U.S. patent application number 14/875285 was filed with the patent office on 2016-02-11 for antibodies specific to cadherin-17.
The applicant listed for this patent is Oxford BioTherapeutics Limited. Invention is credited to Christian ROHLFF, Jonathan Alexander TERRETT.
Application Number | 20160039933 14/875285 |
Document ID | / |
Family ID | 42310689 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160039933 |
Kind Code |
A1 |
ROHLFF; Christian ; et
al. |
February 11, 2016 |
ANTIBODIES SPECIFIC TO CADHERIN-17
Abstract
The present disclosure provides antibodies, including isolated
monoclonal antibodies, which specifically bind to Cadherin-17 with
high affinity. Nucleic acid molecules encoding Cadherin-17
antibodies, expression vectors, host cells and methods for
expressing the Cadherin-17 antibodies are also provided. Bispecific
molecules and pharmaceutical compositions comprising the
Cadherin-17 antibodies are also provided. Methods for detecting
Cadherin-17, as well as methods for treating various cancers,
including colorectal cancer, are disclosed.
Inventors: |
ROHLFF; Christian;
(Abingdon, GB) ; TERRETT; Jonathan Alexander;
(Sunnyvale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oxford BioTherapeutics Limited |
Abingdon |
|
GB |
|
|
Family ID: |
42310689 |
Appl. No.: |
14/875285 |
Filed: |
October 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13265517 |
Jan 9, 2012 |
9181339 |
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PCT/US2010/031719 |
Apr 20, 2010 |
|
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14875285 |
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61170980 |
Apr 20, 2009 |
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Current U.S.
Class: |
424/139.1 ;
424/183.1; 435/252.3; 435/252.31; 435/252.33; 435/254.2;
435/254.21; 435/254.23; 435/320.1; 435/331; 435/419; 435/69.6;
530/387.3; 530/387.9; 530/391.7; 536/23.53 |
Current CPC
Class: |
A61P 11/02 20180101;
A61K 47/6849 20170801; C07K 2317/80 20130101; C07K 2317/565
20130101; C07K 2317/56 20130101; A61K 47/6851 20170801; A61K
47/6817 20170801; A61P 35/00 20180101; C07K 2317/51 20130101; C07K
2317/515 20130101; C07K 16/28 20130101; C07K 2317/77 20130101; A61K
39/00 20130101; C07K 2317/55 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/48 20060101 A61K047/48 |
Claims
1. An isolated anti-Cadherin-17 antibody comprising a heavy chain
variable region CDR1 comprising SEQ ID NO: 1, a heavy chain
variable region CDR2 comprising SEQ ID NO: 5, a heavy chain
variable region CDR3 comprising SEQ ID NO: 14, a light chain
variable region CDR1 comprising SEQ ID NO: 22, a light chain
variable region CDR2 comprising SEQ ID NO: 28, and a light chain
variable region CDR3 comprising SEQ ID NO: 32.
2. An isolated anti-Cadherin-17 antibody comprising heavy and light
chain variable regions, wherein the heavy chain variable region
comprises the mature portion of SEQ ID NO:35.
3. An isolated anti-Cadherin-17 antibody comprising heavy and light
chain variable regions, wherein the light chain variable region
comprises the mature portion of SEQ ID NO:47.
4. An isolated anti-Cadherin-17 antibody comprising a heavy chain
variable region comprising the mature portion of SEQ ID NO:35, or
an amino acid sequence at least 95% identical thereto, and a light
chain variable region comprising the mature portion of SEQ ID
NO:47, or an amino acid sequence at least 95% identical
thereto.
5. The isolated anti-Cadherin-17 antibody of claim 4, wherein the
antibody comprises a heavy chain variable region comprising the
mature portion of SEQ ID NO:35 and a light chain variable region
comprising the mature portion of SEQ ID NO:47.
6. An isolated antibody which binds an epitope on human Cadherin-17
recognized by an antibody comprising a heavy chain variable region
comprising SEQ ID NO:35 and a light chain variable region
comprising SEQ ID NO:47.
7. The antibody of claim 1, wherein the antibody is a full-length
antibody of an IgG1, IgG2, IgG3, or IgG4 isotype.
8. The antibody of claim 1, wherein the antibody is selected from
the group consisting of: a whole antibody, a monoclonal antibody,
an antibody fragment, a humanized antibody, a single chain
antibody, a defucosylated antibody, an antibody mimetic and a
bispecific antibody.
9. An immunoconjugate comprising the antibody of claim 1 conjugated
to a therapeutic agent.
10. The immunoconjugate of claim 9, wherein the therapeutic agent
is a cytotoxin.
11. A composition comprising the antibody of claim 1 and a
pharmaceutically acceptable carrier.
12. An isolated nucleic acid molecule encoding the heavy or light
chain of the antibody of claim 1.
13. An isolated nucleic acid comprising a nucleotide sequence
encoding an antibody variable region, wherein the nucleotide
sequences comprises SEQ ID NO:59 or SEQ ID NO:71.
14. An expression vector comprising the nucleic acid molecule of
claim 12.
15. A host cell comprising the expression vector of claim 14.
16. A method for preparing an anti-Cadherin-17 antibody, said
method comprising the steps of: obtaining a host cell that contains
one or more nucleic acid molecules encoding the antibody of claim
1; growing the host cell in a host cell culture; providing host
cell culture conditions wherein the one or more nucleic acid
molecules are expressed; and recovering the antibody from the host
cell or from the host cell culture.
17. A method for treating cancer associated with target cancer
cells expressing Cadherin-17, said method comprising the step of
administering to a subject the immunoconjugate of claim 9, in an
amount effective to treat the cancer.
18. The method of claim 17, wherein said cancer is a human
cancer.
19. The method of claim 18, wherein said human cancer is colorectal
cancer.
20. The method of claim 18, wherein said human cancer is gastric
cancer or pancreatic cancer.
21. An isolated anti-Cadherin-17 antibody comprising: (a) a heavy
chain variable region CDR1 comprising SEQ ID NO: 2, a heavy chain
variable region CDR2 comprising SEQ ID NO: 6, a heavy chain
variable region CDR3 comprising SEQ ID NO: 15, a light chain
variable region CDR1 comprising SEQ ID NO: 23, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (b) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 7, a heavy chain
variable region CDR3 comprising SEQ ID NO: 16, a light chain
variable region CDR1 comprising SEQ ID NO: 23, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (c) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 7, a heavy chain
variable region CDR3 comprising SEQ ID NO: 18, a light chain
variable region CDR1 comprising SEQ ID NO: 25, a light chain
variable region CDR2 comprising SEQ ID NO: 31, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (d) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 9, a heavy chain
variable region CDR3 comprising SEQ ID NO: 19, a light chain
variable region CDR1 comprising SEQ ID NO: 26, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (e) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 7, a heavy chain
variable region CDR3 comprising SEQ ID NO: 16, a light chain
variable region CDR1 comprising SEQ ID NO: 25, a light chain
variable region CDR2 comprising SEQ ID NO: 31, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (f) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 7, a heavy chain
variable region CDR3 comprising SEQ ID NO: 16, a light chain
variable region CDR1 comprising SEQ ID NO: 25, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (g) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 10, a heavy chain
variable region CDR3 comprising SEQ ID NO: 20, a light chain
variable region CDR1 comprising SEQ ID NO: 27, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (h) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 11, a heavy chain
variable region CDR3 comprising SEQ ID NO: 21, a light chain
variable region CDR1 comprising SEQ ID NO: 25, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; (i) a heavy chain
variable region CDR1 comprising SEQ ID NO: 3, a heavy chain
variable region CDR2 comprising SEQ ID NO: 12, a heavy chain
variable region CDR3 comprising SEQ ID NO: 18, a light chain
variable region CDR1 comprising SEQ ID NO: 25, a light chain
variable region CDR2 comprising SEQ ID NO: 31, and a light chain
variable region CDR3 comprising SEQ ID NO: 33; or (j) a heavy chain
variable region CDR1 comprising SEQ ID NO: 2, a heavy chain
variable region CDR2 comprising SEQ ID NO: 13, a heavy chain
variable region CDR3 comprising SEQ ID NO: 15, a light chain
variable region CDR1 comprising SEQ ID NO: 23, a light chain
variable region CDR2 comprising SEQ ID NO: 29, and a light chain
variable region CDR3 comprising SEQ ID NO:33.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/265,517, filed Jan. 9, 2012, which is a 35 U.S.C. 371
national stage filing of International Application No.
PCT/US2010/031719, filed Apr. 20, 2010, which claims priority to
U.S. Provisional Application No. 61/170,980, filed Apr. 20, 2009.
The contents of the aforementioned applications are hereby
incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Oct. 5,
2015, is named OTJ.sub.--012USDV_Sequence Listing.txt, and is
107,925 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the fields of
immunology and molecular biology. More specifically, provided
herein are antibodies and other therapeutic proteins directed
against cell adhesion molecule Cadherin-17, nucleic acids encoding
such antibodies and therapeutic proteins, methods for preparing
inventive monoclonal antibodies and other therapeutic proteins, and
methods for the treatment of diseases, such as cancers mediated by
Cadherin-17 expression/activity and/or associated with abnormal
expression/activity of ligands therefore.
BACKGROUND OF THE INVENTION
[0004] Cadherins are calcium dependent cell adhesion molecules.
They preferentially interact with themselves in a homophilic manner
in connecting cells; cadherins may thus contribute to the sorting
of heterogeneous cell types. The cadherin molecule Cadherin-17 is
also known as liver-intestine cadherin or intestinal
peptide-associated transporter HPT-1. Cadherin-17 may have a role
in the morphological organization of liver and intestine. It is
also involved in intestinal peptide transport. The Cadherin-17
structure is characterized as having an extracellular domain with 7
cadherin domains, a single hydrophobic transmembrane domain and a
short C-terminal cytoplasmic tail. Only one human Cadherin-17
isoform is known, Genbank.RTM. Accession No. NM 004063. Cadherin-17
has the accession number Q12864 in the SWISS-PROT and trEMBL
databases (held by the Swiss Institute of Bioinformatics (SIB) and
the European Bioinformatics Institute (EBI) which are available at
www.expasy.com). The mouse Cadherin-17 orthologue (Q9R100) shows
76% identity to the human Cadherin-17.
[0005] According to SWISS-PROT, Cadherin-17 is expressed in the
gastrointestinal tract and pancreatic duct. It is not detected in
kidney, lung, liver, brain, adrenal gland or skin.
[0006] Cadherin-17 expression has been reported in gastric cancer
(see, for example, Ito et al., Virchows Arch. 2005 October;
447(4):717-22; Su et al., Mod Pathol. 2008 November;
21(11):1379-86; Ko et al., Biochem Biophys Res Commun. 2004 Jun.
25; 319(2):562-8; and Dong et al., Dig Dis Sci. 2007 February;
52(2):536-42), pancreatic cancer and colorectal cancer (Su et al.,
Mod Pathol. 2008 November; 21(11):1379-86) and hepatocellular
carcinoma (Wong et al., Biochem Biophys Res Commun. 2003 Nov. 21;
311(3):618-24). International Patent Application WO2008/026008
discloses Cadherin-17 as a marker for colorectal cancer and as a
biological target for therapeutic antibodies and other
pharmaceutical agents.
SUMMARY OF THE INVENTION
[0007] The present invention provides antibodies directed against
Cadherin-17, nucleic acids encoding such antibodies and therapeutic
proteins, methods for preparing anti-Cadherin-17 monoclonal
antibodies and other therapeutic proteins, and methods for the
treatment of diseases, such as Cadherin-17 mediated disorders,
e.g., human cancers, including colorectal cancer.
[0008] Thus, the present invention provides isolated monoclonal
antibodies, in particular murine, chimeric, humanized, and
fully-human monoclonal antibodies, that bind to Cadherin-17 and
that exhibit one or more desirable functional property. Such
properties include, for example, high affinity specific binding to
human Cadherin-17. Also provided are methods for treating a variety
of Cadherin-17-mediated diseases using the antibodies, proteins,
and compositions of the present invention.
[0009] The present invention provides an isolated monoclonal
antibody, or an antigen-binding portion thereof, an antibody
fragment, or an antibody mimetic which binds an epitope on human
Cadherin-17 recognized by an antibody comprising a heavy chain
variable region comprising an amino acid sequence set forth in a
SEQ ID NO: selected from the group consisting of 38, 35, 36, 37,
39, 40, 41, 42, 43, 44, 45 and 46 and a light chain variable region
comprising an amino acid sequence set forth in a SEQ ID NO:
selected from the group consisting of 49, 47, 48, 50, 51, 52, 53,
54, 55, 56, 57 and 58. In some embodiments the isolated antibody is
a full-length antibody of an IgG1, IgG2, IgG3, or IgG4 isotype.
[0010] In some embodiments, the antibody of the present invention
is selected from the group consisting of: a whole antibody, an
antibody fragment, a humanized antibody, a single chain antibody,
an immunoconjugate, a defucosylated antibody, and a bispecific
antibody. The antibody fragment may be selected from the group
consisting of: a UniBody.RTM., a domain antibody, and a
Nanobody.RTM.. In some embodiments, the immunoconjugates of the
invention comprise a therapeutic agent. In another aspect of the
invention, the therapeutic agent is a cytotoxin or a radioactive
isotope.
[0011] In some embodiments, the antibody of the present invention
is selected from the group consisting of: an Affibody.RTM., a
DARPin.RTM., an Anticalin.RTM., an Avimer, a Versabody, and a
Duocalin.
[0012] In alternative embodiments, compositions of the present
invention comprise an isolated antibody or antigen-binding portion
and a pharmaceutically acceptable carrier.
[0013] In other aspects, the antibody of the present invention is a
composition comprising the isolated antibody or antigen-binding
portion according to the invention and a pharmaceutically
acceptable carrier.
[0014] In some embodiments, the invention comprises an isolated
nucleic acid molecule encoding the heavy or light chain of the
isolated antibody or antigen-binding portion which binds an epitope
on human Cadherin-17. Other aspects of the invention comprise
expression vectors comprising such nucleic acid molecules, and host
cells comprising such expression vectors.
[0015] In some embodiments, the present invention provides a method
for preparing an anti-Cadherin-17 antibody, said method comprising
the steps of: obtaining a host cell that contains one or more
nucleic acid molecules encoding the antibody of the invention;
growing the host cell in a host cell culture; providing host cell
culture conditions wherein the one or more nucleic acid molecules
are expressed; and recovering the antibody from the host cell or
from the host cell culture.
[0016] In other embodiments, the invention is directed to methods
for treating or preventing a disease associated with target cells
expressing Cadherin-17, said method comprising the step of
administering to a subject an anti-Cadherin-17 antibody, or
antigen-binding portion thereof, in an amount effective to treat or
prevent the disease. In some aspects, the disease treated or
prevented by the antibodies or antigen-binding portion thereof of
the invention, is human cancer. In some embodiments, the disease
treated or prevented by the antibodies of the present invention is
colorectal cancer.
[0017] In other embodiments, the invention is directed to an
anti-Cadherin-17 antibody, or antigen-binding portion thereof, for
use in treating or preventing a disease associated with target
cells expressing Cadherin-17. In some aspects, the disease treated
or prevented by the antibodies or antigen-binding portion thereof
of the invention, is human cancer. In some embodiments, the disease
treated or prevented by the antibodies of the present invention is
colorectal cancer.
[0018] In other embodiments, the invention is directed to the use
of an anti-Cadherin-17 antibody, or antigen-binding portion
thereof, for the manufacture of a medicament for use in treating or
preventing a disease associated with target cells expressing
Cadherin-17. In some aspects, the disease treated or prevented by
the medicament of the invention, is human cancer. In some
embodiments, the disease treated or prevented by the medicament of
the present invention is colorectal cancer.
[0019] In other embodiments, the present invention is an isolated
monoclonal antibody or an antigen binding portion thereof, an
antibody fragment, or an antibody mimetic which binds an epitope on
human Cadherin-17 recognized by an antibody comprising a heavy
chain variable region and a light chain variable region selected
from the group consisting of the heavy chain variable region amino
acid sequence set forth in SEQ ID NO:35 and the light chain
variable region amino acid sequence set forth in SEQ ID NO:47; the
heavy chain variable region amino acid sequence set forth in SEQ ID
NO:36 and the light chain variable region amino acid sequence set
forth in SEQ ID NO:48; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:37 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:48; the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:38
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:49; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:39 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:50; the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:40
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:51; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:40 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:54; the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:40
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:55; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:41 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:52; the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:42
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:53; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:43 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:56; the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:44
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:55; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:45 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:57; and the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:46
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:58. In further aspects, the antibody is selected from
the group consisting of: a whole antibody, an antibody fragment, a
humanized antibody, a single chain antibody, an immunoconjugate, a
defucosylated antibody, and a bispecific antibody. In further
aspects of the invention, the antibody fragment is selected from
the group consisting of: a UniBody.RTM., a domain antibody, and a
Nanobody.RTM.. In some embodiments, the antibody mimetic is
selected from the group consisting of: an Affibody.RTM., a
DARPin.RTM., an Anticalin.RTM., an Avimer, a Versabody, and a
Duocalin. In further embodiments, the composition comprises the
isolated antibody or antigen binding portion thereof and a
pharmaceutically acceptable carrier.
[0020] In some embodiments, the present invention is an isolated
nucleic acid molecule encoding the heavy or light chain of the
isolated antibody or antigen binding portion thereof of antibody of
the invention, and in further aspects may include an expression
vector comprising such nucleic acids, and host cells comprising
such expression vectors.
[0021] Another embodiment of the present invention is a hybridoma
expressing the antibody or antigen binding portion thereof of any
one of antibodies of the invention.
[0022] Other aspects of the invention are directed to methods of
making the antibodies of the invention, comprising the steps of:
[0023] immunizing an animal with a Cadherin-17 peptide; [0024]
recovering mRNA from the B cells of said animal; [0025] converting
said mRNA to cDNA; [0026] expressing said cDNA in phages such that
anti-Cadherin-17 antibodies encoded by said cDNA are presented on
the surface of said phages; [0027] selecting phages that present
anti-Cadherin-17 antibodies; [0028] recovering nucleic acid
molecules from said selected phages that encode said
anti-Cadherin-17 immunoglobulins; [0029] expressing said recovered
nucleic acid molecules in a host cell; and [0030] recovering
antibodies from said host cell that bind Cadherin-17.
[0031] In some aspects of the invention, the isolated monoclonal
antibody, or an antigen binding portion thereof, binds an epitope
on the Cadherin-17 polypeptide having an amino acid sequence of SEQ
ID NOS: 136 or 137 recognized by an antibody comprising a heavy
chain variable region comprising an amino acid sequence set forth
in a SEQ ID NO: selected from the group consisting of 38, 35, 36,
37, 39, 40, 41, 42, 43, 44, 45, or 46 and a light chain variable
region comprising an amino acid sequence set forth in a SEQ ID NO:
selected from the group consisting of 49, 47, 48, 50, 51, 52, 53,
54, 55, 56, 57, or 58.
[0032] Other features and advantages of the instant invention will
be apparent from the following detailed description and examples
which should not be construed as limiting. The contents of all
references, Genbank.RTM. entries, patents and published patent
applications cited throughout this application are expressly
incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows the nucleotide sequence (SEQ ID NO:59) and
amino acid sequence (SEQ ID NO:35) of the heavy chain variable
region of the PTA001_A1 monoclonal antibody. The CDR1 (SEQ ID
NO:1), CDR2 (SEQ ID NO:5) and CDR3 (SEQ ID NO:14) regions are
delineated.
[0034] FIG. 2 shows the nucleotide sequence (SEQ ID NO:60) and
amino acid sequence (SEQ ID NO:36) of the heavy chain variable
region of the PTA001_A2 monoclonal antibody. The CDR1 (SEQ ID
NO:2), CDR2 (SEQ ID NO:6) and CDR3 (SEQ ID NO:15) regions are
delineated.
[0035] FIG. 3 shows the nucleotide sequence (SEQ ID NO:61) and
amino acid sequence (SEQ ID NO:37) of the heavy chain variable
region of the PTA001_A3 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:7) and CDR3 (SEQ ID NO:16) regions are
delineated.
[0036] FIG. 4 shows the nucleotide sequence (SEQ ID NO:62) and
amino acid sequence (SEQ ID NO:38) of the heavy chain variable
region of the PTA001_A4 monoclonal antibody. The CDR1 (SEQ ID
NO:4), CDR2 (SEQ ID NO:8) and CDR3 (SEQ ID NO:17) regions are
delineated.
[0037] FIG. 5 shows the nucleotide sequence (SEQ ID NO:63) and
amino acid sequence (SEQ ID NO:39) of the heavy chain variable
region of the PTA001_A5 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:7) and CDR3 (SEQ ID NO:18) regions are
delineated.
[0038] FIG. 6 shows the nucleotide sequence (SEQ ID NO:64) and
amino acid sequence (SEQ ID NO:40) of the heavy chain variable
region of the PTA001_A6, PTA001_A9 and PTA001_A10 monoclonal
antibodies. The CDR1 (SEQ ID NO:3), CDR2 (SEQ ID NO:7) and CDR3
(SEQ ID NO: 16) regions are delineated.
[0039] FIG. 7 shows the nucleotide sequence (SEQ ID NO:65) and
amino acid sequence (SEQ ID NO:41) of the heavy chain variable
region of the PTA001_A7 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:9) and CDR3 (SEQ ID NO:19) regions are
delineated.
[0040] FIG. 8 shows the nucleotide sequence (SEQ ID NO:66) and
amino acid sequence (SEQ ID NO:42) of the heavy chain variable
region of the PTA001_A8 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:7) and CDR3 (SEQ ID NO:16) regions are
delineated.
[0041] FIG. 9 shows the nucleotide sequence (SEQ ID NO:67) and
amino acid sequence (SEQ ID NO:43) of the heavy chain variable
region of the PTA001_A11 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:10) and CDR3 (SEQ ID NO:20) regions are
delineated.
[0042] FIG. 10 shows the nucleotide sequence (SEQ ID NO:68) and
amino acid sequence (SEQ ID NO:44) of the heavy chain variable
region of the PTA001_A12 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO:21) regions are
delineated.
[0043] FIG. 11 shows the nucleotide sequence (SEQ ID NO:69) and
amino acid sequence (SEQ ID NO:45) of the heavy chain variable
region of the PTA001_A13 monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:12) and CDR3 (SEQ ID NO:18) regions are
delineated.
[0044] FIG. 12 shows the nucleotide sequence (SEQ ID NO:70) and
amino acid sequence (SEQ ID NO:46) of the heavy chain variable
region of the PTA001_A14 monoclonal antibody. The CDR1 (SEQ ID
NO:2), CDR2 (SEQ ID NO:13) and CDR3 (SEQ ID NO:15) regions are
delineated.
[0045] FIG. 13 shows the nucleotide sequence (SEQ ID NO:71) and
amino acid sequence (SEQ ID NO:47) of the light chain variable
region of the PTA001_A1 monoclonal antibody. The CDR1 (SEQ ID
NO:22), CDR2 (SEQ ID NO:28) and CDR3 (SEQ ID NO:32) regions are
delineated.
[0046] FIG. 14 shows the nucleotide sequence (SEQ ID NO:72) and
amino acid sequence (SEQ ID NO:48) of the light chain variable
region of the PTA001_A2 and PTA001_A3 monoclonal antibodies. The
CDR1 (SEQ ID NO:23), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33)
regions are delineated.
[0047] FIG. 15 shows the nucleotide sequence (SEQ ID NO:73) and
amino acid sequence (SEQ ID NO:49) of the light chain variable
region of the PTA001_A4 monoclonal antibody. The CDR1 (SEQ ID
NO:24), CDR2 (SEQ ID NO:30) and CDR3 (SEQ ID NO:34) regions are
delineated.
[0048] FIG. 16 shows the nucleotide sequence (SEQ ID NO:74) and
amino acid sequence (SEQ ID NO:50) of the light chain variable
region of the PTA001_A5 monoclonal antibody. The CDR1 (SEQ ID
NO:25), CDR2 (SEQ ID NO:31) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0049] FIG. 17 shows the nucleotide sequence (SEQ ID NO:75) and
amino acid sequence (SEQ ID NO:51) of the light chain variable
region of the PTA001_A6 monoclonal antibody. The CDR1 (SEQ ID
NO:23), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0050] FIG. 18 shows the nucleotide sequence (SEQ ID NO:76) and
amino acid sequence (SEQ ID NO:52) of the light chain variable
region of the PTA001_A7 monoclonal antibody. The CDR1 (SEQ ID
NO:26), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0051] FIG. 19 shows the nucleotide sequence (SEQ ID NO:77) and
amino acid sequence (SEQ ID NO:53) of the light chain variable
region of the PTA001_A8 monoclonal antibody. The CDR1 (SEQ ID
NO:25), CDR2 (SEQ ID NO:31) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0052] FIG. 20 shows the nucleotide sequence (SEQ ID NO:78) and
amino acid sequence (SEQ ID NO:54) of the light chain variable
region of the PTA001_A9 monoclonal antibody. The CDR1 (SEQ ID
NO:23), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0053] FIG. 21 shows the nucleotide sequence (SEQ ID NOs:79 and 81)
and amino acid sequence (SEQ ID NO:55) of the light chain variable
region of the PTA001_A10 and PTA001_A12 monoclonal antibodies. The
CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33)
regions are delineated.
[0054] FIG. 22 shows the nucleotide sequence (SEQ ID NO:80) and
amino acid sequence (SEQ ID NO:56) of the light chain variable
region of the PTA001_All monoclonal antibody. The CDR1 (SEQ ID
NO:27), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0055] FIG. 23 shows the nucleotide sequence (SEQ ID NO:82) and
amino acid sequence (SEQ ID NO:57) of the light chain variable
region of the PTA001_A13 monoclonal antibody. The CDR1 (SEQ ID
NO:25), CDR2 (SEQ ID NO:31) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0056] FIG. 24 shows the nucleotide sequence (SEQ ID NO:83) and
amino acid sequence (SEQ ID NO:58) of the light chain variable
region of the PTA001_A14 monoclonal antibody. The CDR1 (SEQ ID
NO:23), CDR2 (SEQ ID NO:29) and CDR3 (SEQ ID NO:33) regions are
delineated.
[0057] FIG. 25 shows the alignment of the nucleotide sequences of
the heavy chain CDR1 region of PTA001_A1 (SEQ ID NO:84) with
nucleotides 240-269 of the mouse germline V.sub.H 7-39 nucleotide
sequence (SEQ ID NO:125) and the alignments of the nucleotide
sequences of the heavy chain CDR1 regions of PTA001_A2 and
PTA001_A14 (SEQ ID NO:85); PTA001_A3, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12
and PTA001_A13 (SEQ ID NO:86); and PTA001_A4 (SEQ ID NO:87) with
nucleotides 67-96 of the mouse germline V.sub.H II gene H17
nucleotide sequence (SEQ ID NO:126).
[0058] FIG. 26 shows the alignments of the nucleotide sequences of
the heavy chain CDR2 regions of PTA001_A2 (SEQ ID NO:89);
PTA001_A3, PTA001_A5, PTA001_A6, PTA001_A8, PTA001_A9 and
PTA001_A10 (SEQ ID NO:90); PTA001_A4 (SEQ ID NO:91); PTA001_A7 (SEQ
ID NO:92); PTA001_A11 (SEQ ID NO:93); PTA001_A12 (SEQ ID NO:94);
PTA001_A13 (SEQ ID NO:95); and PTA001_A14 (SEQ ID NO:96) with
nucleotides 1096-1146 of the mouse germline V.sub.H II region VH105
nucleotide sequence (SEQ ID NO:127).
[0059] FIG. 27 shows the alignment of the nucleotide sequence of
the light chain CDR1 region of PTA001_A1 (SEQ ID NO:105) with
nucleotides 1738-1785 of the mouse germline V.sub.K 1-110
nucleotide sequence (SEQ ID NO:128), the alignment of the
nucleotide sequence of the light chain CDR1 region of PTA001_A4
(SEQ ID NO:107) with nucleotides 510-560 of the mouse germline
V.sub.K 8-30 nucleotide sequence (SEQ ID NO: 130) and the
alignments of the nucleotide sequences of the light chain CDR1
regions of PTA001_A2, PTA001_A3, PTA001_A6, PTA001_A9 and
PTA001_A14 (SEQ ID NO:106); PTA001_A5 and PTA001_A13 (SEQ ID
NO:108); PTA001_A7 (SEQ ID NO:109); PTA001_A8 (SEQ ID NO:110);
PTA001_A10 (SEQ ID NO:111); PTA001_A11 (SEQ ID NO:112); and
PTA001_A112 (SEQ ID NO:113) with nucleotides 1807-1854 of the mouse
germline V.sub.K 24-140 nucleotide sequence (SEQ ID NO: 133).
[0060] FIG. 28 shows the alignment of the nucleotide sequence of
the light chain CDR2 region of PTA001_A4 (SEQ ID NO:116) with
nucleotides 606-626 of the mouse germline V.sub.K 8-30 nucleotide
sequence (SEQ ID NO:131) and the alignments of the nucleotide
sequences of the light chain CDR2 regions of PTA001_A2, PTA001_A3,
PTA001_A6, PTA001_A9 and PTA001_A14 (SEQ ID NO:115); PTA001_A5 and
PTA001_A13 (SEQ ID NO:117); PTA001_A7, PTA001_A10, PTA001_A11 and
PTA001_A12 (SEQ ID NO:118); and PTA001_A8 (SEQ ID NO: 119) with
nucleotides 1900-1920 of the mouse germline V.sub.K 24-140
nucleotide sequence (SEQ ID NO: 134).
[0061] FIG. 29 shows the alignment of the nucleotide sequence of
the light chain CDR3 region of PTA001_A1 (SEQ ID NO:120) with
nucleotides 1948-1971 of the mouse germline V.sub.K 1-110
nucleotide sequence (SEQ ID NO:129), the alignment of the
nucleotide sequence of the light chain CDR3 region of PTA001_A4
(SEQ ID NO:122) with nucleotides 723-749 of the mouse germline
V.sub.K 8-30 nucleotide sequence (SEQ ID NO:132) and the alignments
of the nucleotide sequences of the light chain CDR3 regions of
PTA001_A2, PTA001_A3, PTA001_A6, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12 and PTA001_A14 (SEQ ID NO:121); PTA001_A5, PTA001_A8 and
PTA001_A13 (SEQ ID NO:123); and PTA001_A7 (SEQ ID NO: 124) with
nucleotides 2017-2043 of the mouse germline V.sub.K 24-140
nucleotide sequence (SEQ ID NO:135).
[0062] FIG. 30 shows the Western Blotting analysis of Cadherin-17
using the PTA001_A4 monoclonal antibody.
[0063] FIG. 31 shows results of FACS analysis on PTA001_A4 in LoVo
cells.
[0064] FIG. 32 shows results of FACS analysis on PTA001_A4 in LoVo
and LS 174T cells.
[0065] FIG. 33A shows surface binding of PTA001_A4/secondary
antibody FITC conjugate complex to LoVo cells after 60 minutes of
incubation.
[0066] FIG. 33B shows internalization of PTA001_A4/secondary
antibody FITC conjugate complex after 120 minutes of incubation
with LoVo cells.
[0067] FIG. 34A shows results of internalisation of PTA001_A4 by
MabZAP assay in LoVo colon cancer cells.
[0068] FIG. 34B shows results of internalisation of PTA001_A4 by
MabZAP assay in LoVo colon cancer cells.
[0069] FIG. 34C shows results of internalisation of PTA001_A4 by
MabZAP assay in LS174T colon cancer cells.
[0070] FIG. 34D shows results of internalisation of PTA001_A4 by
MabZAP assay in LS174T colon cancer cells.
[0071] FIG. 35A shows results of internalisation of PTA001_A4 by
MabZAP assay in LoVo colon cancer cells.
[0072] FIG. 35B shows results of internalisation of PTA001_A4 by
MabZAP assay in LS174T colon cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The present invention relates to isolated antibodies,
including, but not limited to monoclonal antibodies, for example,
which bind specifically to Cadherin-17 with high affinity. In
certain embodiments, the antibodies of the invention comprise
particular structural features such as CDR regions comprising
particular amino acid sequences. The invention provides isolated
antibodies, defucosylated antibodies, immunoconjugates, bispecific
molecules, affibodies, domain antibodies, Nanobodies.RTM., and
Unibodies.RTM., methods of making said molecules, and
pharmaceutical compositions comprising said molecules and a
pharmaceutical carrier. The invention also relates to methods of
using the molecules, such as to detect Cadherin-17, as well as to
treat diseases associated with expression of Cadherin-17, such as
Cadherin-17 expressed on tumors, including those tumors of
colorectal cancer.
[0074] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0075] The terms "Cadherin-17", "Liver-intestine cadherin",
"LI-cadherin", "Intestinal peptide-associated transported HPT-1"
and "CDH17" are used interchangeably. Cadherin-17 has also been
identified as OGTA001 in International Patent Application
WO2008/026008, which is incorporated herein by reference in its
entirety. Human antibodies of this disclosure may, in certain
cases, cross-react with Cadherin-17 from species other than human.
In certain embodiments, the antibodies may be completely specific
for one or more human Cadherin-17 and may not exhibit species or
other types of non-human cross-reactivity. The complete amino acid
sequence of an exemplary human Cadherin-17 has Genbank.RTM.
accession number NM.sub.--004063.
[0076] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0077] A "signal transduction pathway" refers to the biochemical
relationship between various of signal transduction molecules that
play a role in the transmission of a signal from one portion of a
cell to another portion of a cell. As used herein, the phrase "cell
surface receptor" includes, for example, molecules and complexes of
molecules capable of receiving a signal and the transmission of
such a signal across the plasma membrane of a cell. An example of a
"cell surface receptor" of the present invention is the Cadherin-17
receptor.
[0078] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. An "antibody" refers to a
glycoprotein which may comprise at least two heavy (H) chains and
two light (L) chains inter-connected by disulfide bonds, or an
antigen binding portion thereof. Each heavy chain is comprised of a
heavy chain variable region (abbreviated herein as V.sub.H) and a
heavy chain constant region. The heavy chain constant region is
comprised of three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each
light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L or V.sub.K) and a light chain
constant region. The light chain constant region is comprised of
one domain, C.sub.L. The VH and V.sub.L/V.sub.K regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each V.sub.H and V.sub.L/V.sub.K is composed of three CDRs and four
FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable
regions of the heavy and light chains contain a binding domain that
interacts with an antigen. The constant regions of the antibodies
may mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g.,
effector cells) and the first component (Clq) of the classical
complement system.
[0079] The definition of "antibody" includes, but is not limited
to, full length antibodies, antibody fragments, single chain
antibodies, bispecific antibodies, minibodies, domain antibodies,
synthetic antibodies (sometimes referred to herein as "antibody
mimetics"), chimeric antibodies, humanized antibodies, antibody
fusions (sometimes referred to as "antibody conjugates"), and
fragments of each, respectively.
[0080] In one embodiment, the antibody is an antibody fragment.
Specific antibody fragments include, but are not limited to, (i)
the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the
Fd fragment consisting of the VH and CH1 domains, (iii) the Fv
fragment consisting of the VL and VH domains of a single antibody,
(iv) the dAb fragment, which consists of a single variable domain,
(v) isolated CDR regions, (vi) F(ab')2 fragments, a bivalent
fragment comprising two linked Fab fragments (vii) single chain Fv
molecules (scFv), wherein a VH domain and a VL domain are linked by
a peptide linker which allows the two domains to associate to form
an antigen binding site, (viii) bispecific single chain Fv dimers,
and (ix) "diabodies" or "triabodies", multivalent or multispecific
fragments constructed by gene fusion. The antibody fragments may be
modified. For example, the molecules may be stabilized by the
incorporation of disulfide bridges linking the VH and VL domains.
Examples of antibody formats and architectures are described in
Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136,
and Carter 2006, Nature Reviews Immunology 6:343-357 and references
cited therein, all expressly incorporated by reference. In one
embodiment, an antibody disclosed herein may be a multispecific
antibody, and notably a bispecific antibody, also sometimes
referred to as "diabodies". These are antibodies that bind to two
(or more) different antigens. Diabodies can be manufactured in a
variety of ways known in the art, e.g., prepared chemically or from
hybrid hybridomas. In one embodiment, the antibody is a minibody.
Minibodies are minimized antibody-like proteins comprising a scFv
joined to a CH3 domain. In some cases, the scFv can be joined to
the Fc region, and may include some or all of the hinge region. For
a description of multispecific antibodies see Holliger &
Hudson, 2006, Nature Biotechnology 23(9):1126-1136 and references
cited therein, all expressly incorporated by reference.
[0081] By "CDR" as used herein is meant a Complementarity
Determining Region of an antibody variable domain. Systematic
identification of residues included in the CDRs have been developed
by Kabat (Kabat et al., 1991, Sequences of Proteins of
Immunological Interest, 5th Ed., United States Public Health
Service, National Institutes of Health, Bethesda) and alternately
by Chothia (Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917;
Chothia et al., 1989, Nature 342: 877-883; Al-Lazikani et al.,
1997, J. Mol. Biol. 273: 927-948). For the purposes of the present
invention, CDRs are defined as a slightly smaller set of residues
than the CDRs defined by Chothia. VL CDRs are herein defined to
include residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97
(CDR3), wherein the numbering is according to Chothia. Because the
VL CDRs as defined by Chothia and Kabat are identical, the
numbering of these VL CDR positions is also according to Kabat. VH
CDRs are herein defined to include residues at positions 27-33
(CDR1), 52-56 (CDR2), and 95-102 (CDR3), wherein the numbering is
according to Chothia. These VH CDR positions correspond to Kabat
positions 27-35 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).
[0082] As will be appreciated by those in the art, the CDRs
disclosed herein may also include variants. for example when
backmutating the CDRs disclosed herein into different framework
regions. Generally, the amino acid identity between individual
variant CDRs are at least 80% to the sequences depicted herein, and
more typically with preferably increasing identities of at least
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost
100%. In a similar manner, "percent (%) nucleic acid sequence
identity" with respect to the nucleic acid sequence of the binding
proteins identified herein is defined as the percentage of
nucleotide residues in a candidate sequence that are identical with
the nucleotide residues in the coding sequence of the antigen
binding protein. A specific method utilizes the BLASTN module of
WU-BLAST-2 set to the default parameters, with overlap span and
overlap fraction set to 1 and 0.125, respectively.
[0083] Generally, the nucleic acid sequence identity between the
nucleotide sequences encoding individual variant CDRs and the
nucleotide sequences depicted herein are at least 80%, and more
typically with preferably increasing identities of at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.
[0084] Thus, a "variant CDR" is one with the specified homology,
similarity, or identity to the parent CDR of the invention, and
shares biological function, including, but not limited to, at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or
activity of the parent CDR.
[0085] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed antigen binding
protein CDR variants screened for the optimal combination of
desired activity. Techniques for making substitution mutations at
predetermined sites in DNA having a known sequence are well known,
for example, M13 primer mutagenesis and PCR mutagenesis. Screening
of the mutants is done using assays of antigen binding protein
activities as described herein.
[0086] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about one (1) to
about twenty (20) amino acid residues, although considerably larger
insertions may be tolerated. Deletions range from about one (1) to
about twenty (20) amino acid residues, although in some cases
deletions may be much larger.
[0087] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative or variant.
Generally these changes are done on a few amino acids to minimize
the alteration of the molecule, particularly the immunogenicity and
specificity of the antigen binding protein. However, larger changes
may be tolerated in certain circumstances. By "Fab" or "Fab region"
as used herein is meant the polypeptide that comprises the VH, CH1,
VL, and CL immunoglobulin domains. Fab may refer to this region in
isolation, or this region in the context of a full length antibody,
antibody fragment or Fab fusion protein, or any other antibody
embodiments as outlined herein.
[0088] By "Fv" or "Fv fragment" or "Fv region" as used herein is
meant a polypeptide that comprises the VL and VH domains of a
single antibody.
