affect the weak basal activity of
FGFR3.sup.K652E (data not shown), but nearly abolished
ligand-induced proliferation mediated by this mutant (FIG. 9H).
Hence, R3Mab has a unique capacity to inhibit both WT and prevalent
cancer-associated mutants of FGFR3. Moreover, R3Mab did not display
detectable agonist activity.
[0452] As a separate effort, we generated and characterized
multiple mouse-anti-human FGFR3 hybridoma antibodies. None of the
hybridoma antibodies could inhibit all the cancer-linked FGFR3
mutants we tested (FIG. 17), nor did they share overlapping
epitopes with R3Mab.
[0453] Moreover, all of the hybridoma antibodies showed agonist
activity, strongly stimulating proliferation of cancer-linked FGFR3
mutants R248C and S249C, and showing some stimulation of
proliferation of mutants Y375C and G370C. The hybridoma antibodies
showed differential levels of antagonist and agonism, depending on
the FGFR3 mutant tested, as follows:
TABLE-US-00017 1G6 6G1 15B2 FGFR3-IIIb wildtype inhibition
inhibition inhibition FGFR3-IIIb R248C 2X stimulation 4-5X
stimulation 3-4X stimulation FGFR3-IIIbS249C 2X stimulation 4-5X
stimulation 4-5X stimulation FGFR3-IIIb Y375C 1.2-1.5X 1.2-1.5X
1.2-1.5X stimulation stimulation stimulation FGFR3-IIIb K652E 50%
inhibition 60-70% inhibition inhibition FGFR3-IIIc inhibition
inhibition inhibition FGFR3-IIIc G370C No effect 20-30% inhibition
10-2-% inhibition
Thus, the hybridoma antibodies showed unpredictable differential
effect on Ba/F3 cells cell proliferation driven by various FGFR3
mutants.
Characterization of Mouse-Anti-Human FGFR3 Hybridoma Antibodies
[0454] Mouse anti-human FGFR3 hybridoma antibodies were further
characterized as follows:
[0455] (1) In an assay to test ability of anti-FGFR3 murine
hybridoma antibodies to inhibit FGF1 binding to human FGFR3-IIIb
and IIIc isoforms, antibodies 1G6, 6G1 and 15B2 were able to block
binding of FGF1 to human FGFR3-IIIb and IIIc isoforms in a
dose-dependent manner. When tested across an antibody concentration
range of about 2000 to 0.49 ng/ml, antibodies 1G6, 6G1 and 15B2
blocked FGF1 binding to FGFR3-IIIb with IC50 values of 0.69, 0.87
and 0.72 nM. When tested across an antibody concentration range of
about 5000 to 1.2 ng/ml, antibodies 1G6, 6G1 and 15B2 blocked FGF1
binding to FGFR3-IIIc with IC50 values of 0.57, 3.4 and 0.7 nM,
respectively.
[0456] (2) In an assay to test ability of anti-FGFR3 murine
hybridoma antibodies to inhibit FGF9 binding to human FGFR3-IIIb
and IIIc isoforms, antibodies 1G6, 6G1 and 15B2 efficiently blocked
binding of FGF1 to human FGFR3-IIIb and IIIc isoforms in a
dose-dependent manner. When tested across an antibody concentration
range of about 2000 to 0.49 ng/m, antibodies 1G6, 6G1 and 15B2
blocked FGF9 binding to FGFR3-IIIb with IC50 values of 0.13, 0.16,
and 0.07 nM, respectively. When tested across an antibody
concentration range of about 5000 to 1.2 ng/ml, antibodies 1G6, 6G1
and 15B2 blocked FGF9 binding to FGFR3-IIIc with IC50 values of
0.13, 0.11, and 0.07 nM, respectively.
[0457] (3) The binding affinity of full-length anti-FGFR3 murine
hybridoma antibodies 1G6, 6G1 and 15B2 was determined using Biacore
analysis. The results of this analysis are shown in Table 3.
TABLE-US-00018 TABLE 3 FGFR3-IIIB FGFR3-IIIC Anti- kon koff Kd kon
koff Kd body (10.sup.5M.sup.-1s.sup.-1) (10.sup.-4s.sup.-1) (nM)
(10.sup.5M.sup.-1s.sup.-1) (10.sup.-4s.sup.-1) (nM) 1G6 2.2 3.1 1.4
2.2 2.8 1.3 mIgG 6G1 2.7 3.8 1.4 2.6 3.2 1.2 mIgG 15B2 4.1 29 7.1
3.5 39 11.1 mIgG
[0458] (4) In an assay to test ability of anti-FGFR3 murine
hybridoma antibodies to inhibit Ba/F3 cell proliferation driven by
human FGFR3-IIIb or Mc, antibodies 1G6, 6G1 and 15B2 were able to
block Ba/F3 cell proliferation driven by human FGFR3-IIIb or IIIc
in a dose-dependent manner. When tested across an antibody
concentration range of about 0.01 to 100 ug/ml, antibodies 1G6, 6G1
and 15B2 blocked Ba/F3 cell proliferation driven by FGFR3-IIIb with
IC50 values of 3-5 nM, 3 nM, and 6-8 nM, respectively, and blocked
Ba/F3 cell proliferation driven by FGFR3-IIIc with IC50 values of
10-35 nM, 24 nM, and 60 nM, respectively.
[0459] (5) In an assay to test ability of anti-FGFR3 murine
hybridoma antibodies to inhibit FGF1-induced signaling in Ba/F3
cells expressing human FGFR3-IIIb, antibodies 1G6, 6G1 and 15B2
were able to block FGF1-induced signaling in Ba/F3 cells expressing
human FGFR3-IIIb in a dose-dependent manner when tested across an
antibody concentration range of about 0.25 to 6.75 ug/ml. 25 ng/ml
of FGF1 was used in this experiment. In the absence of FGF1,
antibody treatment had no effect on FGFR3 activation.
[0460] (6) In an assay to test ability of anti-FGFR3 murine
hybridoma antibodies to inhibit FGF1-induced signaling in Ba/F3
cells expressing human FGFR3-IIIc, antibodies 1G6, 6G1 and 15B2
were able to block FGF1-induced signaling in Ba/F3 cells expressing
human FGFR3-IIIc in a dose-dependent manner when tested across an
antibody concentration range of about 0.25 to 6.75 ug/ml. 25 ng/ml
of FGF1 was used in this experiment. In the absence of FGF1,
antibody treatment had no effect on FGFR3 activation.
Structural Basis for the Interaction of R3Mab with FGFR3
[0461] To gain insight into R3Mab's mode of interaction with FGFR3,
we synthesized a panel of 13 overlapping peptides spanning the
FGFR3-IIIb IgD2 and D3 regions and tested their binding to R3Mab.
Peptides 3 (residues 164-178) and 11 (residues 269-283) showed
specific binding to R3Mab, with peptide 3 having a stronger
interaction (FIG. 10A), indicating that the corresponding regions
on FGFR3 are critical for recognition by R3Mab. Previous
crystallographic studies of FGFR1 in complex with FGF2 identified
critical receptor residues engaged in direct binding to FGF and
heparin as well as in receptor dimerization (34). Alignment of
FGFR3 peptides 3 and 11 with the functionally important sites in
FGFR1 revealed that these peptides encompass corresponding FGFR1
residues essential for direct FGF2 binding, receptor dimerization,
as well as interaction with heparin (FIG. 10B). These data indicate
that the epitope of R3Mab on FGFR3 overlaps with receptor residues
engaged in ligand association and receptor-receptor
interaction.
[0462] We next crystallized the complex between the Fab fragment of
R3Mab and the extracellular IgD2-D3 region of human FGFR3-IIIb, and
determined the X-ray structure at 2.1 .ANG. resolution (FIG. 10 C,
D; Table 4). In this complex, approximately 1400 .ANG.2 and 1500
.ANG.2 of solvent-accessible surface areas are buried on FGFR3 and
the Fab, respectively. About 80% of the buried interface involves
IgD2, while the remainder entails the linker and IgD3 regions. On
the Fab side of the complex, about 40% of the buried interface
involve complementarity-determining region (CDR)-H3, 20% CDR-H2,
20% CDR-L2, and minor contributions are from other CDRs and
framework residues. Notably, amino acids (AAs) from CDR-H3 form two
.beta.-strands, which extend the .beta.-sheet of IgD2 (FIG. 10D).
The Fab interacts with AAs that constitute the FGF binding site of
FGFR3 as well as residues that form the receptor dimerization
interface, as previously identified in various dimeric FGF:FGFR
complexes (e.g., PDB code 1CVS, (34); and FIG. 10C, areas in
grey/crosshatched and dark grey). The interaction interfaces
identified by crystallography were fully consistent with the
peptide-based data (FIG. 18 A, B). Together, these results reveal
how R3Mab inhibits ligand binding, and further suggest that binding
of R3Mab to FGFR3 may prevent receptor dimerization. FGFR3 amino
acids that contact R3Mab are shown in Table 5. Crystallographic
coordinates for this structure are deposited in the Protein Data
Bank with accession code 3GRW and shown in Table 6.
