U.S. patent application number 12/663501 was filed with the patent office on 2010-10-07 for polypeptides, antibody variable domains and antagonists.
This patent application is currently assigned to Domantis Limited. Invention is credited to Thil Dinuk Batuwangala, Carolyn Enever, Laurent Jespers, Malgorzata Pupecka, Michael Steward, Ian Thomlinson.
Application Number | 20100254995 12/663501 |
Document ID | / |
Family ID | 39016531 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100254995 |
Kind Code |
A1 |
Steward; Michael ; et
al. |
October 7, 2010 |
POLYPEPTIDES, ANTIBODY VARIABLE DOMAINS AND ANTAGONISTS
Abstract
The invention relates to anti-VEGF polypeptides and antibody
single variable domains (dAbs) that are resistant to degradation by
a protease, as well as antagonists comprising these. The
polypeptides, dAbs and antagonists are useful for pulmonary
administration, oral administration, delivery to the lung and
delivery to the GI tract of a patient, as well as for treating
cancer and inflammatory disease, such as arthritis.
Inventors: |
Steward; Michael;
(Hertfordshire, GB) ; Pupecka; Malgorzata;
(Cambridgeshire, GB) ; Thomlinson; Ian;
(Hertfordshire, GB) ; Enever; Carolyn;
(Cambridgeshire, GB) ; Jespers; Laurent;
(Cambridgeshire, GB) ; Batuwangala; Thil Dinuk;
(Cambridgeshire, GB) |
Correspondence
Address: |
GlaxoSmithKline;GLOBAL PATENTS -US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Assignee: |
Domantis Limited
Brentford, Middlesex
GB
|
Family ID: |
39016531 |
Appl. No.: |
12/663501 |
Filed: |
June 3, 2008 |
PCT Filed: |
June 3, 2008 |
PCT NO: |
PCT/GB2008/050403 |
371 Date: |
June 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60933632 |
Jun 6, 2007 |
|
|
|
Current U.S.
Class: |
424/158.1 ;
530/350; 530/389.2 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 37/08 20180101; C07K 2317/92 20130101; C07K 2317/76 20130101;
A61M 11/005 20130101; C07K 16/005 20130101; A61P 35/00 20180101;
A61P 1/04 20180101; A61M 15/08 20130101; A61P 43/00 20180101; C07K
16/00 20130101; C07K 2317/64 20130101; A61P 31/04 20180101; C07K
2317/567 20130101; A61P 11/06 20180101; C07K 2317/569 20130101;
C07K 2317/73 20130101; A61P 29/00 20180101; A61P 27/02 20180101;
C07K 2317/90 20130101; A61P 11/00 20180101; A61K 9/1641 20130101;
C07K 2317/565 20130101; A61K 9/007 20130101; A61M 16/14 20130101;
A61P 37/06 20180101; A61P 25/00 20180101; C07K 16/2866 20130101;
C07K 16/2878 20130101; A61K 2039/544 20130101; A61P 1/00 20180101;
C07K 2317/94 20130101; A61K 9/0078 20130101; A61K 39/3955 20130101;
A61P 11/02 20180101; C07K 16/22 20130101; A61P 19/02 20180101; A61K
9/1623 20130101 |
Class at
Publication: |
424/158.1 ;
530/389.2; 530/350 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/22 20060101 C07K016/22; C07K 14/00 20060101
C07K014/00; A61P 37/00 20060101 A61P037/00; A61P 35/00 20060101
A61P035/00; A61P 29/00 20060101 A61P029/00; A61P 1/00 20060101
A61P001/00; A61P 31/04 20060101 A61P031/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2007 |
GB |
0724331.4 |
Claims
1. An anti-VEGF immunoglobulin single variable domain comprising an
amino acid sequence selected from the group consisting of (i) an
amino acid sequence that is a variant of the 15-26 lineage selected
from (a) an amino acid sequence that is at least 97% identical to
the amino acid sequence of DOM 15-26-595 (shown in FIG. 5), (b) an
amino acid sequence that is at least 98% identical to the amino
acid sequence of DOM15-26-591 (shown in FIG. 5), (c) an amino acid
sequence that is at least 98% identical to the amino acid sequence
of DOM15-26-589 (shown in FIG. 5), (d) an amino acid sequence that
is at least 98% identical to the amino acid sequence of
DOM15-26-588 (shown in FIG. 5), and (e) an amino acid sequence that
is at least 99% identical to the amino acid sequence of DOM
15-26-555 (shown in FIG. 5); and (ii) an amino acid sequence that
is at least 98% identical to the amino acid sequence of DOM15-10-11
(shown in FIG. 6).
2. The immunoglobulin single variable domain of claim 1 comprising
valine at position 6, wherein numbering is according to Kabat.
3. The immunoglobulin single variable domain of claim 1 comprising
leucine at position 99, wherein numbering is according to
Kabat.
4. The immunoglobulin single variable domain of claim 1, comprising
lysine at position 30, wherein numbering is according to Kabat.
5. An anti-VEGF immunoglobulin single variable domain comprising an
amino acid sequence that is identical to an amino acid sequence
selected from the group consisting of (i) a variant of the 15-26
lineage selected from (a) an amino acid sequence that is identical
to the amino acid sequence of DOM 15-26-595 (shown in FIG. 5), (b)
an amino acid sequence that is identical to the amino acid sequence
of DOM15-26-591 (shown in FIG. 5), (c) an amino acid sequence that
is identical to the amino acid sequence of DOM15-26-589 (shown in
FIG. 5), (d) an amino acid sequence that is identical to the amino
acid sequence of DOM 15-26-588 (shown in FIG. 5), and (e) an amino
acid sequence that is identical to the amino acid sequence of DOM
15-26-555 (shown in FIG. 5); and (ii) an amino acid sequence that
is identical to the amino acid sequence of DOM15-10-11 (shown in
FIG. 6).
6. An anti-VEGF immunoglobulin single variable domain encoded by a
nucleotide sequence that is selected from the group consisting of
(i) a nucleotide sequence that encodes a variant of the 15-26
lineage selected from (a) a nucleic acid sequence that is at least
80% identical to the nucleic acid sequence of DOM15-26-595, (b) a
nucleic acid sequence that is at least 80% identical to the nucleic
acid sequence of DOM 15-26-591 (shown in FIG. 20c), (c) a nucleic
acid sequence that is at least 80% identical to the nucleic acid
sequence of DOM15-26-589 (shown in FIG. 20b), (d) a nucleic acid
sequence that is at least 80% identical to the nucleic acid
sequence of DOM15-26-588 (shown in FIG. 20b), and (e) a nucleic
acid sequence that is at least 80% identical to the nucleic acid
sequence of DOM 15-26-555 (shown in FIG. 20a); and (ii) a nucleic
acid sequence that is at least 80% identical to the nucleic acid
sequence of DOM15-10-11 (shown in FIG. 2Oe).
7-9. (canceled)
10. The anti-VEGF immunoglobulin single variable domain of claim 1,
wherein the anti-VEGF immunoglobulin single variable domain
comprises a monomer or a homodimer of said anti-VEGF immunoglobulin
single variable domain.
11-39. (canceled)
40. A VEGF antagonist comprising a variable domain according to
claim 1.
41-51. (canceled)
52. A method for treating a disease or condition comprising
administering a VEGF antagonist of claim 40 to a patient, wherein
said disease or condition is selected from the group consisting of:
cancer, inflammation, autoimmune disease, Age Related Macular
Degeneration (AMD), a VEGF mediated condition or disease, COPD, and
pneumonia.
53. (canceled)
54. The method of claim 52, wherein the cancer is selected from the
group consisting of: lung, colorectal, head and neck, pancreatic,
breast, prostate and ovarian cancer.
55. The method of claim 52, wherein said condition is AMD.
56. The method of claim 52, wherein said inflammation is selected
from the group consisting of: inflammatory bowel disease, Crohn's
disease and ulcerative colitis.
57. The method of claim 52, wherein said COPD is selected from the
group consisting of: chronic bronchitis, chronic obstructive
bronchitis and emphysema.
58. The method of claim 52, wherein said pneumonia is bacterial
pneumonia.
59. The method of claim 52, wherein said pneumonia is
Staphylococcal pneumonia.
60. A method for treating a disease comprising administering a VEGF
antagonist of claim 40 to a patient, wherein said disease is a
cancer.
61-78. (canceled)
79. A polypeptide comprising an amino acid sequence selected from
the group consisting of: (i) a variant of the 15-26 lineage
selected from (a) a sequence that is at least 97% identical to the
amino acid sequence of DOM 15-26-595 (shown in FIG. 5), (b) an
amino acid sequence that is at least 98% identical to the amino
acid sequence of DOM 15-26-591 (shown in FIG. 5), (c) an amino acid
sequence that is at least 98% identical to the amino acid sequence
of DOM15-26-589 (shown in FIG. 5), (d) an amino acid sequence that
is at least 98% identical to the amino acid sequence of
DOM15-26-588 (shown in FIG. 5), and (e) an amino acid sequence that
is at least 99% identical to the amino acid sequence of DOM
15-26-555 (shown in FIG. 5); and (ii) an amino acid sequence that
is at least 98% identical to the amino acid sequence of DOM15-10-11
(shown in FIG. 6).
80. (canceled)
81. A fusion protein comprising the polypeptide of claim 79.
82. (canceled)
83. The immunoglobulin single variable domain of claim 1 further
comprising an antibody constant domain.
84. (canceled)
85. The antagonist claim 40, further comprising an antibody
constant domain.
Description
[0001] The present invention relates to protease resistant
polypeptides, immunoglobulin (antibody) single variable domains and
vascular endothelial growth factor (VEGF) antagonists comprising
these. The invention further relates to uses, formulations,
compositions and devices comprising such anti-VEGF ligands.
BACKGROUND OF THE INVENTION
[0002] Polypeptides and peptides have become increasingly important
agents in a variety of applications, including industrial
applications and use as medical, therapeutic and diagnostic agents.
However, in certain physiological states, such as Cancer and
inflammatory states (e.g., COPD), the amount of proteases present
in a tissue, organ or animal (e.g., in the lung, in or adjacent to
a tumor) can increase. This increase in proteases can result in
accelerated degradation and inactivation of endogenous proteins and
of therapeutic peptides, polypeptides and proteins that are
administered to treat disease. Accordingly, some agents that have
potential for in vivo use (e.g., use in treating, diagnosing or
preventing disease) have only limited efficacy because they are
rapidly degraded and inactivated by proteases.
[0003] Protease resistant polypeptides provide several advantages.
For example, protease resistant polypeptides remaining active in
vivo longer than protease sensitive agents and, accordingly,
remaining functional for a period of time that is sufficient to
produce biological effects. A need also exists for improved methods
to select polypeptides that are resistant to protease degradation
and also have desirable biological activity.
VEGF:
[0004] VEGF is a secreted, heparin-binding, homodimeric
glycoprotein existing in several alternate forms due to alternative
splicing of its primary transcript (Leung et al., 1989, Science
246: 1306). VEGF is also known as vascular permeability factor
(VPF) due to its ability to induce vascular leakage, a process
important in inflammation.
[0005] An important pathophysiological process that facilitates
tumor formation, metastasis and recurrence is tumor angiogenesis.
This process is mediated by the elaboration of angiogenic factors
expressed by the tumor, such as VEGF, which induce the formation of
blood vessels that deliver nutrients to the tumor. Accordingly, an
approach to treating certain cancers is to inhibit tumor
angiogenesis mediated by VEGF, thereby starving the tumor. AVASTIN
(bevacizumab; Genetech, Inc.) is a humanized antibody that binds
human VEGF that has been approved for treating colorectal cancer.
An antibody referred to as antibody 2C3 (ATCC Accession No. PTA
1595) is reported to bind VEGF and inhibit binding of VEGF to
epidermal growth factor receptor 2.
[0006] Targeting VEGF with currently available therapeutics is not
effective in all patients, or for all cancers. Thus, a need exists
for improved agents for treating cancer and other pathological
conditions nediated by VEGF e.g. vascular proliferative diseases
(e.g. Age related macular degeneration (AMD)).
[0007] VEGF has also been implicated in inflammatpry disorders and
autoimmune diseases. For example, the identification of VEGF in
synovial tissues of RA patients highlighted the potential role of
VEGF in the pathology of RA (Fava et al., 1994, J. Exp. Med. 180:
341: 346; Nagashima et al., 1995, J. Rheumatol. 22: 1624-1630). A
role for VEGF in the pathology of RA was solidified following
studies in which anti-VEGF antibodies were administered in the
murine collagen-induced arthritis (CIA) model. In these studies,
VEGF expression in the joints increased upon induction of the
disease, and the administration of anti-VEGF antisera blocked the
development of arthritic disease and ameliorated established
disease (Sone et al., 2001, Biochem. Biophys. Res. Comm. 281:
562-568; Lu et al., 2000, J. Immunol. 164: 5922-5927). Hence
targeting VEGF may also be of benefit in treating RA, and other
conditions e.g. those associated with inflammation and/or
autoimmune disease.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention provides a polypeptide which
comprises an amino acid sequence that is selected from the
following: (i) an amino acid sequence that is a variant of the
15-26 lineage selected from (a) an amino acid sequence that is at
least 97% identical to the amino acid sequence of DOM15-26-595
(shown in FIG. 5), (b) an amino acid sequence that is at least 98%
identical to the amino acid sequence of DOM15-26-591 (shown in FIG.
5), (c) an amino acid sequence that is at least 98% identical to
the amino acid sequence of DOM15-26-589 (shown in FIG. 5), (d) an
amino acid sequence that is at least 98% identical to the amino
acid sequence of DOM15-26-588 (shown in FIG. 5), (e) an amino acid
sequence that is at least 99% identical to the amino acid sequence
of DOM15-26-555 (shown in FIG. 5); and also (ii) an amino acid
sequence that is at least 98% identical to the amino acid sequence
of DOM15-10-11 (shown in FIG. 6).
[0009] In one embodiment, the polypeptide is selected from:
DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555, and DOM15-10-11. The invention further provides
(substantially) pure monomer of a polypeptide selected from:
DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555, and DOM15-10-11. In one embodiment, the polypeptide
is at least 98,99, 99.5% pure or 100% pure monomer.
[0010] In one aspect, the invention provides a polypeptide (e.g.
that is protease resistant) and that is encoded by an amino acid
sequence that is at least 80% identical to the amino acid sequence
selected from : (i) an amino acid sequence that is a variant of the
15-26 lineage selected from (a) DOM15-26-595 (shown in FIG. 5), (b)
DOM15-26-591 (shown in FIG. 5), (c) DOM15-26-589 (shown in FIG. 5),
(d) DOM15-26-588 (shown in FIG. 5), (e) DOM15-26-555 (shown in FIG.
5); and also (ii) DOM15-10-11 (shown in FIG. 6).
[0011] In one embodiment, the percent identity is at least 70, 80,
85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. In one embodiment
that protease resistant polypeptide is obtainable by the method
described herein for isolating protease resistant polypeptides.
[0012] In one aspect, the invention provides a polypeptide encoded
by an amino acid sequence that is at least 55% identical to the
nucleotide sequence selected from that encoding: DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555; also
DOM15-10-11; and wherein the polypeptide comprises an amino acid
sequence that is at least 97% identical to the respective amino
acid s sequences. In one embodiment, the percent identity of the
nucleotide sequence is at least 60, 65, 70, 75, 80, 85, 90, 91, 92,
93, 94, 95, 96, 97, 98 or 99%. In one embodiment, the percent
identity of the amino acid sequence is at least, 98 or 99% or 100%.
For example, the nucleotide sequence may be a codon-optimised
version of the nucleotide sequence encoding: DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11. Codon optimization of sequences is known in the art.
In one embodiment, the nucleotide sequence is optimized for
expression in a bacterial (eg, E. coli or Pseudomonas, eg P
fluorescens), mammalian (eg, CHO) or yeast host cell (eg. Picchia
or Saccharomyces, eg P. pastoris or S. cerevisiae).
[0013] In one aspect, the invention provides a fusion protein
comprising the polypeptide of the invention.
In one aspect, the invention provides an anti-VEGF immunoglobulin
single variable domain comprising an amino acid sequence that is
selected from the following: (i) an amino acid sequence that is a
variant of the 15-26 lineage selected from (a) an amino acid
sequence that is at least 97% identical to the amino acid sequence
of DOM15-26-595 (shown in FIG. 5), (b) an amino acid sequence that
is at least 98% identical to the amino acid sequence of
DOM15-26-591 (shown in FIG. 5), (c) an amino acid sequence that is
at least 98% identical to the amino acid sequence of DOM15-26-589
(shown in FIG. 5), (d) an amino acid sequence that is at least 98%
identical to the amino acid sequence of DOM15-26-588 (shown in FIG.
5), (e) an amino acid sequence that is at least 99% identical to
the amino acid sequence of DOM15-26-555 (shown in FIG. 5); and also
(ii) an amino acid sequence that is at least 98% identical to the
amino acid sequence of DOM15-10-11 (shown in FIG. 6).
[0014] In one embodiment, the immunoglobulin single variable domain
comprises valine at position 6, wherein numbering is according to
Kabat ("Sequences of Proteins of Immunological Interest", US
Department of Health and Human Services 1991).
[0015] In one embodiment, the immunoglobulin single variable domain
comprises leucine at position 99, wherein numbering is according to
Kabat.
[0016] In one embodiment, the immunoglobulin single variable domain
comprises Lysine at position 30, wherein numbering is according to
Kabat.