[0089] By "framework" as used herein is meant the region of an
antibody variable domain exclusive of those regions defined as
CDRs. Each antibody variable domain framework can be further
subdivided into the contiguous regions separated by the CDRs (FR1,
FR2, FR3 and FR4).
[0090] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., Cadherin-17). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L/V.sub.K, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fab' fragment, which is essentially an Fab
with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul
ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the
V.sub.H and C.sub.H1 domains; (v) a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody; (vi) a
dAb fragment (Ward et al., (1989) Nature 341:544-546), which
consists of a V.sub.H domain; (vii) an isolated complementarity
determining region (CDR); and (viii) a Nanobody.RTM., a heavy chain
variable region containing a single variable domain and two
constant domains. Furthermore, although the two domains of the Fv
fragment, V.sub.L/V.sub.K and V.sub.H, are coded for by separate
genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L/V.sub.K and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies.
[0091] An "isolated antibody" as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds Cadherin-17 is substantially free
of antibodies that specifically bind antigens other than
Cadherin-17). An isolated antibody that specifically binds
Cadherin-17 may, however, have cross-reactivity to other antigens,
such as Cadherin-17 molecules from other species. Moreover, and/or
alternatively an isolated antibody may be substantially free of
other cellular material and/or chemicals in a form not normally
found in nature.
[0092] In some embodiments, the antibodies of the invention are
recombinant proteins, isolated proteins or substantially pure
proteins. An "isolated" protein is unaccompanied by at least some
of the material with which it is normally associated in its natural
state, for example constituting at least about 5%, or at least
about 50% by weight of the total protein in a given sample. It is
understood that the isolated protein may constitute from 5 to 99.9%
by weight of the total protein content depending on the
circumstances. For example, the protein may be made at a
significantly higher concentration through the use of an inducible
promoter or high expression promoter, such that the protein is made
at increased concentration levels. In the case of recombinant
proteins, the definition includes the production of an antibody in
a wide variety of organisms and/or host cells that are known in the
art in which it is not naturally produced.
[0093] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0094] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by the heavy chain constant
region genes.
[0095] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen".
[0096] The term "antibody derivatives" refers to any modified form
of the antibody, e.g., a conjugate of the antibody and another
agent or antibody. For example, antibodies of the present invention
may be conjugated to a toxin, a label, etc. The antibodies of the
present invention may be nonhuman, chimeric, humanized, or fully
human. For a description of the concepts of chimeric and humanized
antibodies see Clark et al., 2000 and references cited therein
(Clark, 2000, Immunol Today 21:397-402). Chimeric antibodies
comprise the variable region of a nonhuman antibody, for example VH
and VL domains of mouse or rat origin, operably linked to the
constant region of a human antibody (see for example U.S. Pat. No.
4,816,567). In a preferred embodiment, the antibodies of the
present invention are humanized. By "humanized" antibody as used
herein is meant an antibody comprising a human framework region
(FR) and one or more complementarity determining regions (CDR's)
from a non-human (usually mouse or rat) antibody. The non-human
antibody providing the CDR's is called the "donor" and the human
immunoglobulin providing the framework is called the "acceptor".
Humanization relies principally on the grafting of donor CDRs onto
acceptor (human) VL and VH frameworks (U.S. Pat. No. 5,225,539).
This strategy is referred to as "CDR grafting". "Backmutation" of
selected acceptor framework residues to the corresponding donor
residues is often required to regain affinity that is lost in the
initial grafted construct (U.S. Pat. No. 5,530,101; U.S. Pat. No.
5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S.
Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No.
5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region, typically that of a human
immunoglobulin, and thus will typically comprise a human Fc region.
Methods for humanizing non-human antibodies are well known in the
art, and can be essentially performed following the method of
Winter and co-workers (Jones et al., 1986, Nature 321:522-525;
Riechmann et al., 1988, Nature 332:323-329; Verhoeyen et al., 1988,
Science, 239:1534-1536). Additional examples of humanized murine
monoclonal antibodies are also known in the art, for example
antibodies binding human protein C (O'Connor et al., 1998, Protein
Eng 11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc
Natl Acad Sci, USA 86:10029-33), and human epidermal growth factor
receptor 2 (Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9).
In an alternate embodiment, the antibodies of the present invention
may be fully human, that is the sequences of the antibodies are
completely or substantially human. A number of methods are known in
the art for generating fully human antibodies, including the use of
transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol
8:455-458) or human antibody libraries coupled with selection
methods (Griffiths et al., 1998, Curr Opin Biotechnol
9:102-108).
[0097] The term "humanized antibody" is intended to refer to
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences. Additional framework region
modifications may be made within the human framework sequences.
[0098] The term "chimeric antibody" is intended to refer to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody.
[0099] The term "specifically binds" (or "immunospecifically
binds") is not intended to indicate that an antibody binds
exclusively to its intended target. Rather, an antibody
"specifically binds" if its affinity for its intended target is
about 5-fold greater when compared to its affinity for a non-target
molecule. Suitably there is no significant cross-reaction or
cross-binding with undesired substances, especially naturally
occurring proteins or tissues of a healthy person or animal. The
affinity of the antibody will, for example, be at least about 5
fold, such as 10 fold, such as 25-fold, especially 50-fold, and
particularly 100-fold or more, greater for a target molecule than
its affinity for a non-target molecule. In some embodiments,
specific binding between an antibody or other binding agent and an
antigen means a binding affinity of at least 10.sup.6 M.sup.-1.
Antibodies may, for example, bind with affinities of at least about
10.sup.7 M.sup.-1, such as between about 10.sup.8 M.sup.-1 to about
10.sup.9 M.sup.-1, about 10.sup.9 M.sup.-1 to about 10.sup.10
M.sup.-1, or about 10.sup.10 M.sup.-1 to about 10.sup.11 M.sup.-1.
Antibodies may, for example, bind with an EC.sub.50 of 50 nM or
less, 10 nM or less, 1 nM or less, 100 pM or less, or more
preferably 10 pM or less.
[0100] The term "does not substantially bind" to a protein or
cells, as used herein, means does not bind or does not bind with a
high affinity to the protein or cells, i.e. binds to the protein or
cells with a K.sub.D of 1.times.10.sup.-6 M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more.
[0101] The term "EC.sub.50" as used herein, is intended to refer to
the potency of a compound by quantifying the concentration that
leads to 50% maximal response/effect. EC.sub.50 may be determined
by Scratchard or FACS.
[0102] The term "K.sub.assoc" or "K.sub.a," as used herein, is
intended to refer to the association rate of a particular
antibody-antigen interaction, whereas the term "K.sub.dis" or
"K.sub.d," as used herein, is intended to refer to the dissociation
rate of a particular antibody-antigen interaction. The term
"K.sub.D," as used herein, is intended to refer to the dissociation
constant, which is obtained from the ratio of K.sub.dto K.sub.a
(i.e., K.sub.d/K.sub.a) and is expressed as a molar concentration
(M). K.sub.D values for antibodies can be determined using methods
well established in the art. A preferred method for determining the
K.sub.D of an antibody is by using surface plasmon resonance,
preferably using a biosensor system such as a Biacore.RTM.
system.
[0103] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D of 1.times.10.sup.-7 M or
less, more preferably 5.times.10-8 M or less, even more preferably
1.times.10.sup.-8 M or less, even more preferably 5.times.10.sup.-9
M or less and even more preferably 1.times.10.sup.-9 M or less for
a target antigen. However, "high affinity" binding can vary for
other antibody isotypes. For example, "high affinity" binding for
an IgM isotype refers to an antibody having a K.sub.D of 10.sup.-6
M or less, more preferably 10.sup.-7 M or less, even more
preferably 10.sup.-8 M or less.
[0104] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include techniques in the art and those described
herein, for example, x-ray crystallography and 2-dimensional
nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in
Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
(1996)).
[0105] Accordingly, also encompassed by the present invention are
antibodies that bind to (i.e., recognize) the same epitope as the
antibodies described herein (i.e., PTA001_A1, PTA001_A2, PTA001_A3,
PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9,
PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 and PTA001_A14).
Antibodies that bind to the same epitope can be identified by their
ability to cross-compete with (i.e., competitively inhibit binding
of) a reference antibody to a target antigen in a statistically
significant manner. Competitive inhibition can occur, for example,
if the antibodies bind to identical or structurally similar
epitopes (e.g., overlapping epitopes), or spatially proximal
epitopes which, when bound, causes steric hindrance between the
antibodies.
[0106] Competitive inhibition can be determined using routine
assays in which the immunoglobulin under test inhibits specific
binding of a reference antibody to a common antigen. Numerous types
of competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label RIA using I-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer
et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0107] Other techniques include, for example, epitope mapping
methods, such as x-ray analyses of crystals of antigen:antibody
complexes which provides atomic resolution of the epitope. Other
methods monitor the binding of the antibody to antigen fragments or
mutated variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component. In
addition, computational combinatorial methods for epitope mapping
can also be used. These methods rely on the ability of the antibody
of interest to affinity isolate specific short peptides from
combinatorial phage display peptide libraries. The peptides are
then regarded as leads for the definition of the epitope
corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been
developed which have been shown to map conformational discontinuous
epitopes.
[0108] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dogs, cats, horses, cows, chickens, amphibians,
reptiles, etc.
[0109] Various aspects of the invention are described in further
detail in the following subsections.
Anti-Cadherin-17 Antibodies
[0110] The antibodies of the invention are characterized by
particular functional features or properties of the antibodies. For
example, the antibodies bind specifically to human Cadherin-17.
Preferably, an antibody of the invention binds to Cadherin-17 with
high affinity, for example with a K.sub.D of 8.times.10.sup.-7 M or
less, even more typically 1.times.10.sup.-8 M or less. The
anti-Cadherin-17 antibodies of the invention preferably exhibit one
or more of the following characteristics: [0111] binds to human
Cadherin-17 with a EC.sub.50 of 50 nM or less, 10 nM or less, 1 nM
or less, 100 pM or less, or more preferably 10 pM or less; [0112]
binds to human cells expressing Cadherin-17.
[0113] In one embodiment, the antibodies preferably bind to an
antigenic epitope present in Cadherin-17, which epitope is not
present in other proteins. The antibodies typically bind
Cadherin-17 but does not bind to other proteins, or binds to
proteins with a low affinity, such as a K.sub.D of
1.times.10.sup.-6 M or more, more preferably 1.times.10.sup.-5 M or
more, more preferably 1.times.10.sup.-4 M or more, more preferably
1.times.10.sup.-3 M or more, even more preferably 1.times.10.sup.-2
M or more. Preferably, the antibodies do not bind to related
proteins, for example, the antibodies do not substantially bind to
other cell adhesion molecules. In one embodiment, the antibody may
be internalized into a cell expressing Cadherin-17. Standard assays
to evaluate antibody internalization are known in the art,
including, for example, a HumZap internalization assay.
[0114] Standard assays to evaluate the binding ability of the
antibodies toward Cadherin-17 are known in the art, including for
example, ELISAs, Western blots, RIAs, and flow cytometry analysis.
Suitable assays are described in detail in the Examples. The
binding kinetics (e.g., binding affinity) of the antibodies also
can be assessed by standard assays known in the art, such as by
Biacore.RTM. system analysis. To assess binding to Raji or Daudi B
cell tumor cells, Raji (ATCC Deposit No. CCL-86) or Daudi (ATCC
Deposit No. CCL-213) cells can be obtained from publicly available
sources, such as the American Type Culture Collection, and used in
standard assays, such as flow cytometric analysis.
Monoclonal Antibodies of the Invention
[0115] Preferred antibodies of the invention are the monoclonal
antibodies PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5,
PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14, isolated and structurally
characterized as described in Examples 1-6. The V.sub.H amino acid
sequences of PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5,
PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ ID
NOs:35-46. The V.sub.K amino acid sequences of PTA001_A1,
PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7,
PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12,
PTA001_A13 and PTA001_A14 are shown in SEQ ID NOs:47-58.
[0116] Given that each of these antibodies can bind to Cadherin-17,
the V.sub.H and V.sub.K sequences can be "mixed and matched" to
create other anti-Cadherin-17 binding molecules of the invention.
Cadherin-17 binding of such "mixed and matched" antibodies can be
tested using the binding assays described above and in the Examples
(e.g., ELISAs). Preferably, when V.sub.H and V.sub.K chains are
mixed and matched, a V.sub.H sequence from a particular
V.sub.H/V.sub.K pairing is replaced with a structurally similar
V.sub.H sequence. Likewise, preferably a V.sub.K sequence from a
particular V.sub.H/V.sub.K pairing is replaced with a structurally
similar V.sub.K sequence.
[0117] Accordingly, in one aspect, the invention provides an
antibody, comprising:
a heavy chain variable region comprising an amino acid sequence set
forth in a SEQ ID NO: selected from the group consisting of 38, 35,
36, 37, 39, 40, 41, 42, 43, 44, 45 and 46 and a light chain
variable region comprising an amino acid sequence set forth in a
SEQ ID NO: selected from the group consisting of 49, 47, 48, 50,
51, 52, 53, 54, 55, 56, 57 and 58; wherein the antibody
specifically binds Cadherin-17, preferably human Cadherin-17.
[0118] Preferred heavy and light chain combinations include:
a heavy chain variable region comprising the amino acid sequence of
SEQ ID NO:35 and a light chain variable region comprising the amino
acid sequence of SEQ ID NO:47; or a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:36; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO:48, or a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:37; and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:48, or a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO:38; and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:49, or a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:39; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO:50, or a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:40; and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:51, or a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO:41; and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:52, or a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:42; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO:53, or a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:40; and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:54, or a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO:40; and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:55, or a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:43; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO:56, or a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:44; and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:55, or a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO:45; and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:57, or a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:46; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO:58.
[0119] In another aspect, the invention provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14, or combinations thereof. The
amino acid sequences of the V.sub.H CDR1s of PTA001_A1, PTA001_A2,
PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8,
PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 and
PTA001_A14 are shown in SEQ ID NOs: 1-4. The amino acid sequences
of the V.sub.H CDR2s of PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4,
PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ
ID NOs: 5-13. The amino acid sequences of the V.sub.H CDR3s of
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ ID
NOs:14-21. The amino acid sequences of the V.sub.K CDR1s of
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ ID
NOs:22-27. The amino acid sequences of the V.sub.K CDR2s of
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ ID
NOs:28-31. The amino acid sequences of the V.sub.K CDR3s of
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ ID
NOs:32-34. The CDR regions are delineated using the Kabat system
(Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242).
[0120] Given that each of these antibodies can bind to Cadherin-17
and that antigen-binding specificity is provided primarily by the
CDR1, CDR2, and CDR3 regions, the V.sub.H CDR1, CDR2, and CDR3
sequences and V.sub.K CDR1, CDR2, and CDR3 sequences can be "mixed
and matched" (i.e., CDRs from different antibodies can be mixed and
matched, although each antibody generally contains a V.sub.H CDR1,
CDR2, and CDR3 and a V.sub.K CDR1, CDR2, and CDR3) to create other
anti-Cadherin-17 binding molecules of the invention. Accordingly,
the invention specifically includes every possible combination of
CDRs of the heavy and light chains.
[0121] Cadherin-17 binding of such "mixed and matched" antibodies
can be tested using the binding assays described above and in the
Examples (e.g., ELISAs, Biacore.RTM. analysis). Preferably, when
V.sub.H CDR sequences are mixed and matched, the CDR1, CDR2 and/or
CDR3 sequence from a particular V.sub.H sequence is replaced with a
structurally similar CDR sequence(s). Likewise, when V.sub.K CDR
sequences are mixed and matched, the CDR1, CDR2 and/or CDR3
sequence from a particular V.sub.K sequence preferably is replaced
with a structurally similar CDR sequence(s). It will be readily
apparent to the ordinarily skilled artisan that novel V.sub.H and
V.sub.K sequences can be created by substituting one or more
V.sub.H and/or V.sub.L/V.sub.K CDR region sequences with
structurally similar sequences from the CDR sequences disclosed
herein for monoclonal antibodies PTA001_A1, PTA001_A2, PTA001_A3,
PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9,
PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 and PTA001_A14.
[0122] Accordingly, in another aspect, the invention provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising:
a heavy chain variable region CDR1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:1-4; a
heavy chain variable region CDR2 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:5-13; a heavy
chain variable region CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:14-21; a light
chain variable region CDR1 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:22-27; a light
chain variable region CDR2 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:28-31; and a light
chain variable region CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:32-34; with all
possible combinations being possible, wherein the antibody
specifically binds Cadherin-17, preferably human Cadherin-17
[0123] In a preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:1; a heavy
chain variable region CDR2 comprising SEQ ID NO:5; a heavy chain
variable region CDR3 comprising SEQ ID NO:14; a light chain
variable region CDR1 comprising SEQ ID NO:22; a light chain
variable region CDR2 comprising SEQ ID NO:28; and a light chain
variable region CDR3 comprising SEQ ID NO:32.
[0124] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:2; a heavy
chain variable region CDR2 comprising SEQ ID NO:6; a heavy chain
variable region CDR3 comprising SEQ ID NO:15; a light chain
variable region CDR1 comprising SEQ ID NO:23; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0125] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:7; a heavy chain
variable region CDR3 comprising SEQ ID NO:16; a light chain
variable region CDR1 comprising SEQ ID NO:23; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0126] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:4; a heavy
chain variable region CDR2 comprising SEQ ID NO:8; a heavy chain
variable region CDR3 comprising SEQ ID NO:17; a light chain
variable region CDR1 comprising SEQ ID NO:24; a light chain
variable region CDR2 comprising SEQ ID NO:30; and a light chain
variable region CDR3 comprising SEQ ID NO:34.
[0127] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:7; a heavy chain
variable region CDR3 comprising SEQ ID NO:18; a light chain
variable region CDR1 comprising SEQ ID NO:25; a light chain
variable region CDR2 comprising SEQ ID NO:31; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0128] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:9; a heavy chain
variable region CDR3 comprising SEQ ID NO:19; a light chain
variable region CDR1 comprising SEQ ID NO:26; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0129] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:7; a heavy chain
variable region CDR3 comprising SEQ ID NO:16; a light chain
variable region CDR1 comprising SEQ ID NO:25; a light chain
variable region CDR2 comprising SEQ ID NO:31; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0130] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:7; a heavy chain
variable region CDR3 comprising SEQ ID NO:16; a light chain
variable region CDR1 comprising SEQ ID NO:25; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0131] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:10; a heavy chain
variable region CDR3 comprising SEQ ID NO:20; a light chain
variable region CDR1 comprising SEQ ID NO:27; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0132] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO: 11; a heavy chain
variable region CDR3 comprising SEQ ID NO:21; a light chain
variable region CDR1 comprising SEQ ID NO:25; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0133] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:3; a heavy
chain variable region CDR2 comprising SEQ ID NO:12; a heavy chain
variable region CDR3 comprising SEQ ID NO:18; a light chain
variable region CDR1 comprising SEQ ID NO:25; a light chain
variable region CDR2 comprising SEQ ID NO:31; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0134] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:2; a heavy
chain variable region CDR2 comprising SEQ ID NO:13; a heavy chain
variable region CDR3 comprising SEQ ID NO:15; a light chain
variable region CDR1 comprising SEQ ID NO:23; a light chain
variable region CDR2 comprising SEQ ID NO:29; and a light chain
variable region CDR3 comprising SEQ ID NO:33.
[0135] It is well known in the art that the CDR3 domain,
independently from the CDR1 and/or CDR2 domain(s), alone can
determine the binding specificity of an antibody for a cognate
antigen and that multiple antibodies can predictably be generated
having the same binding specificity based on a common CDR3
sequence. See, for example, Klimka et al., British J. of Cancer
83(2):252-260 (2000) (describing the production of a humanized
anti-CD30 antibody using only the heavy chain variable domain CDR3
of murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.
296:833-849 (2000) (describing recombinant epithelial
glycoprotein-2 (EGP-2) antibodies using only the heavy chain CDR3
sequence of the parental murine MOC-31 anti-EGP-2 antibody); Rader
et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998)
(describing a panel of humanized anti-integrin
.alpha..sub.v.beta..sub.3 antibodies using a heavy and light chain
variable CDR3 domain of a murine anti-integrin
.alpha..sub.v.beta..sub.3 antibody LM609 wherein each member
antibody comprises a distinct sequence outside the CDR3 domain and
capable of binding the same epitope as the parent murine antibody
with affinities as high or higher than the parent murine antibody);
Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994) (disclosing
that the CDR3 domain provides the most significant contribution to
antigen binding); Barbas et al., Proc. Natl. Acad. Sci. U.S.A.
92:2529-2533 (1995) (describing the grafting of heavy chain CDR3
sequences of three Fabs (SI-1, SI-40, and SI-32) against human
placental DNA onto the heavy chain of an anti-tetanus toxoid Fab
thereby replacing the existing heavy chain CDR3 and demonstrating
that the CDR3 domain alone conferred binding specificity); and
Ditzel et al., J. Immunol. 157:739-749 (1996) (describing grafting
studies wherein transfer of only the heavy chain CDR3 of a parent
polyspecific Fab LNA3 to a heavy chain of a monospecific IgG
tetanus toxoid-binding Fab p313 antibody was sufficient to retain
binding specificity of the parent Fab). Each of these references is
hereby incorporated by reference in its entirety.
[0136] Accordingly, the present invention provides monoclonal
antibodies comprising one or more heavy and/or light chain CDR3
domains from an antibody derived from a human or non-human animal,
wherein the monoclonal antibody is capable of specifically binding
to Cadherin-17. Within certain aspects, the present invention
provides monoclonal antibodies comprising one or more heavy and/or
light chain CDR3 domain from a non-human antibody, such as a mouse
or rat antibody, wherein the monoclonal antibody is capable of
specifically binding to Cadherin-17. Within some embodiments, such
inventive antibodies comprising one or more heavy and/or light
chain CDR3 domain from a non-human antibody (a) are capable of
competing for binding with; (b) retain the functional
characteristics; (c) bind to the same epitope; and/or (d) have a
similar binding affinity as the corresponding parental non-human
antibody.
[0137] Within other aspects, the present invention provides
monoclonal antibodies comprising one or more heavy and/or light
chain CDR3 domains from a human antibody, such as, for example, a
human antibody obtained from a non-human animal, wherein the human
antibody is capable of specifically binding to Cadherin-17. Within
other aspects, the present invention provides monoclonal antibodies
comprising one or more heavy and/or light chain CDR3 domain from a
first human antibody, such as, for example, a human antibody
obtained from a non-human animal, wherein the first human antibody
is capable of specifically binding to Cadherin-17 and wherein the
CDR3 domain from the first human antibody replaces a CDR3 domain in
a human antibody that is lacking binding specificity for
Cadherin-17 to generate a second human antibody that is capable of
specifically binding to Cadherin-17. Within some embodiments, such
inventive antibodies comprising one or more heavy and/or light
chain CDR3 domain from the first human antibody (a) are capable of
competing for binding with; (b) retain the functional
characteristics; (c) bind to the same epitope; and/or (d) have a
similar binding affinity as the corresponding parental first human
antibody.
Antibodies Having Particular Germline Sequences
[0138] In certain embodiments, an antibody of the invention
comprises a heavy chain variable region from a particular germline
heavy chain immunoglobulin gene and/or a light chain variable
region from a particular germline light chain immunoglobulin
gene.
[0139] For example, in a preferred embodiment, the invention
provides an isolated monoclonal antibody, or an antigen-binding
portion thereof, comprising a heavy chain variable region that is
the product of or derived from a murineV.sub.H 7-39 gene, a murine
V.sub.H II region VH105 gene or a murine V.sub.H II gene H17,
wherein the antibody specifically binds Cadherin-17. In yet another
preferred embodiment, the invention provides an isolated monoclonal
antibody, or an antigen-binding portion thereof, comprising a light
chain variable region that is the product of or derived from a
murine V.sub.K 1-110 gene, a murine V.sub.K 8-30 gene or a murine
V.sub.K 24-140 gene, wherein the antibody specifically binds
Cadherin-17.
[0140] In yet another preferred embodiment, the invention provides
an isolated monoclonal antibody, or antigen-binding portion
thereof, wherein the antibody:
comprises a heavy chain variable region that is the product of or
derived from a murine V.sub.H 7-39 gene (which gene includes the
nucleotide sequence set forth in SEQ ID NO: 125); comprises a light
chain variable region that is the product of or derived from a
murine V.sub.K 1-110 gene (which gene includes the nucleotide
sequences set forth in SEQ ID NOs: 128 and 129); and specifically
binds to Cadherin-17, preferably human Cadherin-17. Examples of an
antibody having V.sub.H and V.sub.K of V.sub.H 7-39 and V.sub.K
1-110, respectively, is PTA001_A1.
[0141] In yet another preferred embodiment, the invention provides
an isolated monoclonal antibody, or antigen-binding portion
thereof, wherein the antibody:
comprises a heavy chain variable region that is the product of or
derived from a murine V.sub.H II gene H17 or a murine V.sub.H II
region VH105 gene (which genes include the nucleotide sequences set
forth in SEQ ID NO: 126 and 127 respectively); comprises a light
chain variable region that is the product of or derived from a
murine V.sub.K 8-30 gene (which gene includes the nucleotide
sequences set forth in SEQ ID NOs: 130, 131 and 132); and
specifically binds to Cadherin-17, preferably human
Cadherin-17.
[0142] Examples of an antibody having V.sub.H of V.sub.H II gene
H17 or V.sub.H II region VH105 and V.sub.K of V.sub.K 8-30 is
PTA001_A4.
[0143] In yet another preferred embodiment, the invention provides
an isolated monoclonal antibody, or antigen-binding portion
thereof, wherein the antibody:
comprises a heavy chain variable region that is the product of or
derived from a murine V.sub.H II gene H17 or a murine V.sub.H II
region VH105 gene (which genes include the nucleotide sequences set
forth in SEQ ID NO: 126 and 127 respectively); comprises a light
chain variable region that is the product of or derived from a
murine V.sub.K 24-140 gene (which gene includes the nucleotide
sequences set forth in SEQ ID NOs: 133, 134 and 135); and
specifically binds to Cadherin-17, preferably human Cadherin-17.
Examples of antibodies having V.sub.H of V.sub.H II gene H17 or
V.sub.H II region VH105 and V.sub.K of V.sub.K 24-140 are
PTA001_A2, PTA001_A3, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8,
PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 and
PTA001_A14.
[0144] As used herein, an antibody comprises heavy or light chain
variable regions that is "the product of" or "derived from" a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses murine germline
immunoglobulin genes. Such systems include screening a murine
immunoglobulin gene library displayed on phage with the antigen of
interest. An antibody that is "the product of" or "derived from" a
murine germline immunoglobulin sequence can be identified as such
by comparing the nucleotide or amino acid sequence of the antibody
to the nucleotide or amino acid sequences of murine germline
immunoglobulins and selecting the murine germline immunoglobulin
sequence that is closest in sequence (i.e., greatest % identity) to
the sequence of the antibody. An antibody that is "the product of"
or "derived from" a particular murine germline immunoglobulin
sequence may contain amino acid differences as compared to the
germline sequence, due to, for example, naturally-occurring somatic
mutations or intentional introduction of site-directed mutation.
However, a selected antibody typically is at least 90% identical in
amino acids sequence to an amino acid sequence encoded by a murine
germline immunoglobulin gene and contains amino acid residues that
identify the antibody as being murine when compared to the germline
immunoglobulin amino acid sequences of other species (e.g., human
germline sequences). In certain cases, an antibody may be at least
95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid
sequence to the amino acid sequence encoded by the germline
immunoglobulin gene. Typically, an antibody derived from a
particular murine germline sequence will display no more than 10
amino acid differences from the amino acid sequence encoded by the
murine germline immunoglobulin gene. In certain cases, the antibody
may display no more than 5, or even no more than 4, 3, 2, or 1
amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene.
Homologous Antibodies
[0145] In yet another embodiment, an antibody of the invention
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties of the
anti-Cadherin-17 antibodies of the invention. For example, the
invention provides an isolated monoclonal antibody, or antigen
binding portion thereof, comprising a heavy chain variable region
and a light chain variable region, wherein: [0146] the heavy chain
variable region comprises an amino acid sequence that is at least
80% identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs:35-46; the light chain variable region
comprises an amino acid sequence that is at least 80% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NOs:47-58; and [0147] the antibody binds to human Cadherin-17. Such
antibodies may bind to human Cadherin-17 with an EC.sub.50 of 50 nM
or less, 10 nM or less, 1 nM or less, 100 pM or less, or more
preferably 10 pM or less.
[0148] The antibody may also bind to CHO cells transfected with
human Cadherin-17.
[0149] In various embodiments, the antibody can be, for example, a
human antibody, a humanized antibody or a chimeric antibody.
[0150] In other embodiments, the V.sub.H and/or V.sub.K amino acid
sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to
the sequences set forth above. An antibody having V.sub.H and
V.sub.K regions having high (i.e., 80% or greater) identical to the
V.sub.H and V.sub.K regions of the sequences set forth above, can
be obtained by mutagenesis (e.g., site-directed or PCR-mediated
mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:59-83
followed by testing of the encoded altered antibody for retained
function using the functional assays described herein.
[0151] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences (i.e.,
% homology=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
[0152] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
[0153] Additionally or alternatively, the protein sequences of the
present invention can further be used as a "query sequence" to
perform a search against public databases to, for example, identify
related sequences. Such searches can be performed using the XBLAST
program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.
215:403-10. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the antibody molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See www.ncbi.nlm.nih.gov.
Antibodies with Conservative Modifications
[0154] In certain embodiments, an antibody of the invention
comprises a heavy chain variable region comprising CDR1, CDR2 and
CDR3 sequences and a light chain variable region comprising CDR1,
CDR2 and CDR3 sequences, wherein one or more of these CDR sequences
comprise specified amino acid sequences based on the preferred
antibodies described herein (e.g., PTA001_A1, PTA001_A2, PTA001_A3,
PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9,
PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 or PTA001_A14), or
conservative modifications thereof, and
wherein the antibodies retain the desired functional properties of
the anti-Cadherin-17 antibodies of the invention. Accordingly, the
invention provides an isolated monoclonal antibody, or antigen
binding portion thereof, comprising a heavy chain variable region
comprising CDR1, CDR2, and CDR3 sequences and a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
the heavy chain variable region CDR3 sequence comprises an amino
acid sequence selected from the group consisting of amino acid
sequences of SEQ ID NOs:14-21, and conservative modifications
thereof; the light chain variable region CDR3 sequence comprises an
amino acid sequence selected from the group consisting of amino
acid sequence of SEQ ID NOs:32-34, and conservative modifications
thereof; and the antibody binds to human Cadherin-17. Such
antibodies may bind to human Cadherin-17 with an EC.sub.50 of 50 nM
or less, 10 nM or less, 1 nM or less, 100 pM or less, or more
preferably 10 pM or less.
[0155] The antibody may also bind to CHO cells transfected with
human Cadherin-17.
[0156] In a preferred embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs:5-13, and
conservative modifications thereof; and the light chain variable
region CDR2 sequence comprises an amino acid sequence selected from
the group consisting of amino acid sequences of SEQ ID NOs:28-31,
and conservative modifications thereof. In another preferred
embodiment, the heavy chain variable region CDR1 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOs:1-4, and conservative modifications
thereof; and the light chain variable region CDR1 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequences of SEQ ID NOs:22-27, and conservative
modifications thereof.
[0157] In various embodiments, the antibody can be, for example,
human antibodies, humanized antibodies or chimeric antibodies.
[0158] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of the invention can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function using the functional assays described
herein.
[0159] The heavy chain CDR1 sequence of SEQ ID NO:1-4 may comprise
one or more conservative sequence modification, such as one, two,
three, four, five or more amino acid substitutions, additions or
deletions; the light chain CDR1 sequence of SEQ ID NO:22-27 may
comprise one or more conservative sequence modification, such as
one, two, three, four, five or more amino acid substitutions,
additions or deletions; the heavy chain CDR2 sequence shown in SEQ
ID NO:5-13 may comprise one or more conservative sequence
modification, such as one, two, three, four, five or more amino
acid substitutions, additions or deletions; the light chain CDR2
sequence shown in SEQ ID NO:28-31 may comprise one or more
conservative sequence modification, such as one, two, three, four,
five or more amino acid substitutions, additions or deletions; the
heavy chain CDR3 sequence shown in SEQ ID NO:14-21 may comprise one
or more conservative sequence modification, such as one, two,
three, four, five or more amino acid substitutions, additions or
deletions; and/or the light chain CDR3 sequence shown in SEQ ID
NO:32-34 may comprise one or more conservative sequence
modification, such as one, two, three, four, five or more amino
acid substitutions, additions or deletions.
Antibodies that Bind to the Same Epitope as Anti-Cadherin-17
Antibodies of the Invention
[0160] In another embodiment, the invention provides antibodies
that bind to the same epitope on human Cadherin-17 as any of the
Cadherin-17 monoclonal antibodies of the invention (i.e.,
antibodies that have the ability to cross-compete for binding to
Cadherin-17 with any of the monoclonal antibodies of the
invention). In preferred embodiments, the reference antibody for
cross-competition studies can be the monoclonal antibody PTA001_A1
(having V.sub.H and V.sub.K sequences as shown in SEQ ID NOs:35 and
47, respectively), the monoclonal antibody PTA001_A2 (having
V.sub.H and V.sub.K sequences as shown in SEQ ID NOs:36 and 48
respectively), the monoclonal antibody PTA001_A3 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:37 and 48
respectively), the monoclonal antibody PTA001_A4 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:38 and 49
respectively), the monoclonal antibody PTA001_A5 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:39 and 50
respectively), the monoclonal antibody PTA001_A6 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:40 and 51
respectively), the monoclonal antibody PTA001_A7 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:41 and 52
respectively), the monoclonal antibody PTA001_A8 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:42 and 53
respectively), the monoclonal antibody PTA001_A9 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:40 and 54
respectively), the monoclonal antibody PTA001_A10 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:40 and 55
respectively), the monoclonal antibody PTA001_A11 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:43 and 56
respectively), the monoclonal antibody PTA001_A12 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:44 and 55
respectively), the monoclonal antibody PTA001_A13 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:45 and 57
respectively) or the monoclonal antibody PTA001_A14 (having V.sub.H
and V.sub.K sequences as shown in SEQ ID NOs:46 and 58
respectively). Such cross-competing antibodies can be identified
based on their ability to cross-compete with PTA001_A1, PTA001_A2,
PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8,
PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 or
PTA001_A14 in standard Cadherin-17 binding assays. For example,
BIAcore.RTM. analysis, ELISA assays or flow cytometry may be used
to demonstrate cross-competition with the antibodies of the current
invention. The ability of a test antibody to inhibit the binding
of, for example, PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4,
PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001_A12, PTA001_A13 or PTA001_A14, to human
Cadherin-17 demonstrates that the test antibody can compete with
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 or PTA001_A14 for binding to human
Cadherin-17 and thus binds to the same epitope on human Cadherin-17
as PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5,
PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 or PTA001_A14.
Engineered and Modified Antibodies
[0161] An antibody of the invention further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences disclosed herein which can be used as starting material
to engineer a modified antibody, which modified antibody may have
altered properties as compared to the starting antibody. An
antibody can be engineered by modifying one or more amino acids
within one or both variable regions (i.e., V.sub.H and/or V.sub.L),
for example within one or more CDR regions and/or within one or
more framework regions. Additionally or alternatively, an antibody
can be engineered by modifying residues within the constant
region(s), for example to alter the effector function(s) of the
antibody.
[0162] In certain embodiments, CDR grafting can be used to engineer
variable regions of antibodies. Antibodies interact with target
antigens predominantly through amino acid residues that are located
in the six heavy and light chain complementarity determining
regions (CDRs). For this reason, the amino acid sequences within
CDRs are more diverse between individual antibodies than sequences
outside of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann, L. et al. (1998)
Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525;
Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.)
[0163] Accordingly, another embodiment of the invention pertains to
an isolated monoclonal antibody, or antigen binding portion
thereof, comprising a heavy chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs:1-4, SEQ ID NOs:5-13, and
SEQ ID NOs:14-21, respectively, and a light chain variable region
comprising CDR1, CDR2, and CDR3 sequences comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:22-27,
SEQ ID NOs:28-31, and SEQ ID NOs:32-34, respectively. Thus, such
antibodies contain the V.sub.H and V.sub.K CDR sequences of
monoclonal antibodies PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4,
PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001_A12, PTA001_A13 or PTA001_A114 yet may contain
different framework sequences from these antibodies.
[0164] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for murine
heavy and light chain variable region genes can be found in the
IMGT (international ImMunoGeneTics) murine germline sequence
database (available on the Internet at imgt.cines.fr/), as well as
in Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242; the contents of
each of which are expressly incorporated herein by reference. As
another example, the germline DNA sequences for murine heavy and
light chain variable region genes can be found in the Genbank.RTM.
database.
[0165] Antibody protein sequences are compared against a compiled
protein sequence database using one of the sequence similarity
searching methods called the Gapped BLAST (Altschul et al. (1997)
Nucleic Acids Research 25:3389-3402), which is well known to those
skilled in the art. BLAST is a heuristic algorithm in that a
statistically significant alignment between the antibody sequence
and the database sequence is likely to contain high-scoring segment
pairs (HSP) of aligned words. Segment pairs whose scores cannot be
improved by extension or trimming is called a hit. Briefly, the
nucleotide sequences in the database are translated and the region
between and including FR1 through FR3 framework region is retained.
The database sequences have an average length of 98 residues.
Duplicate sequences which are exact matches over the entire length
of the protein are removed. A BLAST search for proteins using the
program blastp with default, standard parameters except the low
complexity filter, which is turned off, and the substitution matrix
of BLOSUM62, filters for top 5 hits yielding sequence matches. The
nucleotide sequences are translated in all six frames and the frame
with no stop codons in the matching segment of the database
sequence is considered the potential hit. This is in turn confirmed
using the BLAST program tblastx, which translates the antibody
sequence in all six frames and compares those translations to the
nucleotide sequences in the database dynamically translated in all
six frames.
[0166] The identities are exact amino acid matches between the
antibody sequence and the protein database over the entire length
of the sequence. The positives (identities+substitution match) are
not identical but amino acid substitutions guided by the BLOSUM62
substitution matrix. If the antibody sequence matches two of the
database sequences with same identity, the hit with most positives
would be decided to be the matching sequence hit.
[0167] Preferred framework sequences for use in the antibodies of
the invention are those that are structurally similar to the
framework sequences used by selected antibodies of the invention,
e.g., similar to the V.sub.H 7-39 framework sequence, the V.sub.H
II gene H17 framework sequence, the V.sub.H II region VH105
framework sequence, the V.sub.K 1-110 framework sequence, the
V.sub.K 8-30 framework sequence and/or the V.sub.K 24-140 framework
sequences used by preferred monoclonal antibodies of the invention.
The V.sub.H CDR1, CDR2, and CDR3 sequences, and the V.sub.K CDR1,
CDR2, and CDR3 sequences, can be grafted onto framework regions
that have the identical sequence as that found in the germline
immunoglobulin gene from which the framework sequence derive, or
the CDR sequences can be grafted onto framework regions that
contain one or more mutations as compared to the germline
sequences. For example, it has been found that in certain instances
it is beneficial to mutate residues within the framework regions to
maintain or enhance the antigen binding ability of the antibody
(see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to Queen et al.).
[0168] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.K CDR1, CDR2
and/or CDR3 regions to thereby improve one or more binding
properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis can be
performed to introduce the mutation(s) and the effect on antibody
binding, or other functional property of interest, can be evaluated
in in vitro or in vivo assays as described herein and provided in
the Examples. In some embodiments, conservative modifications (as
discussed above) are introduced. Alternatively, non-conservative
modifications can be made. The mutations may be amino acid
substitutions, additions or deletions, but are preferably
substitutions. Moreover, typically no more than one, two, three,
four or five residues within a CDR region are altered, although as
will be appreciated by those in the art, variants in other areas
(framework regions for example) can be greater.