TABLE-US-00019 TABLE 5 Residues in FGFR3 that are in contact with
R3Mab Residue Buried surface of residue in the interface THR 154
0.10 ARG 155 16.50 ARG 158 105.40 MET 159 6.00 LYS 161 52.50 LYS
162 1.70 LEU 163 12.30 LEU 164 55.10 ALA 165 10.10 VAL 166 10.60
PRO 167 45.50 ALA 168 22.60 ALA 169 63.60 ASN 170 75.40 THR 171
83.00 VAL 172 1.70 ARG 173 91.70 PHE 174 1.50 ARG 175 95.60 PRO 177
15.90 GLY 202 2.10 LYS 205 63.40 ARG 207 67.60 GLN 210 31.60 SER
212 0.40 VAL 214 26.40 GLU 216 48.90 SER 217 1.80 TYR 241 15.90 LEU
246 3.10 GLU 247 1.80 ARG 248 46.90 TYR 278 32.20 SER 279 1.80 ASP
280 19.80 ALA 281 3.00 GLN 282 24.80 PRO 283 0.50 SER 314 1.20 GLU
315 82.60 SER 316 33.20 VAL 317 56.60 GLU 318 51.50
TABLE-US-00020 TABLE 4 Summary of crystallographic analysis Data
collection FGFR3-IIIb: R3MAb Fab Space group P2.sub.12.sub.12.sub.1
Cell parameters a = 58.5, b = 99.3, c = 143.7 Resolution (.ANG.)
25-2.1 R.sub.sym.sup.a 0.098 (0.663).sup.b Number of observations
288498 Unique reflections 49851 Completeness (%) 99.99
(100.0).sup.b Refinement Resolution (.ANG.) 20-2.1 Number of
reflections 46714 Final R.sup.c, R.sub.free (F > 0) 0.187, 0.224
Number of non-H atoms 5270 Number of Amino Acids 650 Sulfate 1
Sugar 1 Solvent atoms 274 Rmsd bonds (.ANG.) 0.011 Rmsd angles
(.degree.) 1.4 .sup.aR.sub.sym = .SIGMA.|I-<I>|/.SIGMA. I.
<I> is the average intensity of symmetry related observations
of a unique reflection. .sup.bNumbers in parentheses refer to the
highest resolution shell. .sup.cR =
.SIGMA.|F.sub.o-F.sub.c|/.SIGMA.F.sub.o. R.sub.free is calculated
as R, but for 5% of the reflections excluded from all
refinement.
[0463] We compared the R3Mab-FGFR3 structure with a previously
published structure of FGFR3-IIIc in complex with FGF1 (4, 35)
(FIG. 10E, 10F). Superposition of the antibody-receptor and
ligand-receptor complexes revealed that there are no major
conformational differences within the individual receptor domains,
except in the region that distinguishes FGFR3-IIIc from FGFR3-IIIb;
however, the orientation of IgD3 relative to IgD2 was drastically
different (FIG. 10E, white and grey; FIG. 10F, white and
grey-mesh). Since the relative positions of IgD2 and IgD3 are
critical for ligand binding, the alternate conformation adopted by
IgD3 upon R3Mab binding may provide an additional mechanism to
prevent ligand interaction with FGFR3.
R3Mab Inhibits Endogenous WT and Mutant FGFR3 in Bladder Cancer
Cells
[0464] To assess whether R3Mab could suppress FGFR3 function in
bladder cancer cells, we first examined RT112 and RT4 cell lines,
which express WT FGFR3. R3Mab strongly inhibited
[.sup.3H]-thymidine incorporation by RT112 cells (FIG. 11A) and
exerted a significant, though more moderate suppression of RT4 cell
proliferation (FIG. 19A). To investigate R3Mab's effect on FGFR3
activation, we examined the phosphorylation of FGFR3 in RT112
cells. Consistent with the results in Ba/F3-FGFR3 cells (FIG. 9B),
R3Mab markedly attenuated FGF1-induced FGFR3 phosphorylation (FIG.
11B). We next examined phosphorylation of FRS2.alpha., AKT, and
p44/42 MAPK, three downstream mediators of FGFR3 signaling. FGF1
strongly activated these molecules in RT112 cells, while R3Mab
significantly diminished this activation (FIG. 11B). Similarly,
R3Mab suppressed FGF1-induced phosphorylation of FGFR3 and MAPK in
RT4 cells (FIG. 19B).
[0465] We next investigated whether R3Mab could inhibit activation
of endogenous mutant FGFR3 in human bladder cancer cells. S249C is
the most frequent FGFR3 mutation in bladder cancer (FIG. 9C). Two
available cell lines, UMUC-14 and TCC-97-7, carry a mutated
FGFR3.sup.S249C allele (Ref. 36 and data not shown). Although R3Mab
did not affect the exponential growth of UMUC-14 cells in culture
(data not shown), it significantly reduced the clonal growth of
these cells (FIG. 11C). Specifically, R3Mab decreased the number of
colonies larger than 120 .mu.m in diameter approximately by 77% as
compared with control antibody (FIG. 11D). Furthermore, R3Mab
inhibited [.sup.3H]-thymidine incorporation by TCC-97-7 cells in
culture (FIG. 19C).
[0466] The S249C mutation is reported to result in
ligand-independent activation of FGFR3 (26, 30). Indeed,
FGFR3.sup.S249C was constitutively phosphorylated irrespective of
FGF1 treatment in UMUC-14 cells and TCC-97-7 cells, while R3Mab
reduced constitutive phosphorylation of FGFR3.sup.S249C as compared
with control antibody in both cell lines (FIGS. 11E, 19D).
R3Mab Inhibits Dimer Formation by FGFR3.sup.S249C
[0467] The ability of R3Mab to inhibit constitutive FGFR3.sup.S249C
signaling and proliferation in bladder cancer cells was surprising,
considering that this mutant can undergo disulfide-linked,
ligand-independent dimerization (26, 30). To explore how R3Mab
inhibits FGFR3.sup.S249C, we examined the effect of R3Mab on the
oligomeric state of this mutant in UMUC-14 cells. Under reducing
conditions, FGFR3.sup.S249C migrated as a single band of .about.97
kDa, consistent with monomeric size (FIG. 12A). Under non-reducing
conditions, in cells treated with control antibody a large fraction
of FGFR3.sup.S249C appeared as a band of .about.200 kDa, regardless
of FGF1 addition, indicating a constitutive dimeric state (FIG.
12A). R3Mab treatment substantially decreased the amount of dimers,
with a concomitant increase in monomers (FIG. 12A). Consistently,
R3Mab decreased the level of FGFR3.sup.S249C dimers in TCC-97-7
cells irrespective of FGF1 treatment (FIG. 19E).
[0468] How does R3Mab decrease the FGFR3.sup.S249C dimer levels in
bladder cancer cells? One potential explanation is that it may
disrupt the FGFR3.sup.S249C dimer through antibody-induced FGFR3
internalization and trafficking through endosomes or lysosomes. We
tested this possibility by pharmacologically intervening with
endocytosis. R3Mab nonetheless decreased the amount of dimer in
UMUC-14 cells pre-treated with various endocytosis inhibitors,
despite substantial blockade of FGFR3.sup.S249C internalization
(FIG. 20 A, B). Thus, dimer disruption by R3Mab is independent of
endocytosis. Another possible explanation is that cellular
FGFR3.sup.S249C may exist in a dynamic monomer-dimer equilibrium;
accordingly, binding of R3Mab to monomeric FGFR3.sup.S249C could
prevent dimer formation and thereby shift the equilibrium toward
the monomeric state. To examine this possibility, we used the
non-cell-permeating agent 5,5'Dithiobis 2-nitrobenzoic acid (DTNB),
which selectively reacts with and blocks free sulfhydryl groups of
unpaired cysteines (37). Treatment of UMUC-14 cells with DTNB led
to the accumulation of FGFR3.sup.S249C monomers at the expense of
dimers (FIG. 12B), indicating that FGFR3.sup.S249C exists in a
dynamic equilibrium between monomers and dimers.
[0469] To test whether R3Mab affects this equilibrium, we generated
a soluble recombinant protein comprising the IgD2-D3 domains of
FGFR3.sup.S249C and isolated the dimers by size exclusion
chromatography. We incubated the dimers with buffer or antibodies
in the presence of a very low concentration of reducing agent (25
.mu.M of DTT), and analyzed the oligomeric state of the receptor by
SDS-PAGE under non-reducing conditions. R3Mab significantly
accelerated the appearance of a .about.25 kDa band representing
monomeric FGFR3.sup.S249C at the expense of the .about.50 kDa
dimer, as compared with mock or antibody controls (FIG. 12C);
indeed, by 2 hr the decrease in dimers was substantially more
complete in the presence of R3Mab. These results indicate that
R3Mab shifts the equilibrium between the monomeric and dimeric
states of FGFR3.sup.S249C in favor of the monomer.