[0017] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 and DOM15-10-11.
[0018] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain encoded by a nucleotide
sequence that is at least 80% identical to the nucleotide sequence
of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11. In one embodiment, the percent
identity is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98
or 99%.
[0019] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain encoded by an amino acid
sequence that is at least 55% identical to the nucleotide sequence
of: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11, and wherein the variable domain
comprises an amino acid sequence that is at least 97% identical to
the respective amino acid sequences of DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one
embodiment, the percent identity of the nucleotide sequence is at
least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or
99%. In one embodiment, the percent identity of the amino acid
sequence is at least 98 or 99% or 100%. For example, the nucleotide
sequence may be a codon-optimised version of the nucleotide
sequence of : DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. Codon optimization of
sequences is known in the art. In one embodiment, the nucleotide
sequence is optimized for expression in a bacterial (eg, E. coli or
Pseudomonas, eg P fluorescens), mammalian (eg, CHO) or yeast host
cell (eg. Picchia or Saccharomyces, eg P. pastoris or S.
cerevisiae).
[0020] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain encoded by a sequence that is
identical to the nucleotide sequence selected from:DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11.
[0021] In one aspect, the invention provides an anti-VEGF
antagonist comprising an anti-VEGF immunoglobulin single variable
domain according to the invention. In one embodiment, the
antagonist comprises first and second immunoglobulin single
variable domains, wherein each variable domain is according to
invention. For example, wherein the antagonist comprises a monomer
of said single variable domain or a homodimer of said single
variable domain. In one embodiment, the amino acid sequence of the
(or each) single variable domain is identical to the amino acid
sequence selected from that of : DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11.
[0022] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11, or differs from the amino acid
sequence of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 at no more than 14 amino acid positions
and has a CDR1 sequence that is at least 50% identical to the
respective CDR1 sequences of DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one
embodiment, the difference is no more than 13, 12, 11, 10, 9, 8, 7,
6, 5, 4, 3, 2 or 1 amino acid position. In one embodiment, the CDR
sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90, 95,
96, 97, 98 or 99%.
[0023] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 or differs from the amino acid sequence
of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 at no more than 14 amino acid positions
and has a CDR2 sequence that is at least 50% identical to the
respective CDR2 sequences of either DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one
embodiment, the difference is no more than 13, 12, 11, 10, 9, 8, 7,
6, 5, 4, 3, 2 or 1 amino acid position. In one embodiment, the CDR
sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90, 95,
96, 97, 98 or 99%.
[0024] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11, or differs from the amino acid
sequence of of DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11 at no more than 14 amino
acid positions and has a CDR3 sequence that is at least 50%
identical to the respective CDR3 sequences of either DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11. In one embodiment, the difference is no more than 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position. In
one embodiment, the CDR sequence identity is at least 55, 60, 65,
70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%.
[0025] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11, or differs from the amino acid
sequence of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 at no more than 14 amino acid positions
and has a CDR1 sequence that is at least 50% identical to the
respective CDR1 sequences of either DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11 and has a
CDR2 sequence that is at least 50% identical to the respective CDR2
sequences of either DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
difference is no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 amino acid position. In one embodiment, one or both CDR
sequence identities is respectively at least 55, 60, 65, 70, 75,
80, 85, 90, 95, 96, 97, 98 or 99%.
[0026] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 or differs from the amino acid sequence
of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 at no more than 14 amino acid positions
and has a CDR1 sequence that is at least 50% identical to the
respective CDR1 sequences of DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11 and has a
CDR3 sequence that is at least 50% identical to the respective CDR3
sequences of DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
difference is no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 amino acid position. In one embodiment, one or both CDR
sequence identities is respectively at least 55, 60, 65, 70, 75,
80, 85, 90, 95, 96, 97, 98 or 99%.
[0027] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 or differs from the amino acid sequence
of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 at no more than 14 amino acid positions
and has a CDR2 sequence that is at least 50% identical to the
respective CDR2 sequences of DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11 and has a
CDR3 sequence that is at least 50% identical to the respective CDR3
sequences of DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
difference is no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 amino acid position. In one embodiment, one or both CDR
sequence identities is respectively at least 55, 60, 65, 70, 75,
80, 85, 90, 95, 96, 97, 98 or 99%.
[0028] In one aspect, the invention provides an anti-VEGF
immunoglobulin single variable domain comprising an amino acid
sequence that is identical to the amino acid sequence selected
from: DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 or differs from the amino acid sequence
of DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 at no more than 14 amino acid positions
and has a CDR1 sequence that is at least 50% identical to the
respective CDR1 sequences of DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11, and has a
CDR2 sequence that is at least 50% identical to the respective CDR2
sequences of DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11, and has a CDR3 sequence
that is at least 50% identical to the respective CDR3 sequences of
DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11. In one embodiment, the difference is
no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid
position. In one embodiment, one or two or each CDR sequence
identity is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98
or 99%.
[0029] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR1 sequence that is at least 50% identical to
the CDR1 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,
95, 96, 97, 98 or 99%. The antagonist may be resistant to protease,
for example one or more of the proteases as herein described, for
example under a set of conditions as herein described.
[0030] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR2 sequence that is at least 50% identical to
the CDR1 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,
95, 96, 97, 98 or 99%. The antagonist may be resistant to protease,
for example one or more of the proteases as herein described, for
example under a set of conditions as herein described.
[0031] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR3 sequence that is at least 50% identical to
the CDR1 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,
95, 96, 97, 98 or 99%. The antagonist may be resistant to protease,
for example one or more of the proteases as herein described, for
example under a set of conditions as herein described.
[0032] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR1 sequence that is at least 50% identical to
the CDR1 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11, and a CDR2 sequence that
is at least 50% identical to the CDR2 sequence from: DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11. In one embodiment, the CDR sequence identity of one or
both CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,
98 or 99%. The antagonist may be resistant to protease, for example
one or more of the proteases as herein described, for example under
a set of conditions as herein described.
[0033] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR1 sequence that is at least 50% identical to
the CDR1 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11; and a CDR3 sequence that
is at least 50% identical to the CDR3 sequence from: DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11. In one embodiment, the CDR sequence identity of one or
both CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,
98 or 99%. The antagonist may be resistant to protease, for example
one or more of the proteases as herein described, for example under
a set of conditions as herein described.
[0034] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR2 sequence that is at least 50% identical to
the CDR2 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11; and a CDR3 sequence that
is at least 50% identical to the CDR3 sequence from: DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11. In one embodiment, the CDR sequence identity of one or
both CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,
98 or 99%. The antagonist may be resistant to protease, for example
one or more of the proteases as herein described, for example under
a set of conditions as herein described.
[0035] In one aspect, the invention provides an anti-VEGF
antagonist having a CDR1 sequence that is at least 50% identical to
the CDR1 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11, and a CDR2 sequence that
is at least 50% identical to the CDR2 sequence from: DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11; and a CDR3 sequence that is at least 50% identical to
the CDR3 sequence from: DOM15-26-595, DOM15-26-591, DOM15-26-589,
DOM15-26-588, DOM15-26-555 or DOM15-10-11. In one embodiment, the
CDR sequence identity of one or two or each of the CDRs is at least
55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%. The
antagonist may be resistant to protease, for example one or more of
the proteases as herein described, for example under a set of
conditions as herein described.
[0036] In one aspect, the invention provides an anti-VEGF
antagonist comprising an immunoglobulin single variable domain
comprising the CDR1, CDR2, and/or CDR3 (eg, CDR1, CDR2, CDR3, CDR1
and 2, CDR1 and 3, CDR2 and 3 or CDR1, 2 and 3) sequences from the
respective CDR sequences of: DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 or DOM15-10-11. The
antagonist may be resistant to protease, for example one or more of
the proteases as herein described, for example under a set of
conditions as herein described.
[0037] In one aspect, the invention provides an anti-VEGF
antagonist that competes with an antagonist selected from:
DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11 for binding to VEGF. Thus, the
antagonist may bind the same epitope as either DOM15-26-595,
DOM15-26-591, DOM15-26-589, DOM15-26-588, DOM15-26-555 or
DOM15-10-11, or it may bind an overlapping epitope. In one
embodiment, the antagonist comprises an immunoglobulin single
variable domain having an amino acid sequence selected from the
following: (i) an amino acid sequence that is a variant of the
15-26 lineage selected from (a) an amino acid sequence that is at
least 97% identical to the amino acid sequence of DOM15-26-595, (b)
an amino acid sequence that is at least 98% identical to the amino
acid sequence of DOM15-26-591 , (c) an amino acid sequence that is
at least 98% identical to the amino acid sequence of DOM15-26-589,
(d) an amino acid sequence that is at least 98% identical to the
amino acid sequence of DOM15-26-588 , (e) an amino acid sequence
that is at least 99% identical to the amino acid sequence of
DOM15-26-555, and also (ii) an amino acid sequence that is at least
98% identical to the amino acid sequence of DOM15-10-11.
[0038] In one embodiment, the percent identity is at least 98 or
99%. In one embodiment, the variable domain is selected from:
DOM15-26-595, DOM15-26-591, DOM15-26-589, DOM15-26-588,
DOM15-26-555 or DOM15-10-11. The antagonist may be resistant to
protease, for example one or more of the proteases as herein
described, for example under a set of conditions as herein
described. In one embodiment, the antagonist is an antibody or
antigen-binding fragment thereof, such as a monovalent
antigen-binding fragment (e.g., scFv, Fab, Fab', dAb) that has
binding specificity for VEGF. Other examples of antagonists are
ligands described herein that bind VEGF. The ligands may comprise
an immunoglobulin single variable domain or domain antibody (dAb)
that has binding specificity for VEGF, or the complementarity
determining regions of such a dAb in a suitable format. In some
embodiments, the ligand is a dAb monomer that consists essentially
of, or consists of, an immunoglobulin single variable domain or dAb
that has binding specificity for VEGF. In other embodiments, the
ligand is a polypeptide that comprises a dAb (or the CDRs of a dAb)
in a suitable format, such as an antibody format.
[0039] These VEGF ligands e.g. dAbs, can be formatted to have a
larger hydrodynamic size, for example, by attachment of a PEG
group, serum albumin, transferrin, transferrin receptor or at least
the transferrin-binding portion thereof, an antibody Fc region, or
by conjugation to an antibody domain. For example, an agent (e.g.,
polypeptide, variable domain or antagonist) that i) binds VEGF (ii)
antagonizes the activation of VEGF mediated signal transduction,
and (iii) does not inhibit the binding of VEGF to its receptor,
such as a dAb monomer, can be formatted as a larger antigen-binding
fragment of an antibody (e.g., formatted as a Fab, Fab',
F(ab).sub.2, F(ab').sub.2, IgG, scFv). The hydrodynamic size of a
ligand and its serum half-life can also be increased by conjugating
or linking a VEGF binding agent (antagonist, variable domaiu) to a
binding domain (e.g., antibody or antibody fragment) that binds an
antigen or epitope that increases half-live in vivo, as described
herein (see, Annex 1 of WO2006038027 incorporated herein by
reference in its entirety). For example, the VEGF binding agent
(e.g., polypeptide, E.G. dAb) can be conjugated or linked to an
anti-serum albumin or anti-neonatal Fc receptor antibody or
antibody fragment, eg an anti-SA or anti-neonatal Fc receptor dAb,
Fab, Fab' or scFv, or to an anti-SA affibody or anti-neonatal Fc
receptor affibody.
[0040] Examples of suitable albumin, albumin fragments or albumin
variants for use in a VEGF-binding ligands according to the
invention are described in WO 2005/077042A2 and WO2006038027, which
are incorporated herein by reference in their entirety.
[0041] In other embodiments of the invention described throughout
this disclosure, instead of the use of a "dAb" in an antagonist or
ligand of the invention, it is contemplated that the skilled
addressee can use a domain that comprises the CDRs of a dAb that
binds VEGF (e.g., CDRs grafted onto a suitable protein scaffold or
skeleton, eg an affibody, an SpA scaffold, an LDL receptor class A
domain or an EGF domain) or can be a protein domain comprising a
binding site for VEGF, e.g., wherein the domain is selected from an
affibody, an SpA domain, an LDL receptor class A domain or an EGF
domain. The disclosure as a whole is to be construed accordingly to
provide disclosure of antagonists, ligands and methods using such
domains in place of a dAb.
[0042] Polypeptides, immunoglobulin single variable domains and
antagonists of the invention may be resistant to one or more of the
following: serine protease, cysteine protease, aspartate proteases,
thiol proteases, matrix metalloprotease, carboxypeptidase (e.g.,
carboxypeptidase A, carboxypeptidase B), trypsin, chymotrypsin,
pepsin, papain, elastase, leukozyme, pancreatin, thrombin, plasmin,
cathepsins (e.g., cathepsin G), proteinase (e.g., proteinase 1,
proteinase 2, proteinase 3), thermolysin, chymosin,
enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4,
caspase 5, caspase 9, caspase 12, caspase 13), calpain, ficain,
clostripain, actinidain, bromelain, and separase. In particular
embodiments, the protease is trypsin, elastase or leucozyme. The
protease can also be provided by a biological extract, biological
homogenate or biological preparation. In one embodiment, the
protease is a protease found in sputum, mucus (e.g., gastric mucus,
nasal mucus, bronchial mucus), bronchoalveolar lavage, lung
homogenate, lung extract, pancreatic extract, gastric fluid,
saliva. In one embodiment, the protease is one found in the eye
and/or tears. Examples of such proteases found in the eye include
caspases, calpains, matric metalloproteases, disintegrin,
metalloproteinases (ADAMs) and ADAM with thrombospondin mitifs, the
proteosomes, tissue plasminogen activator, secretases, cathepsin B
and D, cystatin C, serine protease PRSS1, ubiquitin proteosome
pathway (UPP). In one embodiment, the protease is a non-bacterial
protease. In an embodiment, the protease is an animal, eg,
mammalian, eg, human, protease. In an embodiment, the protease is a
GI tract protease or a pulmonary tissue protease, eg, a GI tract
protease or a pulmonary tissue protease found in humans. Such
protease listed here can also be used in the methods described
herein involving exposure of a repertoire of library to a
protease.
[0043] In one aspect, the invention provides a protease resistant
immunoglobulin single variable domain comprising a VEGF binding
site, wherein the variable domain is resistant to protease when
incubated with [0044] (i) a concentration (c) of at least 10
micrograms/ml protease at 37.degree. C. for time (t) of at least
one hour; or [0045] (ii) a concentration (c') of at least 40
micrograms/ml protease at 30.degree. C. for time (t) of at least
one hour. In one embodiment, the ratio (on a mole/mole basis) of
protease, eg trypsin, to variable domain is 8,000 to 80,000
protease:variable domain, eg when C is 10 micrograms/ml, the ratio
is 800 to 80,000 protease:variable domain; or when C or C' is 100
micrograms/ml, the ratio is 8,000 to 80,000 protease:variable
domain. In one embodiment the ratio (on a weight/weight, eg
microgram/microgram basis) of protease (eg, trypsin) to variable
domain is 16,000 to 160,000 protease:variable domain eg when C is
10 micrograms/ml, the ratio is 1,600 to 160,000 protease:variable
domain; or when C or C' is 100 micrograms/ml, the ratio is 1,6000
to 160,000 protease:variable domain. In one embodiment, the
concentration (c or c') is at least 100 or 1000 micrograms/ml
protease. In one embodiment, the concentration (c or c') is at
least 100 or 1000 micrograms/ml protease. Reference is made to the
description herein of the conditions suitable for proteolytic
activity of the protease for use when working with repertoires or
libraries of peptides or polypeptides (eg, w/w parameters). These
conditions can be used for conditions to determine the protease
resistance of a particular immunoglobulin single variable domain.
In one embodiment, time (t) is or is about one, three or 24 hours
or overnight (e.g., about 12-16 hours). In one embodiment, the
variable domain is resistant under conditions (i) and the
concentration (c) is or is about 10 or 100 micrograms/ml protease
and time (t) is 1 hour. In one embodiment, the variable domain is
resistant under conditions (ii) and the concentration (c') is or is
about 40 micrograms/ml protease and time (t) is or is about 3
hours. In one embodiment, the protease is selected from trypsin,
elastase, leucozyme and pancreatin. In one embodiment, the protease
is trypsin. In one embodiment, the protease is a protease found in
sputum, mucus (e.g., gastric mucus, nasal mucus, bronchial mucus),
bronchoalveolar lavage, lung homogenate, lung extract, pancreatic
extract, gastric fluid, saliva or tears or the eye. In one
embodiment, the protease is one found in the eye and/or tears. In
one embodiment, the protease is a non-bacterial protease. In an
embodiment, the protease is an animal, eg, mammalian, eg, human,
protease. In an embodiment, the protease is a GI tract protease or
a pulmonary tissue protease, eg, a GI tract protease or a pulmonary
tissue protease found in humans. Such protease listed here can also
be used in the methods described herein involving exposure of a
repertoire of library to a protease.
[0046] In one embodiment, the variable domain is resistant to
trypsin and/or at least one other protease selected from elastase,
leucozyme and pancreatin. For example, resistance is to trypsin and
elastase; trypsin and leucozyme; trypsin and pacreatin; trypsin,
elastase and leucozyme; trypsin, elastase and pancreatin; trypsin,
elastase, pancreatin and leucozyme; or trypsin, pancreatin and
leucozyme.