[0169] Accordingly, in another embodiment, the instant disclosure
provides isolated anti-Cadherin-17 monoclonal antibodies, or
antigen binding portions thereof, comprising a heavy chain variable
region comprising: (a) a V.sub.H CDR1 region comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:1-4,
or an amino acid sequence having one, two, three, four or five
amino acid substitutions, deletions or additions as compared to SEQ
ID NOs: 1-4; (b) a V.sub.H CDR2 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:5-13, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:5-13; (c) a V.sub.H CDR3 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:14-21, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:14-21; (d) a V.sub.K CDR1 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:22-27, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:22-27; (e) a V.sub.K CDR2 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:28-31, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:28-31; and (f) a V.sub.K CDR3 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:32-34, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:32-34.
[0170] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.K, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived.
[0171] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 2003/0153043 by Carr et al.
[0172] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0173] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0174] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcal protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0175] In another embodiment, the antibody is modified to increase
its biological half life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to
Ward. Alternatively, to increase the biological half life, the
antibody can be altered within the CH1 or C.sub.L region to contain
a salvage receptor binding epitope taken from two loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al.
[0176] In another embodiment, the antibody is produced as a UniBody
as described in WO2007/059782 which is incorporated herein by
reference in its entirety.
[0177] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the antibody. For
example, one or more amino acids selected from amino acid residues
234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a
different amino acid residue such that the antibody has an altered
affinity for an effector ligand but retains the antigen-binding
ability of the parent antibody. The effector ligand to which
affinity is altered can be, for example, an Fc receptor or the C1
component of complement. This approach is described in further
detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et
al.
[0178] In another example, one or more amino acids selected from
amino acid residues 329, 331 and 322 can be replaced with a
different amino acid residue such that the antibody has altered Clq
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. No. 6,194,551 by Idusogie et al.
[0179] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in PCT Publication WO 94/29351 by Bodmer et
al.
[0180] In yet another example, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids at the following positions: 238, 239, 248, 249, 252, 254,
255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283,
285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,
307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,
334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,
414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is
described further in PCT Publication WO 00/42072 by Presta.
Moreover, the binding sites on human IgG1 for Fc.gamma.R1,
Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants
with improved binding have been described (see Shields, R. L. et
al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at
positions 256, 290, 298, 333, 334 and 339 were shown to improve
binding to Fc.gamma.RIII. Additionally, the following combination
mutants were shown to improve Fc.gamma.RIII binding: T256A/S298A,
S298A/E333A, S298A/K224A and S298A/E333A/K334A. Further ADCC
variants are described for example in WO2006/019447.
[0181] In yet another example, the Fc region is modified to
increase the half-life of the antibody, generally by increasing
binding to the FcRn receptor, as described for example in
PCT/US2008/088053, U.S. Pat. No. 7,371,826, U.S. Pat. No. 7,670,600
and WO 97/34631.
[0182] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
antigen. Such carbohydrate modifications can be accomplished by,
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co
et al., and can be accomplished by removing the asparagine at
position 297.
[0183] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. This is sometimes
referred to in the art as a "engineered glycoform". Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can
generally be accomplished in two ways; for example, in some
embodiments, the antibody is expressed in a host cell with altered
glycosylation machinery. Cells with altered glycosylation machinery
have been described in the art and can be used as host cells in
which to express recombinant antibodies of the invention to thereby
produce an antibody with altered glycosylation. Reference is made
to the POTELLIGENT.RTM. technology. For example, the cell lines
Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8
(alpha (1,6) fucosyltransferase), such that antibodies expressed in
the Ms704, Ms705, and Ms709 cell lines lack fucose on their
carbohydrates. The Ms704, Ms705, and Ms709 FUT8.sup.-/- cell lines
were created by the targeted disruption of the FUT8 gene in
CHO/DG44 cells using two replacement vectors (see U.S. Patent
Publication No. 2004/0110704 by Yamane et al., U.S. Pat. No.
7,517,670 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng
87:614-22). As another example, EP 1,176,195 by Hanai et al.
describes a cell line with a functionally disrupted FUT8 gene,
which encodes a fucosyl transferase, such that antibodies expressed
in such a cell line exhibit hypofucosylation by reducing or
eliminating the alpha 1,6 bond-related enzyme. Hanai et al. also
describe cell lines which have a low enzyme activity for adding
fucose to the N-acetylglucosamine that binds to the Fc region of
the antibody or does not have the enzyme activity, for example the
rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO
03/035835 by Presta describes a variant CHO cell line, Lec13 cells,
with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also resulting in hypofucosylation of antibodies
expressed in that host cell (see also Shields, R. L. et al. (2002)
J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by
Umana et al. describes cell lines engineered to express
glycoprotein-modifying glycosyl transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180). Alternatively, the fucose residues of the
antibody may be cleaved off using a fucosidase enzyme. For example,
the fucosidase alpha-L-fucosidase removes fucosyl residues from
antibodies (Tarentino, A. L. et al. (1975) Biochem.
14:5516-23).
[0184] Alternatively, engineered glycoforms, particularly
afucosylation, can be done using small molecule inhibitors of
glycosylation pathway enzymes. See for example Rothman et al., Mol.
Immunol. 26(12):113-1123 (1989); Elbein, FASEB J. 5:3055 (1991);
PCT/US2009/042610 and U.S. Pat. No. 7,700,321.
[0185] Another modification of the antibodies herein that is
contemplated by the invention is pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g., serum)
half life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol
(PEG), such as a reactive ester or aldehyde derivative of PEG,
under conditions in which one or more PEG groups become attached to
the antibody or antibody fragment. Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive PEG molecule (or an analogous reactive
water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended to encompass any of the forms of PEG that have
been used to derivatize other proteins, such as mono (C1-C10)
alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. In certain embodiments, the antibody to be
pegylated is an aglycosylated antibody. Methods for pegylating
proteins are known in the art and can be applied to the antibodies
of the invention. See for example, EP 0 154 316 by Nishimura et al.
and EP 0 401 384 by Ishikawa et al.
[0186] In additional embodiments, for example in the use of the
antibodies of the invention for diagnostic or detection purposes,
the antibodies may comprise a label. By "labeled" herein is meant
that a compound has at least one element, isotope or chemical
compound attached to enable the detection of the compound. In
general, labels fall into three classes: a) isotopic labels, which
may be radioactive or heavy isotopes; b) magnetic, electrical,
thermal; and c) colored or luminescent dyes; although labels
include enzymes and particles such as magnetic particles as well.
Preferred labels include, but are not limited to, fluorescent
lanthanide complexes (including those of Europium and Terbium), and
fluorescent labels including, but not limited to, quantum dots,
fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,
coumarin, methyl-coumarins, pyrene, Malacite green, stilbene,
Lucifer Yellow, Cascade Blue, Texas Red, the Alexa dyes, the Cy
dyes, and others described in the 6th Edition of the Molecular
Probes Handbook by Richard P. Haugland, hereby expressly
incorporated by reference.
Antibody Physical Properties
[0187] The antibodies of the present invention may be further
characterized by the various physical properties of the
anti-Cadherin-17 antibodies. Various assays may be used to detect
and/or differentiate different classes of antibodies based on these
physical properties.
[0188] In some embodiments, antibodies of the present invention may
contain one or more glycosylation sites in either the light or
heavy chain variable region. The presence of one or more
glycosylation sites in the variable region may result in increased
immunogenicity of the antibody or an alteration of the pK of the
antibody due to altered antigen binding (Marshall et al (1972) Annu
Rev Biochem 41:673-702; Gala F A and Morrison SL (2004) J Immunol
172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro R G
(2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N-X-S/T sequence. Variable region glycosylation may be tested using
a Glycoblot assay, which cleaves the antibody to produce a Fab, and
then tests for glycosylation using an assay that measures periodate
oxidation and Schiff base formation. Alternatively, variable region
glycosylation may be tested using Dionex.RTM. light chromatography
(Dionex-LC), which cleaves saccharides from a Fab into
monosaccharides and analyzes the individual saccharide content. In
some instances, it is preferred to have an anti-Cadherin-17
antibody that does not contain variable region glycosylation. This
can be achieved either by selecting antibodies that do not contain
the glycosylation motif in the variable region or by mutating
residues within the glycosylation motif using standard techniques
well known in the art.
[0189] In a preferred embodiment, the antibodies of the present
invention do not contain asparagine isomerism sites. A deamidation
or isoaspartic acid effect may occur on N-G or D-G sequences,
respectively. The deamidation or isoaspartic acid effect results in
the creation of isoaspartic acid which decreases the stability of
an antibody by creating a kinked structure off a side chain carboxy
terminus rather than the main chain. The creation of isoaspartic
acid can be measured using an iso-quant assay, which uses a
reverse-phase HPLC to test for isoaspartic acid.
[0190] Each antibody will have a unique isoelectric point (pI), but
generally antibodies will fall in the pH range of between 6 and
9.5. The pI for an IgG1 antibody typically falls within the pH
range of 7-9.5 and the pI for an IgG4 antibody typically falls
within the pH range of 6-8. Antibodies may have a pI that is
outside this range. Although the effects are generally unknown,
there is speculation that antibodies with a pI outside the normal
range may have some unfolding and instability under in vivo
conditions. The isoelectric point may be tested using a capillary
isoelectric focusing assay, which creates a pH gradient and may
utilize laser focusing for increased accuracy (Janini et al (2002)
Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia
53:S75-89; Hunt et al (1998) J Chromatogr A 800:355-67). In some
instances, it is preferred to have an anti-Cadherin-17 antibody
that contains a pI value that falls in the normal range. This can
be achieved either by selecting antibodies with a pI in the normal
range, or by mutating charged surface residues using standard
techniques well known in the art.
[0191] Each antibody will have a melting temperature that is
indicative of thermal stability (Krishnamurthy R and Manning M C
(2002) Curr Pharm Biotechnol 3:361-71). A higher thermal stability
indicates greater overall antibody stability in vivo. The melting
point of an antibody may be measured using techniques such as
differential scanning calorimetry (Chen et al (2003) Pharm Res
20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). T.sub.M1
indicates the temperature of the initial unfolding of the antibody.
T.sub.M2 indicates the temperature of complete unfolding of the
antibody. Generally, it is preferred that the T.sub.M1 of an
antibody of the present invention is greater than 60.degree. C.,
preferably greater than 65.degree. C., even more preferably greater
than 70.degree. C. Alternatively, the thermal stability of an
antibody may be measure using circular dichroism (Murray et al.
(2002) J. Chromatogr Sci 40:343-9).
[0192] In a preferred embodiment, antibodies are selected that do
not rapidly degrade. Fragmentation of an anti-Cadherin-17 antibody
may be measured using capillary electrophoresis (CE) and MALDI-MS,
as is well understood in the art (Alexander A J and Hughes DE
(1995) Anal Chem 67:3626-32).
[0193] In another preferred embodiment, antibodies are selected
that have minimal aggregation effects. Aggregation may lead to
triggering of an unwanted immune response and/or altered or
unfavorable pharmacokinetic properties. Generally, antibodies are
acceptable with aggregation of 25% or less, preferably 20% or less,
even more preferably 15% or less, even more preferably 10% or less
and even more preferably 5% or less. Aggregation may be measured by
several techniques well known in the art, including size-exclusion
column (SEC) high performance liquid chromatography (HPLC), and
light scattering to identify monomers, dimers, trimers or
multimers.
Methods of Engineering Antibodies
[0194] As discussed above, the anti-Cadherin-17 antibodies having
V.sub.H and V.sub.K sequences disclosed herein can be used to
create new anti-Cadherin-17 antibodies by modifying the V.sub.H
and/or V.sub.K sequences, or the constant region(s) attached
thereto. Thus, in another aspect of the invention, the structural
features of an anti-Cadherin-17 antibody of the invention, e.g.
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 or PTA001_A14, are used to create
structurally related anti-Cadherin-17 antibodies that retain at
least one functional property of the antibodies of the invention,
such as binding to human Cadherin-17. For example, one or more CDR
regions of PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5,
PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 or PTA001_A14, or mutations thereof, can be
combined recombinantly with known framework regions and/or other
CDRs to create additional, recombinantly-engineered,
anti-Cadherin-17 antibodies of the invention, as discussed above.
Other types of modifications include those described in the
previous section. The starting material for the engineering method
is one or more of the V.sub.H and/or V.sub.K sequences provided
herein, or one or more CDR regions thereof. To create the
engineered antibody, it is not necessary to actually prepare (i.e.,
express as a protein) an antibody having one or more of the V.sub.H
and/or V.sub.K sequences provided herein, or one or more CDR
regions thereof. Rather, the information contained in the
sequence(s) is used as the starting material to create a "second
generation" sequence(s) derived from the original sequence(s) and
then the "second generation" sequence(s) is prepared and expressed
as a protein.
[0195] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-Cadherin-17 antibody comprising:
providing: (i) a heavy chain variable region antibody sequence
comprising a CDR1 sequence selected from the group consisting of
SEQ ID NOs:1-4, a CDR2 sequence selected from the group consisting
of SEQ ID NOs:5-13, and/or a CDR3 sequence selected from the group
consisting of SEQ ID NOs:14-21; and/or (ii) a light chain variable
region antibody sequence comprising a CDR1 sequence selected from
the group consisting of SEQ ID NOs:22-27, a CDR2 sequence selected
from the group consisting of SEQ ID NOs:28-31, and/or a CDR3
sequence selected from the group consisting of SEQ ID NOs:32-34;
altering at least one amino acid residue within the heavy chain
variable region antibody sequence and/or the light chain variable
region antibody sequence to create at least one altered antibody
sequence; and expressing the altered antibody sequence as a
protein.
[0196] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence.
[0197] Preferably, the antibody encoded by the altered antibody
sequence(s) is one that retains one, some or all of the functional
properties of the anti-Cadherin-17 antibodies described herein,
which functional properties include, but are not limited to:
binds to human Cadherin-17 with a K.sub.D of 1.times.10.sup.-7 M or
less; binds to human CHO cells transfected with Cadherin-17.
[0198] The functional properties of the altered antibodies can be
assessed using standard assays available in the art and/or
described herein, such as those set forth in the Examples (e.g.,
flow cytometry, binding assays).
[0199] In certain embodiments of the methods of engineering
antibodies of the invention, mutations can be introduced randomly
or selectively along all or part of an anti-Cadherin-17 antibody
coding sequence and the resulting modified anti-Cadherin-17
antibodies can be screened for binding activity and/or other
functional properties as described herein. Mutational methods have
been described in the art. For example, PCT Publication WO
02/092780 by Short describes methods for creating and screening
antibody mutations using saturation mutagenesis, synthetic ligation
assembly, or a combination thereof. Alternatively, PCT Publication
WO 03/074679 by Lazar et al. describes methods of using
computational screening methods to optimize physiochemical
properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of the Invention
[0200] Another aspect of the invention pertains to nucleic acid
molecules that encode the antibodies of the invention. The nucleic
acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid is
"isolated" or "rendered substantially pure" when purified away from
other cellular components or other contaminants, e.g., other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols
in Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of the invention can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0201] Nucleic acids of the invention can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas, cDNAs encoding the light and heavy chains of the
antibody made by the hybridoma can be obtained by standard PCR
amplification or cDNA cloning techniques. For antibodies obtained
from an immunoglobulin gene library (e.g., using phage display
techniques), nucleic acids encoding the antibody can be recovered
from the library.
[0202] Preferred nucleic acids molecules of the invention are those
encoding the V.sub.H and V.sub.K sequences of the PTA001_A1,
PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7,
PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12,
PTA001_A13 or PTA001_A14 monoclonal antibodies. DNA sequences
encoding the V.sub.H sequences of PTA001_A1, PTA001_A2, PTA001_A3,
PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9,
PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 and PTA001_A14 are
shown in SEQ ID NOs: 59-70. DNA sequences encoding the V.sub.K
sequences of PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5,
PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 and PTA001_A14 are shown in SEQ ID NOs:
71-83.
[0203] Other preferred nucleic acids of the invention are nucleic
acids having at least 80% sequence identity, such as at least 85%,
at least 90%, at least 95%, at least 98% or at least 99% sequence
identity, with one of the sequences shown in SEQ ID NOs: 59-83,
which nucleic acids encode an antibody of the invention, or an
antigen-binding portion thereof.
[0204] The percent identity between two nucleic acid sequences is
the number of positions in the sequence in which the nucleotide is
identical, taking into account the number of gaps and the length of
each gap, which need to be introduced for optimal alignment of the
two sequences.
[0205] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, such as the algorithm of Meyers and Miller
or the XBLAST program of Altschul described above.
[0206] Still further, preferred nucleic acids of the invention
comprise one or more CDR-encoding portions of the nucleic acid
sequences shown in SEQ ID NOs:59-83. In this embodiment, the
nucleic acid may encode the heavy chain CDR1, CDR2 and/or CDR3
sequence of PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5,
PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13 or PTA001_A14 or the light chain CDR1, CDR2
and/or CDR3 sequence of PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4,
PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001_A12, PTA001_A13 or PTA001_A14.
[0207] Nucleic acids which have at least 80%, such as at least 85%,
at least 90%, at least 95%, at least 98% or at least 99% sequence
identity, with such a CDR-encoding portion of SEQ ID NO:59-83
(V.sub.H and V.sub.K seqs) are also preferred nucleic acids of the
invention. Such nucleic acids may differ from the corresponding
portion of SEQ ID NO:59-83 in a non-CDR coding region and/or in a
CDR-coding region. Where the difference is in a CDR-coding region,
the nucleic acid CDR region encoded by the nucleic acid typically
comprises one or more conservative sequence modifications as
defined herein compared to the corresponding CDR sequence of
PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13or PTA001_A14.
[0208] Once DNA fragments encoding V.sub.H and V.sub.K segments are
obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes or to a scFv gene. In these manipulations, a
V.sub.K- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA fragment encoding another protein, such as an antibody
constant region or a flexible linker. The term "operatively
linked", as used in this context, is intended to mean that the two
DNA fragments are joined such that the amino acid sequences encoded
by the two DNA fragments remain in-frame.
[0209] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2 and CH3). The sequences of murine
heavy chain constant region genes are known in the art (see e.g.,
Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the V.sub.H-encoding DNA can be operatively
linked to another DNA molecule encoding only the heavy chain CH1
constant region.
[0210] The isolated DNA encoding the V.sub.L/V.sub.K region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of murine light chain constant region genes are known
in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. In preferred embodiments, the light chain
constant region can be a kappa or lambda constant region.
[0211] To create a scFv gene, the V.sub.H- and
V.sub.L/V.sub.K-encoding DNA fragments are operatively linked to
another fragment encoding a flexible linker, e.g., encoding the
amino acid sequence (Gly.sub.4-Ser).sub.3, such that the V.sub.H
and V.sub.L/V.sub.K sequences can be expressed as a contiguous
single-chain protein, with the V.sub.L/V.sub.K and V.sub.H regions
joined by the flexible linker (see e.g., Bird et al. (1988) Science
242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
Production of Monoclonal Antibodies
[0212] According to the invention Cadherin-17 or a fragment or
derivative thereof may be used as an immunogen to generate
antibodies which immunospecifically bind such an immunogen.
[0213] Such immunogens can be isolated by any convenient means. One
skilled in the art will recognize that many procedures are
available for the production of antibodies, for example, as
described in Antibodies, A Laboratory Manual, Ed Harlow and David
Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor,
N.Y. One skilled in the art will also appreciate that binding
fragments or Fab fragments which mimic antibodies can also be
prepared from genetic information by various procedures (Antibody
Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995,
Oxford University Press, Oxford; J. Immunol. 149, 3914-3920
(1992)).
[0214] In one embodiment of the invention, antibodies to a specific
domain of Cadherin-17 are produced. In a specific embodiment,
hydrophilic fragments of Cadherin-17 are used as immunogens for
antibody production.
[0215] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.
ELISA (enzyme-linked immunosorbent assay). For example, to select
antibodies which recognize a specific domain of Cadherin-17, one
may assay generated hybridomas for a product which binds to a
Cadherin-17 fragment containing such domain. For selection of an
antibody that specifically binds a first Cadherin-17 homolog but
which does not specifically bind to (or binds less avidly to) a
second Cadherin-17 homolog, one can select on the basis of positive
binding to the first Cadherin-17 homolog and a lack of binding to
(or reduced binding to) the second Cadherin-17 homolog. Similarly,
for selection of an antibody that specifically binds Cadherin-17
but which does not specifically bind to (or binds less avidly to) a
different isoform of the same protein (such as a different
glycoform having the same core peptide as Cadherin-17), one can
select on the basis of positive binding to Cadherin-17 and a lack
of binding to (or reduced binding to) the different isoform (e.g. a
different glycoform). Thus, the present invention provides an
antibody (such as a monoclonal antibody) that binds with greater
affinity (for example at least 2-fold, such as at least 5-fold,
particularly at least 10-fold greater affinity) to Cadherin-17 than
to a different isoform or isoforms (e.g. glycoforms) of
Cadherin-17.
[0216] Polyclonal antibodies which may be used in the methods of
the invention are heterogeneous populations of antibody molecules
derived from the sera of immunized animals. Unfractionated immune
serum can also be used. Various procedures known in the art may be
used for the production of polyclonal antibodies to Cadherin-17, a
fragment of Cadherin-17, a Cadherin-17-related polypeptide, or a
fragment of a Cadherin-17-related polypeptide. For example, one way
is to purify polypeptides of interest or to synthesize the
polypeptides of interest using, e.g. solid phase peptide synthesis
methods well known in the art. See, e.g. Guide to Protein
Purification, Murray P. Deutcher, ed., Meth. Enzymol. Vol 182
(1990); Solid Phase Peptide Synthesis, Greg B. Fields ed., Meth.
Enzymol. Vol 289 (1997); Kiso et al., Chem. Pharm. Bull. (Tokyo)
38: 1192-99, 1990; Mostafavi et al., Biomed. Pept. Proteins Nucleic
Acids 1: 255-60, 1995; Fujiwara et al., Chem. Pharm. Bull. (Tokyo)
44: 1326-31, 1996. The selected polypeptides may then be used to
immunize by injection various host animals, including but not
limited to rabbits, mice, rats, etc., to generate polyclonal or
monoclonal antibodies. Various adjuvants (i.e. immunostimulants)
may be used to enhance the immunological response, depending on the
host species, including, but not limited to, complete or incomplete
Freund's adjuvant, a mineral gel such as aluminum hydroxide,
surface active substance such as lysolecithin, pluronic polyol, a
polyanion, a peptide, an oil emulsion, keyhole limpet hemocyanin,
dinitrophenol, and an adjuvant such as BCG (bacille
Calmette-Guerin) or corynebacterium parvum. Additional adjuvants
are also well known in the art.
[0217] For preparation of monoclonal antibodies (mAbs) directed
toward Cadherin-17, any technique which provides for the production
of antibody molecules by continuous cell lines in culture may be
used. For example, the hybridoma technique originally developed by
Kohler and Milstein (1975, Nature 256:495-497), as well as the
trioma technique, the human B-cell hybridoma technique (Kozbor et
al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique
to produce human monoclonal antibodies (Cole et al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Such antibodies may be of any immunoglobulin class
including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The
hybridoma producing the monoclonal antibodies may be cultivated in
vitro or in vivo. In an additional embodiment of the invention,
monoclonal antibodies can be produced in germ-free animals
utilizing known technology (PCT/US90/02545, incorporated herein by
reference).
[0218] The preferred animal system for preparing hybridomas is the
murine system. Hybridoma production in the mouse is a very
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known.
[0219] The monoclonal antibodies include but are not limited to
human monoclonal antibodies and chimeric monoclonal antibodies
(e.g. human-mouse chimeras).
[0220] Chimeric or humanized antibodies of the present invention
can be prepared based on the sequence of a non-human monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the non-human
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, murine CDR regions
can be inserted into a human framework using methods known in the
art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0221] Completely human antibodies can be produced using transgenic
or transchromosomic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chain genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.
all or a portion of Cadherin-17.
[0222] Monoclonal antibodies directed against the antigen can be
obtained using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange
during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is
possible to produce therapeutically useful IgG, IgA, IgM and IgE
antibodies. These transgenic and transchromosomic mice include mice
of the HuMAb Mouse (Medarex, Inc.) and KM Mouse.RTM. strains. The
HuMAb Mouse.RTM. strain (Medarex.RTM., Inc.) is described in
Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g. U.S. Pat. No. 5,625,126; U.S.
Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No.
5,661,016; and U.S. Pat. No. 5,545,806. The KM Mouse.RTM. strain
refers to a mouse that carries a human heavy chain transgene and a
human light chain transchromosome and is described in detail in PCT
Publication WO 02/43478 to Ishida et al.
[0223] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-Cadherin-17 antibodies of the invention.
For example, an alternative transgenic system referred to as the
Xenomouse (Amgen, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;
6,150,584 and 6,162,963 to Kucherlapati et al.
[0224] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection". In this approach a selected non-human monoclonal
antibody, e.g. a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al. (1994) Bio/technology 12:899-903).
[0225] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-Cadherin-17 antibodies. For example, mice
carrying both a human heavy chain transchromosome and a human light
chain tranchromosome, referred to as "TC mice" can be used; such
mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci.
USA 97:722-727. Furthermore, cows carrying human heavy and light
chain transchromosomes have been described in the art (Kuroiwa et
al. (2002) Nature Biotechnology 20:889-894) and PCT application No.
WO2002/092812 and can be used to raise anti-Cadherin-17
antibodies.
[0226] Human monoclonal antibodies of the invention can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
[0227] The antibodies of the present invention can be generated by
the use of phage display technology to produce and screen libraries
of polypeptides for binding to a selected target. See, e.g. Cwirla
et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et
al., Science 249, 404-6, 1990, Scott and Smith, Science 249,
386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic
concept of phage display methods is the establishment of a physical
association between DNA encoding a polypeptide to be screened and
the polypeptide. This physical association is provided by the phage
particle, which displays a polypeptide as part of a capsid
enclosing the phage genome which encodes the polypeptide. The
establishment of a physical association between polypeptides and
their genetic material allows simultaneous mass screening of very
large numbers of phage bearing different polypeptides. Phage
displaying a polypeptide with affinity to a target bind to the
target and these phage are enriched by affinity screening to the
target. The identity of polypeptides displayed from these phage can
be determined from their respective genomes. Using these methods a
polypeptide identified as having a binding affinity for a desired
target can then be synthesized in bulk by conventional means. See,
e.g. U.S. Pat. No. 6,057,098, which is hereby incorporated in its
entirety, including all tables, figures, and claims. In particular,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library (e.g.
human or murine). Phage expressing an antigen binding domain that
binds the antigen of interest can be selected or identified with
antigen, e.g. using labeled antigen or antigen bound or captured to
a solid surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Phage display methods that can be used to make the
antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
Application No. PCT/GB91/01134; PCT Publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0228] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g. as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and
F(ab').sub.2 fragments can also be employed using methods known in
the art such as those disclosed in PCT publication WO 92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et
al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043
(1988) (said references incorporated by reference in their
entireties).
[0229] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988).
[0230] The invention provides functionally active fragments,
derivatives or analogs of the anti-Cadherin-17 immunoglobulin
molecules. Functionally active means that the fragment, derivative
or analog is able to elicit anti-anti-idiotype antibodies (i.e.,
tertiary antibodies) that recognize the same antigen that is
recognized by the antibody from which the fragment, derivative or
analog is derived. Specifically, in a particular embodiment the
antigenicity of the idiotype of the immunoglobulin molecule may be
enhanced by deletion of framework and CDR sequences that are
C-terminal to the CDR sequence that specifically recognizes the
antigen. To determine which CDR sequences bind the antigen,
synthetic peptides containing the CDR sequences can be used in
binding assays with the antigen by any binding assay method known
in the art.
[0231] The present invention provides antibody fragments such as,
but not limited to, F(ab').sub.2 fragments and Fab fragments.
Antibody fragments which recognize specific epitopes may be
generated by known techniques. F(ab').sub.2 fragments consist of
the variable region, the light chain constant region and the CH1
domain of the heavy chain and are generated by pepsin digestion of
the antibody molecule. Fab fragments are generated by reducing the
disulfide bridges of the F(ab').sub.2 fragments. The invention also
provides heavy chain and light chain dimers of the antibodies of
the invention, or any minimal fragment thereof such as Fvs or
single chain antibodies (SCAs) (e.g. as described in U.S. Pat. No.
4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-54), or any other molecule with the same specificity
as the antibody of the invention. Single chain antibodies are
formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional Fv fragments
in E. coli may be used (Skerra et al., 1988, Science
242:1038-1041).
[0232] In other embodiments, the invention provides fusion proteins
of the immunoglobulins of the invention (or functionally active
fragments thereof), for example in which the immunoglobulin is
fused via a covalent bond (e.g. a peptide bond), at either the
N-terminus or the C-terminus to an amino acid sequence of another
protein (or portion thereof, preferably at least 10, 20 or 50 amino
acid portion of the protein) that is not the immunoglobulin.
Preferably the immunoglobulin, or fragment thereof, is covalently
linked to the other protein at the N-terminus of the constant
domain. As stated above, such fusion proteins may facilitate
purification, increase half-life in vivo, and enhance the delivery
of an antigen across an epithelial barrier to the immune
system.
[0233] The immunoglobulins of the invention include analogs and
derivatives that are modified, i.e., by the covalent attachment of
any type of molecule as long as such covalent attachment does not
impair immunospecific binding. For example, but not by way of
limitation, the derivatives and analogs of the immunoglobulins
include those that have been further modified, e.g. by
glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications may be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, etc. Additionally, the analog
or derivative may contain one or more non-classical amino
acids.
Immunization of Mice
[0234] Mice can be immunized with a purified or enriched
preparation of Cadherin-17 antigen and/or recombinant Cadherin-17,
or cells expressing Cadherin-17. Preferably, the mice will be 6-16
weeks of age upon the first infusion. For example, a purified or
recombinant preparation (100 .mu.g) of Cadherin-17 antigen can be
used to immunize the mice intraperitoneally.
[0235] Cumulative experience with various antigens has shown that
the mice respond when immunized intraperitoneally (IP) with antigen
in complete Freund's adjuvant. However, adjuvants other than
Freund's are also found to be effective. In addition, whole cells
in the absence of adjuvant are found to be highly immunogenic. The
immune response can be monitored over the course of the
immunization protocol with plasma samples being obtained by
retroorbital bleeds. The plasma can be screened by ELISA (as
described below) to test for satisfactory titres. Mice can be
boosted intravenously with antigen on 3 consecutive days with
sacrifice and removal of the spleen taking place 5 days later. In
one embodiment, A/J mouse strains (Jackson Laboratories, Bar
Harbor, Me.) may be used.
Generation of Transfectomas Producing Monoclonal Antibodies
Antibodies of the invention can be produced in a host cell
transfectoma using, for example, a combination of recombinant DNA
techniques and gene transfection methods as is well known in the
art (e.g., Morrison, S. (1985) Science 229:1202).
[0236] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used.
[0237] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0238] The antibody genes are inserted into the expression vector
by standard methods (e.g., ligation of complementary restriction
sites on the antibody gene fragment and vector, or blunt end
ligation if no restriction sites are present). The light and heavy
chain variable regions of the antibodies described herein can be
used to create full-length antibody genes of any antibody isotype
by inserting them into expression vectors already encoding heavy
chain constant and light chain constant regions of the desired
isotype such that the V.sub.H segment is operatively linked to the
C.sub.H segment(s) within the vector and the V.sub.K segment is
operatively linked to the C.sub.L segment within the vector.
Additionally or alternatively, the recombinant expression vector
can encode a signal peptide that facilitates secretion of the
antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0239] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990)). It will be appreciated by those skilled in the art that
the design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or .beta.-globin
promoter. Still further, regulatory elements composed of sequences
from different sources, such as the SR.alpha. promoter system,
which contains sequences from the SV40 early promoter and the long
terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.
et al. (1988) Mol. Cell. Biol. 8:466-472).
[0240] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0241] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13).
[0242] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese hamster
ovary cells (CHO), in conjunction with a vector such as the major
intermediate early gene promoter element from human cytomegalovirus
(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,
Bio/Technology 8:2), dhfr-CHO cells, described in Urlaub and
Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a
DHFR selectable marker, e.g., as described in R. J. Kaufman and P.
A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS
cells and SP2 cells. In particular, for use with NSO myeloma cells,
another preferred expression system is the GS gene expression
system disclosed in WO 87/04462 (to Wilson), WO 89/01036 (to
Bebbington) and EP 338,841 (to Bebbington).
[0243] A variety of host-expression vector systems may be utilized
to express an antibody molecule of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express the
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g. E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g. Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g. baculovirus) containing the antibody
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g. cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g. Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.
metallothionein promoter) or from mammalian viruses (e.g. the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0244] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions comprising an antibody molecule,
vectors which direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited, to the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the
antibody coding sequence may be ligated individually into the
vector in frame with the lac Z coding region so that a fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption and binding to a matrix glutathione-agarose beads
followed by elution in the presence of free glutathione. The pGEX
vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0245] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodopterafrugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). In mammalian host cells, a number of viral-based
expression systems (e.g. an adenovirus expression system) may be
utilized.
[0246] As discussed above, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g. glycosylation) and processing (e.g. cleavage)
of protein products may be important for the function of the
protein.
[0247] For long-term, high-yield production of recombinant
antibodies, stable expression is preferred. For example, cell lines
that stably express an antibody of interest can be produced by
transfecting the cells with an expression vector comprising the
nucleotide sequence of the antibody and the nucleotide sequence of
a selectable (e.g. neomycin or hygromycin), and selecting for
expression of the selectable marker. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that interact directly or indirectly with the antibody
molecule.
[0248] The expression levels of the antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
[0249] When recombinant expression vectors encoding antibody genes
are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Once the antibody
molecule of the invention has been recombinantly expressed, it may
be purified by any method known in the art for purification of an
antibody molecule, for example, by chromatography (e.g. ion
exchange chromatography, affinity chromatography such as with
protein A or specific antigen, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of proteins.
[0250] Alternatively, any fusion protein may be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-897). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
open reading frame of the gene is translationally fused to an
amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix binding domain for the fusion protein. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
Characterization of Antibody Binding to Antigen
[0251] The antibodies that are generated by these methods may then
be selected by first screening for affinity and specificity with
the purified polypeptide of interest and, if required, comparing
the results to the affinity and specificity of the antibodies with
polypeptides that are desired to be excluded from binding. The
antibodies can be tested for binding to Cadherin-17 by, for
example, standard ELISA. The screening procedure can involve
immobilization of the purified polypeptides in separate wells of
microtiter plates. The solution containing a potential antibody or
groups of antibodies is then placed into the respective microtiter
wells and incubated for about 30 min to 2 h. The microtiter wells
are then washed and a labeled secondary antibody (for example, an
anti-mouse antibody conjugated to alkaline phosphatase if the
raised antibodies are mouse antibodies) is added to the wells and
incubated for about 30 min and then washed. Substrate is added to
the wells and a color reaction will appear where antibody to the
immobilized polypeptide(s) is present. The antibodies so identified
may then be further analyzed for affinity and specificity in the
assay design selected. In the development of immunoassays for a
target protein, the purified target protein acts as a standard with
which to judge the sensitivity and specificity of the immunoassay
using the antibodies that have been selected. Because the binding
affinity of various antibodies may differ; certain antibody pairs
(e.g. in sandwich assays) may interfere with one another
sterically, etc., assay performance of an antibody may be a more
important measure than absolute affinity and specificity of an
antibody.
[0252] Those skilled in the art will recognize that many approaches
can be taken in producing antibodies or binding fragments and
screening and selecting for affinity and specificity for the
various polypeptides, but these approaches do not change the scope
of the invention.
[0253] To determine if the selected anti-Cadherin-17 monoclonal
antibodies bind to unique epitopes, each antibody can be
biotinylated using commercially available reagents (Pierce,
Rockford, Ill.). Competition studies using unlabeled monoclonal
antibodies and biotinylated monoclonal antibodies can be performed
using Cadherin-17 coated-ELISA plates. Biotinylated mAb binding can
be detected with a strep-avidin-alkaline phosphatase probe.
[0254] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for antibodies of a
particular isotype.
[0255] Anti-Cadherin-17 antibodies can be further tested for
reactivity with Cadherin-17 antigen by Western blotting. Briefly,
Cadherin-17 can be prepared and subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis. After electrophoresis, the
separated antigens are transferred to nitrocellulose membranes,
blocked with 10% fetal calf serum, and probed with the monoclonal
antibodies to be tested.
[0256] The binding specificity of an antibody of the invention may
also be determined by monitoring binding of the antibody to cells
expressing Cadherin-17, for example by flow cytometry. Typically, a
cell line, such as a CHO cell line, may be transfected with an
expression vector encoding Cadherin-17. The transfected protein may
comprise a tag, such as a myc-tag, preferably at the N-terminus,
for detection using an antibody to the tag. Binding of an antibody
of the invention to Cadherin-17 may be determined by incubating the
transfected cells with the antibody, and detecting bound antibody.
Binding of an antibody to the tag on the transfected protein may be
used as a positive control.
[0257] The specificity of an antibody of the invention for
Cadherin-17 may be further studied by determining whether or not
the antibody binds to other proteins, such as another member of the
Cadherin family using the same methods by which binding to
Cadherin-17 is determined.
Immunoconjugates
[0258] In another aspect, the present invention features an
anti-Cadherin-17 antibody, or a fragment thereof, conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radiotoxin. Such conjugates are referred to
herein as "immunoconjugates". Immunoconjugates that include one or
more cytotoxins are referred to as "immunotoxins." A cytotoxin or
cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells. Examples include Taxol.RTM., cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Therapeutic agents also include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0259] Other preferred examples of therapeutic cytotoxins that can
be conjugated to an antibody of the invention include duocarmycins,
calicheamicins, maytansines and auristatins, and derivatives
thereof. An example of a calicheamicin antibody conjugate is
commercially available (Mylotarg.RTM.; American Home Products).
[0260] Cytotoxins can be conjugated to antibodies of the invention
using linker technology available in the art. Examples of linker
types that have been used to conjugate a cytotoxin to an antibody
include, but are not limited to, hydrazones, thioethers, esters,
disulfides and peptide-containing linkers. A linker can be chosen
that is, for example, susceptible to cleavage by low pH within the
lysosomal compartment or susceptible to cleavage by proteases, such
as proteases preferentially expressed in tumor tissue such as
cathepsins (e.g., cathepsins B, C, D).
[0261] Examples of cytotoxins are described, for example, in U.S.
Pat. Nos. 6,989,452, 7,087,600, and 7,129,261, and in PCT
Application Nos. PCT/US2002/17210, PCT/US2005/017804,
PCT/US2006/37793, PCT/US2006/060050, PCT/US2006/060711,
WO2006/110476, and in U.S. Patent Application No. 60/891,028, all
of which are incorporated herein by reference in their entirety.
For further discussion of types of cytotoxins, linkers and methods
for conjugating therapeutic agents to antibodies, see also Saito,
G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et
al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003)
Cancer Cell 3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer
2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin.
Investig. Drugs 3:1089-1091; Senter, P. D. and Springer, C. J.
(2001) Adv. Drug Deliv. Rev. 53:247-264.
[0262] Antibodies of the present invention also can be conjugated
to a radioactive isotope to generate cytotoxic
radiopharmaceuticals, also referred to as radioimmunoconjugates.
Examples of radioactive isotopes that can be conjugated to
antibodies for use diagnostically or therapeutically include, but
are not limited to, iodine 131, indium111, yttrium90 and
lutetium177. Method for preparing radioimmunoconjugates are
established in the art. Examples of radioimmunoconjugates are
commercially available, including Zevalin.RTM. (IDEC
Pharmaceuticals) and Bexxar.RTM. (Corixa Pharmaceuticals), and
similar methods can be used to prepare radioimmunoconjugates using
the antibodies of the invention.