R3Mab does not Promote FGFR3 Down-Regulation
[0470] We examined the effect of R3Mab (clone 184.6.1) and
anti-FGFR3 hybridoma antibodies on FGFR3 downregulation by
analyzing FGFR3 internalization and degradation in FGFR3
antibody-treated cells. Bladder cancer cell lines expressing wild
type FGFR3 (RT112) or mutated FGFR3 (S249C in TCC97-7) were treated
with R3Mab or hybridoma antibodies 1G6 or 6G1 for 4 to 24 hours,
then cell lysates were harvested for western blot analysis of total
FGFR3 levels. Treatment with R3Mab did not reduce FGFR3 levels,
while treatment with hybridoma mabs 1G6 and 6G1 significantly
reduced FGFR3 levels. These results suggested that R3Mab did not
promote FGFR3 down-regulation while mabs 1G6 and 6G1 did promote
FGFR3 receptor internalization and down regulation. In a separate
experiment, surface FGFR3 levels were examined using FACS analysis.
After 24 hours of R3Mab (clone 184.6.1) treatment of UMUC-14 cells
(containing FGFR3 S249C mutation), cell surface FGFR3 levels
slightly increased. These results demonstrate that R3Mab treatment
did not promote FGFR3 down-regulation.
R3Mab Inhibits Growth and FGFR3 Signaling in Multiple Tumor
Models
[0471] Next, we examined the effect of R3Mab on the growth of
bladder cancer cells in vivo. We injected nu/nu mice with RT112
cells (which express WT FGFR3), allowed tumors to grow to a mean
volume of .about.150 mm.sup.3, and dosed the animals twice weekly
with vehicle or R3Mab. Compared with vehicle control at day 27,
R3Mab treatment at 5 or 50 mg/kg suppressed tumor growth by about
41% or 73% respectively (FIG. 13A). Analysis of tumor lysates
collected 48 hr or 72 hr after treatment showed that R3Mab markedly
decreased the level of phosphorylated FRS2.alpha. (FIG. 13B).
Intriguingly, total FRS2.alpha. protein levels were also lower in
R3Mab-treated tumors, suggesting that FGFR3 inhibition may further
lead to downregulation of FRS2.alpha.. R3Mab also lowered the
amount of phosphorylated MAPK in tumors, without affecting total
MAPK levels (FIG. 13B). Thus, R3Mab inhibits growth of RT112 tumor
xenografts in conjunction with blocking signaling by WT FGFR3.
[0472] We next investigated the effect of R3Mab on growth of
xenografts expressing mutant FGFR3. R3Mab treatment profoundly
attenuated the progression of Ba/F3-FGFR3.sup.S249C tumors (FIG.
13C). Moreover, R3Mab significantly inhibited growth of UMUC-14
bladder carcinoma xenografts (FIG. 13D). To evaluate whether R3Mab
impacts FGFR3.sup.S249C activation in vivo, we assessed the level
of FGFR3.sup.S249C dimer in tumor lysates collected 24 hr or 72 hr
after treatment. Under non-reducing conditions, the amount of
FGFR3.sup.S249C dimer was substantially lower in R3Mab treated
tumors as compared with control group, whereas total
FGFR3.sup.S249C levels, as judged by the amount detected under
reducing conditions, showed little change (FIG. 13E). No apparent
accumulation of FGFR3.sup.S249C monomer was observed in tumor
lysates, in contrast to the results in cell culture (FIG. 13E vs.
12A). This could be due to the weak detection sensitivity for
monomeric FGFR3 under non-reducing conditions by the rabbit
polyclonal anti-FGFR3 antibody used in this study (FIG. 21).
Importantly, R3Mab also significantly inhibited the phosphorylation
and activation of MAPK in UMUC-14 tumors (FIG. 13E), suggesting
that R3Mab inhibits the activity of FGFR3.sup.S249C in vivo. We did
not observe any significant weight loss or other gross
abnormalities in any the in vivo studies. Furthermore, in a safety
study conducted in mice, R3Mab, which binds with similar affinity
to both human and murine FGFR3, did not exert any discernable
toxicity in any organs, including bladder (data not shown).
Together, these data indicate that multiple exposures to R3Mab are
well tolerated in mouse.
Anti-Tumor Activity of R3Mab in Multiple Myeloma Xenograft Models
Involves ADCC
[0473] To assess whether R3Mab might harbor therapeutic potential
for multiple myeloma, we first tested the effect of R3Mab on the
proliferation and survival of three t(4; 14)+ cell lines in
culture. UTMC-2 cells carry WT FGFR3, while OPM2 and KMS11 harbor a
K650E and Y373C substitution, respectively (7). In culture, R3Mab
abrogated FGF9-induced proliferation of UTMC-2 cells completely
(FIG. 22A). R3Mab modestly inhibited the growth of OPM2 cells, but
had no apparent effect on the proliferation of KMS11 cells (FIG. 22
B, C). Since UTMC-2 cells do not form tumors in mice, we evaluated
the efficacy of R3Mab against OPM2 and KMS11 tumors. R3Mab almost
completely abolished xenograft tumor growth of both cell lines
(FIG. 14 A, B).
[0474] The marked difference in activity of R3Mab against OPM2 and
KMS11 tumor cells in vitro and in vivo suggested the possibility
that R3Mab may be capable of supporting Fc-mediated immune effector
functions against these FGFR3-overexpressing tumors. Both cell
lines express high levels of CD55 and CD59 (data not shown), two
inhibitors of the complement pathway; accordingly, no
complement-dependent cytotoxicity was observed (data not shown). We
then focused on ADCC. ADCC occurs when an antibody binds to its
antigen on a target cell, and via its Fc region, engages Fc.gamma.
receptors (Fc.gamma.Rs) expressed on immune effector cells (38). To
test ADCC in vitro, we incubated KMS11 or OPM2 cells with freshly
isolated human peripheral blood mononuclear cells (PBMC) in the
presence of R3Mab or control antibody. R3Mab mediated significant
PBMC cytolytic activity against both myeloma cell lines (FIG. 14 C,
D). By contrast, R3Mab did not support cytolysis of bladder cancer
RT112 or UMUC-14 cells (FIG. 14 E, F). As measured by Scatchard
analysis, the multiple myeloma cells express substantially more
cell-surface FGFR3 than the bladder carcinoma cell lines
(.about.5-6 fold more receptors per cell; FIG. 23 A, B).
[0475] To address the contribution of ADCC to the activity of R3Mab
in vivo, we introduced the previously characterized D265A/N297A
(DANA) mutation into the antibody's Fc domain. This dual
substitution in the Fc domain of an antibody abolishes its binding
to Fc.gamma.Rs (39), preventing recruitment of immune effector
cells. The DANA mutation did not alter R3Mab binding to FGFR3 or
inhibition of FGFR3 activity in vitro, nor did it change the
pharmacokinetics of R3Mab in mice (data not shown); however, it
substantially abolished in vivo activity against OPM2 or KMS11
xenografts (FIG. 14 G, H). By contrast, the DANA mutation did not
alter the anti-tumor activity of R3Mab towards RT112 and UMUC-14
bladder cancer xenografts (FIG. 24 A, B). Together, these results
suggest that Fc-dependent ADCC plays an important role in the
efficacy of R3Mab against OPM2 and KMS11 multiple myeloma
xenografts.
Additional Xenograft Studies
[0476] R3Mab (clone 184.6.1N54S) was further characterized as
follows: [0477] (a) R3Mab was tested for in vivo efficacy using a
tumor xenograft model based on a liver cancer cell line (Huh7).
When tested at an antibody concentration of 5 mg/kg and 30 mg/kg,
R3Mab significantly inhibited tumor growth in vivo. Tumor growth
was inhibited about 50% compared to tumor growth in control
animals. [0478] (b) R3Mab was tested for in vivo efficacy using a
tumor xenograft model based on a breast cancer cell line (Cal-51)
which expressed FGFR3. Results from this efficacy study showed that
the R3Mab antibody was capable of inhibiting tumors in vivo when
tested at antibody concentration range of about 1 mg/kg to 100
mg/kgs. Tumor growth was inhibited about 30% compared to tumor
growth in control animals.
Discussion
[0479] The association of FGFR3 overexpression with poor prognosis
in t(4; 14)+ multiple myeloma patients and the transforming
activity of activated FGFR3 in several experimental models have
established FGFR3 as an important oncogenic driver and hence a
potential therapeutic target in this hematologic malignancy. By
contrast, despite reports of a high frequency of mutation and/or
overexpression of FGFR3 in bladder carcinoma (24, 25, 40), a
critical role for FGFR3 signaling in this epithelial malignancy has
not been established in vivo. Moreover, the therapeutic potential
of FGFR3 inhibition in bladder cancer has yet to be defined. Here
we show that genetic or pharmacological intervention with FGFR3
inhibits growth of several human bladder cancer xenografts in mice.