[0047] In one embodiment, the variable domain is displayed on
bacteriophage when incubated under condition (i) or (ii) for
example at a phage library size of 10.sup.6 to 10.sup.13, eg
10.sup.8 to 10.sup.12 replicative units (infective virions).
[0048] In one embodiment, the variable domain specifically binds
VEGF following incubation under condition (i) or (ii), eg assessed
using BiaCore.TM. or ELISA, eg phage ELISA or monoclonal phage
ELISA.
[0049] In one embodiment, the variable domains of the invention
specifically bind protein A or protein L. In one embodiment,
specific binding to protein A or L is present following incubation
under condition (i) or (ii).
[0050] In one embodiment, the variable domains of the invention may
have an OD.sub.450 reading in ELISA, eg phage ELISA or monoclonal
phage ELISA) of at least 0.404, eg, following incubation under
condition (i) or (ii).
[0051] In one embodiment, the variable domains of the invention
display (substantially) a single band in gel electrophoresis, eg
following incubation under condition (i) or (ii).
[0052] In certain embodiments, the invention provides a VEGF
antagonist that is a dual-specific ligand that comprises a first
dAb according to the invention that binds VEGF and a second dAb
that has the same or a different binding specificity from the first
dAb. The second dAb may bind a target selected from ApoE, Apo-SAA,
BDNF, Cardiotrophin-1, CEA, CD40, CD40 Ligand, CD56, CD38, CD138,
EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2,
FAP.alpha., FGF-acidic, FGF-basic, fibroblast growth factor-10,
FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1,
human serum albumin, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-1 receptor, IL-1 receptor type 1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),
Inhibin .alpha., Inhibin .beta., IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte
attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1
(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG,
MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin,
Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA,
PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.beta., SCF, SCGF,
stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta., TGF-.beta.2,
TGF-.beta.3, tumour necrosis factor (TNF), TNF-.alpha., TNF-.beta.,
TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF A, VEGF B,
VEGF C, VEGF D, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1, HER
2, HER 3, HER 4, serum albumin, vWF, amyloid proteins (e.g.,
amyloid alpha), MMP12, PDK1, IgE, IL-13R-.alpha.1, IL-13Ra2, IL-15,
IL-15R, IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25,
CD2, CD4, CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD4OL, CD56,
CD138, ALK5, EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12
(SDF-1), chymase, FGF, Furin, Endothelin-1, Eotaxins (e.g.,
Eotaxin, Eotaxin-2, Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE,
IFN.alpha., 1-309, integrins, L-selectin, MIF, MIP4, MDC, MCP-1,
MMPs, neutrophil elastase, osteopontin, OX-40, PARC, PD-1, RANTES,
SCF, SDF-1, siglec8, TARC, TGFb, Thrombin, Tim-1, TNF, TRANCE,
Tryptase, VEGF, VLA-4, VCAM, .alpha.4.beta.7, CCR2, CCR3, CCR4,
CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF, amyloid
proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.
[0053] In one example, the dual-specific ligand comprises a first
dAb that binds a first epitope on VEGF and a second dAb that binds
an epitope on a different target. In another example, the second
dAb binds an epitope on serum albumin.
[0054] In other embodiments, the ligand is a multispecific ligand
that comprises a first epitope binding domain that has binding
specificity for VEGF and at least one other epitope binding domain
that has binding specificity different from the first epitope
binding domain. For example, the first epitope binding domain can
be a dAb that binds VEGF or can be a domain that comprises the CDRs
of a dAb that binds VEGF (e.g., CDRs grafted onto a suitable
protein scaffold or skeleton, e.g., an affibody, an SpA scaffold,
an LDL receptor class A domain or an EGF domain) or can be a domain
that binds VEGF, wherein the domain is selected from an affibody,
an SpA domain, an LDL receptor class A domain or an EGF
domain).
[0055] In certain embodiments, the polypeptide, antagonist, ligand
or anti-VEGF dAb monomer is characterized by one or more of the
following: 1) dissociates from human VEGF with a dissociation
constant (K.sub.d) of 50 nM to 20 pM, and a K.sub.off rate constant
of 5.times.10.sup.-1 to 1.times.10.sup.-7 s.sup.-1; as determined
by surface plasmon resonance; 2) inhibits binding of VEGF to VEGFR2
with an IC50 of 500 nM to 50 pM; 3) neutralizes human VEGF in a
standard HUVEC cell assay with an ND50 of 500 nM to 50 pM; 4)
antagonizes the activity of the VEGF in a standard cell assay with
an ND.sub.50 of .ltoreq.100 nM (5) inhibits or decreases tumour
growth in a mouse xenograft model; 6) resists aggregation; 7) is
secreted in a quantity of at least about 0.5 mg/L when expressed in
E. coli or Pichia species (e.g., P. pastoris) or mammalian cell
expression system such as CHO; 8) unfolds reversibly; or 9) has
efficacy in treating, suppressing or preventing a inflammatory
disease. Reference is made to WO2006038027 and WO 2006059108 and WO
2007049017 for details of assays and tests and parameters
applicable to conditions (1) to (9), and these are incorporated
herein by reference.
[0056] In particular embodiments, the polypeptide, antagonist,
ligand or dAb monomer dissociates from human VEGF with a
dissociation constant (K.sub.d) of 50 nM to 20 pM, and a K.sub.off
rate constant of 5.times.10.sup.-1 to 1.times.10.sup.-7 s.sup.-1 as
determined by surface plasmon resonance'; inhibits binding of
inhibits binding of VEGF to VEGFR2 (VEGF receptor 2) with an IC50
of 500 nM to 50 pM; and neutralizes human VEGF in a standard HUVEC
cell assay with an ND50 of 500 nM to 50 pM. In other particular
embodiments, the polypeptide, antagonist, ligand or dAb monomer
dissociates from human VEGF with a dissociation constant (K.sub.d)
of 50 nM to 20 pM, and a K.sub.off rate constant of
5.times.10.sup.-1 to 1.times.10.sup.-7 s.sup.-1 as determined by
surface plasmon resonance;; inhibits binding of VEGF to VEGFR2 with
an IC50 of 500 nM to 50 pM.
[0057] The protease resistant polypeptides, immunoglobulin single
variable domains and antagonists of the invention have utility in
therapy, prophylaxis and diagnosis of disease or conditions in
mammals, e.g. humans. In particular, they have utility as the basis
of drugs that are likely to encounter proteases when administered
to a patient, such as a human. For example, when administered to
the GI tract (eg, orally, sublingually, rectally administered), in
which case the polypeptides, immunoglobulin single variable domains
and antagonists may be subjected to protease in one or more of the
upper GI tract, lower GI tract, mouth, stomach, small intestine and
large intestine. One embodiment, therefore, provides for a protease
resistant polypeptide, immunoglobulin single variable domain or
antagonist to be administered orally, sublingually or rectally to
the GI tract of a patient to treat and/or prevent a disease or
condition in the patient. For example, oral administration to a
patient (eg, a human patient) for the treatment and/or prevention
of a VEGF-mediated condition or disease such as Cancer e.g. solid
tumours; inflammation and/or autoimmune disease.
[0058] In another example, the polypeptide, variable domain or
antagonist is likely to encounter protease when administered (eg,
by inhalation or intranasally) to pulmonary tissue (eg, the lung or
airways). One embodiment, therefore, provides for administration of
the protease resistant polypeptide, immunoglobulin single variable
domain or antagonist to a patient (eg, to a human) by inhalation or
intranasally to pulmonary tissue of the patient to treat and/or
prevent a disease or condition in the patient. Such condition may
be asthma (eg, allergic asthma), COPD, influenza or any other
pulmonary disease or condition disclosed in WO2006038027,
incorporated herein by reference. In another example, the
polypeptide, variable domain or antagonist is likely to encounter
protease when administered (eg, by intraocular injection or as eye
drops) to an eye of a patient. One embodiment, therefore, provides
for ocular administration of the protease resistant polypeptide,
immunoglobulin single variable domain or antagonist to a patient
(eg, to a human) by to treat and/or prevent a disease or condition
(eg, a disease or condition of the eye) in the patient.
Administration could be topical administration to the eye, in the
form of eye drops or by injection into the eye, eg into the
vitreous humour.
[0059] One embodiment of the invention provides for a protease
resistant polypeptide, immunoglobulin single variable domain or
antagonist to be administered to the eye, e.g. in the form of eye
drops or a gel or e.g. in an implant, e.g. for the treatment and/or
prevention of a VEGF-mediated condition or disease of the eye such
as AMD (Age related macular degeneration).
[0060] In another example, the polypeptide, variable domain or
antagonist is likely to encounter protease when administered (eg,
by inhalation or intranasally) to pulmonary tissue (eg, the lung or
airways). One embodiment, therefore, provides for administration of
any of the polypeptide, variable domain or antagonist described
herein to a patient (eg, to a human) by inhalation or intranasally
to pulmonary tissue of the patient to treat and/or prevent a
disease or condition in the patient. Such condition may be cancer
(e.g. a solid tumour, for example lung, colorectal, head and neck,
pancreatic, breast, prostate, or ovarian cancer), asthma (eg,
allergic asthma), COPD, or any other pulmonary disease or condition
disclosed in WO2006038027, incorporated herein by reference. The
antagonists, polypeptides and immunoglobulin single variable
domains according to the invention may display improved or
relatively high melting temperatures (Tm), providing enhanced
stability. High affinity target binding may also or alternatively
be a feature of the antagonists, polypeptides and variable domains.
One or more of these features, combined with protease resistance,
makes the antagonists, variable domains and polypeptides amenable
to use as drugs in mammals, such as humans, where proteases are
particularly likely to be encountered, eg for GI tract or pulmonary
tissue administration or administration to the eye.
[0061] Thus, in one aspect, the invention provides the VEGF
antagonist for oral delivery. In one aspect, the invention provides
the VEGF antagonist for delivery to the GI tract of a patient. In
one aspect, the invention provides the use of the VEGF antagonist
in the manufacture of a medicament for oral delivery. In one
aspect, the invention provides the use of the VEGF antagonist in
the manufacture of a medicament for delivery to the GI tract of a
patient. In one embodiment, the variable domain is resistant to
trypsin and/or at least one other protease selected from elastase,
leucozyme and pancreatin. For example, resistance is to trypsin and
elastase; trypsin and leucozyme; trypsin and pacreatin; trypsin,
elastase and leucozyme; trypsin, elastase and pancreatin; trypsin,
elastase, pancreatin and leucozyme; or trypsin, pancreatin and
leucozyme.
[0062] In one aspect, the invention provides the VEGF antagonist
for pulmonary delivery. In one aspect, the invention provides the
use of the VEGF antagonist in the manufacture of a medicament for
pulmonary delivery. In one aspect, the invention provides the use
of the VEGF antagonist in the manufacture of a medicament for
delivery to the lung of a patient. In one embodiment, the variable
domain is resistant to leucozyme.
[0063] In one aspect, the invention provides a method of oral
delivery or delivery of a medicament to the GI tract of a patient
or to the lung or pulmonary tissue or eye of a patient, wherein the
method comprises administering to the patient a pharmaceutically
effective amount of a VEGF antagonist of the invention.
[0064] In one aspect, the invention provides the VEGF antagonist of
the invention for treating and/or prophylaxis of a cancer e.g. a
solid tumour. In one embodiment, the solid tumour is selected from
the group consisting of lung, colorectal, head and neck,
pancreatic, breast, prostate, or ovarian cancer.
[0065] In one aspect, the invention provides the VEGF antagonist of
the invention for treating and/or prophylaxis of a vascular
proliferative disease for example angiogenesis, athersclesosis, and
vascular proliferative disease in the eye such as AMD (Age Related
Macular Degeneration).
[0066] In one aspect, the invention provides the VEGF antagonist of
the invention for treating and/or prophylaxis of an inflammatory
condition. In one aspect, the invention provides the use of the
VEGF antagonist in the manufacture of a medicament for treating
and/or prophylaxis of an inflammatory condition. In one embodiment,
the condition is selected from the group consisting of arthritis,
multiple sclerosis, inflammatory bowel disease and chronic
obstructive pulmonary disease. For example, In one aspect, the
invention provides the VEGF antagonist for treating and/or
prophylaxis of a respiratory disease. In one aspect, the invention
provides the use of the VEGF antagonist in the manufacture of a
medicament for treating and/or prophylaxis of a respiratory
disease. For example, said respiratory disease is selected from the
group consisting of lung inflammation, chronic obstructive
pulmonary disease, asthma, pneumonia, hypersensitivity pneumonitis,
pulmonary infiltrate with eosinophilia, environmental lung disease,
pneumonia, bronchiectasis, cystic fibrosis, interstitial lung
disease, primary pulmonary hypertension, pulmonary thromboembolism,
disorders of the pleura, disorders of the mediastinum, disorders of
the diaphragm, hypoventilation, hyperventilation, sleep apnea,
acute respiratory distress syndrome, mesothelioma, sarcoma, graft
rejection, graft versus host disease, lung cancer, allergic
rhinitis, allergy, asbestosis, aspergilloma, aspergillosis,
bronchiectasis, chronic bronchitis, emphysema, eosinophilic
pneumonia, idiopathic pulmonary fibrosis, invasive pneumococcal
disease, influenza, nontuberculous mycobacteria, pleural effusion,
pneumoconiosis, pneumocytosis, pneumonia, pulmonary actinomycosis,
pulmonary alveolar proteinosis, pulmonary anthrax, pulmonary edema,
pulmonary embolus, pulmonary inflammation, pulmonary histiocytosis
X, pulmonary hypertension, pulmonary nocardiosis, pulmonary
tuberculosis, pulmonary veno-occlusive disease, rheumatoid lung
disease, sarcoidosis, and Wegener's granulomatosis. For example,
the disease is chronic obstructive pulmonary disease (COPD). For
example, the disease is asthma.
[0067] An antagonist of the invention comprising an agent that
inhibits VEGF (e.g., wherein the agent is selected from the group
consisting of antibody fragments (e.g, Fab fragment, Fab' fragment,
Fv fragment (e.g., scFv, disulfide bonded Fv), F(ab').sub.2
fragment, dAb), ligands and dAb monomers and multimers (eg, homo-
or heterodimers) can be locally administered to tissue or organs
e.g. to pulmonary tissue (e.g., lung) or eye of a subject using any
suitable method. For example, an agent can be locally administered
to pulmonary tissue via inhalation or intranasal administration.
For inhalation or intranasal administration, the antagonist of VEGF
can be administered using a nebulizer, inhaler, atomizer,
aerosolizer, mister, dry powder inhaler, metered dose inhaler,
metered dose sprayer, metered dose mister, metered dose atomizer,
or other suitable inhaler or intranasal delivery device. Thus, in
one embodiment, the invention provides a pulmonary delivery device
containing the VEGF antagonist. In one embodiment, the device is an
inhaler or an intranasal delivery device.
[0068] In one embodiment, an agent can be locally administered to
the eye via an implantable delivery device. Thus, in one
embodiment, the invention provides a implantable delivery device
containing the VEGF antagonist
[0069] In one aspect, the invention provides an oral formulation
comprising the VEGF antagonist. The formulation can be a tablet,
pill, capsule, liquid or syrup. In one aspect, the invention
provides an ocular formulation for delivery to the eye comprising
the VEGF antagonist e.g. the formulation can be liquid eye drops or
a gel.
[0070] In one embodiment, the invention provides a pulmonary
formulation for delivery to the lung, wherein the formulation
comprise an antagonist, polypeptide or variable domain of the
invention with a particle size range of less than 5 microns, for
example less than 4.5, 4, 3.5 or 3 microns (eg, when in
Britton-Robinson buffer, eg at a pH of 6.5 to 8.0, eg at a pH of 7
to 7.5, eg at pH7 or at pH7.5).
[0071] In one embodiment, the formulations and compositions of the
invention are provided at a pH from 6.5 to 8.0, for example 7 to
7.5, for example 7, for example 7.5.
[0072] Variable domains according to any aspect of the invention
may have a Tm of at least 50.degree. C., or at least 55.degree. C.,
or at least 60.degree. C., or at least 65.degree. C., or at least
70.degree. C. An antagonist, use, method, device or formulation of
the invention may comprise such a variable domain.
[0073] In one aspect of the invention, the polypeptides, variable
domains, antagonists, compositions or formulations of the invention
are substantially stable after incubation (at a concentration of
polypeptide or variable domain of 1 mg/ml) at 37 to 50.degree. C.
for 14 days in Britton Robinson or PBS buffer. In one embodiment,
at least 65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99% of the polypeptide, antagonist or variable domain
remains unaggregated after such incubation at 37 degrees C. In one
embodiment, at least 65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99% of the polypeptide or variable domain
remains monomeric after such incubation at 37 degrees C. In one
embodiment, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% of the polypeptide, antagonist or variable domain remains
unaggregated after such incubation at 50 degrees C. In one
embodiment, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% of the polypeptide or variable domain remains monomeric after
such incubation at 50 degrees C. In one embodiment, no aggregation
of the polypeptides, variable domains, antagonists is seen after
any one of such incubations. In one embodiment, the pI of the
polypeptide or variable domain remains unchanged or substantially
unchanged after incubation at 37 degrees C. at a concentration of
polypeptide or variable domain of 1 mg/ml in Britton-Robinson
buffer.