[0263] The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0264] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Anon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review," in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., Immunol. Rev.,
62:119-58 (1982).
Bispecific Molecules
[0265] In another aspect, the present invention features bispecific
molecules comprising an anti-Cadherin-17 antibody, or a fragment
thereof, of the invention. An antibody of the invention, or
antigen-binding portions thereof, can be derivatized or linked to
another functional molecule, e.g., another peptide or protein
(e.g., another antibody or ligand for a receptor) to generate a
bispecific molecule that binds to at least two different binding
sites or target molecules. The antibody of the invention may in
fact be derivatized or linked to more than one other functional
molecule to generate multispecific molecules that bind to more than
two different binding sites and/or target molecules; such
multispecific molecules are also intended to be encompassed by the
term "bispecific molecule" as used herein. To create a bispecific
molecule of the invention, an antibody of the invention can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other binding
molecules, such as another antibody, antibody fragment, peptide or
binding mimetic, such that a bispecific molecule results.
[0266] Accordingly, the present invention includes bispecific
molecules comprising at least one first binding specificity for
Cadherin-17 and a second binding specificity for a second target
epitope. In a particular embodiment of the invention, the second
target epitope is an Fc receptor, e.g., human Fc.gamma.RI (CD64) or
a human Fc.alpha. receptor (CD89). Therefore, the invention
includes bispecific molecules capable of binding both to Fc.gamma.R
or Fc.alpha.R expressing effector cells (e.g., monocytes,
macrophages or polymorphonuclear cells (PMNs)), and to target cells
expressing Cadherin-17. These bispecific molecules target
Cadherin-17 expressing cells to effector cell and trigger Fc
receptor-mediated effector cell activities, such as phagocytosis of
Cadherin-17 expressing cells, antibody dependent cell-mediated
cytotoxicity (ADCC), cytokine release, or generation of superoxide
anion.
[0267] In an embodiment of the invention in which the bispecific
molecule is multispecific, the molecule can further include a third
binding specificity, in addition to an anti-Fc binding specificity
and an anti-Cadherin-17 binding specificity. In one embodiment, the
third binding specificity is an anti-enhancement factor (EF)
portion, e.g., a molecule which binds to a surface protein involved
in cytotoxic activity and thereby increases the immune response
against the target cell. The "anti-enhancement factor portion" can
be an antibody, functional antibody fragment or a ligand that binds
to a given molecule, e.g., an antigen or a receptor, and thereby
results in an enhancement of the effect of the binding determinants
for the Fc receptor or target cell antigen. The "anti-enhancement
factor portion" can bind an Fc receptor or a target cell antigen.
Alternatively, the anti-enhancement factor portion can bind to an
entity that is different from the entity to which the first and
second binding specificities bind. For example, the
anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.
via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell
that results in an increased immune response against the target
cell).
[0268] In one embodiment, the bispecific molecules of the invention
comprise as a binding specificity at least one antibody, or an
antibody fragment thereof, including, e.g., an Fab, Fab',
F(ab').sub.2, Fv, Fd, dAb or a single chain Fv. The antibody may
also be a light chain or heavy chain dimer, or any minimal fragment
thereof such as a Fv or a single chain construct as described in
U.S. Pat. No. 4,946,778 to Ladner et al., the contents of which is
expressly incorporated by reference.
[0269] In one embodiment, the binding specificity for an Fc.gamma.
receptor is provided by a monoclonal antibody, the binding of which
is not blocked by human immunoglobulin G (IgG). As used herein, the
term "IgG receptor" refers to any of the eight .gamma.-chain genes
located on chromosome 1. These genes encode a total of twelve
transmembrane or soluble receptor isoforms which are grouped into
three Fc.gamma. receptor classes: Fc.gamma.RI (CD64), Fc.gamma.
RII(CD32), and Fc.gamma.RIII (CD16). In one preferred embodiment,
the Fc.gamma. receptor is a human high affinity Fc.gamma.RI. The
human Fc.gamma.RI is a 72 kDa molecule, which shows high affinity
for monomeric IgG (10.sup.8-10.sup.9 M.sup.-1).
[0270] The production and characterization of certain preferred
anti-Fc.gamma. .quadrature..quadrature. monoclonal antibodies are
described in PCT Publication WO 88/00052 and in U.S. Pat. No.
4,954,617 to Fanger et al., the teachings of which are fully
incorporated by reference herein. These antibodies bind to an
epitope of Fc.gamma.RI, Fc.gamma.RII or Fc.gamma.RIII at a site
which is distinct from the Fc.gamma. binding site of the receptor
and, thus, their binding is not blocked substantially by
physiological levels of IgG. Specific anti-Fc.gamma.RI antibodies
useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb
197. The hybridoma producing mAb 32 is available from the American
Type Culture Collection, ATCC Accession No. HB9469. In other
embodiments, the anti-Fc.gamma. receptor antibody is a humanized
form of monoclonal antibody 22 (H22). The production and
characterization of the H22 antibody is described in Graziano, R.
F. et al. (1995) J. Immunol 155 (10): 4996-5002 and PCT Publication
WO 94/10332 to Tempest et al. The H22 antibody producing cell line
was deposited at the American Type Culture Collection under the
designation HA022CL1 and has the accession no. CRL 11177. In still
other preferred embodiments, the binding specificity for an Fc
receptor is provided by an antibody that binds to a human IgA
receptor, e.g., an Fc-alpha receptor (Fc.alpha.RI (CD89)), the
binding of which is preferably not blocked by human immunoglobulin
A (IgA). The term "IgA receptor" is intended to include the gene
product of one .alpha.-gene (Fc.alpha.RI) located on chromosome 19.
This gene is known to encode several alternatively spliced
transmembrane isoforms of 55 to 110 kDa. Fc.alpha.RI (CD89) is
constitutively expressed on monocytes/macrophages, eosinophilic and
neutrophilic granulocytes, but not on non-effector cell
populations. Fc.alpha.RI has medium affinity (=5.times.10.sup.7
M.sup.-1) for both IgA1 and IgA2, which is increased upon exposure
to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996)
Critical Reviews in Immunology 16:423-440). Four
Fc.alpha.RI-specific monoclonal antibodies, identified as A3, A59,
A62 and A77, which bind Fc.alpha.RI outside the IgA ligand binding
domain, have been described (Monteiro, R. C. et al. (1992) J.
Immunol. 148:1764).
[0271] Fc.alpha.RI and Fc.gamma.RI are preferred trigger receptors
for use in the bispecific molecules of the invention because they
are (1) expressed primarily on immune effector cells, e.g.,
monocytes, PMNs, macrophages and dendritic cells; (2) expressed at
high levels (e.g., 5,000-100,000 per cell); (3) mediators of
cytotoxic activities (e.g., ADCC, phagocytosis); and (4) mediate
enhanced antigen presentation of antigens, including self-antigens,
targeted to them.
[0272] Antibodies which can be employed in the bispecific molecules
of the invention are murine, human, chimeric and humanized
monoclonal antibodies.
[0273] The bispecific molecules of the present invention can be
prepared by conjugating the constituent binding specificities,
e.g., the anti-FcR and anti-Cadherin-17 binding specificities,
using methods known in the art. For example, each binding
specificity of the bispecific molecule can be generated separately
and then conjugated to one another. When the binding specificities
are proteins or peptides, a variety of coupling or cross-linking
agents can be used for covalent conjugation. Examples of
cross-linking agents include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0274] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0275] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2 or ligand x
Fab fusion protein. A bispecific molecule of the invention can be a
single chain molecule comprising one single chain antibody and a
binding determinant, or a single chain bispecific molecule
comprising two binding determinants. Bispecific molecules may
comprise at least two single chain molecules. Methods for preparing
bispecific molecules are described for example in U.S. Pat. Nos.
5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;
5,013,653; 5,258,498; and 5,482,858, all of which are expressly
incorporated herein by reference.
[0276] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,
bioassay (e.g., growth inhibition), or Western Blot assay. Each of
these assays generally detects the presence of protein-antibody
complexes of particular interest by employing a labeled reagent
(e.g., an antibody) specific for the complex of interest. For
example, the FcR-antibody complexes can be detected using e.g., an
enzyme-linked antibody or antibody fragment which recognizes and
specifically binds to the antibody-FcR complexes. Alternatively,
the complexes can be detected using any of a variety of other
immunoassays. For example, the antibody can be radioactively
labeled and used in a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of
a.quadrature..gamma..quadrature. counter or a scintillation counter
or by autoradiography.
Antibody Fragments and Antibody Mimetics
[0277] The instant invention is not limited to traditional
antibodies and may be practiced through the use of antibody
fragments and antibody mimetics. As detailed below, a wide variety
of antibody fragment and antibody mimetic technologies have now
been developed and are widely known in the art. While a number of
these technologies, such as domain antibodies, Nanobodies.RTM., and
UniBodies.RTM. make use of fragments of, or other modifications to,
traditional antibody structures, there are also alternative
technologies, such as Affibodies.RTM., DARPins.RTM.,
Anticalins.RTM., Avimers, and Versabodies that employ binding
structures that, while they mimic traditional antibody binding, are
generated from and function via distinct mechanisms.
[0278] Domain Antibodies (dAbs) are the smallest functional binding
units of antibodies, corresponding to the variable regions of
either the heavy (V.sub.H) or light (V.sub.L) chains of human
antibodies. Domain Antibodies have a molecular weight of
approximately 13 kDa. Domantis has developed a series of large and
highly functional libraries of fully human V.sub.H and V.sub.L dAbs
(more than ten billion different sequences in each library), and
uses these libraries to select dAbs that are specific to
therapeutic targets. In contrast to many conventional antibodies,
Domain Antibodies are well expressed in bacterial, yeast, and
mammalian cell systems. Further details of domain antibodies and
methods of production thereof may be obtained by reference to U.S.
Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; US
Serial No. 2004/0110941; European patent application No. 1433846
and European Patents 0368684 & 0616640; WO05/035572,
WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609,
each of which is herein incorporated by reference in its
entirety.
[0279] Nanobodies.RTM. are antibody-derived therapeutic proteins
that contain the unique structural and functional properties of
naturally-occurring heavy-chain antibodies. These heavy-chain
antibodies contain a single variable domain (VHH) and two constant
domains (CH2 and CH3). Importantly, the cloned and isolated VHH
domain is a perfectly stable polypeptide harboring the full
antigen-binding capacity of the original heavy-chain antibody.
Nanobodies.RTM. have a high homology with the VH domains of human
antibodies and can be further humanized without any loss of
activity. Importantly, Nanobodies.RTM. have a low immunogenic
potential, which has been confirmed in primate studies with
Nanobody.RTM. lead compounds.
[0280] Nanobodies.RTM. combine the advantages of conventional
antibodies with important features of small molecule drugs. Like
conventional antibodies, Nanobodies.RTM. show high target
specificity, high affinity for their target and low inherent
toxicity. However, like small molecule drugs they can inhibit
enzymes and readily access receptor clefts. Furthermore,
Nanobodies.RTM. are extremely stable, can be administered by means
other than injection (see e.g. WO 04/041867, which is herein
incorporated by reference in its entirety) and are easy to
manufacture. Other advantages of Nanobodies.RTM. include
recognizing uncommon or hidden epitopes as a result of their small
size, binding into cavities or active sites of protein targets with
high affinity and selectivity due to their unique 3-dimensional,
drug format flexibility, tailoring of half-life and ease and speed
of drug discovery.
[0281] Nanobodies.RTM. are encoded by single genes and are
efficiently produced in almost all prokaryotic and eukaryotic hosts
e.g. E. coli (see e.g. U.S. Pat. No. 6,765,087, which is herein
incorporated by reference in its entirety), molds (for example
Aspergillus or Trichoderma) and yeast (for example Saccharomyces,
Kluyveromyces, Hansenula or Pichia) (see e.g. U.S. Pat. No.
6,838,254, which is herein incorporated by reference in its
entirety). The production process is scalable and multi-kilogram
quantities of Nanobodies.RTM. have been produced. Because
Nanobodies.RTM. exhibit a superior stability compared with
conventional antibodies, they can be formulated as a long
shelf-life, ready-to-use solution.
[0282] The Nanoclone method (see e.g. WO 06/079372, which is herein
incorporated by reference in its entirety) is a proprietary method
for generating Nanobodies.RTM. against a desired target, based on
automated high-throughout selection of B-cells and could be used in
the context of the instant invention.
[0283] UniBodies.RTM. are another antibody fragment technology;
however this one is based upon the removal of the hinge region of
IgG4 antibodies. The deletion of the hinge region results in a
molecule that is essentially half the size of traditional IgG4
antibodies and has a univalent binding region rather than the
bivalent binding region of IgG4 antibodies. It is also well known
that IgG4 antibodies are inert and thus do not interact with the
immune system, which may be advantageous for the treatment of
diseases where an immune response is not desired, and this
advantage is passed onto UniBodies.RTM.. For example,
UniBodies.RTM. may function to inhibit or silence, but not kill,
the cells to which they are bound. Additionally, UniBody.RTM.
binding to cancer cells do not stimulate them to proliferate.
Furthermore, because UniBodies.RTM. are about half the size of
traditional IgG4 antibodies, they may show better distribution over
larger solid tumors with potentially advantageous efficacy.
UniBodies.RTM. are cleared from the body at a similar rate to whole
IgG4 antibodies and are able to bind with a similar affinity for
their antigens as whole antibodies. Further details of
UniBodies.RTM. may be obtained by reference to patent application
WO2007/059782, which is herein incorporated by reference in its
entirety.
[0284] Affibody molecules represent a new class of affinity
proteins based on a 58-amino acid residue protein domain, derived
from one of the IgG-binding domains of staphylococcal protein A.
This three helix bundle domain has been used as a scaffold for the
construction of combinatorial phagemid libraries, from which
Affibody variants that target the desired molecules can be selected
using phage display technology (Nord K, Gunneriusson E, Ringdahl J,
Stahl S, Uhlen M, Nygren P A, Binding proteins selected from
combinatorial libraries of an .alpha.-helical bacterial receptor
domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen
M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands from
combinatorial engineering of protein A, Eur J Biochem 2002;
269:2647-55.). The simple, robust structure of Affibody.RTM.
molecules in combination with their low molecular weight (6 kDa),
make them suitable for a wide variety of applications, for
instance, as detection reagents (Ronmark J, Hansson M, Nguyen T, et
al, Construction and characterization of affibody-Fc chimeras
produced in Escherichia coli, J Immunol Methods 2002; 261:199-211)
and to inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg
G, Nygren P A, Inhibition of the CD28-CD80 co-stimulation signal by
a CD28-binding Affibody ligand developed by combinatorial protein
engineering, Protein Eng 2003; 16:691-7). Further details of
Affibodies and methods of production thereof may be obtained by
reference to U.S. Pat. No. 5,831,012 which is herein incorporated
by reference in its entirety.
[0285] Labelled Affibodies may also be useful in imaging
applications for determining abundance of Isoforms.
[0286] DARPins (Designed Ankyrin Repeat Proteins) are one example
of an antibody mimetic DRP (Designed Repeat Protein) technology
that has been developed to exploit the binding abilities of
non-antibody polypeptides. Repeat proteins such as ankyrin or
leucine-rich repeat proteins, are ubiquitous binding molecules,
which occur, unlike antibodies, intra- and extracellularly. Their
unique modular architecture features repeating structural units
(repeats), which stack together to form elongated repeat domains
displaying variable and modular target-binding surfaces. Based on
this modularity, combinatorial libraries of polypeptides with
highly diversified binding specificities can be generated. This
strategy includes the consensus design of self-compatible repeats
displaying variable surface residues and their random assembly into
repeat domains.
[0287] DARPins can be produced in bacterial expression systems at
very high yields and they belong to the most stable proteins known.
Highly specific, high-affinity DARPins to a broad range of target
proteins, including human receptors, cytokines, kinases, human
proteases, viruses and membrane proteins, have been selected.
DARPins having affinities in the single-digit nanomolar to
picomolar range can be obtained.
[0288] DARPins have been used in a wide range of applications,
including ELISA, sandwich ELISA, flow cytometric analysis (FACS),
immunohistochemistry (IHC), chip applications, affinity
purification or Western blotting. DARPins also proved to be highly
active in the intracellular compartment for example as
intracellular marker proteins fused to green fluorescent protein
(GFP). DARPins were further used to inhibit viral entry with IC50
in the pM range. DARPins are not only ideal to block
protein-protein interactions, but also to inhibit enzymes.
Proteases, kinases and transporters have been successfully
inhibited, most often an allosteric inhibition mode. Very fast and
specific enrichments on the tumor and very favorable tumor to blood
ratios make DARPins well suited for in vivo diagnostics or
therapeutic approaches.
[0289] Additional information regarding DARPins and other DRP
technologies can be found in US Patent Application Publication No.
2004/0132028, and International Patent Application Publication No.
WO 02/20565, both of which are hereby incorporated by reference in
their entirety.
[0290] Anticalins.RTM. are an additional antibody mimetic
technology, however in this case the binding specificity is derived
from lipocalins, a family of low molecular weight proteins that are
naturally and abundantly expressed in human tissues and body
fluids. Lipocalins have evolved to perform a range of functions in
vivo associated with the physiological transport and storage of
chemically sensitive or insoluble compounds. Lipocalins have a
robust intrinsic structure comprising a highly conserved
.beta.-barrel which supports four loops at one terminus of the
protein. These loops form the entrance to a binding pocket and
conformational differences in this part of the molecule account for
the variation in binding specificity between individual
lipocalins.
[0291] While the overall structure of hypervariable loops supported
by a conserved .beta.-sheet framework is reminiscent of
immunoglobulins, lipocalins differ considerably from antibodies in
terms of size, being composed of a single polypeptide chain of
160-180 amino acids which is marginally larger than a single
immunoglobulin domain.
[0292] Lipocalins are cloned and their loops are subjected to
engineering in order to create Anticalins.RTM.. Libraries of
structurally diverse Anticalins.RTM. have been generated and
Anticalin.RTM. display allows the selection and screening of
binding function, followed by the expression and production of
soluble protein for further analysis in prokaryotic or eukaryotic
systems. Studies have successfully demonstrated that
Anticalins.RTM. can be developed that are specific for virtually
any human target protein can be isolated and binding affinities in
the nanomolar or higher range can be obtained.
[0293] Anticalins.RTM. can also be formatted as dual targeting
proteins, so-called Duocalins. A Duocalin binds two separate
therapeutic targets in one easily produced monomeric protein using
standard manufacturing processes while retaining target specificity
and affinity regardless of the structural orientation of its two
binding domains.
[0294] Modulation of multiple targets through a single molecule is
particularly advantageous in diseases known to involve more than a
single causative factor. Moreover, bi- or multivalent binding
formats such as Duocalins have significant potential in targeting
cell surface molecules in disease, mediating agonistic effects on
signal transduction pathways or inducing enhanced internalization
effects via binding and clustering of cell surface receptors.
Furthermore, the high intrinsic stability of Duocalins is
comparable to monomeric Anticalins.RTM., offering flexible
formulation and delivery potential for Duocalins.
[0295] Additional information regarding Anticalins.RTM. can be
found in U.S. Pat. No. 7,250,297 and International Patent
Application Publication No. WO 99/16873, both of which are hereby
incorporated by reference in their entirety.
[0296] Another antibody mimetic technology useful in the context of
the instant invention are Avimers. Avimers are evolved from a large
family of human extracellular receptor domains by in vitro exon
shuffling and phage display, generating multidomain proteins with
binding and inhibitory properties. Linking multiple independent
binding domains has been shown to create avidity and results in
improved affinity and specificity compared with conventional
single-epitope binding proteins. Other potential advantages include
simple and efficient production of multitarget-specific molecules
in Escherichia coli, improved thermostability and resistance to
proteases. Avimers with sub-nanomolar affinities have been obtained
against a variety of targets.
[0297] Additional information regarding Avimers can be found in US
Patent Application Publication Nos. 2006/0286603, 2006/0234299,
2006/0223114, 2006/0177831, 2006/0008844, 2005/0221384,
2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512,
2004/0175756, all of which are hereby incorporated by reference in
their entirety.
[0298] Versabodies are another antibody mimetic technology that
could be used in the context of the instant invention. Versabodies
are small proteins of 3-5 kDa with >15% cysteines, which form a
high disulfide density scaffold, replacing the hydrophobic core
that typical proteins have. The replacement of a large number of
hydrophobic amino acids, comprising the hydrophobic core, with a
small number of disulfides results in a protein that is smaller,
more hydrophilic (less aggregation and non-specific binding), more
resistant to proteases and heat, and has a lower density of T-cell
epitopes, because the residues that contribute most to MHC
presentation are hydrophobic. All four of these properties are
well-known to affect immunogenicity, and together they are expected
to cause a large decrease in immunogenicity.
[0299] The inspiration for Versabodies comes from the natural
injectable biopharmaceuticals produced by leeches, snakes, spiders,
scorpions, snails, and anemones, which are known to exhibit
unexpectedly low immunogenicity. Starting with selected natural
protein families, by design and by screening the size,
hydrophobicity, proteolytic antigen processing, and epitope density
are minimized to levels far below the average for natural
injectable proteins.
[0300] Given the structure of Versabodies, these antibody mimetics
offer a versatile format that includes multi-valency,
multi-specificity, a diversity of half-life mechanisms, tissue
targeting modules and the absence of the antibody Fc region.
Furthermore, Versabodies are manufactured in E. coli at high
yields, and because of their hydrophilicity and small size,
Versabodies are highly soluble and can be formulated to high
concentrations. Versabodies are exceptionally heat stable (they can
be boiled) and offer extended shelf-life.
[0301] Additional information regarding Versabodies can be found in
US Patent Application Publication No. 2007/0191272 which is hereby
incorporated by reference in its entirety.
[0302] The detailed description of antibody fragment and antibody
mimetic technologies provided above is not intended to be a
comprehensive list of all technologies that could be used in the
context of the instant specification. For example, and also not by
way of limitation, a variety of additional technologies including
alternative polypeptide-based technologies, such as fusions of
complimentary determining regions as outlined in Qui et al., Nature
Biotechnology, 25(8) 921-929 (2007), which is hereby incorporated
by reference in its entirety, as well as nucleic acid-based
technologies, such as the RNA aptamer technologies described in
U.S. Pat. Nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566,
6,013,443, 6,376,474, 6,613,526, 6,114,120, 6,261,774, and
6,387,620, all of which are hereby incorporated by reference, could
be used in the context of the instant invention.
Pharmaceutical Compositions
[0303] In another aspect, the present invention provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present invention, formulated together
with a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of (e.g., two or more different)
antibodies, or immunoconjugates or bispecific molecules of the
invention. For example, a pharmaceutical composition of the
invention can comprise a combination of antibodies (or
immunoconjugates or bispecifics) that bind to different epitopes on
the target antigen or that have complementary activities.
[0304] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-Cadherin-17 antibody of the present invention combined with at
least one other anti-tumor agent, or an anti-inflammatory or
immunosuppressant agent. Examples of therapeutic agents that can be
used in combination therapy are described in greater detail below
in the section on uses of the antibodies of the invention.
[0305] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody,
immunoconjugate, or bispecific molecule, may be coated in a
material to protect the compound from the action of acids and other
natural conditions that may inactivate the compound.
[0306] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0307] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0308] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0309] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0310] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0311] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0312] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0313] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0314] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0315] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months.
Preferred dosage regimens for an anti-Cadherin-17 antibody of the
invention include 1 mg/kg body weight or 3 mg/kg body weight via
intravenous administration, with the antibody being given using one
of the following dosing schedules: (i) every four weeks for six
dosages, then every three months; (ii) every three weeks; (iii) 3
mg/kg body weight once followed by 1 mg/kg body weight every three
weeks.
[0316] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml.
[0317] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0318] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0319] A "therapeutically effective dosage" of an anti-Cadherin-17
antibody of the invention preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, or a prevention of impairment or
disability due to the disease affliction. For example, for the
treatment of Cadherin-17.sup.+ tumors, a "therapeutically effective
dosage" preferably inhibits cell growth or tumor growth by at least
about 20%, more preferably by at least about 40%, even more
preferably by at least about 60%, and still more preferably by at
least about 80% relative to untreated subjects. The ability of a
compound to inhibit tumor growth can be evaluated in an animal
model system predictive of efficacy in human tumors. Alternatively,
this property of a composition can be evaluated by examining the
ability of the compound to inhibit cell growth, such inhibition can
be measured in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
[0320] A composition of the present invention can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for antibodies of the invention include intravenous,
intramuscular, intradermal, intraperitoneal, subcutaneous, spinal
or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" as
used herein means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0321] Alternatively, an antibody of the invention can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0322] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0323] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. These patents are incorporated herein by
reference. Many other such implants, delivery systems, and modules
are known to those skilled in the art.
[0324] In certain embodiments, the monoclonal antibodies of the
invention can be formulated to ensure proper distribution in vivo.
For example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic compounds of
the invention cross the BBB (if desired), they can be formulated,
for example, in liposomes. For methods of manufacturing liposomes,
see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134); p.sup.120 (Schreier et al. (1994) J. Biol.
Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS
Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods
4:273.
Uses and Methods
[0325] The antibodies, antibody compositions and methods of the
present invention have numerous in vitro and in vivo diagnostic and
therapeutic utilities involving the diagnosis and treatment of
Cadherin-17 mediated disorders.
[0326] In some embodiments, these molecules can be administered to
cells in culture, in vitro or ex vivo, or to human subjects, e.g.,
in vivo, to treat, prevent and to diagnose a variety of disorders.
As used herein, the term "subject" is intended to include human and
non-human animals. Non-human animals include all vertebrates, e.g.,
mammals and non-mammals, such as non-human primates, sheep, dogs,
cats, cows, horses, chickens, amphibians, and reptiles. Preferred
subjects include human patients having disorders mediated by
Cadherin-17 activity. The methods are particularly suitable for
treating human patients having a disorder associated with aberrant
Cadherin-17 expression. When antibodies to Cadherin-17 are
administered together with another agent, the two can be
administered in either order or simultaneously.
[0327] Given the specific binding of the antibodies of the
invention for Cadherin-17, the antibodies of the invention can be
used to specifically detect Cadherin-17 expression on the surface
of cells and, moreover, can be used to purify Cadherin-17 via
immunoaffinity purification.
[0328] Furthermore, given the expression of Cadherin-17 on tumor
cells, the antibodies, antibody compositions and methods of the
present invention can be used to treat a subject with a tumorigenic
disorder, e.g., a disorder characterized by the presence of tumor
cells expressing Cadherin-17 including, for example, colorectal
cancer. Cadherin-17 has been demonstrated to be internalised on
antibody binding as illustrated in Example 10 below, thus enabling
the antibodies of the invention to be used in any payload mechanism
of action e.g. an ADC approach, radio immuno conjugate, or ADEPT
approach.
[0329] In one embodiment, the antibodies (e.g., monoclonal
antibodies, multispecific and bispecific molecules and
compositions) of the invention can be used to detect levels of
Cadherin-17, or levels of cells which contain Cadherin-17 on their
membrane surface, which levels can then be linked to certain
disease symptoms. Alternatively, the antibodies can be used to
inhibit or block Cadherin-17 function which, in turn, can be linked
to the prevention or amelioration of certain disease symptoms,
thereby implicating Cadherin-17 as a mediator of the disease. This
can be achieved by contacting a sample and a control sample with
the anti-Cadherin-17 antibody under conditions that allow for the
formation of a complex between the antibody and Cadherin-17. Any
complexes formed between the antibody and Cadherin-17 are detected
and compared in the sample and the control.
[0330] In another embodiment, the antibodies (e.g., monoclonal
antibodies, multispecific and bispecific molecules and
compositions) of the invention can be initially tested for binding
activity associated with therapeutic or diagnostic use in vitro.
For example, compositions of the invention can be tested using the
flow cytometric assays described in the Examples below.
[0331] The antibodies (e.g., monoclonal antibodies, multispecific
and bispecific molecules, immunoconjugates and compositions) of the
invention have additional utility in therapy and diagnosis of
Cadherin-17 related diseases. For example, the monoclonal
antibodies, the multispecific or bispecific molecules and the
immunoconjugates can be used to elicit in vivo or in vitro one or
more of the following biological activities: to inhibit the growth
of and/or kill a cell expressing Cadherin-17; to mediate
phagocytosis or ADCC of a cell expressing Cadherin-17 in the
presence of human effector cells, or to block Cadherin-17 ligand
binding to Cadherin-17.
[0332] In a particular embodiment, the antibodies (e.g., monoclonal
antibodies, multispecific and bispecific molecules and
compositions) are used in vivo to treat, prevent or diagnose a
variety of Cadherin-17-related diseases. Examples of
Cadherin-17-related diseases include, among others, human cancer
tissues representing colorectal cancer.
[0333] Suitable routes of administering the antibody compositions
(e.g., monoclonal antibodies, multispecific and bispecific
molecules and immunoconjugates) of the invention in vivo and in
vitro are well known in the art and can be selected by those of
ordinary skill. For example, the antibody compositions can be
administered by injection (e.g., intravenous or subcutaneous).
Suitable dosages of the molecules used will depend on the age and
weight of the subject and the concentration and/or formulation of
the antibody composition.
[0334] As previously described, anti-Cadherin-17 antibodies of the
invention can be co-administered with one or other more therapeutic
agents, e.g., a cytotoxic agent, a radiotoxic agent or an
immunosuppressive agent. The antibody can be linked to the agent
(as an immunocomplex) or can be administered separate from the
agent. In the latter case (separate administration), the antibody
can be administered before, after or concurrently with the agent or
can be co-administered with other known therapies, e.g., an
anti-cancer therapy, e.g., radiation. Such therapeutic agents
include, among others, anti-neoplastic agents such as doxorubicin
(adriamycin), cisplatin bleomycin sulfate, carmustine,
chlorambucil, and cyclophosphamide hydroxyurea which, by
themselves, are only effective at levels which are toxic or
subtoxic to a patient. Cisplatin is intravenously administered as a
100 mg/kg dose once every four weeks and adriamycin is
intravenously administered as a 60-75 mg/ml dose once every 21
days. Other agents suitable for co-administration with the
antibodies of the invention include other agents used for the
treatment of cancers, e.g. pancreatic or colorectal cancer, such as
Avastin.RTM., 5FU and gemcitabine. Co-administration of the
anti-Cadherin-17 antibodies, or antigen binding fragments thereof,
of the present invention with chemotherapeutic agents provides two
anti-cancer agents which operate via different mechanisms which
yield a cytotoxic effect to human tumor cells. Such
co-administration can solve problems due to development of
resistance to drugs or a change in the antigenicity of the tumor
cells which would render them unreactive with the antibody.
[0335] Target-specific effector cells, e.g., effector cells linked
to compositions (e.g., monoclonal antibodies, multispecific and
bispecific molecules) of the invention can also be used as
therapeutic agents. Effector cells for targeting can be human
leukocytes such as macrophages, neutrophils or monocytes. Other
cells include eosinophils, natural killer cells and other IgG- or
IgA-receptor bearing cells. If desired, effector cells can be
obtained from the subject to be treated. The target-specific
effector cells can be administered as a suspension of cells in a
physiologically acceptable solution. The number of cells
administered can be in the order of 10.sup.8-10.sup.9 but will vary
depending on the therapeutic purpose. In general, the amount will
be sufficient to obtain localization at the target cell, e.g., a
tumor cell expressing Cadherin-17, and to affect cell killing by,
e.g., phagocytosis. Routes of administration can also vary.
[0336] Therapy with target-specific effector cells can be performed
in conjunction with other techniques for removal of targeted cells.
For example, anti-tumor therapy using the compositions (e.g.,
monoclonal antibodies, multispecific and bispecific molecules) of
the invention and/or effector cells armed with these compositions
can be used in conjunction with chemotherapy. Additionally,
combination immunotherapy may be used to direct two distinct
cytotoxic effector populations toward tumor cell rejection. For
example, anti-Cadherin-17 antibodies linked to anti-Fc-gamma RI or
anti-CD3 may be used in conjunction with IgG- or IgA-receptor
specific binding agents.
[0337] Bispecific and multispecific molecules of the invention can
also be used to modulate Fc.gamma.R or Fc.gamma.R levels on
effector cells, such as by capping and elimination of receptors on
the cell surface. Mixtures of anti-Fc receptors can also be used
for this purpose.
[0338] The compositions (e.g., monoclonal antibodies, multispecific
and bispecific molecules and immunoconjugates) of the invention
which have complement binding sites, such as portions from IgG1,
-2, or -3 or IgM which bind complement, can also be used in the
presence of complement. In one embodiment, ex vivo treatment of a
population of cells comprising target cells with a binding agent of
the invention and appropriate effector cells can be supplemented by
the addition of complement or serum containing complement.
Phagocytosis of target cells coated with a binding agent of the
invention can be improved by binding of complement proteins. In
another embodiment target cells coated with the compositions (e.g.,
monoclonal antibodies, multispecific and bispecific molecules) of
the invention can also be lysed by complement. In yet another
embodiment, the compositions of the invention do not activate
complement.
[0339] The compositions (e.g., monoclonal antibodies, multispecific
and bispecific molecules and immunoconjugates) of the invention can
also be administered together with complement. In certain
embodiments, the instant disclosure provides compositions
comprising antibodies, multispecific or bispecific molecules and
serum or complement. These compositions can be advantageous when
the complement is located in close proximity to the antibodies,
multispecific or bispecific molecules. Alternatively, the
antibodies, multispecific or bispecific molecules of the invention
and the complement or serum can be administered separately.
[0340] Also within the scope of the present invention are kits
comprising the antibody compositions of the invention (e.g.,
monoclonal antibodies, bispecific or multispecific molecules, or
immunoconjugates) and instructions for use. The kit can further
contain one more more additional reagents, such as an
immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent,
or one or more additional antibodies of the invention (e.g., an
antibody having a complementary activity which binds to an epitope
in the Cadherin-17 antigen distinct from the first antibody).
[0341] Accordingly, patients treated with antibody compositions of
the invention can be additionally administered (prior to,
simultaneously with, or following administration of an antibody of
the invention) with another therapeutic agent, such as a cytotoxic
or radiotoxic agent, which enhances or augments the therapeutic
effect of the antibodies.
[0342] In other embodiments, the subject can be additionally
treated with an agent that modulates, e.g., enhances or inhibits,
the expression or activity of Fc.gamma. or Fc.gamma. receptors by,
for example, treating the subject with a cytokine. Preferred
cytokines for administration during treatment with the
multispecific molecule include of granulocyte colony-stimulating
factor (G-CSF), granulocyte-macrophage colony-stimulating factor
(GM-CSF), interferon-.gamma. (IFN-.gamma.), and tumor necrosis
factor (TNF).
[0343] The compositions (e.g., antibodies, multispecific and
bispecific molecules) of the invention can also be used to target
cells expressing Fc.gamma.R or Cadherin-17, for example for
labeling such cells. For such use, the binding agent can be linked
to a molecule that can be detected. Thus, the invention provides
methods for localizing ex vivo or in vitro cells expressing Fc
receptors, such as Fc.gamma.R, or Cadherin-17. The detectable label
can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or
an enzyme co-factor.
[0344] In a particular embodiment, the invention provides methods
for detecting the presence of Cadherin-17 antigen in a sample, or
measuring the amount of Cadherin-17 antigen, comprising contacting
the sample, and a control sample, with a monoclonal antibody, or an
antigen binding portion thereof, which specifically binds to
Cadherin-17, under conditions that allow for formation of a complex
between the antibody or portion thereof and Cadherin-17. The
formation of a complex is then detected, wherein a difference
complex formation between the sample compared to the control sample
is indicative the presence of Cadherin-17 antigen in the
sample.
[0345] In other embodiments, the invention provides methods for
treating a Cadherin-17 mediated disorder in a subject, e.g., human
cancers, including colorectal cancer.
[0346] In yet another embodiment, immunoconjugates of the invention
can be used to target compounds (e.g., therapeutic agents, labels,
cytotoxins, radiotoxins immunosuppressants, etc.) to cells which
have Cadherin-17 cell surface receptors by linking such compounds
to the antibody. For example, an anti-Cadherin-17 antibody can be
conjugated to any of the toxin compounds described in U.S. Pat.
Nos. 6,281,354 and 6,548,530, US patent publication Nos.
2003/0050331, 2003/0064984, 2003/0073852, and 2004/0087497, or
published in WO 03/022806. Thus, the invention also provides
methods for localizing ex vivo or in vivo cells expressing
Cadherin-17 (e.g., with a detectable label, such as a radioisotope,
a fluorescent compound, an enzyme, or an enzyme co-factor).
Alternatively, the immunoconjugates can be used to kill cells which
have Cadherin-17 cell surface receptors by targeting cytotoxins or
radiotoxins to Cadherin-17.
[0347] The present invention is further illustrated by the
following examples which should not be construed as further
limiting.
[0348] All references cited in this specification, including
without limitation all papers, publications, patents, patent
applications, presentations, texts, reports, manuscripts,
brochures, books, internet postings, journal articles, periodicals,
product fact sheets, and the like, one hereby incorporated by
reference into this specification in their entireties. The
discussion of the references herein is intended to merely summarize
the assertions made by their authors and no admission is made that
any reference constitutes prior art and Applicants' reserve the
right to challenge the accuracy and pertinence of the cited
references. Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be readily apparent to those of
ordinary skill in the art in light of the teachings of this
invention that certain changes and modifications may be made
thereto without departing from the spirit or scope of the dependant
claims.
EXAMPLES
Example 1
Construction of a Phage-Display Library
[0349] A recombinant protein composed of domains 1-2 of the
extracellular domain of Cadherin-17 (SEQ ID NO:136) was generated
in bacteria by standard recombinant methods and used as antigen for
immunization (see below). A recombinant protein composed of the
full length extracellular domain of Cadherin-17 (SEQ ID NO:137) was
also eurkaryotically synthesized by standard recombinant methods
and used for screening.
Immunization and mRNA Isolation
[0350] A phage display library for identification of
Cadherin-17-binding molecules was constructed as follows. A/J mice
(Jackson Laboratories, Bar Harbor, Me.) were immunized
intraperitoneally with recombinant Cadherin-17 antigen (domains 1-2
of the extracellular domain), using 100 .mu.g protein in Freund's
complete adjuvant, on day 0, and with 100 .mu.g antigen on day 28.
Test bleeds of mice were obtained through puncture of the
retro-orbital sinus. If, by testing the titers, they were deemed
high by ELISA using biotinylated Cadherin-17 antigen immobilized
via neutravidin (Reacti-Bind.TM. NeutrAvidin.RTM.-Coated
Polystyrene Plates, Pierce, Rockford, Ill.), the mice were boosted
with 100 .mu.g of protein on day 70, 71 and 72, with subsequent
sacrifice and splenectomy on day 77. If titers of antibody were not
deemed satisfactory, mice were boosted with 100 .mu.g antigen on
day 56 and a test bleed taken on day 63. If satisfactory titers
were obtained, the animals were boosted with 100 .mu.g of antigen
on day 98, 99, and 100 and the spleens harvested on day 105.
[0351] The spleens were harvested in a laminar flow hood and
transferred to a petri dish, trimming off and discarding fat and
connective tissue. The spleens were macerated quickly with the
plunger from a sterile 5 cc syringe in the presence of 1.0 ml of
solution D (25.0 g guanidine thiocyanate (Boehringer Mannheim,
Indianapolis, Ind.), 29.3 ml sterile water, 1.76 ml 0.75 M sodium
citrate pH 7.0, 2.64 ml 10% sarkosyl (Fisher Scientific,
Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (Fisher Scientific,
Pittsburgh, Pa.)). This spleen suspension was pulled through an 18
gauge needle until all cells were lysed and the viscous solution
was transferred to a microcentrifuge tube. The petri dish was
washed with 100 .mu.l of solution D to recover any remaining
spleen. This suspension was then pulled through a 22 gauge needle
an additional 5-10 times.