These results demonstrate that FGFR3 function is critical for tumor
growth in this setting, underscoring the potential importance of
this receptor as an oncogenic driver and therapeutic target in
bladder cancer. Blockade of FGFR3 function inhibited growth of
xenografts expressing either WT or mutant FGFR3 alike, suggesting
that both forms of the receptor may contribute significantly to
bladder tumor progression. Albeit much less frequently than in
bladder cancer, FGFR3 mutations or overexpression have been
identified in other solid tumor malignancies, including cervical
carcinoma (40), hepatocellular carcinoma (41) and non-small cell
lung cancer (42, 43), suggesting a potential contribution of FGFR3
to additional types of epithelial cancer.
[0480] The apparent involvement of FGFR3 in diverse malignancies
identifies this receptor as an intriguing candidate for targeted
therapy. While small molecule compounds that can inhibit FGFR3
kinase activity have been described (18-22, 44), the close homology
of the kinase domains within the FGFR family has hampered the
development of FGFR3-selective inhibitors. The lack of selectivity
of the reported inhibitors makes it difficult to discern the
relative contribution of FGFR3 to the biology of specific cancer
types; further, it may carry safety liabilities, capping maximal
dose levels and thus limiting optimal inhibition of FGFR3.
Therefore, to achieve selective and specific targeting of FGFR3, we
turned to an antibody-based strategy. We reasoned that an optimal
therapeutic antibody should be capable of blocking not only the WT
but also the prevailing cancer-linked mutants of FGFR3.
Furthermore, given that dimerization of FGFR3 is critical for its
activation, an antibody that not only blocks ligand binding but
also interferes with receptor dimerization could be superior.
Additional desirable properties would include the ability to
support Fc-mediated effector function and the long serum half-life
conferred by the natural framework of a full-length antibody. We
focused our screening and engineering efforts to identify an
antibody molecule that combines all of these features, leading to
the generation of R3Mab. Binding studies demonstrated the ability
of R3Mab to compete with FGF ligands for interaction with both the
IIIb and IIIc isoforms of FGFR3. Further experiments with
transfected BaF/3 cell lines confirmed the remarkable ability of
R3Mab to block both WT and prevalent cancer-associated FGFR3
mutants. In addition, R3Mab exerted significant anti-tumor activity
in several xenograft models of bladder cancer expressing either WT
FGFR3 or FGFR3.sup.S249C, which is the most common mutant of the
receptor in this disease. Pharmacodynamic studies suggested that
the anti-tumor activity R3Mab in these models is based on
inhibition of FGFR3 signaling, evident by diminished
phosphorylation of its downstream mediators FRS2.alpha. and MAPK.
These data further reinforce the conclusion that FGFR3 is required
for bladder tumor progression, as demonstrated by our FGFR3 shRNA
studies.
[0481] FGFR3 mutations in bladder cancer represent one of the most
frequent oncogenic alterations of a protein kinase in solid tumor
malignancies, reminiscent of the common mutation of B-Raf in
melanoma (45). Most of the activating mutations in FGFR3 give rise
to an unpaired cysteine, leading to ligand-independent receptor
dimerization and to various degrees of constitutive activation. A
previous study using a monovalent anti-FGFR3 Fab fragment indicated
differential inhibitory activity against specific FGFR3 mutants
(46); however, the molecular basis for this variable effect was not
investigated. Compared with monovalent antibody fragments, bivalent
antibodies have the capacity to induce the clustering of antigens,
and in the case of receptor tyrosine kinases, may cause receptor
oligomerization and activation. Despite its full-length, bivalent
configuration, R3Mab displayed universal inhibition of WT FGFR3 and
of a wide spectrum of FGFR3 mutants, including variants that are
ligand-dependent (FGFR3.sup.G372C, FGFR3.sup.Y375C), constitutively
active (FGFR3.sup.R248C, FGFR3.sup.S249C), or both
(FGFR3.sup.K652E). These results raise the question: How does R3Mab
antagonize both WT and various FGFR3 mutants, including
disulfide-linked variants?
[0482] Based on sequence alignment with FGFR1, the peptide epitope
recognized by R3Mab overlaps with FGFR3 residues involved in
binding to ligand and heparin, as well as receptor dimerization.
This conclusion was confirmed by crystallographic studies of the
complex between R3Mab and the extracellular regions of FGFR3. The
X-ray structure revealed that the antibody binds to regions of IgD2
and IgD3 that are critical for ligand-receptor interaction as well
as receptor-receptor contact. Thus, R3Mab may block WT FGFR3 both
by competing for ligand binding and by preventing receptor
dimerization. R3Mab may employ a similar mechanism to inhibit
FGFR3.sup.K652E, which has low constitutive activity, but requires
ligand for full activation. Furthermore, R3Mab binding changes the
relative orientation of FGFR IgD3 with respect to IgD2. This
finding raises the formal possibility that the antibody might also
inhibit receptor activation by forcing a conformation that is not
conducive to signal transduction--a notion that requires further
study.
[0483] To gain better insight into how R3Mab blocks FGFR3 variants
possessing an unpaired cysteine, we analyzed the most common
mutant, FGFR3.sup.S249C, in greater detail. Experiments with the
free-sulfhydryl blocker DTNB indicated a dynamic equilibrium
between the monomeric and dimeric state of FGFR3.sup.S249C. Similar
equilibrium between oxidized and reduced states modulated by
endogenous redox regulators has been reported for NMDA receptors
(46). Incubation of bladder cancer cells expressing FGFR3.sup.S249C
with R3Mab led to a decline in the amount of receptor dimers and a
concomitant increase in the level of monomers. Moreover, the
purified IgD2-D3 fragment of FGFR3.sup.S249C formed dimers in
solution; when incubated with R3Mab, the dimers steadily
disappeared while monomeric FGFR3.sup.S249C accumulated. Taken
together with the structural analysis, these results suggest that
R3Mab captures monomeric FGFR3.sup.S249C and hinders its
dimerization. Over time, R3Mab shifts the equilibrium towards the
monomeric state, blocking constitutive receptor activity. This
mechanism might also explain how R3Mab inhibits other cysteine
mutants of FGFR3.
[0484] Another important finding of this study was the potent
anti-tumor activity of R3Mab against the t(4; 14)+ multiple myeloma
cell lines OPM2 and KMS11 in vivo. By contrast, R3Mab had modest to
minimal impact on proliferation or survival of these cells in
culture. OPM2 and KMS11 cells express relatively high cell surface
levels of FGFR3 (5-6 fold higher than RT112 and UMUC-14 bladder
carcinoma cells). These higher antigen densities may permit R3Mab
to support efficient recruitment of Fc.gamma.R-bearing immune
effector cells and activation of ADCC. Indeed, in the presence of
human PBMC, R3Mab mediated cytolysis of OPM2 and KMS11 cells, but
not RT112 or UMUC-14 bladder cancer cells. Moreover, the DANA
mutant version of R3Mab, which is incapable of Fc.gamma.R binding,
had no effect on KMS11 or OPM2 growth in vivo, but still suppressed
growth of RT112 and UMUC-14 tumors similarly to R3Mab. Together,
these data indicate that R3Mab has a dual mechanism of anti-tumor
activity: (a) In cells expressing lower surface levels of WT or
mutant FGFR3, it blocks ligand-dependent or constitutive signaling;
(b) In cells expressing relatively high surface FGFR3 levels, it
induces ADCC.
[0485] Our results also raise some new questions. First, it is
unknown why the bladder cancer cell lines tested in this study
display variable sensitivity to R3Mab. Such differential response,
which is common for targeted therapy, may be a reflection of the
distinct genetic make-up of individual tumors. Indeed,
Her2-positive breast cancer cells show variable sensitivity to
anti-Her2 antibody (48), as do various cancer cells in response to
anti-EGFR antibody (49). In this context, development of additional
in vivo models for bladder cancer with WT and mutant FGFR3 is
urgently needed to assess sensitivity to FGFR3 molecules in
animals. Moreover, elucidation of predictive biomarkers may help
identify patients who can optimally benefit from FGFR3-targeted
therapy. Secondly, because R3Mab did not induce tumor regression in
the models we examined, future studies should explore whether R3Mab
can cooperate with established therapeutic agents.
[0486] In conclusion, our findings implicate both WT and mutant
FGFR3 as important for bladder cancer growth, thus expanding the in
vivo oncogenic involvement of this receptor from hematologic to
epithelial malignancy. Furthermore, our results demonstrate that
both WT and mutant FGFR3 can be effectively targeted in tumors with
a full-length antibody that combines the ability to block ligand
binding, receptor dimerization and signaling, as well as to promote
tumor cell lysis by ADCC. These results provide a strong rationale
for investigating antibody-based, FGFR3-targeted therapies in
diverse malignancies associated with this receptor.
PARTIAL REFERENCE LIST
[0487] 1. Eswarakumar, V. P., Lax, I., and Schlessinger, J. 2005.
Cellular signaling by fibroblast growth factor receptors. Cytokine
Growth Factor Rev 16:139-149. [0488] 2. L'Hote, C. G., and Knowles,
M. A. 2005. Cell responses to FGFR3 signalling: growth,
differentiation and apoptosis. Exp Cell Res 304:417-431. [0489] 3.