[0074] In one aspect of the invention, the polypeptides, variable
domains, antagonists, compositions or formulations of the invention
are substantially stable after incubation (at a concentration of
polypeptide or variable domain of 100 mg/ml) at 4.degree. C. for 7
days in Britton Robinson buffer or PBS at a pH of 7 to 7.5 (eg, at
pH7 or pH7.5). In one embodiment, at least 95, 95.5, 96, 96.5, 97,
97.5, 98, 98.5, 99 or 99.5% of the polypeptide, antagonist or
variable domain remains unaggregated after such incubation. In one
embodiment, at least 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or
99.5% of the polypeptide or variable domain remains monomeric after
such incubation. In one embodiment, no aggregation of the
polypeptides, variable domains, antagonists is seen after any one
of such incubations.
[0075] In one aspect of the invention, the polypeptides, variable
domains, antagonists, compositions or formulations of the invention
are substantially stable after nebulisation (e.g. at a
concentration of polypeptide or variable domain of 40mg/m1) eg, at
room temperature, 20 degrees C. or 37.degree. C., for 1 hour, eg
jet nebuliser, eg a in a Pari LC+cup. In one embodiment, at least
65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 95.5, 96,
96.5, 97, 97.5, 98, 98.5, 99 or 99.5% of the polypeptide,
antagonist or variable domain remains unaggregated after such
nebulisation. In one embodiment, at least 65, 70, 75, 80, 85, 86,
87, 88, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5,
99 or 99.5% of the polypeptide or variable domain remains monomeric
after such nebulisation. In one embodiment, no aggregation of the
polypeptides, variable domains, antagonists is seen after any one
of such nebulisation.
[0076] In one aspect, the invention provides an isolated or
recombinant nucleic acid encoding a polypeptide comprising an
immunoglobulin single variable domain according to any aspect of
the invention or encoding a polypeptide, antagonist or variable
domain according to any aspect of the invention. In one aspect, the
invention provides a vector comprising the nucleic acid. In one
aspect, the invention provides a host cell comprising the nucleic
acid or the vector. In one aspect, the invention provides a method
of producing polypeptide comprising an immunoglobulin single
variable domain, the method comprising maintaining the host cell
under conditions suitable for expression of said nucleic acid or
vector, whereby a polypeptide comprising an immunoglobulin single
variable domain is produced. The method may further comprise
isolating the polypeptide, variable domain or antagonist and
optionally producing a variant, eg a mutated variant, having an
improved affinity and/or ND50 than the isolated polypeptide
variable domain or antagonist. Techniques for improving binding
affinity of immunoglobulin single variable domain are known in the
art, eg techniques for affinity maturation.
[0077] In one aspect, the invention provides a pharmaceutical
composition comprising an immunoglobulin single variable domain,
polypeptide or an antagonist of any aspect of the invention, and a
pharmaceutically acceptable carrier, excipient or diluent.
[0078] In one embodiment, the immunoglobulin single variable domain
or the antagonist of any aspect of the invention comprises an
antibody constant domain, for example, an antibody Fc, optionally
wherein the N-terminus of the Fc is linked (optionally directly
linked) to the C-terminus of the variable domain. The amino acid
sequence of a suitable Fc is shown in FIG. 52b.
[0079] The polypeptide or variable domain of the invention can be
isolated and/or recombinant.
[0080] In one aspect, the invention is a method for selecting a
protease resistant peptide or polypeptide, for example an
antagonist of vascular endothelial growth factor (VEGF), e.g. an
anti-VEGF dAb. The method comprises providing a repertoire of
peptides or polypeptides, combining the repertoire and a protease
under conditions suitable for protease activity, and recovering a
peptide or polypeptide that has a desired biological activity,
whereby a protease resistant peptide or polypeptide is
selected.
[0081] The repertoire and the protease are generally incubated for
a period of at least about 30 minutes. Any desired protease can be
used in the method, such as one or more of the following, serine
protease, cysteine protease, aspartate proteases, thiol proteases,
matrix metalloprotease, carboxypeptidase (e.g., carboxypeptidase A,
carboxypeptidase B), trypsin, chymotrypsin, pepsin, papain,
elastase, leukozyme, pancreatin, thrombin, plasmin, cathepsins
(e.g., cathepsin G), proteinase (e.g., proteinase 1, proteinase 2,
proteinase 3), thermolysin, chymosin, enteropeptidase, caspase
(e.g., caspase 1, caspase 2, caspase 4, caspase 5, caspase 9,
caspase 12, caspase 13), calpain, ficain, clostripain, actinidain,
bromelain, and separase. In particular embodiments, the protease is
trypsin, elastase or leucozyme. The protease can also be provided
by a biological extract, biological homogenate or biological
preparation. If desired, the method further comprises adding a
protease inhibitor to the combination of the repertoire and the
protease after incubation is complete.
[0082] In some embodiments, a peptide or polypeptide that has a
desired biological activity is recovered based on a binding
activity. For example, the peptide or polypeptide can be recovered
based on binding a generic ligand, such as protein A, protein G or
protein L. The binding activity can also be specific binding to a
target ligand. Exemplary target ligands include ApoE, Apo-SAA,
BDNF, Cardiotrophin-1, CEA, CD40, CD40 Ligand, CD56, CD38, CD138,
EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2,
FAP.alpha., FGF-acidic, FGF-basic, fibroblast growth factor-10,
FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-.beta.1,
human serum albumin, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-1 receptor, IL-1 receptor type 1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,
IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),
Inhibin .alpha., Inhibin .beta.3, IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte
attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1
(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG,
MIP-1.alpha., MIP-1.beta., MIP-3.alpha., MIP-3.beta., MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin,
Nerve growth factor, .beta.-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA,
PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha., SDF1.alpha., SCF,
SCGF, stem cell factor (SCF), TARC, TGF-.alpha., TGF-.beta.,
TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGF
receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-.beta.,
GRO-.gamma., HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, serum
albumin, vWF, amyloid proteins (e.g., amyloid alpha), MMP12, PDK1,
IgE, IL-13R.alpha.1, IL-13Ra2, IL-15, IL-15R, IL-16, IL-17R, IL-17,
IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2, CD4, CD11a, CD23, CD25,
CD27, CD28, CD30, CD40, CD4OL, CD56, CD138, ALK5, EGFR, FcER1,
TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1), chymase, FGF,
Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2,
Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFN.alpha., I-309,
integrins, L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil
elastase, osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1,
siglec8, TARC, TGFb, Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF,
VLA-4, VCAM, .alpha.4.beta.7, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8,
alphavbeta6, alphavbeta8, cMET, CD8, vWF, amyloid proteins (e.g.,
amyloid alpha), MMP12, PDK1, and IgE.
[0083] In particular embodiments, the peptide or polypeptide is
recovered by panning
[0084] In some embodiments, the repertoire comprises a display
system. For example, the display system can be bacteriophage
display, ribosome display, emulsion compartmentalization and
display, yeast display, puromycin display, bacterial display,
display on plasmid, or covalent display. Exemplary display systems
link coding function of a nucleic acid and functional
characteristics of the peptide or polypeptide encoded by the
nucleic acid. In particular embodiments, the display system
comprises replicable genetic packages.
[0085] In some embodiments, the display system comprises
bacteriophage display. For example, the bacteriophage can be fd,
M13, lambda, MS2 or T7. In particular embodiments, the
bacteriophage display system is multivalent. In some embodiments,
the peptide or polypeptide is displayed as a pIII fusion
protein.
[0086] In other embodiments, the method further comprises
amplifying the nucleic acid encoding a peptide or polypeptide that
has a desired biological activity. In particular embodiments, the
nucleic acid is amplified by phage amplification, cell growth or
polymerase chain reaction.
[0087] In some embodiments, the repertoire is a repertoire of
immunoglobulin single variable domains, which for example are bind
to and are antagonists of vascular endothelial growth factor
(VEGF). In particular embodiments, the immunoglobulin single
variable domain is a heavy chain variable domain. In more
particular embodiments, the heavy chain variable domain is a human
heavy chain variable domain. In other embodiments, the
immunoglobulin single variable domain is a light chain variable
domain. In particular embodiments, the light chain variable domain
is a human light chain variable domain.
[0088] In another aspect, the invention is a method for selecting a
peptide or polypeptide that binds a target ligand e.g. VEGF, with
high affinity from a repertoire of peptides or polypeptides. The
method comprises providing a repertoire of peptides or
polypeptides, combining the repertoire and a protease under
conditions suitable for protease activity, and recovering a peptide
or polypeptide that binds the target ligand.
[0089] The repertoire and the protease are generally incubated for
a period of at least about 30 minutes. Any desired protease can be
used in the method, such as one or more of the following, serine
protease, cysteine protease, aspartate proteases, thiol proteases,
matrix metalloprotease, carboxypeptidase (e.g., carboxypeptidase A,
carboxypeptidase B), trypsin, chymotrypsin, pepsin, papain,
elastase, leukozyme, pancreatin, thrombin, plasmin, cathepsins
(e.g., cathepsin G), proteinase (e.g., proteinase 1, proteinase 2,
proteinase 3), thermolysin, chymosin, enteropeptidase, caspase
(e.g., caspase 1, caspase 2, caspase 4, caspase 5, caspase 9,
caspase 12, caspase 13), calpain, ficain, clostripain, actinidain,
bromelain, and separase. In particular embodiments, the protease is
trypsin, elastase or leucozyme. The protease can also be provided
by a biological extract, biological homogenate or biological
preparation. If desired, the method further comprises adding a
protease inhibitor to the combination of the repertoire and the
protease after incubation is complete.
[0090] The peptide or polypeptide can be recovered based on binding
any desired target ligand, such as the target ligands disclosed
herein. In particular embodiments, the peptide or polypeptide is
recovered by panning
[0091] In some embodiments, the repertoire comprises a display
system. For example, the display system can be bacteriophage
display, ribosome display, emulsion compartmentalization and
display, yeast display, puromycin display, bacterial display,
display on plasmid, or covalent display. Exemplary display systems
link coding function of a nucleic acid and functional
characteristics of the peptide or polypeptide encoded by the
nucleic acid. In particular embodiments, the display system
comprises replicable genetic packages.
[0092] In some embodiments, the display system comprises
bacteriophage display. For example, the bacteriophage can be fd,
M13, lambda, MS2 or T7. In particular embodiments, the
bacteriophage display system is multivalent. In some embodiments,
the peptide or polypeptide is displayed as a pIII fusion
protein.
[0093] In other embodiments, the method further comprises
amplifying the nucleic acid encoding a peptide or polypeptide that
has a desired biological activity. In particular embodiments, the
nucleic acid is amplified by phage amplification, cell growth or
polymerase chain reaction.
[0094] In some embodiments, the repertoire is a repertoire of
immunoglobulin single variable domains, e.g. which bind to and are
antagonists of VEGF. In particular embodiments, the immunoglobulin
single variable domain is a heavy chain variable domain. In more
particular embodiments, the heavy chain variable domain is a human
heavy chain variable domain. In other embodiments, the
immunoglobulin single variable domain is a light chain variable
domain. In particular embodiments, the light chain variable domain
is a human light chain variable domain.
[0095] In another aspect, the invention is a method of producing a
repertoire of protease resistant peptides or polypeptides. The
method comprises providing a repertoire of peptides or
polypeptides, combining the repertoire of peptides or polypeptides
and a protease under suitable conditions for protease activity, and
recovering a plurality of peptides or polypeptides that have a
desired biological activity, whereby a repertoire of protease
resistant peptides or polypeptides is produced.
[0096] In some embodiments, the repertoire and the protease are
incubated for a period of at least about 30 minutes. For example,
the protease used in the method can be one or more of the
following, serine protease, cysteine protease, aspartate proteases,
thiol proteases, matrix metalloprotease, carboxypeptidase (e.g.,
carboxypeptidase A, carboxypeptidase B), trypsin, chymotrypsin,
pepsin, papain, elastase, leukozyme, pancreatin, thrombin, plasmin,
cathepsins (e.g., cathepsin G), proteinase (e.g., proteinase 1,
proteinase 2, proteinase 3), thermolysin, chymosin,
enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4,
caspase 5, caspase 9, caspase 12, caspase 13), calpain, ficain,
clostripain, actinidain, bromelain, and separase. In particular
embodiments, the protease is trypsin, elastase or leucozyme. The
protease can also be provided by a biological extract, biological
homogenate or biological preparation. If desired, the method
further comprises adding a protease inhibitor to the combination of
the repertoire and the protease after incubation is complete.
[0097] In some embodiments, a plurality of peptides or polypeptides
that have a desired biological activity is recovered based on a
binding activity. For example, a plurality of peptides or
polypeptides can be recovered based on binding a generic ligand,
such as protein A, protein G or protein L. The binding activity can
also be specific binding to a target ligand, such as a target
ligand described herein. In particular embodiments, a plurality of
peptides or polypeptides that has the desired biological activity
is recovered by panning
[0098] In some embodiments, the repertoire comprises a display
system. For example, the display system can be bacteriophage
display, ribosome display, emulsion compartmentalization and
display, yeast display, puromycin display, bacterial display,
display on plasmid, or covalent display. In particular embodiments,
the display system links coding function of a nucleic acid and
functional characteristics of the peptide or polypeptide encoded by
the nucleic acid. In particular embodiments, the display system
comprises replicable genetic packages.
[0099] In some embodiments, the display system comprises
bacteriophage display. For example, the bacteriophage can be fd,
M13, lambda, MS2 or T7. In particular embodiments, the
bacteriophage display system is multivalent. In some embodiments,
the peptide or polypeptide is displayed as a pIII fusion
protein.
[0100] In other embodiments, the method further comprises
amplifying the nucleic acids encoding a plurality of peptides or
polypeptides that have a desired biological activity. In particular
embodiments, the nucleic acids are amplified by phage
amplification, cell growth or polymerase chain reaction.
[0101] In some embodiments, the repertoire is a repertoire of
immunoglobulin single variable domains, e.g. which bind to and are
antagonists of VEGF. In particular embodiments, the immunoglobulin
single variable domain is a heavy chain variable domain. In more
particular embodiments, the heavy chain variable domain is a human
heavy chain variable domain. In other embodiments, the
immunoglobulin single variable domain is a light chain variable
domain. In particular embodiments, the light chain variable domain
is a human light chain variable domain.
[0102] In another aspect, the invention is a method for selecting a
protease resistant polypeptide comprising an immunoglobulin single
variable domain (dAb) that binds a target ligand, e.g. VEGF from a
repertoire. In one embodiment, the method comprises providing a
phage display system comprising a repertoire of polypeptides that
comprise an immunoglobulin single variable domain, combining the
phage display system and a protease selected from the group
consisting of elastase, leucozyme and trypsin, under conditions
suitable for protease activity, and recovering a phage that
displays a polypeptide comprising an immunoglobulin single variable
domain that binds the target ligand.
[0103] In some embodiments, the protease is used at 100 .mu.g/ml,
and the combined phage display system and protease are incubated at
about 37.degree. C. overnight.
[0104] In some embodiments, the phage that displays a polypeptide
comprising an immunoglobulin single variable domain that binds the
target ligand is recovered by binding to said target. In other
embodiments, the phage that displays a polypeptide comprising an
immunoglobulin single variable domain that binds the target ligand
is recovered by panning
[0105] The invention also relates to an isolated protease resistant
peptide or polypeptide selectable or selected by the methods
described herein. In a particular embodiment, the invention relates
to an isolated protease (e.g., trypsin, elastase, leucozyme)
resistant immunoglobulin single variable domain (e.g., human
antibody heavy chain variable domain, human antibody light chain
variable domain) selectable or selected by the methods described
herein.
[0106] The invention also relates to an isolated or recombinant
nucleic acid that encodes a protease resistant peptide or
polypeptide (e.g., trypsin-, elastase-, or leucozyme-resistant
immunoglobulin single variable domain) selectable or selected by
the methods described herein, and to vectors (e.g., expression
vectors) and host cells that comprise the nucleic acids.
[0107] The invention also relates to a method for making a protease
resistant peptide or polypeptide (e.g., trypsin-, elastase-, or
leucozyme-resistant immunoglobulin single variable domain)
selectable or selected by the methods described herein, comprising
maintaining a host cell that contains a recombinant nucleic acid
encoding the protease resistant peptide or polypeptide under
conditions suitable for expression, whereby a protease resistant
peptide or polypeptide is produced.
[0108] The invention also relates to a protease resistant peptide
or polypeptide (e.g., trypsin-, elastase-, or leucozyme-resistant
immunoglobulin single variable domain) selectable or selected by
the methods described herein for use in medicine (e.g., for therapy
or diagnosis). The invention also relates to use of a protease
resistant peptide or polypeptide (e.g., trypsin-, elastase-, or
leucozyme-resistant immunoglobulin single variable domain)
selectable or selected by the methods described herein for the
manufacture of a medicament for treating disease. The invention
also relates to a method of treating a disease, comprising
administering to a subject in need thereof, an effective amount of
a protease resistant peptide or polypeptide (e.g., trypsin-,
elastase-, or leucozyme-resistant immunoglobulin single variable
domain) selectable or selected by the methods described herein.
[0109] The invention also relates to a diagnostic kit for determine
whether VEGF is present in a sample or how much VEGF is present in
a sample, comprising a polypeptide, immunoglobulin variable domain
(dAb), or antagonist of the invention and instructions for use
(e.g., to determine the presence and/or quantity of VEGF in the
sample). In some embodiments, the kit further comprises one or more
ancillary reagents, such as a suitable buffer or suitable detecting
reagent (e.g., a detectably labeled antibody or antigen-binding
fragment thereof that binds the polypeptide or dAb of the invention
or a moiety associated or conjugated thereto.