[0352] The sample was divided evenly between two microcentrifuge
tubes and the following added, in order, with mixing by inversion
after each addition: 50 .mu.l 2 M sodium acetate pH 4.0, 0.5 ml
water-saturated phenol (Fisher Scientific, Pittsburgh, Pa.), 100
.mu.l chloroform/isoamyl alcohol 49:1 (Fisher Scientific,
Pittsburgh, Pa.). The solution was vortexed for 10 seconds and
incubated on ice for 15 min. Following centrifugation at 14 krpm
for 20 min at 2-8.degree. C., the aqueous phase was transferred to
a fresh tube. An equal volume of water saturated
phenol:chloroform:isoamyl alcohol (50:49:1) was added, and the tube
vortexed for ten seconds. After a 15 min incubation on ice, the
sample was centrifuged for 20 min at 2-8.degree. C., and the
aqueous phase transferred to a fresh tube and precipitated with an
equal volume of isopropanol at -20.degree. C. for a minimum of 30
min. Following centrifugation at 14 krpm for 20 min at 4.degree.
C., the supernatant was aspirated away, the tubes briefly spun and
all traces of liquid removed from the RNA pellet.
[0353] The RNA pellets were each dissolved in 300 .mu.l of solution
D, combined, and precipitated with an equal volume of isopropanol
at -20.degree. C. for a minimum of 30 min. The sample was
centrifuged 14 krpm for 20 min at 4.degree. C., the supernatant
aspirated as before, and the sample rinsed with 100 .mu.l of
ice-cold 70% ethanol. The sample was again centrifuged 14 krpm for
20 min at 4.degree. C., the 70% ethanol solution aspirated, and the
RNA pellet dried in vacuo. The pellet was resuspended in 100 .mu.l
of sterile diethyl pyrocarbonate-treated water. The concentration
was determined by A260 using an absorbance of 1.0 for a
concentration of 40 .mu.g/ml. The RNAs were stored at -80.degree.
C.
Preparation of Complementary DNA (cDNA)
[0354] The total RNA purified from mouse spleens as described above
was used directly as template for cDNA preparation. RNA (50 .mu.g)
was diluted to 100 .mu.L with sterile water, and 10 .mu.L of 130
ng/.mu.L oligo dT12 (synthesized on Applied Biosystems Model 392
DNA synthesizer) was added. The sample was heated for 10 min at
70.degree. C., then cooled on ice. Forty .mu.L 5* first strand
buffer was added (Gibco/BRL, Gaithersburg, Md.), along with 20
.mu.L 0.1 M dithiothreitol (Gibco/BRL, Gaithersburg, Md.), 10 .mu.L
20 mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim,
Indianapolis, Ind.), and 10 .mu.L water on ice. The sample was then
incubated at 37.degree. C. for 2 min. Ten .mu.L reverse
transcriptase (Superscript.TM. II, Gibco/BRL, Gaithersburg, Md.)
was added and incubation was continued at 37.degree. C. for 1 hr.
The cDNA products were used directly for polymerase chain reaction
(PCR).
Amplification of Antibody Genes by PCR
[0355] To amplify substantially all of the H and L chain genes
using PCR, primers were chosen that corresponded to substantially
all published sequences. Because the nucleotide sequences of the
amino termini of H and L contain considerable diversity, 33
oligonucleotides were synthesized to serve as 5' primers for the H
chains, and 29 oligonucleotides were synthesized to serve as 5'
primers for the kappa L chains as described in U.S. patent
application Ser. No. 08/835,159, filed Apr. 4, 1997. The constant
region nucleotide sequences for each chain required only one 3'
primer for the H chains and one 3' primer for the kappa L
chains.
[0356] A 50 .mu.L reaction was performed for each primer pair with
50 .mu.mol of 5' primer, 50 .mu.mol of 3' primer, 0.25 .mu.L Taq
DNA Polymerase (5 units/.mu.L, Boehringer Mannheim, Indianapolis,
Ind.), 3 .mu.L cDNA (prepared as described), 5 .mu.L 2 mM dNTP's, 5
.mu.L 10*Taq DNA polymerase buffer with MgCl2 (Boehringer Mannheim,
Indianapolis, Ind.), and H2O to 50 .mu.L. Amplification was done
using a GeneAmp.RTM. 9600 thermal cycler (Perkin Elmer, Foster
City, Calif.) with the following thermocycle program: 94.degree. C.
for 1 min; 30 cycles of 94.degree. C. for 20 sec, 55.degree. C. for
30 sec, and 72.degree. C. for 30 sec; 72.degree. C. for 6 min;
4.degree. C.
[0357] The dsDNA products of the PCR process were then subjected to
asymmetric PCR using only a 3' primer to generate substantially
only the anti-sense strand of the target genes. A 100 .mu.L
reaction was done for each dsDNA product with 200 mol of 3' primer,
2 .mu.L of ds-DNA product, 0.5 .mu.L Taq DNA Polymerase, 10 .mu.L 2
mM dNTP's, 10 .mu.L 10*Taq DNA polymerase buffer with MgCl2
(Boehringer Mannheim, Indianapolis, Ind.), and H20 to 100 .mu.L.
The same PCR program as that described above was used to amplify
the single-stranded (ss)-DNA.
[0358] Purification of Single-Stranded DNA by High Performance
Liquid Chromatography and Kinasing Single-Stranded DNA
[0359] The H chain ss-PCR products and the L chain single-stranded
PCR products were ethanol precipitated by adding 2.5 volumes
ethanol and 0.2 volumes 7.5 M ammonium acetate and incubating at
-20.degree. C. for at least 30 min. The DNA was pelleted by
centrifuging in an Eppendorf centrifuge at 14 krpm for 10 min at
2-8.degree. C. The supernatant was carefully aspirated, and the
tubes were briefly spun a 2nd time. The last drop of supernatant
was removed with a pipette. The DNA was dried in vacuo for 10 min
on medium heat. The H chain products were pooled in 210 .mu.L water
and the L chain products were pooled separately in 210 .mu.L water.
The single-stranded DNA was purified by high performance liquid
chromatography (HPLC) using a Hewlett Packard 1090 HPLC and a
GenPak.RTM. FAX anion exchange column (Millipore Corp., Milford,
Mass.). The gradient used to purify the single-stranded DNA is
shown in Table 1, and the oven temperature was 60.degree. C.
Absorbance was monitored at 260 nm. The single-stranded DNA eluted
from the HPLC was collected in 0.5 min fractions. Fractions
containing single-stranded DNA were ethanol precipitated, pelleted
and dried as described above. The dried DNA pellets were pooled in
200 .mu.L sterile water.
TABLE-US-00001 TABLE 1 HPLC gradient for purification of ss-DNA
Time (min) % A % B % C Flow (ml/min) 0 70 30 0 0.75 2 40 60 0 0.75
17 15 85 0 0.75 18 0 100 0 0.75 23 0 100 0 0.75 24 0 0 100 0.75 28
0 0 100 0.75 29 0 100 0 0.75 34 0 100 0 0.75 35 70 30 0 0.75
Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0
Buffer B is 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 8.0
[0360] Buffer C is 40 mm phosphoric acid
[0361] The single-stranded DNA was 5'-phosphorylated in preparation
for mutagenesis. Twenty-four .mu.L 10* kinase buffer (United States
Biochemical, Cleveland, Ohio), 10.4 .mu.L 10 mM
adenosine-5'-triphosphate (Boehringer Mannheim, Indianapolis,
Ind.), and 2 L polynucleotide kinase (30 units/.mu.L, United States
Biochemical, Cleveland, Ohio) was added to each sample, and the
tubes were incubated at 37.degree. C. for 1 hr. The reactions were
stopped by incubating the tubes at 70.degree. C. for 10 min. The
DNA was purified with one extraction of Tris equilibrated phenol
(pH>8.0, United States Biochemical, Cleveland,
Ohio):chloroform:isoamyl alcohol (50:49:1) and one extraction with
chloroform:isoamyl alcohol (49:1). After the extractions, the DNA
was ethanol precipitated and pelleted as described above. The DNA
pellets were dried, then dissolved in 50 .mu.L sterile water. The
concentration was determined by measuring the absorbance of an
aliquot of the DNA at 260 nm using 33 .mu.g/ml for an absorbance of
1.0. Samples were stored at -20.degree. C.
Preparation of Uracil Templates Used in Generation of Spleen
Antibody Phage Libraries
[0362] One ml of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight
culture was added to 50 ml 2*YT in a 250 ml baffled shake flask.
The culture was grown at 37.degree. C. to OD600=0.6, inoculated
with 10 .mu.l of a 1/100 dilution of BS45 vector phage stock
(described in U.S. patent application Ser. No. 08/835,159, filed
Apr. 4, 1997) and growth continued for 6 hr. Approximately 40 ml of
the culture was centrifuged at 12 krpm for 15 minutes at 4.degree.
C. The supernatant (30 ml) was transferred to a fresh centrifuge
tube and incubated at room temperature for 15 minutes after the
addition of 15 .mu.l of 10 mg/ml RNaseA (Boehringer Mannheim,
Indianapolis, Ind.). The phage were precipitated by the addition of
7.5 ml of 20% polyethylene glycol 8000 (Fisher Scientific,
Pittsburgh, Pa.)/3.5M ammonium acetate (Sigma Chemical Co., St.
Louis, Mo.) and incubation on ice for 30 min. The sample was
centrifuged at 12 krpm for 15 min at 2-8.degree. C. The supernatant
was carefully discarded, and the tube briefly spun to remove all
traces of supernatant. The pellet was resuspended in 400 l of high
salt buffer (300 mM NaCl, 100 mM Tris pH 8.0, 1 mM EDTA), and
transferred to a 1.5 ml tube.
[0363] The phage stock was extracted repeatedly with an equal
volume of equilibrated phenol:chloroform:isoamyl alcohol (50:49:1)
until no trace of a white interface was visible, and then extracted
with an equal volume of chloroform:isoamyl alcohol (49:1). The DNA
was precipitated with 2.5 volumes of ethanol and 1/5 volume 7.5 M
ammonium acetate and incubated 30 min at -20.degree. C. The DNA was
centrifuged at 14 krpm for 10 min at 4.degree. C., the pellet
washed once with cold 70% ethanol, and dried in vacuo. The uracil
template DNA was dissolved in 30 .mu.l sterile water and the
concentration determined by A260 using an absorbance of 1.0 for a
concentration of 40 .mu.g/ml. The template was diluted to 250
ng/.mu.L with sterile water, aliquoted, and stored at -20.degree.
C.
Mutagenesis of Uracil Template with Ss-DNA and Electroporation into
E. coli to Generate Antibody Phage Libraries
[0364] Antibody phage display libraries were generated by
simultaneously introducing single-stranded heavy and light chain
genes onto a phage display vector uracil template. A typical
mutagenesis was performed on a 2 .mu.g scale by mixing the
following in a 0.2 ml PCR reaction tube: 8 .mu.l of (250 ng/L)
uracil template, 8 .mu.L of 10* annealing buffer (200 mM Tris pH
7.0, 20 mM MgCl2, 500 mM NaCl), 3.33 .mu.l of kinased
single-stranded heavy chain insert (100 ng/.mu.L), 3.1 .mu.l of
kinased single-stranded light chain insert (100 ng/L), and sterile
water to 80 .mu.l. DNA was annealed in a GeneAmp.RTM. 9600 thermal
cycler using the following thermal profile: 20 sec at 94.degree.
C., 85.degree. C. for 60 sec, 85.degree. C. to 55.degree. C. ramp
over 30 min, hold at 55.degree. C. for 15 min. The DNA was
transferred to ice after the program finished. The
extension/ligation was carried out by adding 8 .mu.l of 10*
synthesis buffer (5 mM each dNTP, 10 mM ATP, 100 mM Tris pH 7.4, 50
mM MgCl2, 20 mM DTT), 8 .mu.L T4 DNA ligase (1 U/.mu.L, Boehringer
Mannheim, Indianapolis, Ind.), 8 .mu.L diluted T7 DNA polymerase (1
U/.mu.L, New England BioLabs, Beverly, Mass.) and incubating at
37.degree. C. for 30 min. The reaction was stopped with 300 .mu.L
of mutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA).
[0365] The mutagenesis DNA was extracted once with equilibrated
phenol (pH>8):chloroform:isoamyl alcohol (50:49:1), once with
chloroform:isoamyl alcohol (49:1), and the DNA was ethanol
precipitated at -20.degree. C. for at least 30 min. The DNA was
pelleted and the supernatant carefully removed as described above.
The sample was briefly spun again and all traces of ethanol removed
with a pipetman. The pellet was dried in vacuo. The DNA was
resuspended in 4 .mu.L of sterile water.
[0366] One microliter of mutagenesis DNA (500 ng) was transferred
into 40 l electrocompetent E. coli DH12S (Gibco/BRL, Gaithersburg,
Md.) using electroporation. The transformed cells were mixed with
approximately 1.0 ml of overnight XL-1 cells which were diluted
with 2*YT broth to 60% the original volume. This mixture was then
transferred to a 15-ml sterile culture tube and 9 ml of top agar
added for plating on a 150-mm LB agar plate. Plates were incubated
for 4 hrs at 37.degree. C. and then transferred to 20.degree. C.
overnight. First round antibody phage were made by eluting phage
off these plates in 10 ml of 2*YT, spinning out debris, and taking
the supernatant. These samples are the antibody phage display
libraries used for selecting antibodies against Cadherin-17.
Efficiency of the electroporations was measured by plating 10 .mu.l
of a 10.sup.-4 dilution of suspended cells on LB agar plates,
follow by overnight incubation of plates at 37.degree. C. The
efficiency was calculated by multiplying the number of plaques on
the 10.sup.-4 dilution plate by 106. Library electroporation
efficiencies are typically greater than 1*10.sup.7 phage under
these conditions.
Transformation of E. coli by Electroporation
[0367] Electrocompetent E. coli cells were thawed on ice. DNA was
mixed with 40 L of these cells by gently pipetting the cells up and
down 2-3 times, being careful not to introduce an air bubble. The
cells were transferred to a Gene Pulser cuvette (0.2 cm gap,
BioRAD, Hercules, Calif.) that had been cooled on ice, again being
careful not to introduce an air bubble in the transfer. The cuvette
was placed in the E. coli Pulser (BioRAD, Hercules, Calif.) and
electroporated with the voltage set at 1.88 kV according to the
manufacturer's recommendation. The transformed sample was
immediately resuspended in 1 ml of 2*YT broth or 1 ml of a mixture
of 400 .mu.l 2*YT/600 .mu.l overnight XL-1 cells and processed as
procedures dictated.
Plating M13 Phage or Cells Transformed with Antibody Phage-Display
Vector Mutagenesis Reaction
[0368] Phage samples were added to 200 .mu.L of an overnight
culture of E. coli XL1-Blue when plating on 100 mm LB agar plates
or to 600 .mu.L of overnight cells when plating on 150 mm plates in
sterile 15 ml culture tubes. After adding LB top agar (3 ml for 100
mm plates or 9 ml for 150 mm plates, top agar stored at 55.degree.
C. (see, Appendix A1, Sambrook et al., supra.), the mixture was
evenly distributed on an LB agar plate that had been pre-warmed
(37.degree. C.-55.degree. C.) to remove any excess moisture on the
agar surface. The plates were cooled at room temperature until the
top agar solidified. The plates were inverted and incubated at
37.degree. C. as indicated.
Preparation of Biotinylated Cadherin-17 and Biotinylated
Antibodies
[0369] Concentrated recombinant Cadherin-17 antigen (full length
extracellular domain) was extensively dialyzed into BBS (20 mM
borate, 150 mM NaCl, 0.1% NaN3, pH 8.0). After dialysis, 1 mg of
Cadherin-17 (1 mg/ml in BBS) was reacted with a 15 fold molar
excess of biotin-XX-NHS ester (Molecular Probes, Eugene, Oreg.,
stock solution at 40 mM in DMSO). The reaction was incubated at
room temperature for 90 min and then quenched with taurine (Sigma
Chemical Co., St. Louis, Mo.) at a final concentration of 20 mM.
The biotinylated reaction mixture was then dialyzed against BBS at
2-8.degree. C. After dialysis, biotinylated Cadherin-17 was diluted
in panning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1%
Tween 20, pH 7.5), aliquoted, and stored at -80.degree. C. until
needed.
[0370] Antibodies were reacted with
3-(N-maleimidylpropionyl)biocytin (Molecular Probes, Eugene, Oreg.)
using a free cysteine located at the carboxy terminus of the heavy
chain.
[0371] Antibodies were reduced by adding DTT to a final
concentration of 1 mM for 30 min at room temperature. Reduced
antibody was passed through a Sephadex.RTM.G50 desalting column
equilibrated in 50 mM potassium phosphate, 10 mM boric acid, 150 mM
NaCl, pH 7.0. 3-(N-maleimidylpropionyl)-biocytin was added to a
final concentration of 1 mM and the reaction allowed to proceed at
room temperature for 60 min. Samples were then dialyzed extensively
against BBS and stored at 2-8.degree. C.
Preparation of Avidin Magnetic Latex
[0372] The magnetic latex (Estapor, 10% solids, Bangs Laboratories,
Fishers, Ind.) was thoroughly resuspended and 2 ml aliquoted into a
15 ml conical tube. The magnetic latex was suspended in 12 ml
distilled water and separated from the solution for 10 min using a
magnet (PerSeptive Biosystems.RTM., Framingham, Mass.). While
maintaining the separation of the magnetic latex with the magnet,
the liquid was carefully removed using a 10 ml sterile pipette.
This washing process was repeated an additional three times. After
the final wash, the latex was resuspended in 2 ml of distilled
water. In a separate 50 ml conical tube, 10 mg of avidin-HS
(NeutrAvidin.RTM., Pierce, Rockford, Ill.) was dissolved in 18 ml
of 40 mM Tris, 0.15 M sodium chloride, pH 7.5 (TBS). While
vortexing, the 2 ml of washed magnetic latex was added to the
diluted avidin-HS and the mixture mixed an additional 30 seconds.
This mixture was incubated at 45.degree. C. for 2 hr, shaking every
30 minutes. The avidin magnetic latex was separated from the
solution using a magnet and washed three times with 20 ml BBS as
described above. After the final wash, the latex was resuspended in
10 ml BBS and stored at 4.degree. C.
[0373] Immediately prior to use, the avidin magnetic latex was
equilibrated in panning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml
BSA, 0.1% Tween 20, pH 7.5). The avidin magnetic latex needed for a
panning experiment (200 .mu.l/sample) was added to a sterile 15 ml
centrifuge tube and brought to 10 ml with panning buffer. The tube
was placed on the magnet for 10 min to separate the latex. The
solution was carefully removed with a 10 ml sterile pipette as
described above. The magnetic latex was resuspended in 10 ml of
panning buffer to begin the second wash. The magnetic latex was
washed a total of 3 times with panning buffer. After the final
wash, the latex was resuspended in panning buffer to the starting
volume.
Example 2
Selection of Recombinant Polyclonal Antibodies to Cadherin-17
Antigen
[0374] Binding reagents that specifically bind to Cadherin-17 were
selected from the phage display libraries created from
hyperimmunized mice as described in Example 1.
Panning
[0375] First round antibody phage were prepared as described in
Example 1 using BS45 uracil template. Electroporations of
mutagenesis DNA were performed yielding phage samples derived from
different immunized mice. To create more diversity in the
recombinant polyclonal library, each phage sample was panned
separately.
[0376] Before the first round of functional panning with
biotinylated Cadherin-17 antigen, antibody phage libraries were
selected for phage displaying both heavy and light chains on their
surface by panning with 7F11-magnetic latex (as described in
Examples 21 and 22 of U.S. patent application Ser. No. 08/835,159,
filed Apr. 4, 1997). Functional panning of these enriched libraries
was performed in principle as described in Example 16 of U.S.
patent application Ser. No. 08/835,159. Specifically, 10 .mu.L of
1*10.sup.-6 M biotinylated Cadherin-17 antigen was added to the
phage samples (approximately 1*10.sup.-8 M Cadherin-17 final
concentration), and the mixture allowed to come to equilibrium
overnight at 2-8.degree. C.
[0377] After reaching equilibrium, samples were panned with avidin
magnetic latex to capture antibody phage bound to Cadherin-17.
Equilibrated avidin magnetic latex (Example 1), 200 .mu.L latex per
sample, was incubated with the phage for 10 min at room
temperature. After 10 min, approximately 9 ml of panning buffer was
added to each phage sample, and the magnetic latex separated from
the solution using a magnet. After a ten minute separation, unbound
phage was carefully removed using a 10 ml sterile pipette. The
magnetic latex was then resuspended in 10 ml of panning buffer to
begin the second wash. The latex was washed a total of three times
as described above. For each wash, the tubes were in contact with
the magnet for 10 min to separate unbound phage from the magnetic
latex. After the third wash, the magnetic latex was resuspended in
1 ml of panning buffer and transferred to a 1.5 mL tube. The entire
volume of magnetic latex for each sample was then collected and
resuspended in 200 .mu.L 2*YT and plated on 150 mm LB plates as
described in Example 1 to amplify bound phage. Plates were
incubated at 37.degree. C. for 4 hr, then overnight at 20.degree.
C.
[0378] The 150 mm plates used to amplify bound phage were used to
generate the next round of antibody phage. After the overnight
incubation, second round antibody phage were eluted from the 150 mm
plates by pipetting 10 mL of 2*YT media onto the lawn and gently
shaking the plate at room temperature for 20 min. The phage samples
were then transferred to 15 ml disposable sterile centrifuge tubes
with a plug seal cap, and the debris from the LB plate pelleted by
centrifuging the tubes for 15 min at 3500 rpm. The supernatant
containing the second round antibody phage was then transferred to
a new tube.
[0379] A second round of functional panning was set up by diluting
100 .mu.L of each phage stock into 900 .mu.L of panning buffer in
15 ml disposable sterile centrifuge tubes. Biotinylated Cadherin-17
antigen was then added to each sample as described for the first
round of panning, and the phage samples incubated for 1 hr at room
temperature. The phage samples were then panned with avidin
magnetic latex as described above. The progress of panning was
monitored at this point by plating aliquots of each latex sample on
100 mm LB agar plates to determine the percentage of kappa
positives. The majority of latex from each panning (99%) was plated
on 150 mm LB agar plates to amplify the phage bound to the latex.
The 100 mm LB agar plates were incubated at 37.degree. C. for 6-7
hr, after which the plates were transferred to room temperature and
nitrocellulose filters (pore size 0.45 mm, BA85 Protran, Schleicher
and Schuell, Keene, N.H.) were overlaid onto the plaques.
[0380] Plates with nitrocellulose filters were incubated overnight
at room temperature and then developed with a goat anti-mouse kappa
alkaline phosphatase conjugate to determine the percentage of kappa
positives as described below. Phage samples with lower percentages
(<70%) of kappa positives in the population were subjected to a
round of panning with 7F11-magnetic latex before performing a third
functional round of panning overnight at 2-8.degree. C. using
biotinylated Cadherin-17 antigen at approximately 2*10.sup.-9 M.
This round of panning was also monitored for kappa positives.
Individual phage samples that had kappa positive percentages
greater than 80% were pooled and subjected to a final round of
panning overnight at 2-8.degree. C. at 5*10.sup.-9 M Cadherin-17.
Antibody genes contained within the eluted phage from this fourth
round of functional panning were subcloned into the expression
vector, pBRncoH3.
[0381] The subcloning process was done generally as described in
Example 18 of U.S. patent application Ser. No. 08/835,159. After
subcloning, the expression vector was electroporated into DH10B
cells and the mixture grown overnight in 2*YT containing 1%
glycerol and 10 .mu.g/ml tetracycline. After a second round of
growth and selection in tetracycline, aliquots of cells were frozen
at -80.degree. C. as the source for Cadherin-17 polyclonal antibody
production.
[0382] Monoclonal antibodies were selected from these polyclonal
mixtures by plating a sample of the mixture on LB agar plates
containing 10 .mu.g/ml tetracycline and screening for antibodies
that recognized Cadherin-17.
Expression and Purification of Recombinant Antibodies Against
Cadherin-17
[0383] A shake flask inoculum was generated overnight from a
-70.degree. C. cell bank in an Innova.RTM. 4330 incubator shaker
(New Brunswick Scientific, Edison, N.J.) set at 37.degree. C., 300
rpm. The inoculum was used to seed a 20 L fermentor (Applikon,
Foster City, Calif.) containing defined culture medium (Pack et al.
(1993) Bio/Technology 11: 1271-1277) supplemented with 3 g/L
L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,
Detroit, Mich.), 12.5 g/L glycerol and 10 .mu.g/ml tetracycline.
The temperature, pH and dissolved oxygen in the fermentor were
controlled at 26.degree. C., 6.0-6.8 and 25% saturation,
respectively. Foam was controlled by addition of polypropylene
glycol (Dow, Midland, Mich.). Glycerol was added to the fermentor
in a fed-batch mode. Fab expression was induced by addition of
L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L during the late
logarithmic growth phase. Cell density was measured by optical
density at 600 nm in an UV-1201 spectrophotometer (Shimadzu,
Columbia, Md.). Following run termination and adjustment of pH to
6.0, the culture was passed twice through an M-210B-EH
Microfluidizer (Microfluidics, Newton, Mass.) at 17,000 psi. The
high pressure homogenization of the cells released the Fab into the
culture supernatant.
[0384] The first step in purification was expanded bed immobilized
metal affinity chromatography (EB-IMAC). Streamline.RTM. chelating
resin (Pharmacia, Piscataway, N.J.) was charged with 0.1 M NiCl2
and was then expanded and equilibrated in 50 mM acetate, 200 mM
NaCl, 10 mM imidazole, 0.01% NaN3, pH 6.0 buffer flowing in the
upward direction. A stock solution was used to bring the culture
homogenate to 10 mM imidazole, following which it was diluted
two-fold or higher in equilibration buffer to reduce the wet solids
content to less than 5% by weight. It was then loaded onto the
Streamline.RTM. column flowing in the upward direction at a
superficial velocity of 300 cm/hr. The cell debris passed through
unhindered, but the Fab was captured by means of the high affinity
interaction between nickel and the hexahistidine tag on the Fab
heavy chain. After washing, the expanded bed was converted to a
packed bed and the Fab was eluted with 20 mM borate, 150 mM NaCl,
200 mM imidazole, 0.01% NaN3, pH 8.0 buffer flowing in the downward
direction.
[0385] The second step in the purification used ion-exchange
chromatography (IEC). Q Sepharose.RTM. FastFlow resin (Pharmacia,
Piscataway, N.J.) was equilibrated in 20 mM borate, 37.5 mM NaCl,
0.01% NaN3, pH 8.0. The Fab elution pool from the EB-IMAC step was
diluted four-fold in 20 mM borate, 0.01% NaN3, pH 8.0 and loaded
onto the IEC column. After washing, the Fab was eluted with a
37.5-200 mM NaCl salt gradient. The elution fractions were
evaluated for purity using an Xcell II.TM. SDS-PAGE system
(Novex.RTM., San Diego, Calif.) prior to pooling. Finally, the Fab
pool was concentrated and diafiltered into 20 mM borate, 150 mM
NaCl, 0.01% NaN3, pH 8.0 buffer for storage. This was achieved in a
Sartocon Slice.TM. system fitted with a 10,000 MWCO cassette
(Sartorius, Bohemia, N.Y.). The final purification yields were
typically 50%. The concentration of the purified Fab was measured
by UV absorbance at 280 nm, assuming an absorbance of 1.6 for a 1
mg/ml solution.
Example 3
Selection of Antibodies to Cadherin-17 Antigen From Tumor Membrane
Preparations
[0386] Antibodies selected in Example 2 were further screened
against tumor membrane preparations to isolate antibodies that
preferentially bind to Cadherin-17 on cancer cells and not to
normal intestinal epithelia.
[0387] Biotinylated plasma membrane preparations from paired
colorectal cancer and normal adjacent tissue samples were used to
pan phage samples with avidin magnetic latex to capture antibody
phage bound to Cadherin-17 as described in Example 2. Antibodies
were selected from these polyclonal mixtures by screening for
antibodies that preferentially bind to Cadherin-17 on the
colorectal cancer cells and not to the normal intestinal epithelia.
These antibodies were then isolated as described in Example 4 and
analyzed for binding to Cadherin-17.
Example 4
Selection of Monoclonal Antibodies to Cadherin-17 from the
Recombinant Polyclonal Antibody Mixtures
[0388] Monoclonal antibodies against Cadherin-17 were isolated from
clones containing the recombinant polyclonal mixtures (Example 3)
by plating a diluted sample of the mixture on LB agar plates
containing 10 .mu.g/ml tetracycline. Individual colonies were then
tested for the ability to produce antibody that recognized
recombinant Cadherin-17 using surface plasmon resonance
(BIACORE.RTM.) (BIACORE.RTM., Uppsala, Sweden). Small scale
production of these monoclonal antibodies was accomplished using a
Ni-chelate batch-binding method (see below). Antibodies isolated
from this method were diluted 1:3 in HBS-EP (0.01 M HEPES, pH 7.4,
0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20 (v/v)), captured with
a goat anti-mouse kappa antibody (Southern Biotechnology
Associates, Inc, Birmingham, Ala.) coupled to a BIACORE.RTM. CM5
sensor chip, and tested for the ability to bind recombinant
Cadherin-17.
Minipreparation of Monoclonal Antibodies by Ni-Chelate
Batch-Binding Method
[0389] Individual colonies were isolated from the recombinant
polyclonal mixtures (Example 3) and used to inoculate 3 ml cultures
of 2*YT medium containing 1% glycerol supplemented with 10 .mu.g/ml
tetracycline. These cultures were grown in an Innova 4330 incubator
shaker (New Brunswick Scientific, Edison, N.J.) set at 37.degree.
C., 300 rpm. The next morning 0.5 ml of each culture was used to
inoculate shake flasks containing 50 ml of defined medium, (Pack et
al. (1993) Bio/Technology 11: 1271-1277) supplemented with 3 g/L
L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,
Detroit, Mich.), 12.5 g/L glycerol and 10 .mu.g/ml tetracycline.
These cultures were shaken at 300 rpm, 37.degree. C. until an
optical density of 4 was reached at 600 nm. Fab expression was then
induced by adding L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L
and shifting the temperature to 23.degree. C. with overnight
shaking. The next day the following was added to the 50 ml
cultures: 0.55 ml of 1 M imidazole, 5 ml B-PER (Pierce, Rockford,
Ill.) and 2 ml Ni-chelating resin (Chelating Sepharose.RTM.
FastFlow resin Pharmacia, Piscataway, N.J.). The mixture was shaken
at 300 rpm, 23.degree. C. for 1 hour after which time shaking was
stopped and the resin allowed to settle to the bottom of the flasks
for 15 minutes.
[0390] The supernatant was then poured off and the resin
resuspended in 40 ml of BBS (20 mM borate, 150 mM NaCl, 0.1% NaN3,
pH 8.0) containing 10 mM imidazole. This suspension was transferred
to a 50 ml conical tube and the resin washed a total of 3 times
with BBS containing 10 mM imidazole. Washing was accomplished by
low speed centrifugation (1100 rpm for 1 minute), removal of
supernatant and, resuspension of the resin in BBS containing 10 mM
imidazole. After the supernatant of the final wash was poured off,
0.5 ml of 1 M imidazole was added to each tube, vortex briefly, and
transferred to a sterile microcentrifuge tube. The samples were
then centrifuged at 14 krpm for 1 minutes and the supernatant
transferred to a new microcentrifuge tube. Antibodies contained in
the supernatant were then analyzed for binding to Cadherin-17 using
a BIACORE.RTM. (BIACORE.RTM., Uppsala, Sweden).
Example 5
Specificity of Monoclonal Antibodies to Cadherin-17 Determined by
Flow Cytometry Analysis
[0391] The specificity of antibodies against Cadherin-17 selected
in Example 4 was tested by flow cytometry. To test the ability of
the antibodies to bind to cell surface Cadherin-17 protein, the
antibodies were incubated with Cadherin-17-expressing cells: LoVo
and LS174T, human colorectal cancer lines. Cells were washed and
resuspended in PBS. Four microliters of the suspensions were
applied to wells of an eight well microscope slide and allowed to
air dry. The slides were lightly heated to fix the smears to the
slide and covered with 0.1 mg/ml of antibody diluted in PBS
containing 1% BSA. The smears were incubated with antibody for 1 h
at 37.degree. C. in a moist chamber. After washing the slides three
times by soaking in PBS for 5 min each, the smears were covered
with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG
(H&L) F(ab')2 (Zymed.RTM. Laboratories, Inc., South San
Francisco, Calif.) diluted 1:80 in PBS, 1% BSA, 0.05% Evans Blue
(Sigma). The slides were incubated for 1 h at 37.degree. C. in a
moist chamber then washed as described above. After a final wash in
deionized water, the slides were allowed to air dry in the dark.
Coverslips were mounted using a 90% glycerol mounting medium
containing 10 mg/ml p-phenylenediamine, pH 8.0.
[0392] The results of the flow cytometry analysis demonstrated that
14 monoclonal antibodies designated PTA001_A1, PTA001_A2,
PTA001_A3, PTA001_A4, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8,
PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 and
PTA001_A14 bind effectively to cell-surface human Cadherin-17.
Example 6
Structural Characterization of Monoclonal Antibodies to
Cadherin-17
[0393] The cDNA sequences encoding the heavy and light chain
variable regions of the PTA001_A1, PTA001_A2, PTA001_A3, PTA001_A4,
PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001_A12, PTA001_A13 and PTA001_A14 monoclonal
antibodies were obtained using standard PCR techniques and were
sequenced using standard DNA sequencing techniques.
[0394] The antibody sequences may be mutagenized to revert back to
germline residues at one or more residues.
[0395] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A1 are shown in FIG. 1 and in SEQ ID
NO:59 and 35, respectively.
[0396] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A1 are shown in FIG. 13 and in SEQ ID
NO:71 and 47, respectively.
[0397] Comparison of the PTA001_A1 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A1 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H 7-39. Further analysis
of the PTA001_A1 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 1, and in SEQ ID
NOs:1, 5 and 14, respectively. The alignment of the PTA001_A1 CDR1
V.sub.H sequence to the germline V.sub.H 7-39 sequence is shown in
FIG. 25.
[0398] Comparison of the PTA001_A1 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A1 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 1-110. Further
analysis of the PTA001_A1 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 13 and in SEQ ID
NOs:22, 28 and 32, respectively. The alignments of the PTA001_A1
CDR1 and CDR3 V.sub.K sequences to the germline V.sub.K 1-110
sequence are shown in FIGS. 27 and 29 respectively.
[0399] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A2 are shown in FIG. 2 and in SEQ ID
NO:60 and 36, respectively.
[0400] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A2 are shown in FIG. 14 and in SEQ ID
NO:72 and 48, respectively.
[0401] Comparison of the PTA001_A2 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A2 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A2 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 2 and in SEQ ID NOs:2, 6 and 15, respectively. The
alignment of the PTA001_A2 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A2 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0402] Comparison of the PTA001_A2 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A2 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A2 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 14, and in SEQ
ID NOs:23, 29 and 33, respectively. The alignments of the PTA001_A2
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0403] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A3 are shown in FIG. 3 and in SEQ ID
NO:61 and 37, respectively.
[0404] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A3 are shown in FIG. 14 and in SEQ ID
NO:72 and 48, respectively.
[0405] Comparison of the PTA001_A3 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A3 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A3 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 3, and in SEQ ID NOs: 3, 7 and 16, respectively. The
alignment of the PTA001_A3 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A3 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0406] Comparison of the PTA001_A3 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A3 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A3 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 14 and in SEQ ID
NOs:23, 29, and 33, respectively. The alignments of the PTA001_A3
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0407] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A4 are shown in FIG. 4 and in SEQ ID
NO:62 and 38, respectively.
[0408] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A4 are shown in FIG. 15 and in SEQ ID
NO:73 and 49, respectively.
[0409] Comparison of the PTA001_A4 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A4 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A4 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 4 and in SEQ ID NOs: 4, 8 and 17, respectively. The
alignment of the PTA001_A4 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A4 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0410] Comparison of the PTA001_A4 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A4 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 8-30. Further analysis
of the PTA001_A4 V.sub.K sequence using the Kabat system of CDR
region determination led to the delineation of the light chain
CDR1, CDR2 and CDR3 regions as shown in FIG. 15 and in SEQ ID
NOs:24, 30, and 34, respectively. The alignments of the PTA001_A4
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K 8-30
sequence are shown in FIGS. 27, 28 and 29 respectively.
[0411] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A5 are shown in FIG. 5 and in SEQ ID
NO:63 and 39, respectively.
[0412] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A5 are shown in FIG. 16 and in SEQ ID
NO:74 and 50, respectively.
[0413] Comparison of the PTA001_A5 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A5 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A5 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 5, and in SEQ ID NOs: 3, 7 and 18, respectively. The
alignment of the PTA001_A5 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A5 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0414] Comparison of the PTA001_A5 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A5 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A5 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 16, and in SEQ
ID NOs:25, 31, and 33, respectively. The alignments of the
PTA001_A5 CDR1, CDR2 and CDR3 V.sub.K sequences to the germline
V.sub.K 24-140 sequence are shown in FIGS. 27, 28 and 29
respectively.
[0415] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A6 are shown in FIG. 6 and in SEQ ID
NO:64 and 40, respectively.
[0416] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A6 are shown in FIG. 17 and in SEQ ID
NO:75 and 51, respectively.
[0417] Comparison of the PTA001_A6 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A6 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A6 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 6 and in SEQ ID NOs: 3, 7 and 16, respectively. The
alignment of the PTA001_A6 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A6 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0418] Comparison of the PTA001_A6 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A6 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A6 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 17 and in SEQ ID
NOs:23, 29, and 33, respectively. The alignments of the PTA001_A6
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0419] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A7 are shown in FIG. 7 and in SEQ ID
NO:65 and 41, respectively.
[0420] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A7 are shown in FIG. 18 and in SEQ ID
NO:76 and 52, respectively.
[0421] Comparison of the PTA001_A7 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A7 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A7 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 7 and in SEQ ID NOs: 3, 9 and 19, respectively. The
alignment of the PTA001_A7 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A7 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0422] Comparison of the PTA001_A7 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A7 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A7 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 18 and in SEQ ID
NOs:26, 29, and 33, respectively. The alignments of the PTA001_A7
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0423] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A8 are shown in FIG. 8 and in SEQ ID
NO:66 and 42, respectively.
[0424] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A8 are shown in FIG. 19 and in SEQ ID
NO:77 and 53, respectively.
[0425] Comparison of the PTA001_A8 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A8 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A8 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 8 and in SEQ ID NOs: 3, 7 and 16, respectively. The
alignment of the PTA001_A8 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A8 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0426] Comparison of the PTA001_A8 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A8 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A8 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 19 and in SEQ ID
NOs:25, 31, and 33, respectively. The alignments of the PTA001_A8
CDR1, CDR2 and CDR3 V.sub.k sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0427] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A9 are shown in FIG. 6 and in SEQ ID
NO:64 and 40, respectively.
[0428] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A9 are shown in FIG. 20 and in SEQ ID
NO:78 and 54, respectively.
[0429] Comparison of the PTA001_A9 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A9 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A9 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 6 and in SEQ ID NOs: 3, 7 and 16, respectively. The
alignment of the PTA001_A9 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A9 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0430] Comparison of the PTA001_A9 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A9 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A9 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 20 and in SEQ ID
NOs:23, 29, and 33, respectively. The alignments of the PTA001_A9
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0431] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A10 are shown in FIG. 6 and in SEQ ID
NO:64 and 40, respectively.
[0432] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A10 are shown in FIG. 21 and in SEQ ID
NO:79 and 55, respectively.