Dailey, L., Ambrosetti, D., Mansukhani, A., and Basilico, C. 2005.
Mechanisms underlying differential responses to FGF signaling.
Cytokine Growth Factor Rev 16:233-247. [0490] 4. Mohammadi, M.,
Olsen, S. K., and Ibrahimi, O. A. 2005. Structural basis for
fibroblast growth factor receptor activation. Cytokine Growth
Factor Rev 16:107-137. [0491] 5. Grose, R., and Dickson, C. 2005.
Fibroblast growth factor signaling in tumorigenesis. Cytokine
Growth Factor Rev 16:179-186. [0492] 6. Chang, H., Stewart, A. K.,
Qi, X. Y., Li, Z. H., Yi, Q. L., and Trudel, S. 2005.
Immunohistochemistry accurately predicts FGFR3 aberrant expression
and t(4; 14) in multiple myeloma. Blood 106:353-355. [0493] 7.
Chesi, M., Nardini, E., Brents, L. A., Schrock, E., Ried, T.,
Kuehl, W. M., and Bergsagel, P. L. 1997. Frequent translocation
t(4; 14) (p16.3;q32.3) in multiple myeloma is associated with
increased expression and activating mutations of fibroblast growth
factor receptor 3. Nat Genet 16:260-264. [0494] 8. Fonseca, R.,
Blood, E., Rue, M., Harrington, D., Oken, M. M., Kyle, R. A.,
Dewald, G. W., Van Ness, B., Van Wier, S. A., Henderson, K. J., et
al. 2003. Clinical and biologic implications of recurrent genomic
aberrations in myeloma. Blood 101:4569-4575. [0495] 9. Moreau, P.,
Facon, T., Leleu, X., Morineau, N., Huyghe, P., Harousseau, J. L.,
Bataille, R., and Avet-Loiseau, H. 2002. Recurrent 14q32
translocations determine the prognosis of multiple myeloma,
especially in patients receiving intensive chemotherapy. Blood
100:1579-1583. [0496] 10. Pollett, J. B., Trudel, S., Stern, D.,
Li, Z. H., and Stewart, A. K. 2002. Overexpression of the
myeloma-associated oncogene fibroblast growth factor receptor 3
confers dexamethasone resistance. Blood 100:3819-3821. [0497] 11.
Bernard-Pierrot, I., Brams, A., Dunois-Larde, C., Caillault, A.,
Diez de Medina, S. G., Cappellen, D., Graff, G., Thiery, J. P.,
Chopin, D., Ricol, D., et al. 2006. Oncogenic properties of the
mutated forms of fibroblast growth factor receptor 3b.
Carcinogenesis 27:740-747. [0498] 12. Agazie, Y. M., Movilla, N.,
Ischenko, I., and Hayman, M. J. 2003. The phosphotyrosine
phosphatase SHP2 is a critical mediator of transformation induced
by the oncogenic fibroblast growth factor receptor 3. Oncogene
22:6909-6918. [0499] 13. Ronchetti, D., Greco, A., Compasso, S.,
Colombo, G., Dell'Era, P., Otsuki, T., Lombardi, L., and Neri, A.
2001. Deregulated FGFR3 mutants in multiple myeloma cell lines with
t(4; 14): comparative analysis of Y373C, K650E and the novel G384D
mutations. Oncogene 20:3553-3562. [0500] 14. Chesi, M., Brents, L.
A., Ely, S. A., Bais, C., Robbiani, D. F., Mesri, E. A., Kuehl, W.
M., and Bergsagel, P. L. 2001. Activated fibroblast growth factor
receptor 3 is an oncogene that contributes to tumor progression in
multiple myeloma. Blood 97:729-736. [0501] 15. Plowright, E. E.,
Li, Z., Bergsagel, P. L., Chesi, M., Barber, D. L., Branch, D. R.,
Hawley, R. G., and Stewart, A. K. 2000. Ectopic expression of
fibroblast growth factor receptor 3 promotes myeloma cell
proliferation and prevents apoptosis. Blood 95:992-998. [0502] 16.
Chen, J., Williams, I. R., Lee, B. H., Duclos, N., Huntly, B. J.,
Donoghue, D. J., and Gilliland, D. G. 2005. Constitutively
activated FGFR3 mutants signal through PLCgamma-dependent and
-independent pathways for hematopoietic transformation. Blood
106:328-337. [0503] 17. Li, Z., Zhu, Y. X., Plowright, E. E.,
Bergsagel, P. L., Chesi, M., Patterson, B., Hawley, T. S., Hawley,
R. G., and Stewart, A. K. 2001. The myeloma-associated oncogene
fibroblast growth factor receptor 3 is transforming in
hematopoietic cells. Blood 97:2413-2419. [0504] 18. Trudel, S.,
Ely, S., Farooqi, Y., Affer, M., Robbiani, D. F., Chesi, M., and
Bergsagel, P. L. 2004 Inhibition of fibroblast growth factor
receptor 3 induces differentiation and apoptosis in t(4; 14)
myeloma. Blood 103:3521-3528. [0505] 19. Trudel, S., Li, Z. H.,
Wei, E., Wiesmann, M., Chang, H., Chen, C., Reece, D., Heise, C.,
and Stewart, A. K. 2005. CHIR-258, a novel, multitargeted tyrosine
kinase inhibitor for the potential treatment of t(4; 14) multiple
myeloma. Blood 105:2941-2948. [0506] 20. Chen, J., Lee, B. H.,
Williams, I. R., Kutok, J. L., Mitsiades, C. S., Duclos, N., Cohen,
S., Adelsperger, J., Okabe, R., Coburn, A., et al. 2005. FGFR3 as a
therapeutic target of the small molecule inhibitor PKC412 in
hematopoietic malignancies. Oncogene 24:8259-8267. [0507] 21.
Paterson, J. L., Li, Z., Wen, X. Y., Masih-Khan, E., Chang, H.,
Pollett, J. B., Trudel, S., and Stewart, A. K. 2004. Preclinical
studies of fibroblast growth factor receptor 3 as a therapeutic
target in multiple myeloma. Br J Haematol 124:595-603. [0508] 22.
Grand, E. K., Chase, A. J., Heath, C., Rahemtulla, A., and Cross,
N. C. 2004. Targeting FGFR3 in multiple myeloma: inhibition of t(4;
14)-positive cells by SU5402 and PD173074. Leukemia 18:962-966.
[0509] 23. Gomez-Roman, J. J., Saenz, P., Molina, M., Cuevas
Gonzalez, J., Escuredo, K., Santa Cruz, S., Junquera, C., Simon,
L., Martinez, A., Gutierrez Banos, J. L., et al. 2005. Fibroblast
growth factor receptor 3 is overexpressed in urinary tract
carcinomas and modulates the neoplastic cell growth. Clin Cancer
Res 11:459-465. [0510] 24. Tomlinson, D. C., Baldo, O., Hamden, P.,
and Knowles, M. A. 2007. FGFR3 protein expression and its
relationship to mutation status and prognostic variables in bladder
cancer. J Pathol 213:91-98. [0511] 25. van Rhijn, B. W., Montironi,
R., Zwarthoff, E. C., Jobsis, A. C., and van der Kwast, T. H. 2002.
Frequent FGFR3 mutations in urothelial papilloma. J Pathol
198:245-251. [0512] 26. Tomlinson, D. C., Hurst, C. D., and
Knowles, M. A. 2007. Knockdown by shRNA identifies S249C mutant
FGFR3 as a potential therapeutic target in bladder cancer. Oncogene
26:5889-5899. [0513] 27. Martinez-Torrecuadrada, J., Cifuentes, G.,
Lopez-Serra, P., Saenz, P., Martinez, A., and Casal, J. I. 2005.
Targeting the extracellular domain of fibroblast growth factor
receptor 3 with human single-chain Fv antibodies inhibits bladder
carcinoma cell line proliferation. Clin Cancer Res 11:6280-6290.
[0514] 28. Martinez-Torrecuadrada, J. L., Cheung, L. H.,
Lopez-Serra, P., Barderas, R., Canamero, M., Ferreiro, S.,
Rosenblum, M. G., and Casal, J. I. 2008. Antitumor activity of
fibroblast growth factor receptor 3-specific immunotoxins in a
xenograft mouse model of bladder carcinoma is mediated by
apoptosis. Mol Cancer Ther 7:862-873. [0515] 29. Ornitz, D. M., and
Leder, P. 1992. Ligand specificity and heparin dependence of
fibroblast growth factor receptors 1 and 3. J Biol Chem
267:16305-16311. [0516] 30. d'Avis, P. Y., Robertson, S. C., Meyer,
A. N., Bardwell, W. M., Webster, M. K., and Donoghue, D. J. 1998.
Constitutive activation of fibroblast growth factor receptor 3 by
mutations responsible for the lethal skeletal dysplasia
thanatophoric dysplasia type I. Cell Growth Differ 9:71-78. [0517]
31. Adar, R., Monsonego-Ornan, E., David, P., and Yayon, A. 2002.
Differential activation of cysteine-substitution mutants of
fibroblast growth factor receptor 3 is determined by cysteine
localization. J Bone Miner Res 17:860-868. [0518] 32. Knowles, M.