The invention also relates to a device comprising a solid surface
on which a polypeptide antagonist or dAb of the invention is
immobilized such that the immobilized polypeptide or dAb binds
VEGF. Any suitable solid surfaces on which an antibody or
antigen-binding fragment thereof can be immobilized can be used,
for example, glass, plastics, carbohydrates (e.g., agarose beads).
If desired the support can contain or be modified to contain
desired functional groups to facilitate immobilization. The device,
and or support, can have any suitable shape, for example, a sheet,
rod, strip, plate, slide, bead, pellet, disk, gel, tube, sphere,
chip, plate or dish, and the like. In some embodiments, the device
is a dipstick. The nucleic acid sequence of DOM15-26-595 referred
to herein (in the description and claims) is as follows:
TABLE-US-00001 DOM15-26-595:
GAGGTGCAGCTGTTGGTTTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAAGGCTTATCCGA
TGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGAG
ATCTCGCCTTCGGGTTCTTATACATACTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCT
CGGAAGATTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
BRIEF DESCRIPTION OF THE DRAWINGS
[0110] FIG. 1 is an illustration of the multiple cloning site of
pDOM13 (aka pDOM33), which was used to prepare a phage display
repertoire.
[0111] FIG. 2 shows several Novex 10-20% Tricene gels run with
samples from different time points of dAbs that were incubated with
trypsin at 40 g/ml at 30.degree. C. Samples were taken immediately
before the addition of trypsin, and then at one hour, three hours
and 24 hours after the addition of trypsin. The proteins were
stained with 1.times. SureBlue. The gels illustrate that both
DOM15-10 and DOM15-26-501 were significantly digested during the
first three hours of incubation with trypsin. Digestion of
DOM15-26, DOM4-130-54 and DOM1h-131-511 only became apparent after
24 hours of incubation with trypsin.
[0112] FIG. 3 is an illustration of the amino acid sequences of
DOM1h-131-511 and 24 selected variants. The amino acids that differ
from the parent sequence in selected clones are highlighted (those
that are identical are marked by dots). The loops corresponding to
CDR1, CDR2 and CDR3 are outlined with boxes.
[0113] FIG. 4 is an illustration of the amino acid sequences of
DOM4-130-54 and 27 selected variants. The amino acids that differ
from the parent sequence in selected clones are highlighted (those
that are identical are marked by dots). The loops corresponding to
CDR1, CDR2 and CDR3 are outlined with boxes.
[0114] FIG. 5 is an illustration of the amino acid sequence of
DOM15-26-555 and 21 selected variants. The amino acids that differ
from the parent sequence in selected clones are highlighted (those
that are identical are marked by dots). The loops corresponding to
CDR1, CDR2 and CDR3 are outlined with boxes.
[0115] FIG. 6 is an illustration of the amino acid sequence of
DOM15-10 and 16 selected variants. The amino acids that differ from
the parent sequence in selected clones are highlighted (those that
are identical are marked by dots). The loops corresponding to CDR1,
CDR2 and CDR3 are outlined with boxes.
[0116] FIGS. 7A-7D are BIAcore traces showing bind of a parent dAb,
DOM1h-131-511 (FIG. 7A) and three variant dAbs, DOM1h-131-203 (FIG.
7B), DOM1h-131-204 (FIG. 7C) and DOM1h-131-206 (FIG. 7D), to
immoblized TNFR1 after incubation with different concentrations of
trypsin (ranging from 0 to 100 .mu.g/ml) overnight at 37.degree. C.
The results show that all three variants are more resistant than
the parent to proteolysis at high concentrations of trypsin (100
ug/ml).
[0117] FIGS. 8A-8C are BIAcore traces showing binding of dAbs
DOM1h-131-511 (FIG. 8A), DOM1h-131-202 (FIG. 8B) and DOM1h-131-206
(FIG. 8C) to immobilized TNFR1 after incubation with elastase and
leucozyme overnight. The dAbs showed increased resistance to
proteolysis compared to the parent against both elastase and
leucozyme.
[0118] FIG. 9 shows two 4-12% Novex Bis-Tris gels run with samples
of dAbs DOM1h-131-511, DOM1h-131-203, DOM1h-131-204, DOM1h-131-206,
DOM1h-131-54, DOM1h-131-201, and DOM1h-131-202 before incubation
with trypsin and samples after incubation with 100 .mu.g/ml of
trypsin for 1 hour, 3 hours and 24 hours. FIGS. 10A-10C are BIAcore
traces showing binding of DOM4-130-54 (FIG. 10A), DOM4-130-201
(FIG. 10B) and DOM4-130-202 (FIG. 10C) to immobilized IL-1R1 fusion
protein after incubation with different concentrations of trypsin
(ranging from 0 to 100 .mu.g/ml) overnight at 37.degree. C. The
results show that both variants are more resistant than their
parent to proteolysis at high concentrations of trypsin (100
.mu.g/ml).
[0119] FIGS. 11A-11C are BIAcore traces showing binding of
DOM4-130-54 (FIG. 11A), DOM4-130-201 (FIG. 11B) and DOM4-130-202
(FIG. 11C) to immobilized IL-1R1 fusion protein after incubation
with elastase and leucozyme overnight. The dAbs showed increased
resistance to proteolysis compared to parent against both proteases
tested.
[0120] FIG. 12 is an illustration of the amino acid sequence of
DOM15-26-555and 6 variants. The amino acids that differ from the
parent sequence in selected clones are highlighted (those that are
identical are marked by dots).
[0121] FIGS. 13A and 13B are BIAcore traces showing binding of the
parent dAb, DOM15-26-555 (FIG. 13A) and the most protease resistant
variant, DOM15-26-593 (FIG. 13B) to immobilized VEGF. The parent
and the variant were compared on the BIAcore for hVEGF binding at
the dAb concentration of 100 nM after incubation with trypsin at a
concentration of 200 .mu.g/ml. The reaction was carried out for
three hours or 24 hours at 37.degree. C. The results show that the
variant is more resistant than the parent to proteolysis after 24
hours of trypsin treatment.
[0122] FIG. 14 is a graph showing effects of trypsin treatment on
hVEGF binding by DOM15-26-555 variants. The results clearly show
that all variants are more resistant than the parent (DOM15-26-555)
to proteolysis after 24 hours of trypsin treatment.
[0123] FIG. 15 shows two Novex 10-20% Tricine gels that were loaded
with 15 .mu.g of treated and untreated samples of DOM15-26-555 or
DOM15-26-593. Samples were taken immediately before the addition of
trypsin, and then at one hour, three hours and 24 hours after the
addition of trypsin. The proteins were stained with 1.times.
SureBlue. The gels illustrate that the trypsin resistance profile
of DOM15-26-593 varied from the profile shown by the BIAcore
experiment.
[0124] FIG. 16 is an illustration of the amino acid sequence of
DOM15-10 and a variant, DOM15-10-11. The amino acids that differ
from the parent sequence in the variant are highlighted (those that
are identical are marked by dots).
[0125] FIGS. 17A and 17B are BIAcore traces showing binding of the
parent, DOM15-10 (FIG. 17A) and the variant, DOM15-10-11 (FIG.
17B), to immobilized VEGF. The parent and the variant were compared
on the BIAcore for hVEGF binding at the dAb concentration of 100 nM
after incubation with trypsin at a concentration of 200 .mu.g/ml.
The reaction was carried out for one hour, three hours and 24 hours
at 37.degree. C. The results show that the variant is more
resistant than the parent to proteolysis after 24 hours of trypsin
treatment.
[0126] FIG. 18 shows two Novex 10-20% Tricene gels that were loaded
with 15 .mu.g of samples of DOM15-10 and DOM15-10-11. Samples were
taken immediately before the addition of trypsin, and then at one
hour, three hours, and 24 hours after the addition of trypsin. The
proteins were stained with SureBlue (1.times.). The results show
that the binding activity seen in the BIAcore study directly
reflects the protein's integrity.
[0127] FIGS. 19A-19L illustrate the nucleotide sequences of several
nucleic acids encoding dAbs that are variants of DOM1h-131-511 or
DOM4-130-54. The nucleotide sequences encode the amino acid
sequences presented in FIG. 3 and FIG. 4, respectively.
[0128] FIGS. 20A-20E illustrate the nucleotide sequences of several
nucleic acids encoding dAbs that are variants of DOM15-26-555 or
DOM15-10. The nucleotide sequences encode the amino acid sequences
presented in FIG. 5 and FIG. 6, respectively.
[0129] FIG. 21 shows a vector map of pDOM 38.
[0130] FIG. 22: Shows a Gel run on Labchip of DOM10-53-474 and
DOM15-26-593 proteins treated with trypsin at 25:1 dAb:trypsin
ratio at 30.degree. C. for different time points. Arrows show full
length protein.
[0131] FIG. 23: Is a Size exclusion chromatography trace showing
the high level of purity obtained for each sample after
purification by MMC chromatography followed by anion exchange. The
UV was monitored at 225 nm and the column was run in 1.times. PBS
with 10% ethanol (v/v). The percentage monomer was calculated by
integration of the peak area with baseline correction.
[0132] FIG. 24: Shows Protease stability data for DOM1h-131-511,
DOM1h-131-202 and DOM1h-131-206.
[0133] FIG. 25: Is an SEC which illustrates 14 day stability data
of DOM1.times.h-131-202, DOM1h-131-206 and DOM1h-131-511 in
Britton-Robinson buffer at 37 and 50.degree. C. The protein
concentration for all the dAbs was 1 mg/ml. SEC was used to
determine if any changes had occurred in the protein during thermal
stress and the amount of monomer left in solution relative to the
time=0 (T0) sample.
[0134] FIGS. 26A to I: Show SEC traces showing the effect of
thermal stress (37 and 50.degree. C.) on DOM1h-131-511 (A to C),
-202 (D to F) and -206 (G to I). Also shown is the percentage of
monomer left in solution relative to the T=0 at the given time
point.
[0135] FIG. 27: Shows IEF analysis of DOM1h-131-202, DOM1h-131-206
and DOM1h-131-511 at 24 hr, 48 hr and 7 and 14 days thermal stress.
The samples had been incubated at either 37 or 50.degree. C. in
Britton-Robinson buffer.
[0136] FIG. 28: TNFR-1 RBA showing the effect of 14 days incubation
of DOM1h-131-202, DOM1h-131-206 and DOM1h-131-511 at 50.degree. C.
The protein concentration was assumed to be 1 mg/ml. A negative
control dAb (VH dummy) which does not bind antigen is also
shown.
[0137] FIG. 29: Illustrates Effects of storing A: DOM1h-131-202, B:
DOM1h-131-206 and C: DOM1h-131-511 at .about.100 mg/ml for 7 days
in Britton-Robinson buffer at +4.degree. C. The UV was monitored at
280 nm.
[0138] FIG. 30: Shows data from Nebuliser testing of DOM1h-131-202,
DOM1h-131-206 and DOM1h-131-511 in the Pari E-flow and LC+. The
protein concentration was 5 mg/ml in either Britton-Robinson
buffer.
[0139] FIG. 31: Illustrates the Relative percentage changes in
monomer concentrations during nebulisation of DOM1h-131-202,
DOM1h-131-206 and DOM1h-131-511 in Britton-Robinson buffer at 5
mg/ml.
[0140] FIG. 32: Shows SEC traces of DOM1h-131-206 and DOM1h-131-511
in Britton-Robinson buffer post nebulisation from the Pari LC+.
[0141] FIG. 33: Shows SEC traces of DOM1h-131-206 during the
nebulisation process over 1 hour at 40 mg/ml in PBS. The protein in
both the nebuliser cup and aerosol are highly resistance to the
effects of shear and thermal stress that may be experienced by the
dAb during nebulisation.
[0142] FIG. 34: Shows the sedimentation velocity curves for each of
the three lead proteins (DOM1h-131-206 and DOM1h-131-511 and
DOM1h-131-202) The bimodal peak observed for the lower
concentration sample of DOM1h-131-206 is an artefact owing to a
sample leak from the cell in this instance.
[0143] FIG. 35: Shows the effect of buffer and device on nebulised
droplet size of GSK 1995056A (DOM1h-131-511).
[0144] FIG. 36: Stability of GSK1995056A (DOM1h-131-511) after
nebulisation in various devices assessed by dimer formation as
measured by SEC.
[0145] FIG. 37: Shows Nebuliser testing of GSK1922567A (202),
GSK1995057A (206) and GSK1995056A (511) in the Pari E-flow and LC+.
A) testing in Britton-Robinson buffer, B) testing in
PEG1000/sucrose buffer.
[0146] FIG. 38: Depicts a TNF-.alpha. dose curve in the human TNFR1
receptor binding assay. Each sample was tested as four
replicates.
[0147] FIG. 39: Shows Inhibition by GSK1922567A(DOM1h-131-202),
GSK1995057A (DOM1h-131-206) and GSK1995056A (DOM1h-131-511) in the
human TNFRI receptor binding assay. Each sample was tested as four
replicates.
[0148] FIG. 40: Illustrates potency of the DOM15-26 and
DOM15-26-593 dAbs in the VEGF RBA.
[0149] FIG. 41: Shows pharmacokinetics of DMS1529 (DOM 15-26-593)
and DMS1545 (DOM15-26-501) after single bolus dose i.v.
administration to rats at 5 mg/mg
[0150] FIG. 42a: Shows SEC-MALLs (Size exclusion
chromatograph-multi-angle laser light scattering) analysis of
DMS1529 Fc fusion (DOM 15-26-593 Fc fusion) confirming monomeric
properties. Two different batches are shown that demonstrate
similar properties with regard to refractive index (i.e.
concentration; broken lines) and light scattering (solid lines).
The line marked with the arrow signifies the molecular mass
calculation.
[0151] FIG. 42b: Shows AUC (analytical ultracentrifugation)
analysis of DMS 1529 Fc fusion (DOM 15-26-593 Fc fusion) confirming
monomeric properties. One batch of material was tested at three
different concentrations, approximating to 0.2, 0.5 & 1.0 mg/ml
in PBS buffer. The analysis of the sedimentation rate confirmed a
molecular mass of approx. 80 kDa.
[0152] FIG. 43: Shows DSC traces of DMS1529 (DOM15-26-593) and
DOM15-26-501.
[0153] FIG. 44: Is a VEGF Binding ELISA for DMS1529 (DOM 15-26-593)
before and after, 10 freeze-thaw cycles on two different batches of
material.
[0154] FIG. 45: Shows the consistency of DOM 15-26-593 SEC profile
before and after 10 freeze thaw cycles.
[0155] FIG. 46: Illustrates results from an accelerated stability
study of the DMS 1529 fusion (DOM 15-26-593 Fc fusion); binding
ELISA demonstrating activity after 7 days incubation at the
temperature shown.
[0156] FIG. 47A: Shows stability of DMS1529 (DOM 15-26-593) in
human cynomolgus after 14 & 15 days incubation at 37.degree.
C.
[0157] FIG. 47B: Shows stability of DMS1529 (DOM 15-26-593) in
human serum after 14 & 15 days incubation at 37.degree. C.
[0158] FIG. 48: Shows potency of DOM15-26 & DOM15-26-593 dAbs
as Fc fusions (DMS1564 & 1529 respectively) in the VEGF
RBA.
[0159] FIG. 49: Illustrates inhibition of HUVEC cell proliferation
by the DMS 1529 fusion (DOM15-26-593 FC fusion).
[0160] FIG. 50: pDom33 vector map.
[0161] FIG. 51a: Depicts amino acid sequences of dAbs that bind
serum albumin.
[0162] FIG. 51b: Depicts nucleic acid sequences of dAbs that bind
serum albumin.
[0163] FIG. 52a: Depicts the amino acid sequence of DOM15-26-593-Fc
fusion
[0164] FIG. 52b: Depicts the amino acid sequence of an antibody
Fc
[0165] FIG. 52c: Depicts the nucleic acid sequence of
DOM15-26-593-Fc fusion.
DETAILED DESCRIPTION OF THE INVENTION
[0166] Within this specification the invention has been described,
with reference to embodiments, in a way which enables a clear and
concise specification to be written. It is intended and should be
appreciated that embodiments may be variously combined or separated
without parting from the invention.
[0167] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods.
[0168] As used herein, the term "antagonist of vascular endothelial
growth factor (VEGF)" or "anti-VEGF antagonist" or the like refers
to an agent (e.g., a molecule, a compound) which binds VEGF and can
inhibit a (i.e., one or more) function of VEGF.
[0169] As used herein, "peptide" refers to about two to about 50
amino acids that are joined together via peptide bonds.
[0170] As used herein, "polypeptide" refers to at least about 50
amino acids that are joined together by peptide bonds. Polypeptides
generally comprise tertiary structure and fold into functional
domains.
[0171] As used herein, a peptide or polypeptide (e.g. a domain
antibody (dAb)) that is "resistant to protease degradation" is not
substantially degraded by a protease when incubated with the
protease under conditions suitable for protease activity. A
polypeptide (e.g., a dAb) is not substantially degraded when no
more than about 25%, no more than about 20%, no more than about
15%, no more than about 14%, no more than about 13%, no more than
about 12%, no more than about 11%, no more than about 10%, no more
than about 9%, no more than about 8%, no more than about 7%, no
more than about 6%, no more than about 5%, no more than about 4%,
no more than about 3%, no more that about 2%, no more than about
1%, or substantially none of the protein is degraded by protease
after incubation with the protease for about one hour at a
temperature suitable for protease activity. For example at 37 or 50
degrees C. Protein degradation can be assessed using any suitable
method, for example, by SDS-PAGE or by functional assay (e.g.,
ligand binding) as described herein.