[0433] Comparison of the PTA001_A10 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A10 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A10 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 6 and in SEQ ID NOs: 3, 7 and 16, respectively. The
alignment of the PTA001_A10 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A10 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0434] Comparison of the PTA001_A10 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A10 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A10 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 21 and in SEQ ID
NOs:25, 29, and 33, respectively. The alignments of the PTA001_A10
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0435] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A11 are shown in FIG. 9 and in SEQ ID
NO:67 and 43, respectively.
[0436] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A11 are shown in FIG. 22 and in SEQ ID
NO:80 and 56, respectively.
[0437] Comparison of the PTA001_A11 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A11 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A11 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 9 and in SEQ ID NOs: 3, 10 and 20, respectively. The
alignment of the PTA001_A11 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A11 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0438] Comparison of the PTA001_A11 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A11 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A11 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 22 and in SEQ ID
NOs:27, 29, and 33, respectively. The alignments of the PTA001_A11
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0439] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A12 are shown in FIG. 10 and in SEQ ID
NO:68 and 44, respectively.
[0440] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A12 are shown in FIG. 21 and in SEQ ID
NO:81 and 55, respectively.
[0441] Comparison of the PTA001_A12 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A12 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and VH
II gene H17. Further analysis of the PTA001_A12 VH sequence using
the Kabat system of CDR region determination led to the delineation
of the heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 10
and in SEQ ID NOs: 3, 11 and 21, respectively.
[0442] The alignment of the PTA001_A12 CDR1 VH sequence to the
germline V.sub.H II gene H17 sequence is shown in FIG. 25 and the
alignment of the PTA001_A12 CDR2 V.sub.H sequence to the germline
V.sub.H II region VH105 is shown in FIG. 26.
[0443] Comparison of the PTA001_A12 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A12 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A12 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 21 and in SEQ ID
NOs:25, 29, and 33, respectively. The alignments of the PTA001_A12
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0444] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A13 are shown in FIG. 11 and in SEQ ID
NO:69 and 45, respectively.
[0445] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A13 are shown in FIG. 23 and in SEQ ID
NO:82 and 57, respectively.
[0446] Comparison of the PTA001_A13 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A13 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and VH
II gene H17. Further analysis of the PTA001_A13 V.sub.H sequence
using the Kabat system of CDR region determination led to the
delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown
in FIG. 11 and in SEQ ID NOs: 3, 12 and 18, respectively. The
alignment of the PTA001_A13 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A13 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0447] Comparison of the PTA001_A13 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A13 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A13 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 23 and in SEQ ID
NOs:25, 31, and 33, respectively. The alignments of the PTA001_A13
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
[0448] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA001_A14 are shown in FIG. 12 and in SEQ ID
NO:70 and 46, respectively.
[0449] The nucleotide and amino acid sequences of the light chain
variable region of PTA001_A14 are shown in FIG. 24 and in SEQ ID
NO:83 and 58, respectively.
[0450] Comparison of the PTA001_A14 heavy chain immunoglobulin
sequence to the known murine germline immunoglobulin heavy chain
sequences demonstrated that the PTA001_A14 heavy chain utilizes a
V.sub.H segment from murine germline V.sub.H II region VH105 and
V.sub.H II gene H17. Further analysis of the PTA001_A14 V.sub.H
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as
shown in FIG. 12 and in SEQ ID NOs: 2, 13 and 15, respectively. The
alignment of the PTA001_A14 CDR1 V.sub.H sequence to the germline
V.sub.H II gene H17 sequence is shown in FIG. 25 and the alignment
of the PTA001_A14 CDR2 V.sub.H sequence to the germline V.sub.H II
region VH105 is shown in FIG. 26.
[0451] Comparison of the PTA001_A14 light chain immunoglobulin
sequence to the known murine germline immunoglobulin light chain
sequences demonstrated that the PTA001_A14 light chain utilizes a
V.sub.K segment from murine germline V.sub.K 24-140. Further
analysis of the PTA001_A14 V.sub.K sequence using the Kabat system
of CDR region determination led to the delineation of the light
chain CDR1, CDR2 and CDR3 regions as shown in FIG. 24 and in SEQ ID
NOs:23, 29, and 33, respectively. The alignments of the PTA001_A14
CDR1, CDR2 and CDR3 V.sub.K sequences to the germline V.sub.K
24-140 sequence are shown in FIGS. 27, 28 and 29 respectively.
Example 7
Immunohistochemistry on FFPE Sections Using Anti-Cadherin-17
Antibodies
[0452] Immunohistochemistry was performed on FFPE sections of
colorectal tumor and normal adjacent tissue using the
anti-Cadherin-17 antibodies PTA001_A3, PTA001_A4, PTA001_A6,
PTA001_A8 and PTA001_A9.
[0453] EX-De-Wax was from BioGenex.RTM., CA, USA. Tissue sections
and arrays were from Biomax, Md., USA.
[0454] Slides were heated for 2 h at 60.degree. C. in 50 ml Falcons
in a water bath with no buffer. Each Falcon had one slide or two
slides back-to back with long gel loading tip between them to
prevent slides from sticking to each other. Slides were
deparaffinised in EZ-DeWax for 5 min in black slide rack, then
rinsed well with the same DeWax solution using 1 ml pipette, then
washed with water from the wash bottle. Slides were placed in a
coplin jar filled with water until the pressure cooker was ready;
the water was changed a couple of times.
[0455] Water was exchanged for antigen retrieval
solution=1.times.citrate buffer, pH 6 (DAKO). Antigen was retrieved
by the pressure cooker method. The slides in the plastic coplin jar
in antigen retrieval solution were placed into a pressure cooker
which was then heated up to position 6 (the highest setting). 15-20
min into the incubation, the temperature was reduced to position 3
and left at that (when the temperature inside the pressure cooker
was 117.degree. C.) for another 20-25 minutes. Then the hob was
switched off and the cooker was placed onto the cold hob and the
pressure was released by carefully moving the handle into the
position between "open" and "closed". The whole system was left to
release the pressure and to cool down for another 20 minutes. The
lid was opened and samples taken out to rest on the bench. The
slides were washed 1.times.5 min with PBS-3T (0.5 L PBS+3 drops of
Tween-20) and placed in PBS.
[0456] After antigen retrieval, slides were mounted in the Shandon
Coverplate system. Trapping of air bubbles between the slide and
plastic coverplate was prevented by placing the coverplate into the
coplin jar filled with PBS and gently sliding the slide with tissue
sections into the coverplate. The slide was pulled out of the
coplin jar while holding it tightly together with the coverplate.
The assembled slide was placed into the rack, letting PBS trapped
in the funnel and between the slide and coverplate to run through.
Slides were washed with 2.times.2 ml (or 4.times.1 ml) PBS-3T,
1.times.2 ml PBS, waiting until all PBS had gone through the slide
and virtually no PBS was left in the funnel.
[0457] Endogenous peroxide blockade was performed using 1-4 drops
of peroxide solution per slide; the incubation time was 5 minutes.
The slides were rinsed with water and then once with 2 ml PBS-3T
and once with 2 ml PBS; it was important to wait until virtually no
liquid was left in the funnel before adding a new portion of wash
buffer.
[0458] The primary antibody was diluted with an Antibody diluent
reagent (DAKO). Optimal dilution was determined to be 1:400. Up to
200 .mu.l of diluted primary antibody was applied to each slide and
incubated for 45 minutes at room temperature. Slides were washed
with 2.times.2 ml (or 4.times.1 ml) PBS-3T and then 1.times.2 ml
PBS.
[0459] The goat anti-mouse kappa HRP secondary (1 mg/ml, cat.
1050-05, Southern Biotech) was applied 2.times.2 drops per slide
and incubated for 35 min at room temperature. The slides were
washed as above.
[0460] The DAB substrate was made up in dilution buffer; 2 ml
containing 2 drops of substrate was enough for 10 slides. The DAB
reagent was applied to the slides by applying a few drops at a time
and left for 10 min. The slides were washed 1.times.2 ml (or
2.times.1 ml) with PBS-3T and 1.times.2 ml (or 2.times.1 ml) with
PBS.
[0461] Hematoxylin (DAKO) was applied; 1 ml was enough for 10
slides and slides were incubated for 1 min at room temperature. The
funnels of the Shandon Coverplate system were filled with 2 ml of
water and let to run through. When slides were clear of the excess
of hematoxylin, the system was disassembled, tissue sections and/or
arrays were washed with water from the wash bottle and placed into
black slide rack. Tissues were dehydrated by incubating in EZ-DeWax
for 5 min and then in 95% ethanol for 2-5 min.
[0462] Slides were left to dry on the bench at room temperature and
then mounted in mounting media and covered with coverslip.
[0463] Immunohistochemical analysis on antibodies PTA001_A3,
PTA001_A4, PTA001_A6, PTA001_A8 and PTA001_A9 revealed specific
membrane staining of tumor cells in colorectal cancer and no
appreciable staining of normal adjacent tissue in all cases.
Antibody PTA001_A4, in particular, showed clear specific membrane
staining of tumor cells.
Example 8
Immunohistochemistry on Frozen Sections Using Anti-Cadherin-17
Antibodies
[0464] Immunohistochemistry was performed on frozen paired tumor
and normal adjacent tissues using the anti-Cadherin-17 antibodies
PTA001_A4, PTA001_A6, PTA001_A8 and PTA001_A9.
[0465] Tissue sections were from BioChain.RTM. Institute Inc., CA,
USA.
[0466] Frozen sections were washed with PBS twice for 3 minutes
each and were then placed in PBS.
[0467] Endogenous peroxide blockade was performed using Peroxidase
Blocker (S2001, DAKO). 1-4 drops of peroxidase blocker was added to
each slide and incubated for 5 minutes. The slides were rinsed
three times with 3 ml PBS.
[0468] The primary antibody was diluted with an Antibody diluent
reagent (DAKO). 150 l of diluted primary antibody was applied to
each slide and incubated for 45 minutes at room temperature. Slides
were washed with twice for 3 minutes with PBS-3T (500 ml PBS+3
drops of Tween-20) and then once for 3 minutes with PBS.
[0469] The goat anti-mouse kappa HRP secondary was applied at
1:1000 (1 mg/ml, cat.1050-05, Southern Biotech) and incubated for
35 min at room temperature. The slides were washed as above.
[0470] The DAB substrate was made up in dilution buffer; 2 ml
containing 2 drops of substrate was enough for 10 slides. The DAB
reagent was applied to the slides by applying a few drops at a time
and incubated for 10 min. The slides were washed once for 3 minutes
with PBS-3T and twice for 3 minutes with water.
[0471] Hematoxylin (DAKO) was applied; 1 ml was enough for 10
slides and slides were incubated for 1 min at room temperature.
[0472] Slides were left to dry on the bench at room temperature and
then mounted in water-based mounting media from Vector and covered
with coverslip.
[0473] Immunohistochemical analysis on antibodies PTA001_A4,
PTA001_A6, PTA001_A8 and PTA001_A9 on three colorectal cancer
samples along with the paired normal adjacent tissue samples
revealed strong specific membrane staining of tumor cells in
colorectal cancer and some weak staining of normal adjacent tissue.
Antibody PTA001_A4, in particular, showed clear specific membrane
staining of tumor cells.
Example 9
Western Blotting Using Anti-Cadherin-17 Antibodies
[0474] Western blotting was performed using anti-Cadherin-17
antibodies PTA001_A3, PTA001_A4, PTA001_A6, PTA001_A8 and PTA001_A9
to detect Cadherin-17 in a panel of genetically characterized
colorectal cancer cell lines representing combinations of three
critical mutation phenotypes (p53, APC and RER+/).
[0475] Snap frozen cell pellets were lysed in modified RIPA buffer
(50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM
NaCl, 1 mM EDTA) containing protease inhibitors (Roche) and cleared
by spinning down at 18,000 g for 15 min at 4 C. The 4.times.LDS
sample loading buffer (Invitrogen Inc.) was added to the cleared
lysate to a final concentration of 1.times.; sample was heated for
10 min at 70.degree. C. and kept at -80.degree. C. afterwards.
Before loading on a gel samples were re-heated.
[0476] Proteins from 10 .mu.g (PC/JW) and 20 .mu.g (other cell
lines) of lysate per lane were separated by mini-gel
electrophoresis on NuPAGE.RTM. Novex.RTM. precast mini-gel
(Invitrogen, UK). Gels were blotted onto nitrocellulose membrane
with iBlot.RTM. Dry Blotting System (Invitrogen, UK).
[0477] Membrane was incubated with animal-free blocker (Vector) and
probed with anti-Cadherin-17 antibody in animal-free blocker at
1:500 dilution, at 4 C, for 14-18 h, rotating. The secondary was
anti-mouse DyLight.RTM. 488 conjugate (Pierce).
[0478] A clear signal was detected at a size corresponding to the
Cadherin-17 protein (92 kDa) in 11 of the 14 chosen colorectal cell
lines for all 5 Cadherin-17 antibodies (PTA001_A3, PTA001_A4,
PTA001_A6, PTA001_A8 and PTA001_A9). FIG. 25 shows the Western
blotting analysis of Cadherin-17 for antibody PTA001_A4. Cell lines
were carefully selected for different genetic backgrounds relevant
to the initiation and progression of CRC (RER+=replication error
positive tumor phenotype; RER-=replication error deficient tumor
phenotype; p53 wild type or mutant and APC wild type or mutant
genotype). Table 2 below shows the phenotype of the colorectal
tumor derived cell lines.
TABLE-US-00002 TABLE 2 Phenotype of colorectal tumor derived cell
lines Cell line Characteristics LS411 Colorectal carcinoma, Tumor
stage: Dukes' type B; Tumorigenic in nude mice. LoVo Colorectal
adenocarcinoma, Dukes' type C, grade IV derived from metastatic
site; Tumorigenic in nude mice. Vaco 5 Colorectal carcinoma. DLD-1
Colorectal adenocarcinoma, Dukes' type C; Tumorigenic in nude mice.
LS 174T Colorectal adenocarcinoma, Dukes' type B, Tumorigenic in
nude mice. HCT 116 Colorectal carcinoma, Tumorigenic in nude mice.
PC/JW Adenocarcinoma. Tumorigenic in ethylic nude mice. C99
Colorectal adenocarcinoma. C84 Colorectal adenocarcinoma. HT29
Colorectal adenocarcinoma, Tumorigenic in nude mice. LS513
Colorectal carcinoma, Dukes' type C; Tumorigenic in nude mice.
NCI-H716 Colorectal adenocarcinoma; Tumorigenic in nude mice. Caco2
Colorectal adenocarcinoma Colo205 Colorectal adenocarcinoma
Example 10
Internalization of Anti-Cadherin-17 Antibodies
[0479] PTA001_A4 was shown to be internalized by LoVo cells upon
binding to the cells using a Immunofluorescence microscopy assay.
The Immunofluorescence microscopy assay showed internalization of
the anti-Cadherin-17 monoclonal antibodies through binding of an
anti-human IgG secondary antibody conjugated to Fluorescein
isothiocyanate (GamK-FITC). First, PTA001_A4 were bound to the
surface of the LoVo cells. Then, the secondary antibody conjugated
to Fluorescein isothiocyanate were bound to the primary antibodies.
Next, the PTA001_A4/secondary antibody FITC conjugate complex was
internalized by the cells.
[0480] The Immunofluorescence microscopy assay was conducted as
follows. LoVo cell were incubated at 37.degree. C. for 12 hours for
cells to adhere to each other. PTA001_A4 and secondary antibody
conjugated to Fluorescein isothiocyanate were serially diluted,
washed with FACS buffer (PBS, 2% FBS) and then added to the culture
media. The media was then washed again with FACS buffer (PBS, 2%
FBS) and incubated at 37%, after which 200 ul 2% PFA was added.
Coverslips were mounted using a 9 ul aqeous mountaing media and the
cells were then visualized at regular time intervals using Leica
fluorescent microscope. FIG. 32A and FIG. 32B shows surface binding
of PTA001_A4/secondary antibody FITC conjugate complex to LoVo
cells after 60 minutes of incubation and internalization of
PTA001_A4/secondary antibody FITC conjugate complex after 120
minutes.
[0481] The monoclonal antibody, PTA001_A4, was shown to be
internalized by LS147T and LoVo cells upon binding to the cells
using a MabZap assay. The MabZAP assay showed internalization of
the anti-CDH 17 monoclonal antibodies through binding of an
anti-human IgG secondary antibody conjugated to the toxin saporin.
(Advanced Targeting System, San Diego, Calif., IT-22-100). First,
PTA001_A4 was bound to the surface of the LS147T and LoVo cells.
Then, the MabZAP antibodies were bound to the primary antibodies.
Next, the MabZAP complex was internalized by the cells. The
entrance of Saporin into the cells resulted in protein synthesis
inhibition and eventual cell death.
[0482] The MabZAP assay was conducted as follows. Each of the cells
was seeded at a density of 5.times.10.sup.3 cells per well. The
anti-CDH17 monoclonal antibodies or an isotype control human IgG
were serially diluted then added to the cells. The MabZAP was then
added at a concentration of 50 .mu.g/ml and the plates allowed to
incubate for 48 and 72 hours. Cell viability in the plates was
detected by CellTiter-Glo.RTM. Luminescent Cell Viability Assay kit
(Promega, G7571) and the plates were read at 490 nM by a
Luminomitor (Tuner BioSystems, Sunnyvale, Calif.). The data was
analyzed by Prism (Graphpad). Cell death was proportional to the
concentration of PTA001_A4 and monoclonal antibody. FIGS. 35A and
35B show that the anti-CDH17 monoclonal antibodies were efficiently
internalized by LS174T and LoVo cells respectively as compared to
the anti-human IgG isotype control antibody.
TABLE-US-00003 SEQUENCE LISTING SEQ ID SEQUENCE NO DESCRIPTION
SEQUENCE 1 VH CDR1 amino acid GFTFSNYGMS PTA001_A1 2 VH CDR1 amino
acid GYTFSDHAIH PTA001_A2, PTA001_A14 3 VH CDR1 amino acid
GYTFTDHAIH PTA001_A3, PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8,
PTA001_A9, PTA001_A10, PTA001_A11, PTA001_A12, PTA001_A13 4 VH CDR1
amino acid GYTLTDHTIH PTA001_A4 5 VH CDR2 amino acid
AINRDGGTTYYTDNVKG PTA001_A1 6 VH CDR2 amino acid YIYPRHGTTNYNENFKG
PTA001_A2 7 VH CDR2 amino acid YIYPEHGTIKYNEKFKG PTA001_A3,
PTA001_A5, PTA001_A6, PTA001_A8, PTA001_A9, PTA001_A10 8 VH CDR2
amino acid YIYPRDGITGYNEKFKG PTA001_A4 9 VH CDR2 amino acid
YIYPRDGFTKYNEKFKG PTA001_A7 10 VH CDR2 amino acid YIYPEHGTITYNEKFKG
PTA001_A11 11 VH CDR2 amino acid YIYPRDDFAKVNEKFKG PTA001_A12 12 VH
CDR2 amino acid YIYPEHGTITYNEKFKG PTA001_A13 13 VH CDR2 amino acid
YIFPRDAFSLNNEKFKG PTA001_A14 14 VH CDR3 amino acid FLLWDGWYFDV
PTA001_A1 15 VH CDR3 amino acid RNYFYVMDY PTA001_A2, PTA001_A14 16
VH CDR3 amino acid TNYFYVMEY PTA001_A3, PTA001_A6, PTA001_A8,
PTA001_A9, PTA001_A10 17 VH CDR3 amino acid GYSYRNYAYYYDY PTA001_A4
18 VH CDR3 amino acid RNYLYIMDY PTA001_A5, PTA001_A13 19 VH CDR3
amino acid TNYFYTMDY PTA001_A7 20 VH CDR3 amino acid RNYLYVMDY
PTA001_A11 21 VH CDR3 amino acid TNYLYIMDY PTA001_A12 22 VK CDR1
amino acid RSSQSLLHSNGNTYLH PTA001_A1 23 VK CDR1 amino acid
TSSKSLLRSNGNTYLY PTA001_A2, PTA001_A3, PTA001_A6, PTA001_A9,
PTA001_A14 24 VK CDR1 amino acid KSSQSLLHSSNQKNYLA PTA001_A4 25 VK
CDR1 amino acid RSSKSLLRSNGNTYLY PTA001_A5, PTA001_A8, PTA001_A10,
PTA001_A12, PTA001_A13 26 VK CDR1 amino acid RSSKSLLRTNGNTYLH
PTA001_A7 27 VK CDR1 amino acid RSTKSLLRSNGNTYLY PTA001_A11 28 VK
CDR2 amino acid KVSNRFS PTA001_A1 29 VK CDR2 amino acid RMSNLAS
PTA001_A2, PTA001_A3, PTA001_A6, PTA001_A7, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001_A12, PTA001_A14 30 VK CDR2 amino acid WASTRES
PTA001_A4 31 VK CDR2 amino acid RLSNLAS PTA001_A5, PTA001_A8,
PTA001_A13 32 VK CDR3 amino acid SQSTHVLT PTA001_A1 33 VK CDR3
amino acid MQHLEYPFT PTA001_A2, PTA001_A3, PTA001_A5, PTA001_A6,
PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10, PTA001_A11,
PTA001_A12, PTA001_A13, PTA001_A14 34 VK CDR3 amino acid QQYYSYPWT
PTA001_A4 35 VH amino acid PTA001_A1
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAEVQLLETGGGVVKPG
GSLKLSCAASGFTFSNYGMSWVRQTPEKRLEWVAAINRDGGTTYYTDNVKG
RFTISRDNAKNSLYLQMSSLRSEDTALYYCARQFLLWDGWYFDVWGAGTTV
TVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSG
VHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 36 VH amino
acid PTA001_A2 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPG
ASVKISCKVSGYTFSDHAIHWMSQRPGQGLKWIGYIYPRHGTTNYNENFKGK
ATLTADTSSSTAYMQLNSLTSEDSAVYFCARMRNYFYVMDYWGQGTSVTVS
SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH
TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 37 VH amino
acid PTA001_A3 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVLLQQSDAELVKPG
ASVKISCKASGYTFTDHAIHWVKQRPEQGLEWIGYIYPEHGTIKYNEKFKGKA
TLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYFYVMEYWGQGTSVTVSSA
KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFP
AVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTICVDKKIVPRDC 38 VH amino acid
PTA001_A4 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAEVQLQQSVAELVKPG
(with leader sequence)
ASVKMSCKVSGYTLTDHTIHWMKQRPEQGLEWIGYIYPRDGITGYNEKFKGK
ATLTADTSSSTAYMQLNSLTSEDSAVYFCARWGYSYRNYAYYYDYWGQGT
TLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLS
SGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DC 39 VH
amino acid PTA001_A5
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDADLVKPG (with leader
sequence) ASVKISCICASGYTFTDHAIHWVKQRPEQGLEWIGYIYPEHGTIKYNEKFKGICA
TLTADKSSSTAYMQLNSLTSEDSAVYFCARLRNYLYIMDYWGQGTSVTVSSA
KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFP
AVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTICVDKKIVPRDC 40 VH amino acid
PTA001_A6, LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPG
PTA001_A9, PTA001_A10
ASVKISCICASGYTFTDHAIHWVKQRPEQGLEWIGYIYPEHGTIKYNEKFKGICA
TLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYFYVMEYWGQGTSVTVSSA
KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFP
AVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTICVDKKIVPRDC 41 VH amino acid
PTA001_A7 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPG
ASVKISCKVSGYTFTDHAIHWMKQRPEQGLEWIGYIYPRDGFTICYNEICFKGK
ATLTADTSSSTAYMQLNSLTSEDSTVYFCARMTNYFYTMDYWGQGTSVTVS
SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH
TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 42 VH amino
acid PTA001_A8 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDADLVKPG
ASVKISCICASGYTFTDHAIHWVKQRPEQGLEWIGYIYPEHGTIKYNEKFKGICA
TLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYFYVMEYWGQGTSVTVSSA
KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFP
AVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTICVDKKIVPRDC 43 VH amino acid
PTA001_A11 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPG
ASVKISCICASGYTFTDHAIHWVKQRPEQGLEWIGYIYPEHGSITYNEKFKGKA
TLTADKSSSTVYMHLNSLTSEDSAVYFCARLRNYLYVMDYWGQGTSVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT
FPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 44 VH amino acid
PTA001 _A12 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSEAELVKPG
ASVKLSCKASGYTFTDHAIHWMKQRPEQGLEWIGYIYPRDDFAKVNEKFKG
KATLTADTSSSTAYMQLNSLTSEDSAVYFCARMTNYLY1MDYWGQGTSVTV
SSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGV
HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 45 VH amino
acid PTA001_A13
LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPG
ASVKISCICASGYTFTDHAIHWVKQRPEQGLEWIGYIYPEHGTITYNEICFKGKA
TLTADKSSSTVYMHLNSLTSEDSAVYFCARLRNYLYIMDYWGQGTSVTVSSA
KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFP
AVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTICVDKKIVPRDC 46 VH amino acid
PTA001_A14 LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAALVKPG
ASVKISCKVSGYTFSDHAIHWMICQRPEQGLEWIGYIFPRDAFSLNNEICFKGK
ATLSADTSSSTAYMELTSLTFEDSAVYFCARMRNYFYVMDYWGQGTSVTVS
SAKTTPPSVYTLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH
TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 47 VK amino
acid PTA001_A1 RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADVVLT
QTPLSLPVTLGDQASISCRSSQSLLHSNGNTYLHWYLLKPGQSPKLLIYKVSN
RFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYFCSQSTHVLTFGAGTKLELK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
48 VK amino acid PTA001_A2,
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT PTA001_A3
QAAPSVPVTPGESVSISCTSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRMSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
49 VK amino acid PTA001_A4
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMS
QSPSSLAVSVGEKVTMSCKSSQSLLHSSNQKNYLAWYQQKPGQSPKVLIYWA
STRESGVPDRFTGSGSGTDFTLTITSVKSEDLAVYYCQQYYSYPWTFGGGTRL
EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNG
VLNSWTDQDSICDSTYSMSSTLTLTICDEYERHNSYTCEATHKTSTSPIVKSFNR
NESYPYDVPDYAS 50 VK amino acid PTA001_AS
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVPVTPGESVSISCRSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRLSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
51 VK amino acid PTA001_A6
RILPDAFYRNSLLFLHTRFFGWSETIKYLLPTAAAGLLLLAAQPAMADIVMTQ
AAPSVPVTPGESVSISCTSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRMSNLA
SGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKR
ADAAPTVSILPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLN
SWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNES YPYDVPDYAS 52
VK amino acid PTA001_A7
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVPVTPGESVSISCRSSKSLLRTNGNTYLHWFLQRPGQSPQLLIYRMSNL
ASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
53 VK amino acid PTA001_A8
RILPYAFYRNSLLFLITTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVPVTPGESVSISCRSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRLSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
54 VK amino acid PTA001_A9
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVPVTPGESVSISCTSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRMSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
RADAAPTVSISPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
55 VK amino acid PTA001_A10,
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT PTA001_A12
QAAPSVPVTPGESVSISCRSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRMSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
56 VK amino acid PTA001_A11
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVSVTPGESVSISCRSTKSLLRSNGNTYLYWFLQRPGQSPQLLIYRMSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
57 VK amino acid PTA001 A13
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVPVTPGESVSISCRSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRLSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
RADAAPTVSIFPQYSEQLTTGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL
NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE SYPYDVPDYAS
58 VK amino acid PTA001 _A14
RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMT
QAAPSVPVTPGESVSISCTSSKSLLRSNGNTYLYWFLQRPGQSPQLLIYRMSNL
ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTNLEIK
RADAAPTVSIFTTSREQLTSGGASVVCFLNNFYPKDINVK 59 VH n.t. PTA001_A1
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCGAAGTGCAGCTGTTGGAGACTGGGGGAGGCGTAGTG
AAGCCCGGAGGGTCCCTTAAACTCTCCTGTGCAGCCTCTGGATTCACTTTC
AGTAACTATGGCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGA
GTGGGTCGCAGCCATTAATCGTGATGGTGGTACCACCTACTATACAGACA
ATGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTG
TACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCTTGTATTACTG
TGCAAGACAGTTCCTTCTCTGGGACGGCTGGTACTTCGATGTCTGGGGCGC
AGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCT
ATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGG
GATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAAC
TCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT
GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCC
AGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGT
GGACAAGAAAATTGTGCCCAGGGATTGT 60 VH n.t. PTA001_A2
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGA
AACCTGGAGCTTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCA
GTGACCATGCTATTCACTGGATGAGTCAGAGACCTGGACAGGGCCTGAAA
TGGATTGGATATATTTATCCTAGACATGGGACTACTAACTACAATGAGAA
CTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCT
ACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCCGTCTATTTCTGTG
CAAGAATGAGAAACTACTTCTATGTTATGGACTACTGGGGTCAAGGAACC
TCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTG
GTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATC
CCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTA
CACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGA
CCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG
AAAATTGTGCCCAGGGATTGT 61 VH n.t. PTA001_A3
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCTGCTGCAACAGTCTGACGCTGAGTTGGTGA
AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAA
GTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCT
ATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTT
CAAGACTCACTAACTACTTCTATGTTATGGAGTATTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG
TCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCC
CTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGAC
CGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGA
AAATTGTGCCCAGGGATTGT 62 VH n.t. PTA001_A4
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCGAGGTTCAGCTGCAGCAGTCTGTCGCTGAGTTGGTGA
AACCTGGAGCTTCAGTGAAGATGTCATGCAAGGTTTCTGGCTACACCCTC
ACTGACCATACTATTCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGA
ATGGATTGGATATATTTACCCTAGAGATGGAATAACTGGGTACAATGAGA
AGTTCAAGGGCAAGGCCACACTGACTGCAGACACTTCTTCCAGCACAGCC
TACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGT
GCCAGATGGGGCTATAGTTACAGGAATTACGCGTACTACTATGACTACTG
GGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCAT
CTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGA
CCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACC
TGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTG
CAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACC
TGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCAC
CAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 63 VH n.t. PTA001_A5
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGACTTGGTGA
AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAA
GTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCT
ATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTG
CAAGACTCAGGAACTATTTGTATATTATGGACTACTGGGGTCAAGGAACC
TCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTG
GTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATC
CCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTA
CACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGA
CCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG
AAAATTGTGCCCAGGGATTGT 64 VH n.t. PTA001_A6,
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA PTA001_A9,
PTA001_A10 AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGA
AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAA
GTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCT
ATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTT
CAAGACTCACTAACTACTTCTATGTTATGGAGTATTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG
TCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCC
CTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGAC
CGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGA
AAATTGTGCCCAGGGATTGT 65 VH n.t. PTA001_A7
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGA
AACCTGGAGCCTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTAGAGATGGTTTTACTAAGTACAATGAGAAG
TTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTA
CATGCAGCTCAACAGCCTGACATCTGAGGATTCTACAGTCTATTTCTGTGC
AAGAATGACTAACTACTTCTATACTATGGACTACTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG
TCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCC
CTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGAC
CGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGA
AAATTGTGCCCAGGGATTGT 66 VH n.t. PTA001_A8
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGACTTGGTGA
AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAA
GTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCT
ATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTT
CAAGACTCACTAACTACTTCTATGTTATGGAGTATTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG
TCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCC
CTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGAC
CGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGA
AAATTGTGCCCAGGGATTGT 67 VH n.t. PTA001_A11
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGA
AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTGAACATGGTAGTATTACGTATAATGAGAAG
TTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGTACTGTCTA
TATGCACCTCAATAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGC
AAGACTCAGGAACTACTTGTATGTTATGGACTACTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG
TCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCC
CTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGAC
CGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGA
AAATTGTGCCCAGGGATTGT 68 VH n.t. PTA001_A12
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGAGGCTGAGCTTGTGA
AGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATCTACCCCAGAGATGATTTTGCTAAGGTGAATGAGAA
GTTCAAGGGCAAGGCCACACTGACAGCAGACACATCCTCCAGCACAGCCT
ACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGTG
CAAGAATGACTAACTACCTCTATATTATGGACTACTGGGGTCAAGGAACC
TCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTG
GCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTG
GTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATC
CCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTA
CACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGA
CCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG
AAAATTGTGCCCAGGGATTGT 69 VH n.t. PTA001_A13
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGA
AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCA
CTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTATCCTGAACATGGTACTATTACGTATAATGAGAAG
TTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGTACTGTCTA
TATGCACCTCAATAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGC
AAGACTCAGGAACTATTTGTATATTATGGACTACTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGG
CCCCCGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTG
GTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATC
CCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTA
CACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGA
CCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG
AAAATTGTGCCCAGGGATTGT 70 VH n.t. PTA001_A14
TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAA
AGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGCTCTTATTTACCCC
TGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCCGCGTTGGTGA
AACCTGGAGCTTCAGTGAAGATATCGTGCAAGGTTTCTGGCTACACCTTCA
GTGACCATGCTATTCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAA
TGGATTGGATATATTTTTCCTAGAGATGCTTTTAGTTTGAACAATGAGAAG
TTCAAGGGCAAGGCCACACTGAGTGCAGACACATCCTCCAGCACAGCCTA
CATGGAGCTCACCAGCCTGACATTTGAGGATTCTGCAGTCTATTTCTGTGC
AAGAATGAGAAACTACTTCTATGTTATGGACTACTGGGGTCAAGGAACCT
CAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATACACTGG
CCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGG
TCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCC
CTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGAC
CGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGA
AAATTGTGCCCAGGGATTGT 71 VK n.t. PTA001_A1
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TGTTGTGCTGACCCAGACTCCACTCTCCCTGCCTGTCACTCTTGGAGATCA
AGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTTTACACAGTAATGGAAA
CACCTATTTACATTGGTACCTGCTGAAGCCAGGCCAGTCTCCAAAGCTCCT
GATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGG
CAGTGGATCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTG
AGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTGCTCACGTTCG
GTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTA
TCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTC
GTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAA
GATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATC
AGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACC
AAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAA
GACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCC
ATATGATGTGCCAGATTATGCGAGCTAA 72 VK n.t. PTA001_A2,
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT PTA001_A3
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAA
CACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCT
GATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACG
TTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAAC
TGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 73 VK n.t. PTA001_A4
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
CATCGTTATGTCTCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAA
GGTTACTATGAGCTGCAAGTCCAGCCAGAGCCTTTTACATAGTAGCAATC
AAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAA
GTGCTGATTTACTGGGCATCCACTAGAGAATCTGGGGTCCCTGATCGCTTC
ACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTGTGAA
GTCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCGTG
GACGTTCGGTGGCGGCACCAGGCTGGAAATCAAACGGGCTGATGCTGCAC
CAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTG
CCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCA
AGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGG
ACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCAC
GTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCA
CTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAG
TCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 74 VK n.t. PTA001_A5
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCA
ACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCC
TGATATATCGGCTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCTGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACA
TTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAAC
TGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 75 VK n.t. PTA001_A6
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATAAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAA
CACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCT
GATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACG
TTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAAC
TGTATCCATCCTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 76 VK n.t. PTA001_A7
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTTTCCATCTCCTGCAGGTCTTCTAAGAGTCTCCTGCGTACTAATGGCAA
CACTTACTTGCATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCT
GATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGTTTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCATTCACG
TTCGGCTCGGGGACAAAGTTGGAAATAAAAAGGGCTGATGCTGCACCAAC
TGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 77 VK n.t. PTA001_A8
TAAGATTAGCGGATCCTACCTTACGCTTTTTATCGCAACTCTCTACTGTTTC
TCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCTA
CGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGAT
ATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAATCA
GTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAAC
ACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTG
ATATATCGGCTGTCTAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGC
AGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGA
GGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACATT
CGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTG
TATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAG
TCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGA
AGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGAT
CAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGAC
CAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACA
AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTAT
CCATATGATGTGCCAGATTATGCGAGCTAA 78 VK n.t. PTA001_A9
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAA
CACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCT
GATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACG
TTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAAC
TGTATCCATCTCCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 79 VK n.t. PTA001_A10
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCAGGTCCAGTAAGAGTCTCCTGCGTAGTAATGGCA
ACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCC
TCATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGCCTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACG
TTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAA
CTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCT
CAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGT
GGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACT
GATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTT
GACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTC
ACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCT
TATCCATATGATGTGCCAGATTATGCGAGCTAA 80 VK n.t. PTA001_A11
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTTCTGTTTC
TCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCTA
CGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGAT
ATTGTGATGACCCAGGCTGCACCCTCTGTATCTGTCACTCCTGGAGAGTCA
GTATCCATCTCCTGCAGGTCTACTAAGAGTCTCCTGCGTAGTAATGGCAAC
ACTTACTTGTATTGGTTCCTCCAGAGGCCAGGCCAGTCTCCTCAGCTCCTG
ATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGG
CAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTG
AGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGT
TCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAAC
TGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 81 VK n.t. PTA001_A12
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACGTAGTAATGGCAA
CACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCT
GATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGCCTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACG
TTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAA
CTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCT
CAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGT
GGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACT
GATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTT
GACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTC
ACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCT
TATCCATATGATGTGCCAGATTATGCGAGCTAA 82 VK n.t. PTA001_A13
TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTT
CTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCT
ACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGA
TATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC
AGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCA
ACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCC
TGATATATCGGCTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTG
GCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCT
GAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACA
TTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAAC
TGTATCCATCTTCCCACAATACAGTGAGCAGTTAACAACTGGAGGTGCCTC
AGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG
GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTG
ATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG
ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCA
CAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTT
ATCCATATGATGTGCCAGATTATGCGAGCTAA 83 VK n.t. PTA001_A14
CGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCC
GTTTTTTTGGATGGAGTGAAACGATGAAATACCTATTGCCTACGGCAGCC
GCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATG
ACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATC
TCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTG
TATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGG
ATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTC
AGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGG
GTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGCTCGG
GGACAAATTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC
TTCACAACATCCAGAGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTG
CTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAG 84 VH CDR1 n.t. PTA001_A1
GGATTCACTTTCAGTAACTATGGCATGTCT 85 VH CDR1 n.t. PTA001_A2,
GGCTACACCTTCAGTGACCATGCTATTCAC PTA001_A14
86 VH CDR1 n.t. PTA001_A3, GGCTACACCTTCACTGACCATGCTATTCAC
PTA001_A5, PTA001_A6, PTA001_A7, PTA001_A8, PTA001_A9, PTA001_A10,
PTA001_A11, PTA001 A12, PTA001_A13 87 VH CDR1 n.t. PTA001_A4
GGCTACACCCTCACTGACCATACTATTCAC 88 VH CDR2 n.t. PTA001_A1
GCCATTAATCGTGATGGTGGTACCACCTACTATACAGACAATGTGAAGGG C 89 VH CDR2
n.t. PTA001_A2 TATATTTATCCTAGACATGGGACTACTAACTACAATGAGAACTTCAAGGG C
90 VH CDR2 n.t. PTA001_A3,
TATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAAGTTCAAGGG PTA001_A5,
PTA001_A6, C PTA001_A8, PTA001_A9, PTA001_A10 91 VH CDR2 n.t.
PTA001_A4 TATATTTACCCTAGAGATGGAATAACTGGGTACAATGAGAAGTTCAAGGG C 92
VH CDR2 n.t. PTA001_A7
TATATTTATCCTAGAGATGGTTTTACTAAGTACAATGAGAAGTTCAAGGGC 93 VH CDR2 n.t.
PTA001_A11 TATATTTATCCTGAACATGGTAGTATTACGTATAATGAGAAGTTCAAGGGC 94
VH CDR2 n.t. PTA001_A12
TATATCTACCCCAGAGATGATTTTGCTAAGGTGAATGAGAAGTTCAAGGG C 95 VH CDR2
n.t. PTA001_A13 TATATTTATCCTGAACATGGTACTATTACGTATAATGAGAAGTTCAAGGGC
96 VH CDR2 n.t. PTA001_A14
TATATTTTTCCTAGAGATGCTTTTAGTTTGAACAATGAGAAGTTCAAGGGC 97 VH CDR3 n.t.