A. 2008. Novel therapeutic targets in bladder cancer: mutation and
expression of FGF receptors. Future Oncol 4:71-83. [0519] 33.
Naski, M. C., Wang, Q., Xu, J., and Ornitz, D. M. 1996. Graded
activation of fibroblast growth factor receptor 3 by mutations
causing achondroplasia and thanatophoric dysplasia. Nat Genet
13:233-237. [0520] 34. Plotnikov, A. N., Schlessinger, J., Hubbard,
S. R., and Mohammadi, M. 1999. Structural basis for FGF receptor
dimerization and activation. Cell 98:641-650. [0521] 35. Olsen, S.
K., Ibrahimi, O. A., Raucci, A., Zhang, F., Eliseenkova, A. V.,
Yayon, A., Basilico, C., Linhardt, R. J., Schlessinger, J., and
Mohammadi, M. 2004. Insights into the molecular basis for
fibroblast growth factor receptor autoinhibition and ligand-binding
promiscuity. Proc Natl Acad Sci USA 101:935-940. [0522] 36. Jebar,
A. H., Hurst, C. D., Tomlinson, D. C., Johnston, C., Taylor, C. F.,
and Knowles, M. A. 2005. FGFR3 and Ras gene mutations are mutually
exclusive genetic events in urothelial cell carcinoma. Oncogene
24:5218-5225. [0523] 37. Ellman, G. L. 1959. Tissue sulfhydryl
groups. Arch Biochem Biophys 82:70-77. [0524] 38. Adams, G. P., and
Weiner, L. M. 2005. Monoclonal antibody therapy of cancer. Nat
Biotechnol 23:1147-1157. [0525] 39. Gong, Q., Ou, Q., Ye, S., Lee,
W. P., Cornelius, J., Diehl, L., Lin, W. Y., Hu, Z., Lu, Y., Chen,
Y., et al. 2005. Importance of cellular microenvironment and
circulatory dynamics in B cell immunotherapy. J Immunol
174:817-826. [0526] 40. Cappellen, D., De Oliveira, C., Ricol, D.,
de Medina, S., Bourdin, J., Sastre-Garau, X., Chopin, D., Thiery,
J. P., and Radvanyi, F. 1999. Frequent activating mutations of
FGFR3 in human bladder and cervix carcinomas. Nat Genet 23:18-20.
[0527] 41. Qiu, W. H., Zhou, B. S., Chu, P. G., Chen, W. G., Chung,
C., Shih, J., Hwu, P., Yeh, C., Lopez, R., and Yen, Y. 2005.
Over-expression of fibroblast growth factor receptor 3 in human
hepatocellular carcinoma. World J Gastroenterol 11:5266-5272.
[0528] 42. Cortese, R., Hartmann, O., Berlin, K., and Eckhardt, F.
2008. Correlative gene expression and DNA methylation profiling in
lung development nominate new biomarkers in lung cancer. Int J
Biochem Cell Biol 40:1494-1508. [0529] 43. Woenckhaus, M.,
Klein-Hitpass, L., Grepmeier, U., Merk, J., Pfeifer, M., Wild, P.,
Bettstetter, M., Wuensch, P., Blaszyk, H., Hartmann, A., et al.
2006. Smoking and cancer-related gene expression in bronchial
epithelium and non-small-cell lung cancers. J Pathol 210:192-204.
[0530] 44. Xin, X., Abrams, T. J., Hollenbach, P. W., Rendahl, K.
G., Tang, Y., Oei, Y. A., Embry, M. G., Swinarski, D. E., Garrett,
E. N., Pryer, N. K., et al. 2006. CHIR-258 is efficacious in a
newly developed fibroblast growth factor receptor 3-expressing
orthotopic multiple myeloma model in mice. Clin Cancer Res
12:4908-4915. [0531] 45. Davies, H., Bignell, G. R., Cox, C.,
Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H.,
Garnett, M. J., Bottomley, W., et al. 2002. Mutations of the BRAF
gene in human cancer. Nature 417:949-954. [0532] 46. Trudel, S.,
Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer, S., Chumakov,
I., Singer, Y., Chang, H., Liang, S. B., et al. 2006. The
inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t(4; 14)
multiple myeloma cells. Blood 107:4039-4046. [0533] 47. Gozlan, H.,
and Ben-Ari, Y. 1995. NMDA receptor redox sites: are they targets
for selective neuronal protection? Trends Pharmacol Sci 16:368-374.
[0534] 48. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M.,
Shepard, H. M., and Ullrich, A. 1989. p185HER2 monoclonal antibody
has antiproliferative effects in vitro and sensitizes human breast
tumor cells to tumor necrosis factor. Mol Cell Biol 9:1165-1172.
[0535] 49. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato,
G., and Mendelsohn, J. 1984. Growth inhibition of human tumor cells
in athymic mice by anti-epidermal growth factor receptor monoclonal
antibodies. Cancer Res 44:1002-1007. [0536] 50. Pai, R., Dunlap,
D., Qing, J., Mohtashemi, I., Hotzel, K., and French, D. M. 2008.
Inhibition of fibroblast growth factor 19 reduces tumor growth by
modulating beta-catenin signaling. Cancer Res 68:5086-5095. [0537]
51. Pegram, M., Hsu, S., Lewis, G., Pietras, R., Beryt, M.,
Sliwkowski, M., Coombs, D., Baly, D., Kabbinavar, F., and Slamon,
D. 1999 Inhibitory effects of combinations of HER-2/neu antibody
and chemotherapeutic agents used for treatment of human breast
cancers. Oncogene 18:2241-2251. [0538] 52. Lee, C. V., Liang, W.
C., Dennis, M. S., Eigenbrot, C., Sidhu, S. S., and Fuh, G. 2004.
High-affinity human antibodies from phage-displayed synthetic Fab
libraries with a single framework scaffold. J Mol Biol
340:1073-1093. [0539] 53. Liang, W. C., Dennis, M. S., Stawicki,
S., Chanthery, Y., Pan, Q., Chen, Y., Eigenbrot, C., Yin, J., Koch,
A. W., Wu, X., et al. 2007. Function blocking antibodies to
neuropilin-1 generated from a designed human synthetic antibody
phage library. J Mol Biol 366:815-829. [0540] 54. Carter, P.,
Presta, L., Gorman, C. M., Ridgway, J. B., Henner, D., Wong, W. L.,
Rowland, A. M., Kotts, C., Carver, M. E., and Shepard, H. M. 1992.
Humanization of an anti-p185HER2 antibody for human cancer therapy.
Proc Natl Acad Sci USA 89:4285-4289. [0541] 55. Sidhu, S. S., Li,
B., Chen, Y., Fellouse, F. A., Eigenbrot, C., and Fuh, G. 2004.
Phage-displayed antibody libraries of synthetic heavy chain
complementarity determining regions. J Mol Biol 338:299-310. [0542]
56. Otwinowski, Z. a. M., W. 1997. Processing of X-ray diffraction
data collected in oscillation mode. Methods in Enzymology
276:307-326. [0543] 57. McCoy, A. J., Grosse-Kunstleve, R. W.,
Storoni, L. C., and Read, R. J. 2005. Likelihood-enhanced fast
translation functions. Acta Crystallogr D Biol Crystallogr
61:458-464. [0544] 58. Emsley, P., and Cowtan, K. 2004. Coot:
model-building tools for molecular graphics. Acta Crystallogr D
Biol Crystallogr 60:2126-2132. [0545] 59. Murshudov, G. N., Vagin,
A. A., and Dodson, E. J. 1997. Refinement of macromolecular
structures by the maximum-likelihood method. Acta Crystallogr D
Biol Crystallogr 53:240-255.
[0546] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention.