[0172] As used herein, "display system" refers to a system in which
a collection of polypeptides or peptides are accessible for
selection based upon a desired characteristic, such as a physical,
chemical or functional characteristic. The display system can be a
suitable repertoire of polypeptides or peptides (e.g., in a
solution, immobilized on a suitable support). The display system
can also be a system that employs a cellular expression system
(e.g., expression of a library of nucleic acids in, e.g.,
transformed, infected, transfected or transduced cells and display
of the encoded polypeptides on the surface of the cells) or an
acellular expression system (e.g., emulsion compartmentalization
and display). Exemplary display systems link the coding function of
a nucleic acid and physical, chemical and/or functional
characteristics of a polypeptide or peptide encoded by the nucleic
acid. When such a display system is employed, polypeptides or
peptides that have a desired physical, chemical and/or functional
characteristic can be selected and a nucleic acid encoding the
selected polypeptide or peptide can be readily isolated or
recovered. A number of display systems that link the coding
function of a nucleic acid and physical, chemical and/or functional
characteristics of a polypeptide or peptide are known in the art,
for example, bacteriophage display (phage display, for example
phagemid display), ribosome display, emulsion compartmentalization
and display, yeast display, puromycin display, bacterial display,
display on plasmid, covalent display and the like. (See, e.g., EP
0436597 (Dyax), U.S. Pat. No. 6,172,197 (McCafferty et al.), U.S.
Pat. No. 6,489,103 (Griffiths et al.).)
[0173] As used herein, "repertoire" refers to a collection of
polypeptides or peptides that are characterized by amino acid
sequence diversity. The individual members of a repertoire can have
common features, such as common structural features (e.g., a common
core structure) and/or common functional features (e.g., capacity
to bind a common ligand (e.g., a generic ligand or a target
ligand)).
[0174] As used herein, "functional" describes a polypeptide or
peptide that has biological activity, such as specific binding
activity. For example, the term "functional polypeptide" includes
an antibody or antigen-binding fragment thereof that binds a target
antigen through its antigen-binding site.
[0175] As used herein, "generic ligand" refers to a ligand that
binds a substantial portion (e.g., substantially all) of the
functional members of a given repertoire. A generic ligand (e.g., a
common generic ligand) can bind many members of a given repertoire
even though the members may not have binding specificity for a
common target ligand. In general, the presence of a functional
generic ligand-binding site on a polypeptide (as indicated by the
ability to bind a generic ligand) indicates that the polypeptide is
correctly folded and functional. Suitable examples of generic
ligands include superantigens, antibodies that bind an epitope
expressed on a substantial portion of functional members of a
repertoire, and the like.
[0176] "Superantigen" is a term of art that refers to generic
ligands that interact with members of the immunoglobulin
superfamily at a site that is distinct from the target
ligand-binding sites of these proteins. Staphylococcal enterotoxins
are examples of superantigens which interact with T-cell receptors.
Superantigens that bind antibodies include Protein G, which binds
the IgG constant region (Bjorck and Kronvall, J. Immunol., 133:969
(1984)); Protein A which binds the IgG constant region and V.sub.H
domains (Forsgren and Sjoquist, J. Immunol., 97:822 (1966)); and
Protein L which binds V.sub.L domains (Bjorck, J. Immunol.,
140:1194 (1988)).
[0177] As used herein, "target ligand" refers to a ligand which is
specifically or selectively bound by a polypeptide or peptide. For
example, when a polypeptide is an antibody or antigen-binding
fragment thereof, the target ligand can be any desired antigen or
epitope. Binding to the target antigen is dependent upon the
polypeptide or peptide being functional.
[0178] As used herein an antibody refers to IgG, IgM, IgA, IgD or
IgE or a fragment (such as a Fab , F(ab').sub.2, Fv, disulphide
linked Fv, scFv, closed conformation multispecific antibody,
disulphide-linked scFv, diabody) whether derived from any species
naturally producing an antibody, or created by recombinant DNA
technology; whether isolated from serum, B-cells, hybridomas,
transfectomas, yeast or bacteria.
[0179] As used herein, "antibody format" refers to any suitable
polypeptide structure in which one or more antibody variable
domains can be incorporated so as to confer binding specificity for
antigen on the structure. A variety of suitable antibody formats
are known in the art, such as, chimeric antibodies, humanized
antibodies, human antibodies, single chain antibodies, bispecific
antibodies, antibody heavy chains, antibody light chains,
homodimers and heterodimers of antibody heavy chains and/or light
chains, antigen-binding fragments of any of the foregoing (e.g., a
Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv),
a Fab fragment, a Fab' fragment, a F(ab').sub.2 fragment), a single
antibody variable domain (e.g., a dAb, V.sub.H, V.sub.HH, V.sub.L),
and modified versions of any of the foregoing (e.g., modified by
the covalent attachment of polyethylene glycol or other suitable
polymer or a humanized V.sub.HH).
[0180] The phrase "immunoglobulin single variable domain" refers to
an antibody variable domain (V.sub.H, V.sub.HH, V.sub.L) that
specifically binds an antigen or epitope independently of other V
regions or domains. An immunoglobulin single variable domain can be
present in a format (e.g., homo- or hetero-multimer) with other
variable regions or variable domains where the other regions or
domains are not required for antigen binding by the single
immunoglobulin variable domain (i.e., where the immunoglobulin
single variable domain binds antigen independently of the
additional variable domains). A "domain antibody" or "dAb" is the
same as an "immunoglobulin single variable domain" as the term is
used herein. A "single immunoglobulin variable domain" is the same
as an "immunoglobulin single variable domain" as the term is used
herein. A "single antibody variable domain" is the same as an
"immunoglobulin single variable domain" as the term is used herein.
An immunoglobulin single variable domain is in one embodiment a
human antibody variable domain, but also includes single antibody
variable domains from other species such as rodent (for example, as
disclosed in WO 00/29004, the contents of which are incorporated
herein by reference in their entirety), nurse shark and Camelid
V.sub.HH dAbs. Camelid V.sub.HH are immunoglobulin single variable
domain polypeptides that are derived from species including camel,
llama, alpaca, dromedary, and guanaco, which produce heavy chain
antibodies naturally devoid of light chains. The V.sub.HH may be
humanized.
[0181] A "domain" is a folded protein structure which has tertiary
structure independent of the rest of the protein. Generally,
domains are responsible for discrete functional properties of
proteins, and in many cases may be added, removed or transferred to
other proteins without loss of function of the remainder of the
protein and/or of the domain. A "single antibody variable domain"
is a folded polypeptide domain comprising sequences characteristic
of antibody variable domains. It therefore includes complete
antibody variable domains and modified variable domains, for
example, in which one or more loops have been replaced by sequences
which are not characteristic of antibody variable domains, or
antibody variable domains which have been truncated or comprise N-
or C-terminal extensions, as well as folded fragments of variable
domains which retain at least the binding activity and specificity
of the full-length domain.
[0182] The term "library" refers to a mixture of heterogeneous
polypeptides or nucleic acids. The library is composed of members,
each of which has a single polypeptide or nucleic acid sequence. To
this extent, "library" is synonymous with "repertoire." Sequence
differences between library members are responsible for the
diversity present in the library. The library may take the form of
a simple mixture of polypeptides or nucleic acids, or may be in the
form of organisms or cells, for example bacteria, viruses, animal
or plant cells and the like, transformed with a library of nucleic
acids. In one embodiment, each individual organism or cell contains
only one or a limited number of library members. In one embodiment,
the nucleic acids are incorporated into expression vectors, in
order to allow expression of the polypeptides encoded by the
nucleic acids. In an aspect, therefore, a library may take the form
of a population of host organisms, each organism containing one or
more copies of an expression vector containing a single member of
the library in nucleic acid form which can be expressed to produce
its corresponding polypeptide member. Thus, the population of host
organisms has the potential to encode a large repertoire of diverse
polypeptides.
[0183] A "universal framework" is a single antibody framework
sequence corresponding to the regions of an antibody conserved in
sequence as defined by Kabat ("Sequences of Proteins of
Immunological Interest", US Department of Health and Human
Services) or corresponding to the human germline immunoglobulin
repertoire or structure as defined by Chothia and Lesk, (1987) J.
Mol. Biol. 196:910-917. Libraries and repertoires can use a single
framework, or a set of such frameworks, which has been found to
permit the derivation of virtually any binding specificity though
variation in the hypervariable regions alone.
[0184] As used herein, the term "dose" refers to the quantity of
ligand administered to a subject all at one time (unit dose), or in
two or more administrations over a defined time interval. For
example, dose can refer to the quantity of ligand (e.g., ligand
comprising an immunoglobulin single variable domain that binds
target antigen) administered to a subject over the course of one
day (24 hours) (daily dose), two days, one week, two weeks, three
weeks or one or more months (e.g., by a single administration, or
by two or more administrations). The interval between doses can be
any desired amount of time.
[0185] The phrase, "half-life," refers to the time taken for the
serum concentration of the ligand (eg, dAb, polypeptide or
antagonist) to reduce by 50%, in vivo, for example due to
degradation of the ligand and/or clearance or sequestration of the
ligand by natural mechanisms. The ligands of the invention are
stabilized in vivo and their half-life increased by binding to
molecules which resist degradation and/or clearance or
sequestration. Typically, such molecules are naturally occurring
proteins which themselves have a long half-life in vivo. The
half-life of a ligand is increased if its functional activity
persists, in vivo, for a longer period than a similar ligand which
is not specific for the half-life increasing molecule. For example,
a ligand specific for human serum albumin (HAS) and a target
molecule is compared with the same ligand wherein the specificity
to HSA is not present, that is does not bind HSA but binds another
molecule. For example, it may bind a third target on the cell.
Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50% or
more. Increases in the range of 2.times., 3.times., 4.times.,
5.times., 10.times., 20.times., 30.times., 40.times., 50.times. or
more of the half-life are possible. Alternatively, or in addition,
increases in the range of up to 30.times., 40.times., 50.times.,
60.times., 70.times., 80.times., 90.times., 100.times., 150.times.
of the half-life are possible.
[0186] As used herein, "hydrodynamic size" refers to the apparent
size of a molecule (e.g., a protein molecule, ligand) based on the
diffusion of the molecule through an aqueous solution. The
diffusion, or motion of a protein through solution can be processed
to derive an apparent size of the protein, where the size is given
by the "Stokes radius" or "hydrodynamic radius" of the protein
particle. The "hydrodynamic size" of a protein depends on both mass
and shape (conformation), such that two proteins having the same
molecular mass may have differing hydrodynamic sizes based on the
overall conformation of the protein.
[0187] As referred to herein, the term "competes" means that the
binding of a first target to its cognate target binding domain is
inhibited in the presence of a second binding domain that is
specific for said cognate target. For example, binding may be
inhibited sterically, for example by physical blocking of a binding
domain or by alteration of the structure or environment of a
binding domain such that its affinity or avidity for a target is
reduced. See WO2006038027 for details of how to perform competition
ELISA and competition BiaCore experiments to determine competition
between first and second binding domains.
[0188] Calculations of "homology" or "identity" or "similarity"
between two sequences (the terms are used interchangeably herein)
are performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In an embodiment, the length of a
reference sequence aligned for comparison purposes is at least 30%,
or at least 40%, or at least 50%, or at least 60%, or at least 70%,
80%, 90%, 100% of the length of the reference sequence. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "homology" is equivalent to amino acid
or nucleic acid "identity"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, 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. Amino acid and nucleotide sequence
alignments and homology, similarity or identity, as defined herein
may be prepared and determined using the algorithm BLAST 2
Sequences, using default parameters (Tatusova, T. A. et al., FEMS
Microbiol Lett, 174:187-188 (1999).
Selection Methods
[0189] The invention in one embodiment relates to polypeptides and
dAbs, e.g. anti-VEGF dAbs, selected by a method of selection of
protease resistant peptides and polypeptides that have a desired
biological activity e.g. binding to VEGF. Two selective pressures
are used in the method to produce an efficient process for
selecting polypeptides that are highly stable and resistant to
protease degradation, and that have desired biological activity. As
described herein, protease resistant peptides and polypeptides
generally retain biological activity. In contrast, protease
sensitive peptides and polypeptides are cleaved or digested by
protease in the methods described herein, and therefore, lose their
biological activity. Accordingly, protease resistant peptides or
polypeptides are generally selected based on their biological
activity, such as binding activity.
[0190] The methods described herein provide several advantages. For
example, as disclosed and exemplified herein, variable domains,
antagonists, peptides or polypeptides that are selected for
resistance to proteolytic degradation by one protease (e.g.,
trypsin), are also resistant to degradation by other proteases
(e.g., elastase, leucozyme). In one embodiment protease resistance
correlates with a higher melting temperature (Tm) of the peptide or
polypeptide. Higher melting temperatures are indicative of more
stable variable domains, antagonists, peptides and polypeptides.
Resistance to protease degradation also correlates in one
embodiment with high affinity binding to target ligands. Thus, the
methods described herein provide an efficient way to select,
isolate and/or recover variable domains, antagonists, peptides,
polypeptides that have a desired biological activity and that are
well suited for in vivo therapeutic and/or diagnostic uses because
they are protease resistant and stable. In one embodiment protease
resistance correlates with an improved PK, for example improved
over n variable domain, antagonist, peptide or polypeptide that is
not protease resistant. Improved PK may be an improved AUC (area
under the curve) and/or an improved half-life. In one embodiment
protease resistance correlates with an improved stability of the
variable domain, antagonist, peptide or polypeptide to shear and/or
thermal stress and/or a reduced propensity to aggregate during
nebulisation, for example improved over an variable domain,
antagonist, peptide or polypeptide that is not protease resistant.
In one embodiment protease resistance correlates with an improved
storage stability, for example improved over an variable domain,
antagonist, peptide or polypeptide that is not protease resistant.
In one aspect, one, two, three, four or all of the advantages are
provided, the advantages being resistance to protease degradation,
higher Tm and high affinity binding to target ligand.
Selection Methods
[0191] In one aspect, there is provided a method for selecting,
isolating and/or recovering a peptide or polypeptide from a library
or a repertoire of peptides and polypeptides (e.g., a display
system) that is resistant to degradation by a protease (e.g., one
or more proteases). In one embodiment, the method is a method for
selecting, isolating and/or recovering a polypeptide from a library
or a repertoire of peptides and polypeptides (e.g., a display
system) that is resistant to degradation by a protease (e.g., one
or more proteases). Generally, the method comprises providing a
library or repertoire of peptides or polypeptides, combining the
library or repertoire with a protease (e.g., trypsin, elastase,
leucozyme, pancreatin, sputum) under conditions suitable for
protease activity, and selecting, isolating and/or recovering a
peptide or polypeptide that is resistant to degradation by the
protease and has a desired biological activity. Peptides or
polypeptides that are degraded by a protease generally have reduced
biological activity or lose their biological activity due to the
activity of protease. Accordingly, peptides or polypeptides that
are resistant to protease degradation can be selected, isolated
and/or recovered using the method based on their biological
activity, such as binding activity (e.g., binding a general ligand,
binding a specific ligand, binding a substrate), catalytic activity
or other biological activity.
[0192] The library or repertoire of peptides or polypeptides is
combined with a protease (e.g., one or more proteases) under
conditions suitable for proteolytic activity of the protease.