PTA001_A1 TTCCTTCTCTGGGACGGCTGGTACTTCGATGTC 98 VH CDR3 n.t.
PTA001_A2, AGAAACTACTTCTATGTTATGGACTAC PTA001_A14 99 VH CDR3 n.t.
PTA001_A3, ACTAACTACTTCTATGTTATGGAGTAT PTA001_A6, PTA001_A8,
PTA001_A9, PTA001_A10 100 VH CDR3 n.t. PTA001_A4
GGCTATAGTTACAGGAATTACGCGTACTACTATGACTAC 101 VH CDR3 n.t. PTA001_A5,
AGGAACTATTTGTATATTATGGACTAC PTA001_A13 102 VH CDR3 n.t. PTA001_A7
ACTAACTACTTCTATACTATGGACTAC 103 VH CDR3 n.t. PTA001_A11
AGGAACTACTTGTATGTTATGGACTAC 104 VH CDR3 n.t. PTA001_A12
ACTAACTACCTCTATATTATGGACTAC 105 VK CDR1 n.t. PTA001_A1
AGATCTAGTCAGAGCCTTTTACACAGTAATGGAAACACCTATTTACAT 106 VK CDR1 n.t.
PTA001_A2, ACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTAT
PTA001_A3, PTA001_A6, PTA001_A9, PTA001_A14 107 VK CDR1 n.t.
PTA001_A4 AAGTCCAGCCAGAGCCTTTTACATAGTAGCAATCAAAAGAACTACTTGGC C 108
VK CDR1 n.t. PTA001_A5,
AGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCAACACTTACTTGTAT PTA001_A13 109 VK
CDR1 n.t. PTA001_A7
AGGTCTTCTAAGAGTCTCCTGCGTACTAATGGCAACACTTACTTGCAT 110 VK CDR1 n.t.
PTA001_A8 AGGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTAT 111 VK
CDR1 n.t. PTA001_A10
AGGTCCAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTAT 112 VK CDR1 n.t.
PTA001_A11 AGGTCTACTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTAT 113 VK
CDR1 n.t. PTA001_A12
AGGTCTAGTAAGAGTCTCCTACGTAGTAATGGCAACACTTACTTGTAT 114 VK CDR2 n.t.
PTA001_A1 AAAGTTTCCAACCGATTTTCT 115 VK CDR2 n.t. PTA001_A2,
CGGATGTCCAACCTTGCCTCG PTA001_A3, PTA001_A6, PTA001_A9, PTA001_A14
116 VK CDR2 n.t. PTA001_A4 TGGGCATCCACTAGAGAATCT 117 VK CDR2 n.t.
PTA001_A5, CGGCTGTCCAACCTTGCCTCA PTA001_A13 118 VK CDR2 n.t.
PTA001_A7, CGGATGTCCAACCTTGCCTCA PTA001_A10, PTA001_A11, PTA001_A12
119 VK CDR2 n.t. PTA001_A8 CGGCTGTCTAACCTTGCCTCA 120 VK CDR3 n.t.
PTA001_A1 TCTCAAAGTACACATGTGCTCACG 121 VK CDR3 n.t. PTA001_A2,
ATGCAACATCTAGAATATCCTTTCACG PTA001_A3, PTA001_A6, PTA001_A9,
PTA001_A10, PTA001_A11, PTA001 A12, PTA001_A14 122 VK CDR3 n.t.
PTA001_A4 CAGCAATATTATAGCTATCCGTGGACG 123 VK CDR3 n.t. PTA001_A5,
ATGCAACATCTAGAATATCCTTTCACA PTA001_A8, PTA001_A13 124 VK CDR3 n.t.
PTA001_A7 ATGCAACATCTAGAATATCCATTCACG 125 VH7-39 (Genbank
GGATTCACTTTCAGTAGCTATGGCATGTCT AJ851868.3) n.t. 240-269 126 VHII
gene H17 (GenBank GGCTACACCTTCACTGACCATACTATTCAC X02466.1) n.t.
67-96 127 VHII region VH105 (Genbank
TATATTTATCCTAGAGATGGTAGTACTAAGTACAATGAGAAGTTCAAGGG J00507)
n.t.1096-1146 C 128 VK1-110 (GenBank
AGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT AY591695.1) n.t.
1738-1785 129 VK1-110 (GenBank TCTCAAAGTACACATGTTCCTCCC AY591695.1)
n.t. 1948-1971 130 VK8-30 (GenBank
AAGTCCAGTCAGAGCCTTITATATAGTAGCAATCAAAAGAACTACTTGGC AJ235948.1) n.t.
510-560 C 131 VK8-30 (GenBank TGGGCATCCACTAGGGAATCT AJ235948.1)
n.t. 606-626 132 VK8-30 (GenBank CAGCAATATTATAGCTATCCTCCCACA
AJ235948.1) n.t. 723-749 133 VK24-140 (GenBank
AGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTAT AY591710.1) n.t.
1807-1854 134 VK24-140 (GenBank CGGATGTCCAACCTTGCCTCA AY591710.1)
n.t. 1900-1920 135 VK24-140 (GenBank ATGCAACATCTAGAATATCCTTTCACA
AY591710.1) n.t. 2017-2043 136 Cadherin-17 ECD domains
QEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLL 1-2
YYNRALDRETRSTHNLQVAALDANGIIVEGPVPITIKVKDINDNRPTFLQSKYE
GSVRQNSRPGKPFLYVNATDLDDPATPNGQLYYQIVIQLPMINNVMYFQINN
KTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENI WKAPKP 137
Cadherin-17 ECD
QEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLL
YYNRALDRETRSTHNLQVAALDANGIIVEGPVPITIKVKDINDNRPTFLQSKYE
GSVRQNSRPGKPFLYVNATDLDDPATPNGQLYYQIVIQLPMINNVMYFQINN
KTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENI
WKAPKPVEMVENSTDPHPIKITQVRWNDPGAQYSLVDKEKLPRFPFSIDQEG
DIYVTQPLDREEKDAYVFYAVAKDEYGKPLSYPLEIHVKVKDINDNPPTCPSP
VTVFEVQENERLGNSIGTLTAHDRDEENTANSFLNYRIVEQTPKLPMDGLFLI
QTYAGMLQLAKQSLKKQDTPQYNLTIEVSDKDFKTLCFVQINVIDINDQIPIFE
KSDYGNLTLAEDTNIGST1LTIQATDADEPFTGSSKILYIIIIKGDSEGRLGVDTD
PHTNTGYVIIKKPLDFETAAVSNIVFKAENPEPLVFGVKYNASSFAKFTLIVTD
VNEAPQFSQHVFQAKVSEDVAIGTKVGNVTAKDPEGLDISYSLRGDTRGWLK
IDHVTGEIFSVAPLDREAGSPYRVQVVATEVGGSSLSSVSEFHLILMDVNDNPP
RLAKDYTGLFFCHPLSAPGSL1FEATDDDQHLFRGPHFTFSLGSGSLQNDWEV
SIGNGTHARLSTRHTDFEEREYVVLIRINDGGRPPLEGIVSLPVTFCSCVEGSCF
RPAGHQTGIPTVGM 138 Mature VH amino acid
EVQLQQSVAELVKPGASVKMSCKVSGYTLTDHTIHWMKQRPEQGLEWIGYI PTA001_A4
(without 1eader
YPRDGITGYNEKFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARWGYSY sequence)
RNYAYYYDYWGQGTTLTVSS 139 Mature VK amino acid
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLHSSNQKNYLAWYQQKPGQSPKV PTA001_A4
(without 1eader LIYWASTR sequence)
ESGVPDRFTGSGSGTDFTLTITSVKSEDLAVYYCQQYYSYPWTFGGGTRLEIK RA
Sequence CWU 1
1
139110PRTHomo sapiens 1Gly Phe Thr Phe Ser Asn Tyr Gly Met Ser 1 5
10 210PRTHomo sapiens 2Gly Tyr Thr Phe Ser Asp His Ala Ile His 1 5
10 310PRTHomo sapiens 3Gly Tyr Thr Phe Thr Asp His Ala Ile His 1 5
10 410PRTHomo sapiens 4Gly Tyr Thr Leu Thr Asp His Thr Ile His 1 5
10 517PRTHomo sapiens 5Ala Ile Asn Arg Asp Gly Gly Thr Thr Tyr Tyr
Thr Asp Asn Val Lys 1 5 10 15 Gly 617PRTHomo sapiens 6Tyr Ile Tyr
Pro Arg His Gly Thr Thr Asn Tyr Asn Glu Asn Phe Lys 1 5 10 15 Gly
717PRTHomo sapiens 7Tyr Ile Tyr Pro Glu His Gly Thr Ile Lys Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Gly 817PRTHomo sapiens 8Tyr Ile Tyr Pro
Arg Asp Gly Ile Thr Gly Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
917PRTHomo sapiens 9Tyr Ile Tyr Pro Arg Asp Gly Phe Thr Lys Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Gly 1017PRTHomo sapiens 10Tyr Ile Tyr Pro
Glu His Gly Ser Ile Thr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
1117PRTHomo sapiens 11Tyr Ile Tyr Pro Arg Asp Asp Phe Ala Lys Val
Asn Glu Lys Phe Lys 1 5 10 15 Gly 1217PRTHomo sapiens 12Tyr Ile Tyr
Pro Glu His Gly Thr Ile Thr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
1317PRTHomo sapiens 13Tyr Ile Phe Pro Arg Asp Ala Phe Ser Leu Asn
Asn Glu Lys Phe Lys 1 5 10 15 Gly 1411PRTHomo sapiens 14Phe Leu Leu
Trp Asp Gly Trp Tyr Phe Asp Val 1 5 10 159PRTHomo sapiens 15Arg Asn
Tyr Phe Tyr Val Met Asp Tyr 1 5 169PRTHomo sapiens 16Thr Asn Tyr
Phe Tyr Val Met Glu Tyr 1 5 1713PRTHomo sapiens 17Gly Tyr Ser Tyr
Arg Asn Tyr Ala Tyr Tyr Tyr Asp Tyr 1 5 10 189PRTHomo sapiens 18Arg
Asn Tyr Leu Tyr Ile Met Asp Tyr 1 5 199PRTHomo sapiens 19Thr Asn
Tyr Phe Tyr Thr Met Asp Tyr 1 5 209PRTHomo sapiens 20Arg Asn Tyr
Leu Tyr Val Met Asp Tyr 1 5 219PRTHomo sapiens 21Thr Asn Tyr Leu
Tyr Ile Met Asp Tyr 1 5 2216PRTHomo sapiens 22Arg Ser Ser Gln Ser
Leu Leu His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 2316PRTHomo
sapiens 23Thr Ser Ser Lys Ser Leu Leu Arg Ser Asn Gly Asn Thr Tyr
Leu Tyr 1 5 10 15 2417PRTHomo sapiens 24Lys Ser Ser Gln Ser Leu Leu
His Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 2516PRTHomo
sapiens 25Arg Ser Ser Lys Ser Leu Leu Arg Ser Asn Gly Asn Thr Tyr
Leu Tyr 1 5 10 15 2616PRTHomo sapiens 26Arg Ser Ser Lys Ser Leu Leu
Arg Thr Asn Gly Asn Thr Tyr Leu His 1 5 10 15 2716PRTHomo sapiens
27Arg Ser Thr Lys Ser Leu Leu Arg Ser Asn Gly Asn Thr Tyr Leu Tyr 1
5 10 15 287PRTHomo sapiens 28Lys Val Ser Asn Arg Phe Ser 1 5
297PRTHomo sapiens 29Arg Met Ser Asn Leu Ala Ser 1 5 307PRTHomo
sapiens 30Trp Ala Ser Thr Arg Glu Ser 1 5 317PRTHomo sapiens 31Arg
Leu Ser Asn Leu Ala Ser 1 5 328PRTHomo sapiens 32Ser Gln Ser Thr
His Val Leu Thr 1 5 339PRTHomo sapiens 33Met Gln His Leu Glu Tyr
Pro Phe Thr 1 5 349PRTHomo sapiens 34Gln Gln Tyr Tyr Ser Tyr Pro
Trp Thr 1 5 35260PRTHomo sapiens 35Leu Gly Lys Pro Trp Arg Tyr Pro
Arg Phe Val His Gly Glu Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile
Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys
Ala Glu Val Gln Leu Leu Glu Thr Gly Gly Gly Val 35 40 45 Val Lys
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 50 55 60
Thr Phe Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys 65
70 75 80 Arg Leu Glu Trp Val Ala Ala Ile Asn Arg Asp Gly Gly Thr
Thr Tyr 85 90 95 Tyr Thr Asp Asn Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala 100 105 110 Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser
Leu Arg Ser Glu Asp Thr 115 120 125 Ala Leu Tyr Tyr Cys Ala Arg Gln
Phe Leu Leu Trp Asp Gly Trp Tyr 130 135 140 Phe Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys 145 150 155 160 Thr Thr Pro
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln 165 170 175 Thr
Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro 180 185
190 Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val
195 200 205 His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
Ser Ser 210 215 220 Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu
Thr Val Thr Cys 225 230 235 240 Asn Val Ala His Pro Ala Ser Ser Thr
Lys Val Asp Lys Lys Ile Val 245 250 255 Pro Arg Asp Cys 260
36258PRTHomo sapiens 36Leu Gly Lys Pro Trp Arg Tyr Pro Arg Phe Val
His Gly Glu Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile Ala Leu Ala
Leu Leu Pro Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys Ala Gln Val
Gln Leu Gln Gln Ser Asp Ala Glu Leu 35 40 45 Val Lys Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr 50 55 60 Thr Phe Ser
Asp His Ala Ile His Trp Met Ser Gln Arg Pro Gly Gln 65 70 75 80 Gly
Leu Lys Trp Ile Gly Tyr Ile Tyr Pro Arg His Gly Thr Thr Asn 85 90
95 Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser
100 105 110 Ser Ser Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu
Asp Ser 115 120 125 Ala Val Tyr Phe Cys Ala Arg Met Arg Asn Tyr Phe
Tyr Val Met Asp 130 135 140 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser Ala Lys Thr Thr 145 150 155 160 Pro Pro Ser Val Tyr Pro Leu
Ala Pro Gly Ser Ala Ala Gln Thr Asn 165 170 175 Ser Met Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 180 185 190 Val Thr Val
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 195 200 205 Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 210 215
220 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val
225 230 235 240 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
Val Pro Arg 245 250 255 Asp Cys 37258PRTHomo sapiens 37Leu Gly Lys
Pro Trp Arg Tyr Pro Arg Phe Val His Gly Glu Asn Lys 1 5 10 15 Val
Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 20 25
30 Pro Val Ala Lys Ala Gln Val Leu Leu Gln Gln Ser Asp Ala Glu Leu
35 40 45 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr 50 55 60 Thr Phe Thr Asp His Ala Ile His Trp Val Lys Gln
Arg Pro Glu Gln 65 70 75 80 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro
Glu His Gly Thr Ile Lys 85 90 95 Tyr Asn Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser 100 105 110 Ser Ser Thr Ala Tyr Met
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser 115 120 125 Ala Val Tyr Phe
Cys Ser Arg Leu Thr Asn Tyr Phe Tyr Val Met Glu 130 135 140 Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 145 150 155
160 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn
165 170 175 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro 180 185 190 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr 195 200 205 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val 210 215 220 Thr Val Pro Ser Ser Thr Trp Pro
Ser Glu Thr Val Thr Cys Asn Val 225 230 235 240 Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 245 250 255 Asp Cys
38262PRTHomo sapiens 38Leu Gly Lys Pro Trp Arg Tyr Pro Arg Phe Val
His Gly Glu Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile Ala Leu Ala
Leu Leu Pro Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys Ala Glu Val
Gln Leu Gln Gln Ser Val Ala Glu Leu 35 40 45 Val Lys Pro Gly Ala
Ser Val Lys Met Ser Cys Lys Val Ser Gly Tyr 50 55 60 Thr Leu Thr
Asp His Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln 65 70 75 80 Gly
Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Ile Thr Gly 85 90
95 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser
100 105 110 Ser Ser Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu
Asp Ser 115 120 125 Ala Val Tyr Phe Cys Ala Arg Trp Gly Tyr Ser Tyr
Arg Asn Tyr Ala 130 135 140 Tyr Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser 145 150 155 160 Ala Lys Thr Thr Pro Pro Ser
Val Tyr Pro Leu Ala Pro Gly Ser Ala 165 170 175 Ala Gln Thr Asn Ser
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 180 185 190 Phe Pro Glu
Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 195 200 205 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 210 215
220 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
225 230 235 240 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
Asp Lys Lys 245 250 255 Ile Val Pro Arg Asp Cys 260 39258PRTHomo
sapiens 39Leu Gly Lys Pro Trp Arg Tyr Pro Arg Phe Val His Gly Glu
Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro
Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys Ala Gln Val Gln Leu Gln
Gln Ser Asp Ala Asp Leu 35 40 45 Val Lys Pro Gly Ala Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr 50 55 60 Thr Phe Thr Asp His Ala
Ile His Trp Val Lys Gln Arg Pro Glu Gln 65 70 75 80 Gly Leu Glu Trp
Ile Gly Tyr Ile Tyr Pro Glu His Gly Thr Ile Lys 85 90 95 Tyr Asn
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 100 105 110
Ser Ser Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser 115
120 125 Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Tyr Leu Tyr Ile Met
Asp 130 135 140 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala
Lys Thr Thr 145 150 155 160 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
Ser Ala Ala Gln Thr Asn 165 170 175 Ser Met Val Thr Leu Gly Cys Leu
Val Lys Gly Tyr Phe Pro Glu Pro 180 185 190 Val Thr Val Thr Trp Asn
Ser Gly Ser Leu Ser Ser Gly Val His Thr 195 200 205 Phe Pro Ala Val
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 210 215 220 Thr Val
Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val 225 230 235
240 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg
245 250 255 Asp Cys 40258PRTHomo sapiens 40Leu Gly Lys Pro Trp Arg
Tyr Pro Arg Phe Val His Gly Glu Asn Lys 1 5 10 15 Val Lys Gln Ser
Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 20 25 30 Pro Val
Ala Lys Ala Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu 35 40 45
Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 50
55 60 Thr Phe Thr Asp His Ala Ile His Trp Val Lys Gln Arg Pro Glu
Gln 65 70 75 80 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Glu His Gly
Thr Ile Lys 85 90 95 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu
Thr Ala Asp Lys Ser 100 105 110 Ser Ser Thr Ala Tyr Met Gln Leu Asn
Ser Leu Thr Ser Glu Asp Ser 115 120 125 Ala Val Tyr Phe Cys Ser Arg
Leu Thr Asn Tyr Phe Tyr Val Met Glu 130 135 140 Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 145 150 155 160 Pro Pro
Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 165 170 175
Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 180
185 190 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His
Thr 195 200 205 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser
Ser Ser Val 210 215 220 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr
Val Thr Cys Asn Val 225 230 235 240 Ala His Pro Ala Ser Ser Thr Lys
Val Asp Lys Lys Ile Val Pro Arg 245 250 255 Asp Cys 41258PRTHomo
sapiens 41Leu Gly Lys Pro Trp Arg Tyr Pro Arg Phe Val His Gly Glu
Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro
Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys Ala Gln Val Gln Leu Gln
Gln Ser Asp Ala Glu Leu 35 40 45 Val Lys Pro Gly Ala Ser Val Lys
Ile Ser Cys Lys Val Ser Gly Tyr 50 55 60 Thr Phe Thr Asp His Ala
Ile His Trp Met Lys Gln Arg Pro Glu Gln 65 70 75 80 Gly Leu Glu Trp
Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Phe Thr Lys 85 90 95 Tyr Asn
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser 100 105 110
Ser Ser Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser 115
120 125 Thr Val Tyr Phe Cys Ala Arg Met Thr Asn Tyr Phe Tyr Thr Met
Asp 130 135 140 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala
Lys Thr Thr 145 150 155 160 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
Ser Ala Ala Gln Thr Asn 165 170 175 Ser Met Val Thr Leu Gly Cys Leu
Val Lys Gly Tyr Phe Pro Glu Pro 180 185 190 Val Thr Val Thr Trp Asn
Ser Gly Ser Leu Ser Ser Gly Val His Thr 195 200 205 Phe Pro Ala Val
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 210 215
220 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val
225 230 235 240 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
Val Pro Arg 245 250 255 Asp Cys 42258PRTHomo sapiens 42Leu Gly Lys
Pro Trp Arg Tyr Pro Arg Phe Val His Gly Glu Asn Lys 1 5 10 15 Val
Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 20 25
30 Pro Val Ala Lys Ala Gln Val Gln Leu Gln Gln Ser Asp Ala Asp Leu
35 40 45 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr 50 55 60 Thr Phe Thr Asp His Ala Ile His Trp Val Lys Gln
Arg Pro Glu Gln 65 70 75 80 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro
Glu His Gly Thr Ile Lys 85 90 95 Tyr Asn Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser 100 105 110 Ser Ser Thr Ala Tyr Met
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser 115 120 125 Ala Val Tyr Phe
Cys Ser Arg Leu Thr Asn Tyr Phe Tyr Val Met Glu 130 135 140 Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 145 150 155
160 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn
165 170 175 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro 180 185 190 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr 195 200 205 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val 210 215 220 Thr Val Pro Ser Ser Thr Trp Pro
Ser Glu Thr Val Thr Cys Asn Val 225 230 235 240 Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 245 250 255 Asp Cys
43258PRTHomo sapiens 43Leu Gly Lys Pro Trp Arg Tyr Pro Arg Phe Val
His Gly Glu Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile Ala Leu Ala
Leu Leu Pro Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys Ala Gln Val
Gln Leu Gln Gln Ser Asp Ala Glu Leu 35 40 45 Val Lys Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 50 55 60 Thr Phe Thr
Asp His Ala Ile His Trp Val Lys Gln Arg Pro Glu Gln 65 70 75 80 Gly
Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Glu His Gly Ser Ile Thr 85 90
95 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
100 105 110 Ser Ser Thr Val Tyr Met His Leu Asn Ser Leu Thr Ser Glu
Asp Ser 115 120 125 Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Tyr Leu
Tyr Val Met Asp 130 135 140 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser Ala Lys Thr Thr 145 150 155 160 Pro Pro Ser Val Tyr Pro Leu
Ala Pro Gly Ser Ala Ala Gln Thr Asn 165 170 175 Ser Met Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 180 185 190 Val Thr Val
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 195 200 205 Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 210 215
220 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val
225 230 235 240 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
Val Pro Arg 245 250 255 Asp Cys 44258PRTHomo sapiens 44Leu Gly Lys
Pro Trp Arg Tyr Pro Arg Phe Val His Gly Glu Asn Lys 1 5 10 15 Val
Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 20 25
30 Pro Val Ala Lys Ala Gln Val Gln Leu Gln Gln Ser Glu Ala Glu Leu
35 40 45 Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr 50 55 60 Thr Phe Thr Asp His Ala Ile His Trp Met Lys Gln
Arg Pro Glu Gln 65 70 75 80 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro
Arg Asp Asp Phe Ala Lys 85 90 95 Val Asn Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ala Asp Thr Ser 100 105 110 Ser Ser Thr Ala Tyr Met
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser 115 120 125 Ala Val Tyr Phe
Cys Ala Arg Met Thr Asn Tyr Leu Tyr Ile Met Asp 130 135 140 Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 145 150 155
160 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn
165 170 175 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro 180 185 190 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr 195 200 205 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val 210 215 220 Thr Val Pro Ser Ser Thr Trp Pro
Ser Glu Thr Val Thr Cys Asn Val 225 230 235 240 Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 245 250 255 Asp Cys
45258PRTHomo sapiens 45Leu Gly Lys Pro Trp Arg Tyr Pro Arg Phe Val
His Gly Glu Asn Lys 1 5 10 15 Val Lys Gln Ser Thr Ile Ala Leu Ala
Leu Leu Pro Leu Leu Phe Thr 20 25 30 Pro Val Ala Lys Ala Gln Val
Gln Leu Gln Gln Ser Asp Ala Glu Leu 35 40 45 Val Lys Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 50 55 60 Thr Phe Thr
Asp His Ala Ile His Trp Val Lys Gln Arg Pro Glu Gln 65 70 75 80 Gly
Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Glu His Gly Thr Ile Thr 85 90
95 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
100 105 110 Ser Ser Thr Val Tyr Met His Leu Asn Ser Leu Thr Ser Glu
Asp Ser 115 120 125 Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Tyr Leu
Tyr Ile Met Asp 130 135 140 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser Ala Lys Thr Thr 145 150 155 160 Pro Pro Ser Val Tyr Pro Leu
Ala Pro Gly Ser Ala Ala Gln Thr Asn 165 170 175 Ser Met Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 180 185 190 Val Thr Val
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 195 200 205 Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 210 215
220 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val
225 230 235 240 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
Val Pro Arg 245 250 255 Asp Cys 46258PRTHomo sapiens 46Leu Gly Lys
Pro Trp Arg Tyr Pro Arg Phe Val His Gly Glu Asn Lys 1 5 10 15 Val
Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 20 25
30 Pro Val Ala Lys Ala Gln Val Gln Leu Gln Gln Ser Asp Ala Ala Leu
35 40 45 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser
Gly Tyr 50 55 60 Thr Phe Ser Asp His Ala Ile His Trp Met Lys Gln
Arg Pro Glu Gln 65 70 75 80 Gly Leu Glu Trp Ile Gly Tyr Ile Phe Pro
Arg Asp Ala Phe Ser Leu 85 90 95 Asn Asn Glu Lys Phe Lys Gly Lys
Ala Thr Leu Ser Ala Asp Thr Ser 100 105 110 Ser Ser Thr Ala Tyr Met
Glu Leu Thr Ser Leu Thr Phe Glu Asp Ser 115 120 125 Ala Val Tyr Phe
Cys Ala Arg Met Arg Asn Tyr Phe Tyr Val Met Asp 130 135 140 Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 145 150 155
160 Pro Pro Ser Val Tyr Thr Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn
165 170 175 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro 180 185 190 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr 195 200 205 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val 210 215 220 Thr Val Pro Ser Ser Thr Trp Pro
Ser Glu Thr Val Thr Cys Asn Val 225 230 235 240 Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 245 250 255 Asp Cys
47275PRTHomo sapiens 47Arg Ile Leu Pro Asp Ala Phe Tyr Arg Asn Ser
Leu Leu Phe Leu His 1 5 10 15 Thr Arg Phe Phe Gly Trp Ser Glu Thr
Met Lys Tyr Leu Leu Pro Thr 20 25 30 Ala Ala Ala Gly Leu Leu Leu
Leu Ala Ala Gln Pro Ala Met Ala Asp 35 40 45 Val Val Leu Thr Gln
Thr Pro Leu Ser Leu Pro Val Thr Leu Gly Asp 50 55 60 Gln Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn 65 70 75 80 Gly
Asn Thr Tyr Leu His Trp Tyr Leu Leu Lys Pro Gly Gln Ser Pro 85 90
95 Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp
100 105 110 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile Thr 115 120 125 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
Ser Gln Ser Thr 130 135 140 His Val Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys Arg Ala 145 150 155 160 Asp Ala Ala Pro Thr Val Ser
Ile Phe Pro Pro Ser Ser Glu Gln Leu 165 170 175 Thr Ser Gly Gly Ala
Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro 180 185 190 Lys Asp Ile
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn 195 200 205 Gly
Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr 210 215
220 Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His
225 230 235 240 Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr
Ser Pro Ile 245 250 255 Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr Pro
Tyr Asp Val Pro Asp 260 265 270 Tyr Ala Ser 275 48276PRTHomo
sapiens 48Arg Ile Leu Pro Asp Ala Phe Tyr Arg Asn Ser Leu Leu Phe
Leu His 1 5 10 15 Thr Arg Phe Phe Gly Trp Ser Glu Thr Met Lys Tyr
Leu Leu Pro Thr 20 25 30 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala
Gln Pro Ala Met Ala Asp 35 40 45 Ile Val Met Thr Gln Ala Ala Pro
Ser Val Pro Val Thr Pro Gly Glu 50 55 60 Ser Val Ser Ile Ser Cys
Thr Ser Ser Lys Ser Leu Leu Arg Ser Asn 65 70 75 80 Gly Asn Thr Tyr
Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro 85 90 95 Gln Leu
Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp 100 105 110
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser 115
120 125 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His
Leu 130 135 140 Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
Ile Lys Arg 145 150 155 160 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro Ser Ser Glu Gln 165 170 175 Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe Tyr 180 185 190 Pro Lys Asp Ile Asn Val
Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 195 200 205 Asn Gly Val Leu
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 210 215 220 Tyr Ser
Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 225 230 235
240 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
245 250 255 Ile Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr Pro Tyr Asp
Val Pro 260 265 270 Asp Tyr Ala Ser 275 49277PRTHomo sapiens 49Arg
Ile Leu Pro Asp Ala Phe Tyr Arg Asn Ser Leu Leu Phe Leu His 1 5 10
15 Thr Arg Phe Phe Gly Trp Ser Glu Thr Met Lys Tyr Leu Leu Pro Thr
20 25 30 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met
Ala Asp 35 40 45 Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val
Ser Val Gly Glu 50 55 60 Lys Val Thr Met Ser Cys Lys Ser Ser Gln
Ser Leu Leu His Ser Ser 65 70 75 80 Asn Gln Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser 85 90 95 Pro Lys Val Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val Pro 100 105 110 Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 115 120 125 Thr Ser
Val Lys Ser Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr 130 135 140
Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 145
150 155 160 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser
Ser Glu 165 170 175 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe
Leu Asn Asn Phe 180 185 190 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys
Ile Asp Gly Ser Glu Arg 195 200 205 Gln Asn Gly Val Leu Asn Ser Trp
Thr Asp Gln Asp Ser Lys Asp Ser 210 215 220 Thr Tyr Ser Met Ser Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 225 230 235 240 Arg His Asn
Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 245 250 255 Pro
Ile Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr Pro Tyr Asp Val 260 265
270 Pro Asp Tyr Ala Ser 275 50276PRTHomo sapiens 50Arg Ile Leu Pro
Asp Ala Phe Tyr Arg Asn Ser Leu Leu Phe Leu His 1 5 10 15 Thr Arg
Phe Phe Gly Trp Ser Glu Thr Met Lys Tyr Leu Leu Pro Thr 20 25 30
Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Asp 35
40 45 Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly
Glu 50 55 60 Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu
Arg Ser Asn 65 70 75 80 Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg
Pro Gly Gln Ser Pro 85 90 95 Gln Leu Leu Ile Tyr Arg Leu Ser Asn
Leu Ala Ser Gly Val Pro Asp 100 105 110 Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ala Phe Thr Leu Arg Ile Ser 115 120 125
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu 130
135 140 Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
Arg 145 150 155 160 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro
Ser Ser Glu Gln 165 170 175 Leu Thr Ser Gly Gly Ala Ser Val Val Cys
Phe Leu Asn Asn Phe Tyr 180 185 190 Pro Lys Asp Ile Asn Val Lys Trp
Lys Ile Asp Gly Ser Glu Arg Gln 195 200 205 Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 210 215 220 Tyr Ser Met Ser
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 225 230 235 240 His
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 245 250
255 Ile Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr Pro Tyr Asp Val Pro
260 265 270 Asp Tyr Ala Ser 275 51276PRTHomo sapiens 51Arg Ile Leu
Pro Asp Ala Phe Tyr Arg Asn Ser Leu Leu Phe Leu His 1 5 10 15 Thr
Arg Phe Phe Gly Trp Ser Glu Thr Ile Lys Tyr Leu Leu Pro Thr 20 25
30 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Asp
35 40 45 Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro
Gly Glu 50 55 60 Ser Val Ser Ile Ser Cys Thr Ser Ser Lys Ser Leu
Leu Arg Ser Asn 65 70 75 80 Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln
Arg Pro Gly Gln Ser Pro 85 90 95 Gln Leu Leu Ile Tyr Arg Met Ser
Asn Leu Ala Ser Gly Val Pro Asp 100 105 110 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser 115 120 125 Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu 130 135 140 Glu Tyr
Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 145 150 155
160 Ala Asp Ala Ala Pro Thr Val Ser Ile Leu Pro Pro Ser Ser Glu Gln
165 170 175 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn
Phe Tyr 180 185 190 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly
Ser Glu Arg Gln 195 200 205 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser Thr 210 215 220 Tyr Ser Met Ser Ser Thr Leu Thr
Leu Thr Lys Asp Glu Tyr Glu Arg 225 230 235 240 His Asn Ser Tyr Thr
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 245 250 255 Ile Val Lys
Ser Phe Asn Arg Asn Glu Ser Tyr Pro Tyr Asp Val Pro 260 265 270 Asp
Tyr Ala Ser 275 52276PRTHomo sapiens 52Arg Ile Leu Pro Asp Ala Phe
Tyr Arg Asn Ser Leu Leu Phe Leu His 1 5 10 15 Thr Arg Phe Phe Gly
Trp Ser Glu Thr Met Lys Tyr Leu Leu Pro Thr 20 25 30 Ala Ala Ala
Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Asp 35 40 45 Ile
Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly Glu 50 55
60 Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Arg Thr Asn
65 70 75 80 Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Arg Pro Gly Gln
Ser Pro 85 90 95 Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser
Gly Val Pro Asp 100 105 110 Arg Phe Ser Gly Ser Gly Ser Gly Thr Val
Phe Thr Leu Arg Ile Ser 115 120 125 Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Met Gln His Leu 130 135 140 Glu Tyr Pro Phe Thr Phe
Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 145 150 155 160 Ala Asp Ala
Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 165 170 175 Leu
Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 180 185
190 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
195 200 205 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp
Ser Thr 210 215 220 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp
Glu Tyr Glu Arg 225 230 235 240 His Asn Ser Tyr Thr Cys Glu Ala Thr
His Lys Thr Ser Thr Ser Pro 245 250 255 Ile Val Lys Ser Phe Asn Arg
Asn Glu Ser Tyr Pro Tyr Asp Val Pro 260 265 270 Asp Tyr Ala Ser 275
53276PRTHomo sapiens 53Arg Ile Leu Pro Tyr Ala Phe Tyr Arg Asn Ser
Leu Leu Phe Leu His 1 5 10 15 Thr Arg Phe Phe Gly Trp Ser Glu Thr
Met Lys Tyr Leu Leu Pro Thr 20 25 30 Ala Ala Ala Gly Leu Leu Leu
Leu Ala Ala Gln Pro Ala Met Ala Asp 35 40 45 Ile Val Met Thr Gln
Ala Ala Pro Ser Val Pro Val Thr Pro Gly Glu 50 55 60 Ser Val Ser
Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Arg Ser Asn 65 70 75 80 Gly
Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro 85 90
95 Gln Leu Leu Ile Tyr Arg Leu Ser Asn Leu Ala Ser Gly Val Pro Asp
100 105 110 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg
Ile Ser 115 120 125 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln His Leu 130 135 140 Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Arg 145 150 155 160 Ala Asp Ala Ala Pro Thr Val
Ser Ile Phe Pro Pro Ser Ser Glu Gln 165 170 175 Leu Thr Ser Gly Gly
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 180 185 190 Pro Lys Asp
Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 195 200 205 Asn
Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 210 215
220 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
225 230 235 240 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser
Thr Ser Pro 245 250 255 Ile Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr
Pro Tyr Asp Val Pro 260 265 270 Asp Tyr Ala Ser 275 54276PRTHomo
sapiens 54Arg Ile Leu Pro Asp Ala Phe Tyr Arg Asn Ser Leu Leu Phe
Leu His 1 5 10 15 Thr Arg Phe Phe Gly Trp Ser Glu Thr Met Lys Tyr
Leu Leu Pro Thr 20 25 30 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala
Gln Pro Ala Met Ala Asp 35 40 45 Ile Val Met Thr Gln Ala Ala Pro
Ser Val Pro Val Thr Pro Gly Glu 50 55 60 Ser Val Ser Ile Ser Cys
Thr Ser Ser Lys Ser Leu Leu Arg Ser Asn 65 70 75 80 Gly Asn Thr Tyr
Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro 85 90 95 Gln Leu
Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp 100 105 110
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser 115
120 125 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His
Leu 130 135 140 Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
Ile Lys Arg 145 150 155 160 Ala Asp Ala Ala Pro Thr Val Ser Ile Ser
Pro Pro Ser Ser Glu Gln 165 170 175 Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe Tyr 180 185 190 Pro Lys Asp Ile Asn Val
Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 195 200 205 Asn Gly Val Leu
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 210 215 220 Tyr Ser
Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 225 230 235
240 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
245 250 255 Ile Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr Pro Tyr Asp
Val Pro 260 265 270 Asp Tyr Ala Ser 275 55276PRTHomo sapiens 55Arg
Ile Leu Pro Asp Ala Phe Tyr Arg Asn Ser Leu Leu Phe Leu His 1 5 10
15 Thr Arg Phe Phe Gly Trp Ser Glu Thr Met Lys Tyr Leu Leu Pro Thr
20 25 30 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met
Ala Asp 35 40 45 Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val