Sequence CWU 1
1
277110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 320PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 411PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala 1 5 10 57PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Ser Ala Ser Phe Leu Tyr Ser
1 5 69PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Gln Gln Ser Tyr Thr Thr Pro Pro Thr 1 5
710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 920PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 1011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Arg Ala Ser Gln Asp Val Asp
Thr Ser Leu Ala 1 5 10 117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Ser Ala Ser Phe Leu Tyr Ser
1 5 129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Gln Gln Ser Thr Gly His Pro Gln Thr 1 5
1310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
1418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 1520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 1611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Arg Ala Ser Gln Asp Val Asp
Ile Ser Leu Ala 1 5 10 177PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Ser Ala Ser Ser Leu Ala Ser
1 5 189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gln Gln Gly Ala Gly Asn Pro Tyr Thr 1 5
1930PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr 20 25 30 2025PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 20Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser 20 25 2125PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser 20 25 2225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser 20 25 2330PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Val Ser 20 25 30 2425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 2525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 2625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 2730PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20
25 30 2825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25
2925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25
3025PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25
3130PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asn Ile Lys 20 25 30 3225PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 32Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser 20 25 3325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser 20 25 3430PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 34Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30 3525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 3625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 3725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 3823PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys 20 3923PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys 20 4023PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys 20 4123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Glu Arg Ala Thr Ile Asn Cys 20 4223PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys 20 4315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
4432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 44Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser
Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 4510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 4625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 4713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
4810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Gly Phe Thr Phe Ser Thr Thr Gly Ile Ser 1 5 10
4918PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Gly Arg Ile Tyr Pro Leu Tyr Gly Ser Thr His Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 5020PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 50Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 5111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 51Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala 1 5 10 527PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 52Ser Ala Ser Phe Leu Tyr Ser
1 5 539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Gln Gln Thr Tyr Thr Thr Ser Leu Thr 1 5
5410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
5518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Gly Arg Ile Tyr Pro Tyr Asp Asp Ser Phe Tyr Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 5620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 5711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 57Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala 1 5 10 587PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 58Ser Ala Ser Phe Leu Tyr Ser
1 5 599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Gln Gln Ser Tyr Thr Thr Pro Leu Thr 1 5
6010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
6118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 6220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 62Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 6311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 63Arg Ala Ser Gln Val Ile Asp
Ile Ser Leu Ala 1 5 10 647PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 64Gly Ala Ser Thr Leu Ala Ser
1 5 659PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Gln Gln Ser Ala Ala Asp Pro Tyr Thr 1 5
6610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Gly Phe Ser Phe Thr Gly Thr Gly Ile Ser 1 5 10
6718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 67Gly Ser Ile Tyr Pro Tyr Phe Ala Thr Lys Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 6820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 6911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 69Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala 1 5 10 707PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Ser Ala Ser Phe Leu Tyr Ser
1 5 719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 71Gln Gln Ser Tyr Thr Thr Pro Pro Thr 1 5
7210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Gly Phe Thr Phe Tyr Thr Thr Gly Ile Ser 1 5 10
7318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Gly Arg Ile Tyr Pro Ala Phe Gly Ser Ser Ile Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 7420PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 7511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 75Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala 1 5 10 767PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 76Ser Ala Ser Phe Leu Tyr Ser
1 5 779PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Gln Gln Thr Tyr Ser Ala Gln Pro Thr 1 5
7810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 78Gly Phe Ser Phe Trp Ser Thr Gly Ile Ser 1 5 10
7918PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Gly Arg Ile Tyr Pro Ser Ser Ala Thr Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 8020PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 8111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 81Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala 1 5
10 827PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Ser Ala Ser Phe Leu Tyr Ser 1 5
839PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Gln Gln Ser Tyr Ser His Gln Ser Thr 1 5
8410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
8518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 85Gly Arg Ile Tyr Pro Thr Ser Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 8620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 8711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 87Arg Ala Ser Gln Asp Val Asp
Thr Ser Leu Ala 1 5 10 887PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 88Ser Ala Ser Phe Leu Tyr Ser
1 5 899PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 89Gln Gln Ser Thr Gly His Pro Gln Thr 1 5
9010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
9118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 91Gly Arg Ile Tyr Pro Thr Gly Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 9220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 9311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 93Arg Ala Ser Gln Asp Val Asp
Thr Ser Leu Ala 1 5 10 947PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 94Ser Ala Ser Phe Leu Tyr Ser
1 5 959PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Gln Gln Ser Thr Gly His Pro Gln Thr 1 5
9610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
9718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Gly Arg Ile Tyr Pro Thr Ala Gly Ser Thr Asn Tyr
Ala Asp Ser Val 1 5 10 15 Lys Gly 9820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 98Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 9911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 99Arg Ala Ser Gln Asp Val Asp
Thr Ser Leu Ala 1 5 10 1007PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 100Ser Ala Ser Phe Leu Tyr
Ser 1 5 1019PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 101Gln Gln Ser Thr Gly His Pro Gln Thr 1
5 10210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 102Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
10318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 103Gly Arg Ile Tyr Pro Thr Gln Gly Ser Thr Asn
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 10420PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 10511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 105Arg Ala Ser Gln Asp Val
Asp Thr Ser Leu Ala 1 5 10 1067PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 106Ser Ala Ser Phe Leu Tyr
Ser 1 5 1079PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 107Gln Gln Ser Thr Gly His Pro Gln Thr 1
5 10810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
10918PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Gly Arg Ile Tyr Pro Thr Ser Gly Ser Thr Asn
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 11020PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 11111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 111Arg Ala Ser Gln Val Val
Asp Thr Ser Leu Ala 1 5 10 1127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 112Ser Ala Ser Ser Leu Ala
Ser 1 5 1139PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 113Gln Gln Gly Ala Gly Asn Pro Tyr Thr 1
5 11410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
11518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Gly Arg Ile Tyr Pro Thr Gly Gly Ser Thr Asn
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 11620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 116Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 11711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 117Arg Ala Ser Gln Val Val
Asp Thr Ser Leu Ala 1 5 10 1187PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 118Ser Ala Ser Ser Leu Ala
Ser 1 5 1199PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 119Gln Gln Gly Ala Gly Asn Pro Tyr Thr 1
5 12010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
12118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 121Gly Arg Ile Tyr Pro Thr Ala Gly Ser Thr Asn
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 12220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 122Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 12311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 123Arg Ala Ser Gln Val Val
Asp Thr Ser Leu Ala 1 5 10 1247PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 124Ser Ala Ser Ser Leu Ala
Ser 1 5 1259PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 125Gln Gln Gly Ala Gly Asn Pro Tyr Thr 1
5 12610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 126Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1 5 10
12718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 127Gly Arg Ile Tyr Pro Thr Gln Gly Ser Thr Asn
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 12820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 128Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 12911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 129Arg Ala Ser Gln Val Val
Asp Thr Ser Leu Ala 1 5 10 1307PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 130Ser Ala Ser Ser Leu Ala
Ser 1 5 1319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 131Gln Gln Gly Ala Gly Asn Pro Tyr Thr 1
5 132125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 132Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Thr Ser Thr 20 25 30 Gly Ile Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Tyr
Pro Thr Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr
Thr 100 105 110 Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
115 120 125 133108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 133Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Asp Thr Ser 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Thr Gly His
Pro Gln 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 134125PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 134Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr 20 25 30 Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr
Val Asp Tyr Thr 100 105 110 Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly
Thr Leu Val 115 120 125 135108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 135Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Ile Ser 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ala
Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 136125PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 136Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Thr 20 25 30 Gly
Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Tyr Pro Leu Tyr Gly Ser Thr His Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg Thr Tyr Gly Ile Tyr Asp
Leu Tyr Val Asp Tyr Thr 100 105 110 Glu Tyr Val Met Asp Tyr Trp Gly
Gln Gly Thr Leu Val 115 120 125 137108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
137Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser
Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Tyr Thr Thr Ser Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 138125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
138Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr
Ser Thr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Tyr Pro Thr Ser Gly Ser Thr
Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg
Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr 100 105 110 Glu Tyr
Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 115 120 125
139108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 139Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Glu Thr Ser 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Thr Gly His Pro Gln 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
14011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Arg Ala Ser Gln Asp Val Glu Thr Ser Leu Ala 1
5 10 1417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Ser Ala Ser Phe Leu Tyr Ser 1 5
1429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Gln Gln Ser Thr Gly His Pro Gln Thr 1 5
14310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 1
5 10 14418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Gly Arg Ile Tyr Pro Thr Ser Gly Ser Thr Asn
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 14520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 145Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 14611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 146Arg Ala Ser Gln Xaa Xaa
Xaa Xaa Xaa Xaa Ala 1 5 10 1477PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 147Xaa Ala Ser Phe Leu Xaa
Ser 1 5 1489PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 148Gln Gln Xaa Xaa Xaa Xaa Xaa Xaa Thr 1
5 14910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Gly Phe Xaa Phe Xaa Xaa Thr Gly Ile Ser 1 5 10
15018PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Gly Arg Ile Tyr Pro Xaa Xaa Xaa Xaa Xaa Xaa
Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly 15120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 151Ala
Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 1 5 10
15 Val Met Asp Tyr 20 15211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 152Arg Ala Ser Gln Xaa Val
Xaa Xaa Xaa Val Ala 1 5 10 1539PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 153Gln Gln Xaa Xaa Xaa Xaa
Xaa Xaa Thr 1 5 15418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 154Gly Arg Ile Tyr Pro Xaa
Xaa Gly Ser Thr Xaa Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly
15510PRTMus musculus 155Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5
10 15610PRTMus musculus 156Ser Ala Ser Ser Ser Val Ser Tyr Met His
1 5 10 15711PRTMus musculus 157Leu Ala Ser Gln Thr Ile Gly Thr Trp
Leu Ala 1 5 10 15811PRTMus musculus 158Thr Trp Ile Tyr Asp Thr Ser
Ile Leu Ala Ser 1 5 10 15911PRTMus musculus 159Arg Trp Ile Tyr Asp
Thr Ser Lys Leu Ala Ser 1 5 10 16011PRTMus musculus 160Leu Leu Ile
Tyr Ala Ala Thr Ser Leu Ala Asp 1 5 10 1619PRTMus musculus 161Gln