Conditions that are suitable for proteolytic activity of protease,
and biological preparations or mixtures that contain proteolytic
activity, are well-known in the art or can be readily determined by
a person of ordinary skill in the art. If desired, suitable
conditions can be identified or optimized, for example, by
assessing protease activity under a range of pH conditions,
protease concentrations, temperatures and/or by varying the amount
of time the library or repertoire and the protease are permitted to
react. For example, in some embodiments, the ratio (on a mole/mole
basis) of protease, eg trypsin, to peptide or polypeptide (eg,
variable domain) is 800 to 80,00 (eg, 8,000 to 80,000)
protease:peptide or polypeptide, eg when 10 micrograms/ml of
protease is used, the ratio is 800 to 80,000 protease:peptide or
polypeptide; or when 100 micrograms/ml of protease is used, the
ratio is 8,000 to 80,000 protease:peptide or polypeptide. In one
embodiment the ratio (on a weight/weight, eg microgram/microgram
basis) of protease (eg, trypsin) to peptide or polypeptide (eg,
variable domain) is 1,600 to 160,000 (eg, 16,000 to 160,000)
protease:peptide or polypeptide eg when 10 micrograms/ml of
protease is used, the ratio is 1,600 to 160,000 protease:peptide or
polypeptide; or when 100 micrograms/ml of protease is used, the
ratio is 16,000 to 160,000 protease:peptide or polypeptide. In one
embodiment, the protease is used at a concentration of at least 100
or 1000 micrograms/ml and the protease: peptide ratio (on a
mole/mole basis) of protease, eg trypsin, to peptide or polypeptide
(eg, variable domain) is 8,000 to 80,000 protease:peptide or
polypeptide. In one embodiment, the protease is used at a
concentration of at least 10 micrograms/ml and the protease:
peptide ratio (on a mole/mole basis) of protease, eg trypsin, to
peptide or polypeptide (eg, variable domain) is 800 to 80,000
protease:peptide or polypeptide. In one embodiment the ratio (on a
weight/weight, eg microgram/microgram basis) of protease (eg,
trypsin) to peptide or polypeptide (eg, variable domain) is 1600 to
160,000 protease:peptide or polypeptide eg when C is 10
micrograms/ml; or when C or C' is 100 micrograms/ml, the ratio is
16,000 to 160,000 protease:peptide or polypeptide. In one
embodiment, the concentration (c or c') is at least 100 or 1000
micrograms/ml protease. For testing an individual or isolated
peptide or polypeptide (eg, an immunoglobulin variable domain), eg
one that has already been isolated from a repertoire or library, a
protease can be added to a solution of peptide or polypeptide in a
suitable buffer (e.g., PBS) to produce a peptide or
polypeptide/protease solution, such as a solution of at least about
0.01% (w/w) protease/peptide or polypeptide, about 0.01% to about
5% (w/w) protease/peptide or polypeptide, about 0.05% to about 5%
(w/w) protease/peptide or polypeptide, about 0.1% to about 5% (w/w)
protease/peptide or polypeptide, about 0.5% to about 5% (w/w)
protease/peptide or polypeptide, about 1% to about 5% (w/w)
protease/peptide or polypeptide, at least about 0.01% (w/w)
protease/peptide or polypeptide, at least about 0.02% (w/w)
protease/peptide or polypeptide, at least about 0.03% (w/w)
protease/peptide or polypeptide, at least about 0.04% (w/w)
protease/peptide or polypeptide, at least about 0.05% (w/w)
protease/peptide or polypeptide, at least about 0.06% (w/w)
protease/peptide or polypeptide, at least about 0.07% (w/w)
protease/peptide or polypeptide, at least about 0.08% (w/w)
protease/peptide or polypeptide, at least about 0.09% (w/w)
protease/peptide or polypeptide, at least about 0.1% (w/w)
protease/peptide or polypeptide, at least about 0.2% (w/w)
protease/peptide or polypeptide, at least about 0.3% (w/w)
protease/peptide or polypeptide, at least about 0.4% (w/w)
protease/peptide or polypeptide, at least about 0.5% (w/w)
protease/peptide or polypeptide, at least about 0.6% (w/w)
protease/peptide or polypeptide, at least about 0.7% (w/w)
protease/peptide or polypeptide, at least about 0.8% (w/w)
protease/peptide or polypeptide, at least about 0.9% (w/w)
protease/peptide or polypeptide, at least about 1% (w/w)
protease/peptide or polypeptide, at least about 2% (w/w)
protease/peptide or polypeptide, at least about 3% (w/w)
protease/peptide or polypeptide, at least about 4% (w/w)
protease/peptide or polypeptide, or about 5% (w/w) protease/peptide
or polypeptide. The mixture can be incubated at a suitable
temperature for protease activity (e.g., room temperature, about
37.degree. C.) and samples can be taken at time intervals (e.g., at
1 hour, 2 hours, 3 hours, etc.). The samples can be analyzed for
protein degradation using any suitable method, such as SDS-PAGE
analysis or ligand binding , and the results can be used to
establish a time course of degradation.
[0193] Any desired protease or proteases can be used in the methods
described herein. For example, a single protease, any desired
combination of different proteases, or any biological preparation,
biological extract, or biological homogenate that contains
proteolytic activity can be used. It is not necessary that the
identity of the protease or proteases that are used be known.
Suitable examples of proteases that can be used alone or in any
desired combination include serine protease, cysteine protease,
aspartate proteases, thiol proteases, matrix metalloprotease,
carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),
trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme,
pancreatin, thrombin, plasmin, cathepsins (e.g., cathepsin G),
proteinase (e.g., proteinase 1, proteinase 2, proteinase 3),
thermolysin, chymosin, enteropeptidase, caspase (e.g., caspase 1,
caspase 2, caspase 4, caspase 5, caspase 9, caspase 12, caspase
13), calpain, ficain, clostripain, actinidain, bromelain, separase
and the like. Suitable biological extracts, homogenates and
preparations that contains proteolytic activity include sputum,
mucus (e.g., gastric mucus, nasal mucus, bronchial mucus),
bronchoalveolar lavage, lung homogenate, lung extract, pancreatic
extract, gastric fluid, saliva, tears and the like. The protease is
used in an amount suitable for proteolytic degradation to occur.
For example, as described herein, protease can be used at about
0.01% to about 5% (w/w, protease/peptide or polypeptide). When
protease is combined with a display system that comprises the
repertoire of peptides or polypeptides (e.g., a phage display
system), for example, the protease can be used at a concentration
of about 10 .mu.g/ml to about 3 mg/ml, about 10 .mu.g/ml, about 20
.mu.g/ml, about 30 .mu.g/ml, about 40 .mu.g/ml, about 50 .mu.g/ml,
about 60 .mu.g/ml, about 70 .mu.g/ml, about 80 .mu.g/ml, about 90
.mu.g/ml, about 100 .mu.g/ml, about 200 .mu.g/ml, about 300
.mu.g/ml, about 400 .mu.g/ml, about 500 .mu.g/ml, about 600
.mu.g/ml, about 700 .mu.g/ml, about 800 .mu.g/ml, about 900
.mu.g/ml, about 1000 .mu.g/ml, about 1.5 mg/ml, about 2 mg/ml,
about 2.5 mg/ml or about 3 mg/ml.
[0194] The protease is incubated with the collection of peptides or
polypeptides (library or repertoire) at a temperature that is
suitable for activity of the protease. For example, the protease
and collection of peptides or polypeptides can be incubated at a
temperature of about 20.degree. C. to about 40.degree. C. (e.g., at
room temperature, about 20.degree. C., about 21.degree. C., about
22.degree. C., about 23.degree. C., about 24.degree. C., about
25.degree. C., about 26.degree. C., about 27.degree. C., about
28.degree. C., about 29.degree. C., about 30.degree. C., about
31.degree. C., about 32.degree. C., about 33.degree. C., about
34.degree. C., about 35.degree. C., about 36.degree. C., about
37.degree. C., about 38.degree. C., about 39.degree. C., about
40.degree. C.). The protease and the collection of peptides or
polypeptides are incubated together for a period of time sufficient
for proteolytic degradation to occur. For example, the collection
of peptides or polypeptides can be incubated together with protease
for about 30 minutes to about 24 or about 48 hours. In some
examples, the collection of peptides or polypeptides is incubated
together with protease overnight, or for at least about 30 minutes,
about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, about 24
hours, about 48 hours, or longer.
[0195] It is generally desirable, at least in early selection
rounds (e.g. when a display system is used), that the protease
results in a reduction in the number of clones that have the
desired biological activity that is selected for by at least one
order of magnitude, in comparison to selections that do not include
incubation with protease. In particular examples, the amount of
protease and conditions used in the methods are sufficient to
reduce the number of recovered clones by at least about one log (a
factor of 10), at least about 2 logs (a factor of 100), at least
about 3 logs (a factor of 1000) or at least about 4 logs (a factor
of 10,000). Suitable amounts of protease and incubation conditions
that will result in the desired reduction in recovered clones can
be easily determined using conventional methods and/or the guidance
provided herein.
[0196] The protease and collection of peptides or polypeptides can
be combined and incubated using any suitable method (e.g., in
vitro, in vivo or ex vivo). For example, the protease and
collection of peptides or polypeptides can be combined in a
suitable container and held stationary, rocked, shaken, swirled or
the like, at a temperature suitable for protease activity. If
desired, the protease and collection of peptides or polypeptides
can be combined in an in vivo or ex vivo system, such as by
introducing the collection of polypeptides (e.g., a phage display
library or repertoire) into a suitable animal (e.g., a mouse), and
after sufficient time for protease activity has passed, recovering
the collection of peptides or polypeptides. In another example, an
organ or tissue is perfused with the collection of polypeptides
(e.g., a phage display library or repertoire), and after sufficient
time for protease activity has passed, the collection of
polypeptides is recovered.
[0197] Following incubation, a protease resistant peptide or
polypeptide can be selected based on a desired biological activity,
such as a binding activity. If desired, a protease inhibitor can be
added before selection. Any suitable protease inhibitor (or
combination of two or more protease inhibitors) that will not
substantially interfere with the selection method can be used.
Examples of suitable protease inhibitors include,
.alpha.1-anti-trypsin, .alpha.2-macroglobulin, amastatin, antipain,
antithrombin III, aprotinin, 4-(2-Aminoethyl)benzenesulfonyl
fluoride hydrochloride (AEBSF), (4-Amidino-Phenyl)-Methane-Sulfonyl
Fluoride (APMSF), bestatin, benzamidine, chymostatin,
3,4-Dichloroisocoumarin, diisoproply fluorophosphate (DIFP), E-64,
ethylenediamine tetraacedic acid (EDTA), elastatinal, leupeptin,
N-Ethylmaleimide, phenylmethylsulfonylfluoride (PMSF), pepstatin,
1,10-Phenanthroline, phosphoramidon, serine protease inhibitors,
N-tosyl-L-lysine-chloromethyl ketone (TLCK),
Na-Tosyl-Phe-chloromethylketone (TPCK) and the like. In addition,
many preparations that contain inhibitors of several classes of
proteases are commercially available (e.g., Roche Complete Protease
Inhibitor Cocktail Tablets.TM. (Roche Diagnostics Corporation;
Indianapolis, Ind., USA), which inhibits chymotrypsin, thermolysin,
papain, pronase, pancreatic extract and trypsin).
[0198] A protease resistant peptide or polypeptide can be selected
using a desired biological activity selection method, which allows
peptides and polypeptides that have the desired biological activity
to be distinguished from and selected over peptides and
polypeptides that do not have the desired biological activity.
Generally, peptides or polypeptides that have been digested or
cleaved by protease loose their biological activity, while protease
resistant peptides or polypeptides remain functional. Thus,
suitable assays for biological activity can be used to select
protease resistant peptides or polypeptides. For example, a common
binding function (e.g., binding of a general ligand, binding of a
specific ligand, or binding of a substrate) can be assessed using a
suitable binding assay (e.g., ELISA, panning) For example,
polypeptides that bind a target ligand or a generic ligand, such as
protein A, protein L or an antibody, can be selected, isolated,
and/or recovered by panning or using a suitable affinity matrix.
Panning can be accomplished by adding a solution of ligand (e.g.,
generic ligand, target ligand) to a suitable vessel (e.g., tube,
petri dish) and allowing the ligand to become deposited or coated
onto the walls of the vessel. Excess ligand can be washed away and
polypeptides (e.g., a phage display library) can be added to the
vessel and the vessel maintained under conditions suitable for the
polypeptides to bind the immobilized ligand. Unbound polypeptide
can be washed away and bound polypeptides can be recovered using
any suitable method, such as scraping or lowering the pH, for
example.
[0199] When a phage display system is used, binding can be tested
in a phage ELISA. Phage ELISA may be performed according to any
suitable procedure. In one example, populations of phage produced
at each round of selection can be screened for binding by ELISA to
the selected target ligand or generic ligand, to identify phage
that display protease resistant peptides or polypeptides. If
desired, soluble peptides and polypeptides can be tested for
binding to target ligand or generic ligand, for example by ELISA
using reagents, for example, against a C- or N-terminal tag (see
for example Winter et al. (1994) Ann. Rev. Immunology 12, 433-55
and references cited therein). The diversity of the selected phage
may also be assessed by gel electrophoresis of PCR products (Marks
et al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson
et al., 1992) J. Mol. Biol. 227, 776) or by sequencing of the
vector DNA.
[0200] In addition to specificity for VEGF, an antagonist or
polypeptide (eg, a dual specific ligand) comprising an anti-VEGF
protease resistant polypeptide (e.g., single antibody variable
domain) can have binding specificity for a generic ligand or any
desired target ligand, such as human or animal proteins, including
cytokines, growth factors, cytokine receptors, growth factor
receptors, enzymes (e.g., proteases), co-factors for enzymes, DNA
binding proteins, lipids and carbohydrates.
[0201] In some embodiments, the protease resistant peptide or
polypeptide (eg, dAb) or antagonist binds VEGF in pulmonary tissue.
In one embodiment, the antagonist or polypeptide also binds a
further target in pulmonary tissue.
[0202] When a display system (e.g., a display system that links
coding function of a nucleic acid and functional characteristics of
the peptide or polypeptide encoded by the nucleic acid) is used in
the methods described herein it may be frequently advantageous to
amplify or increase the copy number of the nucleic acids that
encode the selected peptides or polypeptides. This provides an
efficient way of obtaining sufficient quantities of nucleic acids
and/or peptides or polypeptides for additional rounds of selection,
using the methods described herein or other suitable methods, or
for preparing additional repertoires (e.g., affinity maturation
repertoires). Thus, in some embodiments, the methods comprise using
a display system (e.g., that links coding function of a nucleic
acid and functional characteristics of the peptide or polypeptide
encoded by the nucleic acid, such as phage display) and further
comprises amplifying or increasing the copy number of a nucleic
acid that encodes a selected peptide or polypeptide. Nucleic acids
can be amplified using any suitable methods, such as by phage
amplification, cell growth or polymerase chain reaction.
[0203] The methods described herein can be used as part of a
program to isolate protease resistant peptides or polypeptides, eg
dAbs, that can comprise, if desired, other suitable selection
methods. In these situations, the methods described herein can be
employed at any desired point in the program, such as before or
after other selection methods are used. The methods described
herein can also be used to provide two or more rounds of selection,
as described and exemplified herein.
[0204] In one example, there is provided a method for selecting a
peptide or polypeptide (eg, a dAb) that specifically binds VEGF and
is resistant to degradation by trypsin, comprising providing a
library or repertoire of the peptides or polypeptides, combining
the library or repertoire with trypsin under conditions suitable
for proteolytic digestion by trypsin, and selecting, isolating
and/or recovering a peptide or polypeptide that is resistant to
degradation by trypsin and specifically binds VEGF.
[0205] In particular embodiments, there is provided a method for
selecting an immunoglobulin single variable domain (a dAb) that is
resistant to degradation by trypsin and specifically binds VEGF. In
these embodiments, a library or repertoire comprising dAbs is
provided and combined with trypsin (or a biological preparation,
extract or homogenate comprising trypsin) under conditions suitable
for proteolytic digestion by trypsin. Trypsin resistant dAbs are
selected that bind VEGF. For example, the trypsin resistant dAb is
not substantially degraded when incubated at 37.degree. C. in a
0.04% (w/w) solution of trypsin for a period of at least about 2
hours. In one embodiment, the trypsin resistant dAb is not
substantially degraded when incubated at 37.degree. C. in a 0.04%
(w/w) solution of trypsin for a period of at least about 3 hours.
In one embodiment, the trypsin resistant dAb is not substantially
degraded when incubated at 37.degree. C. in a 0.04% (w/w) solution
of trypsin for a period of at least about 4 hours, at least about 5
hours, at least about 6 hours, at least about 7 hours, at least
about 8 hours, at least about 9 hours, at least about 10 hours, at
least about 11 hours, or at least about 12 hours.
[0206] In an exemplary embodiment, there is provided a method for
selecting an immunoglobulin single variable domain (a dAb) that is
resistant to degradation by trypsin and specifically binds VEGF.
The method comprises providing a phage display system comprising a
repertoire of polypeptides that comprise an immunoglobulin single
variable domain, combining the phage display system with trypsin
(100 .mu.g/ml) and incubating the mixture at about 37.degree. C.,
for example overnight (e.g., about 12-16 hours), and then selecting
phage that display a dAb that specifically bind VEGF.
[0207] In another example, the method is for selecting a peptide or
polypeptide, eg a dAb, that is resistant to degradation by
elastase, comprising providing a library or repertoire of peptides
or polypeptides, combining the library or repertoire with elastase
(or a biological preparation, extract or homogenate comprising
elastase) under conditions suitable for proteolytic digestion by
elastase, and selecting, isolating and/or recovering a peptide or
polypeptide that is resistant to degradation by elastase and has
VEGF binding activity.
[0208] In particular embodiments, there is provided a method for
selecting an immunoglobulin single variable domain (a dAb) that is
resistant to degradation by elastase and binds VEGF. In these
embodiments, a library or repertoire comprising dAbs is provided
and combined with elastase (or a biological preparation, extract or
homogenate comprising elastase) under conditions suitable for
proteolytic digestion by elastase. Elastase resistant dAbs are
selected that specifically bind VEGF. For example, the elastase
resistant dAb is not substantially degraded when incubated at
37.degree. C. in a 0.04% (w/w) solution of elastase for a period of
at least about 2 hours. In one embodiment, the elastase resistant
dAb is not substantially degraded when incubated at 37.degree. C.
in a 0.04% (w/w) solution of elastase for a period of at least
about 12 hours. In one embodiment, the elastase resistant dAb is
not substantially degraded when incubated at 37.degree. C. in a
0.04% (w/w) solution of elastase for a period of at least about 24
hours, at least about 36 hours, or at least about 48 hours.
[0209] In an embodiment, there is provided a method for selecting
an immunoglobulin single variable domain (a dAb) that is resistant
to degradation by elastase and binds VEGF. The method comprises
providing a phage display system comprising a repertoire of
polypeptides that comprise an immunoglobulin single variable
domain, combining the phage display system with elastase (about 100
.mu.g/ml) and incubating the mixture at about 37.degree. C., for
example, overnight (e.g., about 12-16 hours), and then selecting
phage that display a dAb that specifically bind VEGF.