Thr Pro Gly Glu 50 55 60 Ser Val Ser Ile Ser Cys Arg Ser Ser Lys
Ser Leu Leu Arg Ser Asn 65 70 75 80 Gly Asn Thr Tyr Leu Tyr Trp Phe
Leu Gln Arg Pro Gly Gln Ser Pro 85 90 95 Gln Leu Leu Ile Tyr Arg
Met Ser Asn Leu Ala Ser Gly Val Pro Asp 100 105 110 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser 115 120 125 Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu 130 135 140
Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 145
150 155 160 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser
Glu Gln 165 170 175 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu
Asn Asn Phe Tyr 180 185 190 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile
Asp Gly Ser Glu Arg Gln 195 200 205 Asn Gly Val Leu Asn Ser Trp Thr
Asp Gln Asp Ser Lys Asp Ser Thr 210 215 220 Tyr Ser Met Ser Ser Thr
Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 225 230 235 240 His Asn Ser
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 245 250 255 Ile
Val Lys Ser Phe Asn Arg Asn Glu Ser Tyr Pro Tyr Asp Val Pro 260 265
270 Asp Tyr Ala Ser 275 56276PRTHomo sapiens 56Arg Ile Leu Pro Asp
Ala Phe Tyr Arg Asn Ser Leu Leu Phe Leu His 1 5 10 15 Thr Arg Phe
Phe Gly Trp Ser Glu Thr Met Lys Tyr Leu Leu Pro Thr 20 25 30 Ala
Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Asp 35 40
45 Ile Val Met Thr Gln Ala Ala Pro Ser Val Ser Val Thr Pro Gly Glu
50 55 60 Ser Val Ser Ile Ser Cys Arg Ser Thr Lys Ser Leu Leu Arg
Ser Asn 65 70 75 80 Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro
Gly Gln Ser Pro 85 90 95 Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu
Ala Ser Gly Val Pro Asp 100 105 110 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ala Phe Thr Leu Arg Ile Ser 115 120 125 Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Met Gln His Leu 130 135 140 Glu Tyr Pro Phe
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 145 150 155 160 Ala
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 165 170
175 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
180 185 190 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
Arg Gln 195 200 205 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser
Lys Asp Ser Thr 210 215 220 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr
Lys Asp Glu Tyr Glu Arg 225 230 235 240 His Asn Ser Tyr Thr Cys Glu
Ala Thr His Lys Thr Ser Thr Ser Pro 245 250 255 Ile Val Lys Ser Phe
Asn Arg Asn Glu Ser Tyr Pro Tyr Asp Val Pro 260 265 270 Asp Tyr Ala
Ser 275 57276PRTHomo sapiens 57Arg Ile Leu Pro Asp Ala Phe Tyr Arg
Asn Ser Leu Leu Phe Leu His 1 5 10 15 Thr Arg Phe Phe Gly Trp Ser
Glu Thr Met Lys Tyr Leu Leu Pro Thr 20 25 30 Ala Ala Ala Gly Leu
Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Asp 35 40 45 Ile Val Met
Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly Glu 50 55 60 Ser
Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Arg Ser Asn 65 70
75 80 Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser
Pro 85 90 95 Gln Leu Leu Ile Tyr Arg Leu Ser Asn Leu Ala Ser Gly
Val Pro Asp 100 105 110 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
Thr Leu Arg Ile Ser 115 120 125 Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln His Leu 130 135 140 Glu Tyr Pro Phe Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys Arg 145 150 155 160 Ala Asp Ala Ala
Pro Thr Val Ser Ile Phe Pro Gln Tyr Ser Glu Gln 165 170 175 Leu Thr
Thr Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 180 185 190
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 195
200 205 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
Thr 210 215 220 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu
Tyr Glu Arg 225 230 235 240 His Asn Ser Tyr Thr Cys Glu Ala Thr His
Lys Thr Ser Thr Ser Pro 245 250 255 Ile Val Lys Ser Phe Asn Arg Asn
Glu Ser Tyr Pro Tyr Asp Val Pro 260 265 270 Asp Tyr Ala Ser 275
58199PRTHomo sapiens 58Arg Ile Leu Pro Asp Ala Phe Tyr Arg Asn Ser
Leu Leu Phe Leu His 1 5 10 15 Thr Arg Phe Phe Gly Trp Ser Glu Thr
Met Lys Tyr Leu Leu Pro Thr 20 25 30 Ala Ala Ala Gly Leu Leu Leu
Leu Ala Ala Gln Pro Ala Met Ala Asp 35 40 45 Ile Val Met Thr Gln
Ala Ala Pro Ser Val Pro Val Thr Pro Gly Glu 50 55 60 Ser Val Ser
Ile Ser Cys Thr Ser Ser Lys Ser Leu Leu Arg Ser Asn 65 70 75 80 Gly
Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro 85 90
95 Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp
100 105 110 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg
Ile Ser 115 120 125 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln His Leu 130 135 140 Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr
Asn Leu Glu Ile Lys Arg 145 150 155 160 Ala Asp Ala Ala Pro Thr Val
Ser Ile Phe Thr Thr Ser Arg Glu Gln 165 170 175 Leu Thr Ser Gly Gly
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 180 185 190 Pro Lys Asp
Ile Asn Val Lys 195 59783DNAHomo sapiens
59tgactgggaa aaccctggcg ttacccacgc tttgtacatg gagaaaataa agtgaaacaa
60agcactattg cactggcact cttaccgctc ttatttaccc ctgtggcaaa agccgaagtg
120cagctgttgg agactggggg aggcgtagtg aagcccggag ggtcccttaa
actctcctgt 180gcagcctctg gattcacttt cagtaactat ggcatgtctt
gggttcgcca gactccggag 240aagaggctgg agtgggtcgc agccattaat
cgtgatggtg gtaccaccta ctatacagac 300aatgtgaagg gccgattcac
catctccaga gacaatgcca agaacagcct gtacctgcaa 360atgagcagtc
tgaggtctga ggacacagcc ttgtattact gtgcaagaca gttccttctc
420tgggacggct ggtacttcga tgtctggggc gcagggacca cggtcaccgt
ctcctcagcc 480aaaacgacac ccccatctgt ctatccactg gcccctggat
ctgctgccca aactaactcc 540atggtgaccc tgggatgcct ggtcaagggc
tatttccctg agccagtgac agtgacctgg 600aactctggat ccctgtccag
cggtgtgcac accttcccag ctgtcctgca gtctgacctc 660tacactctga
gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc
720tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt
gcccagggat 780tgt 78360777DNAHomo sapiens 60tgactgggaa aaccctggcg
ttacccacgc tttgtacatg gagaaaataa agtgaaacaa 60agcactattg cactggcact
cttaccgctc ttatttaccc ctgtggcaaa agcccaggtt 120cagctgcaac
agtctgacgc tgagttggtg aaacctggag cttcagtgaa gatatcctgc
180aaggtttctg gctacacctt cagtgaccat gctattcact ggatgagtca
gagacctgga 240cagggcctga aatggattgg atatatttat cctagacatg
ggactactaa ctacaatgag 300aacttcaagg gcaaggccac actgactgca
gacacatcct ccagcacagc ctacatgcag 360ctcaacagcc tgacatctga
agattctgcc gtctatttct gtgcaagaat gagaaactac 420ttctatgtta
tggactactg gggtcaagga acctcagtca ccgtctcctc agccaaaacg
480acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa
ctccatggtg 540accctgggat gcctggtcaa gggctatttc cctgagccag
tgacagtgac ctggaactct 600ggatccctgt ccagcggtgt gcacaccttc
ccagctgtcc tgcagtctga cctctacact 660ctgagcagct cagtgactgt
cccctccagc acctggccca gcgagaccgt cacctgcaac 720gttgcccacc
cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgt 77761777DNAHomo
sapiens 61tgactgggaa aaccctggcg ttacccacgc tttgtacatg gagaaaataa
agtgaaacaa 60agcactattg cactggcact cttaccgctc ttatttaccc ctgtggcaaa
agcccaggtt 120ctgctgcaac agtctgacgc tgagttggtg aaacctgggg
cttcagtgaa gatatcctgc 180aaggcttctg gctacacctt cactgaccat
gctattcact gggtgaagca gaggcctgaa 240cagggcctgg aatggattgg
atatatttat cctgaacatg gaactattaa gtataatgag 300aagttcaagg
gcaaggccac attgactgca gataaatcct ccagcactgc ctatatgcag
360ctcaacagcc tgacatctga ggattcagca gtgtatttct gttcaagact
cactaactac 420ttctatgtta tggagtattg gggtcaagga acctcagtca
ccgtctcctc agccaaaacg 480acacccccat ctgtctatcc actggcccct
ggatctgctg cccaaactaa ctccatggtg 540accctgggat gcctggtcaa
gggctatttc cctgagccag tgacagtgac ctggaactct 600ggatccctgt
ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact
660ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt
cacctgcaac 720gttgcccacc cggccagcag caccaaggtg gacaagaaaa
ttgtgcccag ggattgt 77762789DNAHomo sapiens 62tgactgggaa aaccctggcg
ttacccacgc tttgtacatg gagaaaataa agtgaaacaa 60agcactattg cactggcact
cttaccgctc ttatttaccc ctgtggcaaa agccgaggtt 120cagctgcagc
agtctgtcgc tgagttggtg aaacctggag cttcagtgaa gatgtcatgc
180aaggtttctg gctacaccct cactgaccat actattcact ggatgaagca
gaggcctgaa 240cagggcctgg aatggattgg atatatttac cctagagatg
gaataactgg gtacaatgag 300aagttcaagg gcaaggccac actgactgca
gacacttctt ccagcacagc ctacatgcag 360ctcaacagcc tgacatctga
ggattctgca gtctatttct gtgccagatg gggctatagt 420tacaggaatt
acgcgtacta ctatgactac tggggccaag gcaccactct cacagtctcc
480tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc
tgcccaaact 540aactccatgg tgaccctggg atgcctggtc aagggctatt
tccctgagcc agtgacagtg 600acctggaact ctggatccct gtccagcggt
gtgcacacct tcccagctgt cctgcagtct 660gacctctaca ctctgagcag
ctcagtgact gtcccctcca gcacctggcc cagcgagacc 720gtcacctgca
acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 780agggattgt
78963777DNAHomo sapiens 63tgactgggaa aaccctggcg ttacccacgc
tttgtacatg gagaaaataa agtgaaacaa 60agcactattg cactggcact cttaccgctc
ttatttaccc ctgtggcaaa agcccaggtt 120cagctgcaac agtctgacgc
tgacttggtg aaacctgggg cttcagtgaa gatatcctgc 180aaggcttctg
gctacacctt cactgaccat gctattcact gggtgaaaca gaggcctgaa
240cagggcctgg aatggattgg atatatttat cctgaacatg gaactattaa
gtataatgag 300aagttcaagg gcaaggccac attgactgca gataaatcct
ccagcactgc ctatatgcag 360ctcaacagcc tgacatctga ggattcagca
gtgtatttct gtgcaagact caggaactat 420ttgtatatta tggactactg
gggtcaagga acctcagtca ccgtctcctc agccaaaacg 480acacccccat
ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg
540accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac
ctggaactct 600ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc
tgcagtctga cctctacact 660ctgagcagct cagtgactgt cccctccagc
acctggccca gcgagaccgt cacctgcaac 720gttgcccacc cggccagcag
caccaaggtg gacaagaaaa ttgtgcccag ggattgt 77764777DNAHomo sapiens
64tgactgggaa aaccctggcg ttacccacgc tttgtacatg gagaaaataa agtgaaacaa
60agcactattg cactggcact cttaccgctc ttatttaccc ctgtggcaaa agcccaggtt
120cagctgcaac agtctgacgc tgagttggtg aaacctgggg cttcagtgaa
gatatcctgc 180aaggcttctg gctacacctt cactgaccat gctattcact
gggtgaagca gaggcctgaa 240cagggcctgg aatggattgg atatatttat
cctgaacatg gaactattaa gtataatgag 300aagttcaagg gcaaggccac
attgactgca gataaatcct ccagcactgc ctatatgcag 360ctcaacagcc
tgacatctga ggattcagca gtgtatttct gttcaagact cactaactac
420ttctatgtta tggagtattg gggtcaagga acctcagtca ccgtctcctc
agccaaaacg 480acacccccat ctgtctatcc actggcccct ggatctgctg
cccaaactaa ctccatggtg 540accctgggat gcctggtcaa gggctatttc
cctgagccag tgacagtgac ctggaactct 600ggatccctgt ccagcggtgt
gcacaccttc ccagctgtcc tgcagtctga cctctacact 660ctgagcagct
cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac
720gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgt
77765777DNAHomo sapiens 65tgactgggaa aaccctggcg ttacccacgc
tttgtacatg gagaaaataa agtgaaacaa 60agcactattg cactggcact cttaccgctc
ttatttaccc ctgtggcaaa agcccaggtt 120cagctgcaac agtctgacgc
tgagttggtg aaacctggag cctcagtgaa gatatcctgc 180aaggtttctg
gctacacctt cactgaccat gctattcact ggatgaaaca gaggcctgaa
240cagggcctgg aatggattgg atatatttat cctagagatg gttttactaa
gtacaatgag 300aagttcaagg gcaaggccac actgactgca gacacatcct
ccagcacagc ctacatgcag 360ctcaacagcc tgacatctga ggattctaca
gtctatttct gtgcaagaat gactaactac 420ttctatacta tggactactg
gggtcaagga acctcagtca ccgtctcctc agccaaaacg 480acacccccat
ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg
540accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac
ctggaactct 600ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc
tgcagtctga cctctacact 660ctgagcagct cagtgactgt cccctccagc
acctggccca gcgagaccgt cacctgcaac 720gttgcccacc cggccagcag
caccaaggtg gacaagaaaa ttgtgcccag ggattgt 77766777DNAHomo sapiens
66tgactgggaa aaccctggcg ttacccacgc tttgtacatg gagaaaataa agtgaaacaa
60agcactattg cactggcact cttaccgctc ttatttaccc ctgtggcaaa agcccaggtt
120cagctgcaac agtctgacgc tgacttggtg aaacctgggg cttcagtgaa
gatatcctgc 180aaggcttctg gctacacctt cactgaccat gctattcact
gggtgaaaca gaggcctgaa 240cagggcctgg aatggattgg atatatttat
cctgaacatg gaactattaa gtataatgag 300aagttcaagg gcaaggccac
attgactgca gataaatcct ccagcactgc ctatatgcag 360ctcaacagcc
tgacatctga ggattcagca gtgtatttct gttcaagact cactaactac
420ttctatgtta tggagtattg gggtcaagga acctcagtca ccgtctcctc
agccaaaacg 480acacccccat ctgtctatcc actggcccct ggatctgctg
cccaaactaa ctccatggtg 540accctgggat gcctggtcaa gggctatttc
cctgagccag tgacagtgac ctggaactct 600ggatccctgt ccagcggtgt
gcacaccttc ccagctgtcc tgcagtctga cctctacact 660ctgagcagct
cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac
720gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgt
77767777DNAHomo sapiens 67tgactgggaa aaccctggcg ttacccacgc
tttgtacatg gagaaaataa agtgaaacaa 60agcactattg cactggcact cttaccgctc
ttatttaccc ctgtggcaaa agcccaggtt 120cagctgcaac agtctgacgc
tgagttggtg aaacctgggg cttcagtgaa gatatcctgc 180aaggcttctg
gctacacctt cactgaccat gctattcact gggtgaagca gaggcctgaa
240cagggcctgg aatggattgg atatatttat cctgaacatg gtagtattac
gtataatgag 300aagttcaagg gcaaggccac attgactgca gataaatcct
ccagtactgt ctatatgcac 360ctcaatagcc tgacatctga ggattcagca
gtgtatttct gtgcaagact caggaactac 420ttgtatgtta tggactactg
gggtcaagga acctcagtca ccgtctcctc agccaaaacg 480acacccccat
ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg
540accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac
ctggaactct 600ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc
tgcagtctga cctctacact 660ctgagcagct cagtgactgt cccctccagc
acctggccca gcgagaccgt cacctgcaac 720gttgcccacc cggccagcag
caccaaggtg gacaagaaaa ttgtgcccag ggattgt 77768777DNAHomo sapiens
68tgactgggaa aaccctggcg ttacccacgc tttgtacatg gagaaaataa agtgaaacaa
60agcactattg cactggcact cttaccgctc ttatttaccc ctgtggcaaa agcccaggtt
120cagctgcaac agtctgaggc tgagcttgtg aagcctgggg cttcagtgaa
gctgtcctgc 180aaggcttctg gctacacctt cactgaccat gctattcact
ggatgaaaca gaggcctgaa 240cagggcctgg aatggattgg atatatctac
cccagagatg attttgctaa ggtgaatgag 300aagttcaagg gcaaggccac
actgacagca gacacatcct ccagcacagc ctacatgcag 360ctcaacagcc
tgacatctga ggattctgca gtctatttct gtgcaagaat gactaactac
420ctctatatta tggactactg gggtcaagga acctcagtca ccgtctcctc
agccaaaacg 480acacccccat ctgtctatcc actggcccct ggatctgctg
cccaaactaa ctccatggtg 540accctgggat gcctggtcaa gggctatttc
cctgagccag tgacagtgac ctggaactct 600ggatccctgt ccagcggtgt
gcacaccttc ccagctgtcc tgcagtctga cctctacact 660ctgagcagct
cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac
720gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgt
77769777DNAHomo sapiens 69tgactgggaa aaccctggcg ttacccacgc
tttgtacatg gagaaaataa agtgaaacaa 60agcactattg cactggcact cttaccgctc
ttatttaccc ctgtggcaaa agcccaggtt 120cagctgcaac agtctgacgc
tgagttggtg aaacctgggg cttcagtgaa gatatcctgc 180aaggcttctg
gctacacctt cactgaccat gctattcact gggtgaagca gaggcctgaa
240cagggcctgg aatggattgg atatatttat cctgaacatg gtactattac
gtataatgag 300aagttcaagg gcaaggccac attgactgca gataaatcct
ccagtactgt ctatatgcac 360ctcaatagcc tgacatctga ggattcagca
gtgtatttct gtgcaagact caggaactat 420ttgtatatta tggactactg
gggtcaagga acctcagtca ccgtctcctc agccaaaacg 480acacccccat
ctgtctatcc actggccccc ggatctgctg cccaaactaa ctccatggtg
540accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac
ctggaactct 600ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc
tgcagtctga cctctacact 660ctgagcagct cagtgactgt cccctccagc
acctggccca gcgagaccgt cacctgcaac 720gttgcccacc cggccagcag
caccaaggtg gacaagaaaa ttgtgcccag ggattgt 77770777DNAHomo sapiens
70tgactgggaa aaccctggcg ttacccacgc tttgtacatg gagaaaataa agtgaaacaa
60agcactattg cactggcact cttaccgctc ttatttaccc ctgtggcaaa agcccaggtt
120cagctgcaac agtctgacgc cgcgttggtg aaacctggag cttcagtgaa
gatatcgtgc 180aaggtttctg gctacacctt cagtgaccat gctattcact
ggatgaagca gaggcctgaa 240cagggcctgg aatggattgg atatattttt
cctagagatg cttttagttt gaacaatgag 300aagttcaagg gcaaggccac
actgagtgca gacacatcct ccagcacagc ctacatggag 360ctcaccagcc
tgacatttga ggattctgca gtctatttct gtgcaagaat gagaaactac
420ttctatgtta tggactactg gggtcaagga acctcagtca ccgtctcctc
agccaaaacg 480acacccccat ctgtctatac actggcccct ggatctgctg
cccaaactaa ctccatggtg 540accctgggat gcctggtcaa gggctatttc
cctgagccag tgacagtgac ctggaactct 600ggatccctgt ccagcggtgt
gcacaccttc ccagctgtcc tgcagtctga cctctacact 660ctgagcagct
cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac
720gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgt
77771837DNAHomo sapiens 71taagattagc ggatcctacc tgacgctttt
tatcgcaact ctctactgtt tctccatacc 60cgtttttttg gatggagtga aacgatgaaa
tacctattgc ctacggcagc cgctggattg 120ttattactcg ctgcccaacc
agccatggcc gatgttgtgc tgacccagac tccactctcc 180ctgcctgtca
ctcttggaga tcaagcctcc atctcttgca gatctagtca gagcctttta
240cacagtaatg gaaacaccta tttacattgg tacctgctga agccaggcca
gtctccaaag 300ctcctgatct acaaagtttc caaccgattt tctggggtcc
cagacaggtt cagtggcagt 360ggatcaggga cagatttcac actcaagatc
accagagtgg aggctgagga tctgggagtt 420tatttctgct ctcaaagtac
acatgtgctc acgttcggtg ctgggaccaa gctggagctg 480aaacgggctg
atgctgcacc aactgtatcc atcttcccac catccagtga gcagttaaca
540tctggaggtg cctcagtcgt gtgcttcttg aacaacttct accccaaaga
catcaatgtc 600aagtggaaga ttgatggcag tgaacgacaa aatggcgtcc
tgaacagttg gactgatcag 660gacagcaaag acagcaccta cagcatgagc
agcaccctca cgttgaccaa ggacgagtat 720gaacgacata acagctatac
ctgtgaggcc actcacaaga catcaacttc acccattgtc 780aagagcttca
acaggaatga gtcttatcca tatgatgtgc cagattatgc gagctaa 83772840DNAHomo
sapiens 72taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt
tctccatacc 60cgtttttttg gatggagtga aacgatgaaa tacctattgc ctacggcagc
cgctggattg 120ttattactcg ctgcccaacc agccatggcc gatattgtga
tgacccaggc tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc
atctcctgca cgtctagtaa gagtctcctg 240cgtagtaatg gcaacactta
cttgtattgg ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat
atcggatgtc caaccttgcc tcgggagtcc cagacaggtt cagtggcagt
360gggtcaggaa ctgctttcac actgagaatc agtagagtgg aggctgagga
tgtgggtgtt 420tattactgta tgcaacatct agaatatcct ttcacgttcg
gctcggggac aaagttggaa 480ataaaacggg ctgatgctgc accaactgta
tccatcttcc caccatccag tgagcagtta 540acatctggag gtgcctcagt
cgtgtgcttc ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga
agattgatgg cagtgaacga caaaatggcg tcctgaacag ttggactgat
660caggacagca aagacagcac ctacagcatg agcagcaccc tcacgttgac
caaggacgag 720tatgaacgac ataacagcta tacctgtgag gccactcaca
agacatcaac ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat
ccatatgatg tgccagatta tgcgagctaa 84073843DNAHomo sapiens
73taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc
60cgtttttttg gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gacatcgtta tgtctcagtc
tccatcctcc 180ctagctgtgt cagttggaga gaaggttact atgagctgca
agtccagcca gagcctttta 240catagtagca atcaaaagaa ctacttggcc
tggtaccagc agaaaccagg gcagtctcct 300aaagtgctga tttactgggc
atccactaga gaatctgggg tccctgatcg cttcacaggc 360agtggatctg
ggacagattt cactctcacc atcaccagtg tgaagtctga agacctggca
420gtttattact gtcagcaata ttatagctat ccgtggacgt tcggtggcgg
caccaggctg 480gaaatcaaac gggctgatgc tgcaccaact gtatccatct
tcccaccatc cagtgagcag 540ttaacatctg gaggtgcctc agtcgtgtgc
ttcttgaaca acttctaccc caaagacatc 600aatgtcaagt ggaagattga
tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact 660gatcaggaca
gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac
720gagtatgaac gacataacag ctatacctgt gaggccactc acaagacatc
aacttcaccc 780attgtcaaga gcttcaacag gaatgagtct tatccatatg
atgtgccaga ttatgcgagc 840taa 84374840DNAHomo sapiens 74taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc atctcctgca
ggtctagtaa gagtctcctg 240cgcagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggctgtc
caaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtctggaa
ctgctttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacattcg gctcggggac
aaagttggaa 480ataaaacggg ctgatgctgc accaactgta tccatcttcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84075840DNAHomo sapiens 75taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgataaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc atctcctgca
cgtctagtaa gagtctcctg 240cgtagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggatgtc
caaccttgcc tcgggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgctttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacgttcg gctcggggac
aaagttggaa 480ataaaacggg ctgatgctgc accaactgta tccatcctcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84076840DNAHomo sapiens 76taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtttcc atctcctgca
ggtcttctaa gagtctcctg 240cgtactaatg gcaacactta cttgcattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggatgtc
caaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgttttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcca ttcacgttcg gctcggggac
aaagttggaa 480ataaaaaggg ctgatgctgc accaactgta tccatcttcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84077840DNAHomo sapiens 77taagattagc
ggatcctacc ttacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga atcagtatcc atctcctgca
ggtctagtaa gagtctcctg 240cgtagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggctgtc
taaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgctttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacattcg gctcggggac
aaagttggaa 480ataaaacggg ctgatgctgc accaactgta tccatcttcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84078840DNAHomo sapiens 78taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc atctcctgca
cgtctagtaa gagtctcctg 240cgtagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggatgtc
caaccttgcc tcgggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgctttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacgttcg gctcggggac
aaagttggaa 480ataaaacggg ctgatgctgc accaactgta tccatctccc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84079840DNAHomo sapiens 79taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc atctcctgca
ggtccagtaa gagtctcctg 240cgtagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctcatat atcggatgtc
caaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgccttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacgttcg gaggggggac
caagctggaa 480ataaaacggg ctgatgctgc accaactgta tccatcttcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84080840DNAHomo sapiens 80taagattagc
ggatcctacc tgacgctttt tatcgcaact ctcttctgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtatctgtca ctcctggaga gtcagtatcc atctcctgca
ggtctactaa gagtctcctg 240cgtagtaatg gcaacactta cttgtattgg
ttcctccaga ggccaggcca gtctcctcag 300ctcctgatat atcggatgtc
caaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgctttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacgttcg gaggggggac
caagctggaa 480ataaaacggg ctgatgctgc accaactgta tccatcttcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84081840DNAHomo sapiens 81taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc atctcctgca
ggtctagtaa gagtctccta 240cgtagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggatgtc
caaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgccttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacgttcg gaggggggac
caagctggaa 480ataaaacggg ctgatgctgc accaactgta tccatcttcc
caccatccag tgagcagtta 540acatctggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84082840DNAHomo sapiens 82taagattagc
ggatcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc 60cgtttttttg
gatggagtga aacgatgaaa tacctattgc ctacggcagc cgctggattg
120ttattactcg ctgcccaacc agccatggcc gatattgtga tgacccaggc
tgcaccctct 180gtacctgtca ctcctggaga gtcagtatcc atctcctgca
ggtctagtaa gagtctcctg 240cgcagtaatg gcaacactta cttgtattgg
ttcctgcaga ggccaggcca gtctcctcag 300ctcctgatat atcggctgtc
caaccttgcc tcaggagtcc cagacaggtt cagtggcagt 360gggtcaggaa
ctgctttcac actgagaatc agtagagtgg aggctgagga tgtgggtgtt
420tattactgta tgcaacatct agaatatcct ttcacattcg gctcggggac
aaagttggaa 480ataaaacggg ctgatgctgc accaactgta tccatcttcc
cacaatacag tgagcagtta 540acaactggag gtgcctcagt cgtgtgcttc
ttgaacaact tctaccccaa agacatcaat 600gtcaagtgga agattgatgg
cagtgaacga caaaatggcg tcctgaacag ttggactgat 660caggacagca
aagacagcac ctacagcatg agcagcaccc tcacgttgac caaggacgag
720tatgaacgac ataacagcta tacctgtgag gccactcaca agacatcaac
ttcacccatt 780gtcaagagct tcaacaggaa tgagtcttat ccatatgatg
tgccagatta tgcgagctaa 84083597DNAHomo sapiens 83cggatcctac
ctgacgcttt ttatcgcaac tctctactgt ttctccatac ccgttttttt 60ggatggagtg
aaacgatgaa atacctattg cctacggcag ccgctggatt gttattactc
120gctgcccaac cagccatggc cgatattgtg atgacccagg ctgcaccctc
tgtacctgtc 180actcctggag agtcagtatc catctcctgc acgtctagta
agagtctcct gcgtagtaat 240ggcaacactt acttgtattg gttcctgcag
aggccaggcc agtctcctca gctcctgata 300tatcggatgt ccaaccttgc
ctcgggagtc ccagacaggt tcagtggcag tgggtcagga 360actgctttca
cactgagaat cagtagagtg gaggctgagg atgtgggtgt ttattactgt
420atgcaacatc tagaatatcc tttcacgttc ggctcgggga caaatttgga
aataaaacgg 480gctgatgctg caccaactgt atccatcttc acaacatcca
gagagcagtt aacatctgga 540ggtgcctcag tcgtgtgctt cttgaacaac
ttctacccca aagacatcaa tgtcaag 5978430DNAHomo sapiens 84ggattcactt
tcagtaacta tggcatgtct 308530DNAHomo sapiens 85ggctacacct tcagtgacca
tgctattcac 308630DNAHomo sapiens 86ggctacacct tcactgacca tgctattcac
308730DNAHomo sapiens 87ggctacaccc tcactgacca tactattcac
308851DNAHomo sapiens 88gccattaatc gtgatggtgg taccacctac tatacagaca
atgtgaaggg c 518951DNAHomo sapiens 89tatatttatc ctagacatgg
gactactaac tacaatgaga acttcaaggg c 519051DNAHomo sapiens
90tatatttatc ctgaacatgg aactattaag tataatgaga agttcaaggg c
519151DNAHomo sapiens 91tatatttacc ctagagatgg aataactggg tacaatgaga
agttcaaggg c 519251DNAHomo sapiens 92tatatttatc ctagagatgg
ttttactaag tacaatgaga agttcaaggg c 519351DNAHomo sapiens
93tatatttatc ctgaacatgg tagtattacg tataatgaga agttcaaggg c
519451DNAHomo sapiens 94tatatctacc ccagagatga ttttgctaag gtgaatgaga
agttcaaggg c 519551DNAHomo sapiens 95tatatttatc ctgaacatgg
tactattacg tataatgaga agttcaaggg c 519651DNAHomo sapiens
96tatatttttc ctagagatgc ttttagtttg aacaatgaga agttcaaggg c
519733DNAHomo sapiens 97ttccttctct gggacggctg gtacttcgat gtc
339827DNAHomo sapiens 98agaaactact tctatgttat ggactac 279927DNAHomo
sapiens 99actaactact tctatgttat ggagtat 2710039DNAHomo sapiens
100ggctatagtt acaggaatta cgcgtactac tatgactac 3910127DNAHomo
sapiens 101aggaactatt tgtatattat ggactac 2710227DNAHomo sapiens
102actaactact tctatactat ggactac 2710327DNAHomo sapiens
103aggaactact tgtatgttat ggactac 2710427DNAHomo sapiens
104actaactacc tctatattat ggactac 2710548DNAHomo sapiens
105agatctagtc agagcctttt acacagtaat ggaaacacct atttacat
4810648DNAHomo sapiens 106acgtctagta agagtctcct gcgtagtaat
ggcaacactt acttgtat 4810751DNAHomo sapiens 107aagtccagcc agagcctttt
acatagtagc aatcaaaaga actacttggc c 5110848DNAHomo sapiens
108aggtctagta agagtctcct gcgcagtaat ggcaacactt acttgtat
4810948DNAHomo sapiens 109aggtcttcta agagtctcct gcgtactaat
ggcaacactt acttgcat 4811048DNAHomo sapiens 110aggtctagta agagtctcct
gcgtagtaat ggcaacactt acttgtat 4811148DNAHomo sapiens 111aggtccagta
agagtctcct gcgtagtaat ggcaacactt acttgtat 4811248DNAHomo sapiens
112aggtctacta agagtctcct gcgtagtaat ggcaacactt acttgtat
4811348DNAHomo sapiens 113aggtctagta agagtctcct acgtagtaat
ggcaacactt acttgtat 4811421DNAHomo sapiens 114aaagtttcca accgattttc
t 2111521DNAHomo sapiens 115cggatgtcca accttgcctc g 2111621DNAHomo
sapiens 116tgggcatcca ctagagaatc t 2111721DNAHomo sapiens
117cggctgtcca accttgcctc a 2111821DNAHomo sapiens 118cggatgtcca
accttgcctc a 2111921DNAHomo sapiens 119cggctgtcta accttgcctc a
2112024DNAHomo sapiens 120tctcaaagta cacatgtgct cacg 2412127DNAHomo
sapiens 121atgcaacatc tagaatatcc tttcacg 2712227DNAHomo sapiens
122cagcaatatt atagctatcc gtggacg 2712327DNAHomo sapiens
123atgcaacatc tagaatatcc tttcaca 2712427DNAHomo sapiens
124atgcaacatc tagaatatcc attcacg 2712530DNAHomo sapiens
125ggattcactt tcagtagcta tggcatgtct 3012630DNAHomo sapiens
126ggctacacct tcactgacca tactattcac 3012751DNAHomo sapiens
127tatatttatc ctagagatgg tagtactaag tacaatgaga agttcaaggg c
5112848DNAHomo sapiens 128agatctagtc agagccttgt acacagtaat
ggaaacacct atttacat 4812924DNAHomo sapiens 129tctcaaagta cacatgttcc
tccc 2413051DNAHomo sapiens 130aagtccagtc agagcctttt atatagtagc
aatcaaaaga actacttggc c 5113121DNAHomo sapiens 131tgggcatcca
ctagggaatc t 2113227DNAHomo sapiens 132cagcaatatt atagctatcc
tcccaca 2713348DNAHomo sapiens 133aggtctagta agagtctcct gcatagtaat
ggcaacactt acttgtat 4813421DNAHomo sapiens 134cggatgtcca accttgcctc
a 2113527DNAHomo sapiens 135atgcaacatc tagaatatcc tttcaca
27136222PRTHomo sapiens 136Gln Glu Gly Lys Phe Ser Gly Pro Leu Lys
Pro Met Thr Phe Ser Ile 1 5 10 15 Tyr Glu Gly Gln Glu Pro Ser Gln
Ile Ile Phe Gln Phe Lys Ala Asn 20 25 30 Pro Pro Ala Val Thr Phe
Glu Leu Thr Gly Glu Thr Asp Asn Ile Phe 35 40 45 Val Ile Glu Arg
Glu Gly Leu Leu Tyr Tyr Asn Arg Ala Leu Asp Arg 50 55 60 Glu Thr
Arg Ser Thr His Asn Leu Gln Val Ala Ala Leu Asp Ala Asn 65 70 75 80
Gly Ile Ile Val Glu Gly Pro Val Pro Ile Thr Ile Lys Val Lys Asp 85
90 95 Ile Asn Asp Asn Arg Pro Thr Phe Leu Gln Ser Lys Tyr Glu Gly
Ser 100 105 110 Val Arg Gln Asn Ser Arg Pro Gly Lys Pro Phe Leu Tyr
Val Asn Ala 115 120 125 Thr Asp Leu Asp Asp Pro Ala Thr Pro Asn Gly
Gln Leu Tyr Tyr Gln 130 135 140 Ile Val Ile Gln Leu Pro Met Ile Asn
Asn Val Met Tyr Phe Gln Ile 145 150 155 160 Asn Asn Lys Thr Gly Ala
Ile Ser Leu Thr Arg Glu Gly Ser Gln Glu 165 170 175 Leu Asn Pro Ala
Lys Asn Pro Ser Tyr Asn Leu Val Ile Ser Val Lys 180 185 190 Asp Met
Gly Gly Gln Ser Glu Asn Ser Phe Ser Asp Thr Thr Ser Val 195 200 205
Asp Ile Ile Val Thr Glu Asn Ile Trp Lys Ala Pro Lys Pro 210 215 220
137765PRTHomo sapiens 137Gln Glu Gly Lys Phe Ser Gly Pro Leu Lys
Pro Met Thr Phe Ser Ile 1 5 10 15 Tyr Glu Gly Gln Glu Pro Ser Gln
Ile Ile Phe Gln Phe Lys Ala Asn 20 25 30 Pro Pro Ala Val Thr Phe
Glu Leu Thr Gly Glu Thr Asp Asn Ile Phe 35 40 45 Val Ile Glu Arg
Glu Gly Leu Leu Tyr Tyr Asn Arg Ala Leu Asp Arg 50 55 60 Glu Thr
Arg Ser Thr His Asn Leu Gln Val Ala Ala Leu Asp Ala Asn 65 70 75 80
Gly Ile Ile Val Glu Gly Pro Val Pro Ile Thr Ile Lys Val Lys Asp 85
90 95 Ile Asn Asp Asn Arg Pro Thr Phe Leu Gln Ser Lys Tyr Glu Gly
Ser 100 105 110 Val Arg Gln Asn Ser Arg Pro Gly Lys Pro Phe Leu Tyr
Val Asn Ala 115 120 125 Thr Asp Leu Asp Asp Pro Ala Thr Pro Asn Gly
Gln Leu Tyr Tyr Gln 130 135 140 Ile Val Ile Gln Leu Pro Met Ile Asn
Asn Val Met Tyr Phe Gln Ile 145 150 155 160 Asn Asn Lys Thr Gly Ala
Ile Ser Leu Thr Arg Glu Gly Ser Gln Glu 165 170 175 Leu Asn Pro Ala
Lys Asn Pro Ser Tyr Asn Leu Val Ile Ser Val Lys 180 185 190 Asp Met
Gly Gly Gln Ser Glu Asn Ser Phe Ser Asp Thr Thr Ser Val 195 200 205
Asp Ile Ile Val Thr Glu Asn Ile Trp Lys Ala Pro Lys Pro Val Glu 210
215 220 Met Val Glu Asn Ser Thr Asp Pro His Pro Ile Lys Ile Thr Gln
Val 225 230 235 240 Arg Trp Asn Asp Pro Gly Ala Gln Tyr Ser Leu Val
Asp Lys Glu Lys 245 250 255 Leu Pro Arg Phe Pro Phe Ser Ile Asp Gln
Glu Gly Asp Ile Tyr Val 260 265 270 Thr Gln Pro Leu Asp Arg Glu Glu
Lys Asp Ala Tyr Val Phe Tyr Ala 275 280 285 Val Ala Lys Asp Glu Tyr
Gly Lys Pro Leu Ser Tyr Pro Leu Glu Ile 290 295 300 His Val Lys Val
Lys Asp Ile Asn Asp Asn Pro Pro Thr Cys Pro Ser 305 310 315 320 Pro
Val Thr Val Phe Glu Val Gln Glu Asn Glu Arg Leu Gly Asn Ser 325 330
335 Ile Gly Thr Leu Thr Ala His Asp Arg Asp Glu Glu Asn Thr Ala Asn
340 345 350 Ser Phe Leu Asn Tyr Arg Ile Val Glu Gln Thr Pro Lys Leu
Pro Met 355 360 365 Asp Gly Leu Phe Leu Ile Gln Thr Tyr Ala Gly Met
Leu Gln Leu Ala 370 375 380 Lys Gln Ser Leu Lys Lys Gln Asp Thr Pro
Gln Tyr Asn Leu Thr Ile 385 390 395 400 Glu Val Ser Asp Lys Asp Phe
Lys Thr Leu Cys Phe Val Gln Ile Asn 405 410 415 Val Ile Asp Ile Asn
Asp Gln Ile Pro Ile Phe Glu Lys Ser Asp Tyr 420 425 430 Gly Asn Leu
Thr Leu Ala Glu Asp Thr Asn Ile Gly Ser Thr Ile Leu 435 440 445 Thr
Ile Gln Ala Thr Asp Ala Asp Glu Pro Phe Thr Gly Ser Ser Lys 450 455
460 Ile Leu Tyr His Ile Ile Lys Gly Asp Ser Glu Gly Arg Leu Gly Val
465 470 475 480 Asp Thr Asp Pro His Thr Asn Thr Gly Tyr Val Ile Ile
Lys Lys Pro 485 490 495 Leu Asp Phe Glu Thr Ala Ala Val Ser Asn Ile
Val Phe Lys Ala Glu 500 505 510 Asn Pro Glu Pro Leu Val Phe Gly Val
Lys Tyr Asn Ala Ser Ser Phe 515 520 525 Ala Lys Phe Thr Leu Ile Val
Thr Asp Val Asn Glu Ala Pro Gln Phe 530 535 540 Ser Gln His Val Phe
Gln Ala Lys Val Ser Glu Asp Val Ala Ile Gly 545 550 555 560 Thr Lys
Val Gly Asn Val Thr Ala Lys Asp Pro Glu Gly Leu Asp Ile 565 570 575
Ser Tyr
Ser Leu Arg Gly Asp Thr Arg Gly Trp Leu Lys Ile Asp His 580 585 590
Val Thr Gly Glu Ile Phe Ser Val Ala Pro Leu Asp Arg Glu Ala Gly 595
600 605 Ser Pro Tyr Arg Val Gln Val Val Ala Thr Glu Val Gly Gly Ser
Ser 610 615 620 Leu Ser Ser Val Ser Glu Phe His Leu Ile Leu Met Asp
Val Asn Asp 625 630 635 640 Asn Pro Pro Arg Leu Ala Lys Asp Tyr Thr
Gly Leu Phe Phe Cys His 645 650 655 Pro Leu Ser Ala Pro Gly Ser Leu
Ile Phe Glu Ala Thr Asp Asp Asp 660 665 670 Gln His Leu Phe Arg Gly
Pro His Phe Thr Phe Ser Leu Gly Ser Gly 675 680 685 Ser Leu Gln Asn
Asp Trp Glu Val Ser Lys Ile Asn Gly Thr His Ala 690 695 700 Arg Leu
Ser Thr Arg His Thr Asp Phe Glu Glu Arg Glu Tyr Val Val 705 710 715
720 Leu Ile Arg Ile Asn Asp Gly Gly Arg Pro Pro Leu Glu Gly Ile Val
725 730 735 Ser Leu Pro Val Thr Phe Cys Ser Cys Val Glu Gly Ser Cys
Phe Arg 740 745 750 Pro Ala Gly His Gln Thr Gly Ile Pro Thr Val Gly
Met 755 760 765 138123PRTHomo sapiens 138Glu Val Gln Leu Gln Gln
Ser Val Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met
Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Asp His 20 25 30 Thr Ile
His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Tyr Pro Arg Asp Gly Ile Thr Gly Tyr Asn Glu Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Trp Gly Tyr Ser Tyr Arg Asn Tyr Ala
Tyr Tyr Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Leu Thr Val
Ser Ser 115 120 139115PRTHomo sapiens 139Asp Ile Val Met Ser Gln
Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr
Met Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Asn
Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45
Ser Pro Lys Val Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50
55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 65 70 75 80 Ile Thr Ser Val Lys Ser Glu Asp Leu Ala Val Tyr Tyr
Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly
Thr Arg Leu Glu Ile 100 105 110 Lys Arg Ala 115
* * * * *
References