Gln Trp Thr Ser Asn Pro Leu Thr 1 5 1629PRTMus musculus 162Gln Gln
Trp Ser Ser Tyr Pro Pro Thr 1 5 1639PRTMus musculus 163Gln Gln Leu
Tyr Ser Pro Pro Trp Thr 1 5 16410PRTMus musculus 164Gly Tyr Ser Phe
Thr Asp Tyr Asn Met Tyr 1 5 10 16510PRTMus musculus 165Gly Tyr Val
Phe Thr His Tyr Asn Met Tyr 1 5 10 16610PRTMus musculus 166Gly Tyr
Ala Phe Thr Ser Tyr Asn Met Tyr 1 5 10 16720PRTMus musculus 167Trp
Ile Gly Tyr Ile Glu Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln 1 5 10
15 Lys Phe Lys Gly 20 16820PRTMus musculus 168Trp Ile Gly Tyr Ile
Glu Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln 1 5 10 15 Lys Phe Lys
Gly 20 16920PRTMus musculus 169Trp Ile Gly Tyr Ile Asp Pro Tyr Ile
Gly Gly Thr Ser Tyr Asn Gln 1 5 10 15 Lys Phe Lys Gly 20
17013PRTMus musculus 170Ala Ser Pro Asn Tyr Tyr Asp Ser Ser Pro Phe
Ala Tyr 1 5 10 17110PRTMus musculus 171Ala Arg Gly Gln Gly Pro Asp
Phe Asp Val 1 5 10 17213PRTMus musculus 172Ala Arg Trp Gly Asp Tyr
Asp Val Gly Ala Met Asp Tyr 1 5 10 173107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
173Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn
Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 174107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
174Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser
Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 17530PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
175Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
20 25 30 17611PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 176Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 1 5 10 17732PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 177Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 17830PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
178Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
20 25 30 17915PRTHomo sapiens 179Leu Ala Val Pro Ala Ala Asn Thr
Val Arg Phe Arg Cys Pro Ala 1 5 10 15 18015PRTHomo sapiens 180Ser
Asp Val Glu Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro 1 5 10 15
18115PRTHomo sapiens 181His Ala Val Pro Ala Ala Lys Thr Val Lys Phe
Lys Cys Pro Ser 1 5 10 15 18215PRTHomo sapiens 182Ser Asn Val Glu
Phe Met Cys Lys Val Tyr Ser Asp Pro Gln Pro 1 5 10 15
18311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 183Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1
5 10 18425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 184Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25 18513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 185Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 1 5 10 18630PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 186Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 18711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 187Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 18823PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 188Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys 20 18915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 189Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
19032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 190Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 19110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 191Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 19265DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 192gatccccgca tcaagctgcg gcatcattca agagatgatg
ccgcagcttg atgctttttt 60ggaaa 6519365DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 193gatcccctgc acaacctcga ctactattca agagatagta
gtcgaggttg tgcatttttt 60ggaaa 6519465DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 194gatccccaac ctcgactact acaagattca agagatcttg
tagtagtcga ggtttttttt 60ggaaa 6519530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
195gtcagatatc gtkctsacmc artctccwgc 3019637DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
196gatcgacgta cgctgagatc carytgcarc artctgg 3719727DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
197gtcagatatc gtgctgacmc artctcc 2719837DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
198gatcgacgta cgctgagatc carytgcarc artctgg 3719927DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
199gtacgatatc cagatgacmc artctcc 2720037DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
200gatcgacgta cgctgagatc carytgcarc artctgg 3720121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
201tttdakytcc agcttggtac c 2120243DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 202acagtgggcc cttggtggag
gctgmrgaga cdgtgashrd rgt 4320314PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 203Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 20432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
204Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu
1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg 20 25 30 20511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 205Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 1 5 10 20613PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 206Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 1 5 10 20732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
207Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu
1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg 20 25 30 20811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 208Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 1 5 10 20913PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 209Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 1 5 10 21031PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
210Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu
1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 20 25 30 21111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 211Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 1 5 10 21213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 212Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 1 5 10 21330PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 213Arg Val Thr Ile Thr
Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30
21411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 214Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1
5 10 21514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 215Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile Gly 1 5 10 21632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 216Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
21711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 217Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1
5 10 21813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 218Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 1 5 10 21932PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 219Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 22011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 220Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 22113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 221Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1 5 10
22231PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 222Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 20 25 30 22311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 223Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 22413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 224Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1 5 10
22530PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 225Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 20 25 30 22611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 226Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 22714PRTArtificial
SequenceDescription of Artificial Sequence
Synthetic peptide 227Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser 1 5 10 22832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 228Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
22911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 229Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1
5 10 23013PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 230Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 1 5 10 23132PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 231Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 23211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 232Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 23313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 233Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
23431PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 234Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 20 25 30 23511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 235Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 23613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 236Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
23730PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 237Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 20 25 30 23811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 238Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 23914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 239Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10
24032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 240Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30 24111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 241Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 24213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 242Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
24332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 243Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30 24411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 244Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 24513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 245Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
24631PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 246Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ser 20 25 30 24711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 247Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 24814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 248Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10
24932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 249Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 25011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 250Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 25113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 251Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
25232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 252Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 25311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 253Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 25413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 254Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
25531PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 255Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 20 25 30 25611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 256Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 25713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 257Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10
25830PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 258Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 20 25 30 25911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 259Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 26015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 260Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
26132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 261Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 26210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 262Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 26315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 263Trp
Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15
26432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 264Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 26510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 265Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 26615PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 266Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15
26732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 267Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Arg Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 26810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 268Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 26915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 269Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15
27032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 270Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ala
Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30 27110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 271Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 272235PRTHomo sapiens
272Ala Asp Pro Asp Thr Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg
1 5 10 15 Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val Pro Ala Ala
Asn Thr 20 25 30 Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro Thr
Pro Ser Ile Ser 35 40 45 Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly
Glu His Arg Ile Gly Gly 50 55 60 Ile Lys Leu Arg His Gln Gln Trp
Ser Leu Val Met Glu Ser Val Val 65 70 75 80 Pro Ser Asp Arg Gly Asn
Tyr Thr Cys Val Val Glu Asn Lys Phe Gly 85 90 95 Ser Ile Arg Gln
Thr Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His 100 105 110 Arg Pro
Ile Leu Gln Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu 115 120 125
Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro 130
135 140 His Ile Gln Trp Leu Lys His Val Glu Val Asn Gly Ser Lys Val
Gly 145 150 155 160 Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys Ser
Trp Ile Ser Glu 165 170 175 Ser Val Glu Ala Asp Val Arg Leu Arg Leu
Ala Asn Val Ser Glu Arg 180 185 190 Asp Gly Gly Glu Tyr Leu Cys Arg
Ala Thr Asn Phe Ile Gly Val Ala 195 200 205 Glu Lys Ala Phe Trp Leu
Ser Val His Gly Pro Arg Ala Ala Glu Glu 210 215 220 Glu Leu Val Glu
Ala Asp Glu Ala Gly Ser Val 225 230 235 273241PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
273Ala Asp Pro Asp Thr Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg
1 5 10 15 Pro Glu Arg Met Asp Lys Lys Leu Leu Ala Val Pro Ala Ala
Asn Thr 20 25 30 Val Arg Phe Arg Cys Pro Ala Ala Gly Asn Pro Thr
Pro Ser Ile Ser 35 40 45 Trp Leu Lys Asn Gly Arg Glu Phe Arg Gly
Glu His Arg Ile Gly Gly 50 55 60 Ile Lys Leu Arg His Gln Gln Trp
Ser Leu Val Met Glu Ser Val Val 65 70 75 80 Pro Ser Asp Arg Gly Asn
Tyr Thr Cys Val Val Glu Asn Lys Phe Gly 85 90 95 Ser Ile Arg Gln
Thr Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His 100 105 110 Arg Pro
Ile Leu Gln Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu 115 120 125
Gly Ser Asp Val Glu Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro 130
135 140 His Ile Gln Trp Leu Lys His Val Glu Val Asn Gly Ser Lys Val
Gly 145 150 155 160 Pro Asp Gly Thr Pro Tyr Val Thr Val Leu Lys Ser
Trp Ile Ser Glu 165 170 175 Ser Val Glu Ala Asp Val Arg Leu Arg Leu
Ala Asn Val Ser Glu Arg 180 185 190 Asp Gly Gly Glu Tyr Leu Cys Arg
Ala Thr Asn Phe Ile Gly Val Ala 195 200 205 Glu Lys Ala Phe Trp Leu
Ser Val His Gly Pro Arg Ala Ala Glu Glu 210 215 220 Glu Leu Val Glu
Ala Asp Glu Ala Gly Ser Val His His His His His 225 230 235 240 His
274108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 274Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
275125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 275Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Thr Ser Thr 20 25 30 Gly Ile Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Tyr
Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr
Thr 100 105 110 Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
115 120 125 276214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 276Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210 277232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
277Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr
Ser Thr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr
Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg
Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr 100 105 110 Glu Tyr
Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Ala Ser Thr 115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130
135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys
Asp Lys Thr His 225 230
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References