[0210] In one example, there is provided a method for selecting a
peptide or polypeptide (eg, a dAb) that is resistant to degradation
by leucozyme, comprising providing a library or repertoire of
peptides or polypeptides, combining the library or repertoire with
leucozyme (or a biological preparation, extract or homogenate
comprising leucozyme) under conditions suitable for proteolytic
digestion by leucozyme, and selecting, isolating and/or recovering
a peptide or polypeptide that is resistant to degradation by
leucozyme and has specific VEGF binding activity.
[0211] In particular embodiments, there is provided a method for
selecting an immunoglobulin single variable domain (a dAb) that is
resistant to degradation by leucozyme and binds VEGF. In these
embodiments, a library or repertoire comprising dAbs is provided
and combined with leucozyme (or a biological preparation, extract
or homogenate comprising leucozyme) under conditions suitable for
proteolytic digestion by leucozyme. Leucozyme resistant dAbs are
selected that specifically bind VEGF. For example, the leucozyme
resistant dAb is not substantially degraded when incubated at
37.degree. C. in a 0.04% (w/w) solution of leucozyme for a period
of at least about 2 hours. In one embodiment, the leucozyme
resistant dAb is not substantially degraded when incubated at
37.degree. C. in a 0.04% (w/w) solution of leucozyme for a period
of at least about 12 hours. In one embodiment, the leucozyme
resistant dAb is not substantially degraded when incubated at
37.degree. C. in a 0.04% (w/w) solution of leucozyme for a period
of at least about 24 hours, at least about 36 hours, or at least
about 48 hours.
[0212] In an embodiment, there is provided a method for selecting
an immunoglobulin single variable domain (a dAb) that is resistant
to degradation by leucozyme and specifically binds VEGF. The method
comprises providing a phage display system comprising a repertoire
of polypeptides that comprise an immunoglobulin single variable
domain, combining the phage display system with leucozyme (about
100 .mu.g/ml) and incubating the mixture at about 37.degree. C.,
for example, overnight (e.g., about 12-16 hours), and then
selecting phage that display a dAb that specifically bind VEGF.
[0213] In another aspect, there is provided a method of producing a
repertoire of protease resistant peptides or polypeptides (eg,
dAbs). The method comprises providing a repertoire of peptides or
polypeptides; combining the repertoire of peptides or polypeptides
and a protease under suitable conditions for protease activity; and
recovering a plurality of peptides or polypeptides that
specifically bind VEGF, whereby a repertoire of protease resistant
peptides or polypeptides is produced. Proteases, display systems,
conditions for protease activity, and methods for selecting
peptides or polypeptides that are suitable for use in the method
are described herein with respect to the other methods.
[0214] In some embodiments, a display system (e.g., a display
system that links coding function of a nucleic acid and functional
characteristics of the peptide or polypeptide encoded by the
nucleic acid) that comprises a repertoire of peptides or
polypeptides is used, and the method further comprises amplifying
or increasing the copy number of the nucleic acids that encode the
plurality of selected peptides or polypeptides. Nucleic acids can
be amplified using any suitable method, such as by phage
amplification, cell growth or polymerase chain reaction.
[0215] In particular embodiment, there is provided a method of
producing a repertoire of protease resistant polypeptides that
comprise anti-VEGF dAbs. The method comprises providing a
repertoire of polypeptides that comprise anti-VEGF dAbs; combining
the repertoire of peptides or polypeptides and a protease (e.g.,
trypsin, elastase, leucozyme) under suitable conditions for
protease activity; and recovering a plurality of polypeptides that
comprise dAbs that have binding specificity for VEGF. The method
can be used to produce a nave repertoire, or a repertoire that is
biased toward a desired binding specificity, such as an affinity
maturation repertoire based on a parental dAb that has binding
specificity for VEGF.
Polypeptide Display Systems
[0216] In one embodiment, the repertoire or library of peptides or
polypeptides provided for use in the methods described herein
comprise a suitable display system. The display system may resist
degradation by protease (e.g., a single protease or a combination
of proteases, and any biological extract, homogenate or preparation
that contains proteolytic activity (e.g., sputum, mucus (e.g.,
gastric mucus, nasal mucus, bronchial mucus), bronchoalveolar
lavage, lung homogenate, lung extract, pancreatic extract, gastric
fluid, saliva, tears and the like). The display system and the link
between the display system and the displayed polypeptide is in one
embodiment at least as resistant to protease as the most stable
peptides or polypeptides of the repertoire. This allows a nucleic
acid that encodes a selected displayed polypeptide to be easily
isolated and/or amplified.
[0217] In one example, a protease resistant peptide or polypeptide,
eg a dAb, can be selected, isolated and/or recovered from a
repertoire of peptides or polypeptides that is in solution, or is
covalently or noncovalently attached to a suitable surface, such as
plastic or glass (e.g., microtiter plate, polypeptide array such as
a microarray). For example an array of peptides on a surface in a
manner that places each distinct library member (e.g., unique
peptide sequence) at a discrete, predefined location in the array
can be used. The identity of each library member in such an array
can be determined by its spatial location in the array. The
locations in the array where binding interactions between a target
ligand, for example, and reactive library members occur can be
determined, thereby identifying the sequences of the reactive
members on the basis of spatial location. (See, e.g., U.S. Pat. No.
5,143,854, WO 90/15070 and WO 92/10092.)
[0218] In one embodiment, the methods employ a display system that
links the coding function of a nucleic acid and physical, chemical
and/or functional characteristics of the polypeptide encoded by the
nucleic acid. Such a display system can comprise a plurality of
replicable genetic packages, such as bacteriophage or cells
(bacteria). In one embodiment, the display system comprises a
library, such as a bacteriophage display library.
[0219] A number of suitable bacteriophage display systems (e.g.,
monovalent display and multivalent display systems) have been
described. (See, e.g., Griffiths et al., U.S. Pat. No. 6,555,313 B1
(incorporated herein by reference); Johnson et al., U.S. Pat. No.
5,733,743 (incorporated herein by reference); McCafferty et al.,
U.S. Pat. No. 5,969,108 (incorporated herein by reference);
Mulligan-Kehoe, U.S. Pat. No.
[0220] 5,702,892 (Incorporated herein by reference); Winter, G. et
al., Annu. Rev. Immunol. 12:433-455 (1994); Soumillion, P. et al.,
Appl. Biochem. Biotechnol. 47(2-3):175-189 (1994); Castagnoli, L.
et al., Comb. Chem. High Throughput Screen, 4(2):121-133 (2001).)
The peptides or polypeptides displayed in a bacteriophage display
system can be displayed on any suitable bacteriophage, such as a
filamentous phage (e.g., fd, M13, F1), a lytic phage (e.g., T4, T7,
lambda), or an RNA phage (e.g., MS2), for example.
[0221] Generally, a library of phage that displays a repertoire of
peptides or phage polypeptides, as fusion proteins with a suitable
phage coat protein (e.g., fd pIII protein), is produced or
provided. The fusion protein can display the peptides or
polypeptides at the tip of the phage coat protein, or if desired at
an internal position. For example, the displayed peptide or
polypeptide can be present at a position that is amino-terminal to
domain 1 of pIII. (Domain 1 of pIII is also referred to as N1.) The
displayed polypeptide can be directly fused to pIII (e.g., the
N-terminus of domain 1 of pIII) or fused to pIII using a linker. If
desired, the fusion can further comprise a tag (e.g., myc epitope,
His tag). Libraries that comprise a repertoire of peptides or
polypeptides that are displayed as fusion proteins with a phage
coat protein can be produced using any suitable methods, such as by
introducing a library of phage vectors or phagemid vectors encoding
the displayed peptides or polypeptides into suitable host bacteria,
and culturing the resulting bacteria to produce phage (e.g., using
a suitable helper phage or complementing plasmid if desired). The
library of phage can be recovered from the culture using any
suitable method, such as precipitation and centrifugation.
[0222] The display system can comprise a repertoire of peptides or
polypeptides that contains any desired amount of diversity. For
example, the repertoire can contain peptides or polypeptides that
have amino acid sequences that correspond to naturally occurring
polypeptides expressed by an organism, group of organisms (eg, a
repertoire of sequences of V.sub.HH dAbs isolated from a Camelid),
desired tissue or desired cell type, or can contain peptides or
polypeptides that have random or randomized amino acid sequences.
If desired, the polypeptides can share a common core or scaffold.
The polypeptides in such a repertoire or library can comprise
defined regions of random or randomized amino acid sequence and
regions of common amino acid sequence. In certain embodiments, all
or substantially all polypeptides in a repertoire are of a desired
type, such as a desired enzyme (e.g., a polymerase) or a desired
antigen-binding fragment of an antibody (e.g., human V.sub.H or
human V.sub.L). In embodiments, the polypeptide display system
comprises a repertoire of polypeptides wherein each polypeptide
comprises an antibody variable domain. For example, each
polypeptide in the repertoire can contain a V.sub.H, a V.sub.L or
an Fv (e.g., a single chain Fv).
[0223] Amino acid sequence diversity can be introduced into any
desired region of a peptide or polypeptide or scaffold using any
suitable method. For example, amino acid sequence diversity can be
introduced into a target region, such as a complementarity
determining region of an antibody variable domain or a hydrophobic
domain, by preparing a library of nucleic acids that encode the
diversified polypeptides using any suitable mutagenesis methods
(e.g., low fidelity PCR, oligonucleotide-mediated or site directed
mutagenesis, diversification using NNK codons) or any other
suitable method. If desired, a region of a polypeptide to be
diversified can be randomized.
[0224] The size of the polypeptides that make up the repertoire is
largely a matter of choice and uniform polypeptide size is not
required. In one embodiment, the polypeptides in the repertoire
have at least tertiary structure (form at least one domain).
Selection/Isolation/Recovery
[0225] A protease resistant peptide or polypeptide (e.g., a
population of protease resistant polypeptides) can be selected,
isolated and/or recovered from a repertoire or library (e.g., in a
display system) using any suitable method. In one embodiment, a
protease resistant polypeptide is selected or isolated based on a
selectable characteristic (e.g., physical characteristic, chemical
characteristic, functional characteristic). Suitable selectable
functional characteristics include biological activities of the
peptides or polypeptides in the repertoire, for example, binding to
a generic ligand (e.g., a superantigen), binding to a target ligand
(e.g., an antigen, an epitope, a substrate), binding to an antibody
(e.g., through an epitope expressed on a peptide or polypeptide),
and catalytic activity. (See, e.g., Tomlinson et al., WO 99/20749;
WO 01/57065; WO 99/58655). In one embodiment, the selection is
based on specific binding to VEGF. In another embodiment, selection
is on the basis of the selected functional characteristic to
produce a second repertoire in which members are protease
resistant, followed by selection of a member from the second
repertoire that specifically binds VEGF.
[0226] In some embodiments, the protease resistant peptide or
polypeptide is selected and/or isolated from a library or
repertoire of peptides or polypeptides in which substantially all
protease resistant peptides or polypeptides share a common
selectable feature. For example, the protease resistant peptide or
polypeptide can be selected from a library or repertoire in which
substantially all protease resistant peptides or polypeptides bind
a common generic ligand, bind a common target ligand, bind (or are
bound by) a common antibody, or possess a common catalytic
activity. This type of selection is particularly useful for
preparing a repertoire of protease resistant peptides or
polypeptides that are based on a parental peptide or polypeptide
that has a desired biological activity, for example, when
performing affinity maturation of an immunoglobulin single variable
domain.
[0227] Selection based on binding to a common generic ligand can
yield a collection or population of peptides or polypeptides that
contain all or substantially all of the protease resistant peptides
or polypeptides that were components of the original library or
repertoire. For example, peptides or polypeptides that bind a
target ligand or a generic ligand, such as protein A, protein L or
an antibody, can be selected, isolated and/or recovered by panning
or using a suitable affinity matrix. Panning can be accomplished by
adding a solution of ligand (e.g., generic ligand, target ligand)
to a suitable vessel (e.g., tube, petri dish) and allowing the
ligand to become deposited or coated onto the walls of the vessel.
Excess ligand can be washed away and peptides or polypeptides
(e.g., a repertoire that has been incubated with protease) can be
added to the vessel and the vessel maintained under conditions
suitable for peptides or polypeptides to bind the immobilized
ligand. Unbound peptides or polypeptides can be washed away and
bound peptides or polypeptides can be recovered using any suitable
method, such as scraping or lowering the pH, for example.
[0228] Suitable ligand affinity matrices generally contain a solid
support or bead (e.g., agarose) to which a ligand is covalently or
noncovalently attached. The affinity matrix can be combined with
peptides or polypeptides (e.g., a repertoire that has been
incubated with protease) using a batch process, a column process or
any other suitable process under conditions suitable for binding of
peptides or polypeptides to the ligand on the matrix. Peptides or
polypeptides that do not bind the affinity matrix can be washed
away and bound peptides or polypeptides can be eluted and recovered
using any suitable method, such as elution with a lower pH buffer,
with a mild denaturing agent (e.g., urea), or with a peptide that
competes for binding to the ligand. In one example, a biotinylated
target ligand is combined with a repertoire under conditions
suitable for peptides or polypeptides in the repertoire to bind the
target ligand (VEGF). Bound peptides or polypeptides are recovered
using immobilized avidin or streptavidin (e.g., on a bead).
[0229] In some embodiments, the generic ligand is an antibody or
antigen binding fragment thereof. Antibodies or antigen binding
fragments that bind structural features of peptides or polypeptides
that are substantially conserved in the peptides or polypeptides of
a library or repertoire are particularly useful as generic ligands.
Antibodies and antigen binding fragments suitable for use as
ligands for isolating, selecting and/or recovering protease
resistant peptides or polypeptides can be monoclonal or polyclonal
and can be prepared using any suitable method.
Libraries/Repertoires
[0230] In other aspects, there are provided repertoires of protease
resistant peptides and polypeptides, to libraries that encode
protease resistant peptides and polypeptides, and to methods for
producing such libraries and repertoires.
[0231] Libraries that encode and/or contain protease resistant
peptides and polypeptides can be prepared or obtained using any
suitable method. The library can be designed to encode protease
resistant peptides or polypeptides based on a peptide or
polypeptide of interest (e.g., an anti-VEGF peptide or polypeptide
selected from a library) or can be selected from another library
using the methods described herein. For example, a library enriched
in protease resistant polypeptides can be prepared using a suitable
polypeptide display system.
[0232] In one example, a phage display library comprising a
repertoire of displayed polypeptides comprising immunoglobulin
single variable domains (e.g., V.sub.H, Vk, V.lamda.) is combined
with a protease under conditions suitable for protease activity, as
described herein. Protease resistant polypeptides are recovered
based on a desired biological activity, such as a binding activity
(e.g., binding generic ligand, binding target ligand) thereby
yielding a phage display library enriched in protease resistant
polypeptides. In one embodiment, the recovery is on the basis of
binding generic ligand to yield an enriched library, followed by
selection of an anti-VEGF member of that library based on specific
binding to VEGF.
[0233] In another example, a phage display library comprising a
repertoire of displayed polypeptides comprising immunoglobulin
single variable domains (e.g., V.sub.H, V.kappa., V.lamda.) is
first screened to identify members of the repertoire that have
binding specificity for a desired target antigen (e.g. VEGF). A
collection of polypeptides having the desired binding specificity
are recovered and the collection is combined with protease under
conditions suitable for proteolytic activity, as described herein.
A collection of protease resistant polypeptides that have the
desired target binding specificity is recovered, yielding a library
enriched in protease resistant and high affinity polypeptides. As
described herein in an embodiment, protease resistance in this
selection method correlates with high affinity binding.
[0234] Libraries that encode a repertoire of a desired type of
polypeptides can readily be produced using any suitable method. For
example, a nucleic acid sequence that encodes a desired type of
polypeptide (e.g., a polymerase, an immunoglobulin variable domain)
can be obtained and a collection of nucleic acids that each contain
one or more mutations can be prepared, for example by amplifying
the nucleic acid using an error-prone polymerase chain reaction
(PCR) system, by chemical mutagenesis (Deng et al., J. Biol. Chem.,
269:9533 (1994)) or using bacterial mutator strains (Low et al., J.
Mol. Biol., 260:359 (1996)).
[0235] In other embodiments, particular regions of the nucleic acid
can be targeted for diversification. Methods for mutating selected
positions are also well known in the art and include, for example,
the use of mismatched oligonucleotides or degenerate
oligonucleotides, with or without the use of PCR. For example,
synthetic antibody libraries have been created by targeting
mutations to the antigen binding loops. Random or semi-random
antibody H3 and L3 regions have been appended to germline
immunoblulin V gene segments to produce large libraries with
unmutated framework regions (Hoogenboom and Winter (1992) supra;
Nissim et al. (1994) supra; Griffiths et al. (1994) supra; DeKruif
et al. (1995) supra). Such diversification has been extended to
include some or all of the other antigen binding loops (Crameri et
al. (1996) Nature Med., 2:100; Riechmann et al. (1995)
Bio/Technology, 13:475; Morphosys, WO 97/08320, supra). In other
embodiments, particular regions of the nucleic acid can be targeted
for diversification by, for example, a two-step PCR strategy
employing the product of the first PCR as a "mega-primer." (See,
e.g., Landt, O. et al., Gene 96:125-128 (1990).) Targeted
diversification can also be accomplished, for example, by SOE PCR.
(See, e.g., Horton, R. M. et al., Gene 77:61-68 (1989).)
[0236] Sequence diversity at selected positions can be achieved by
altering the coding sequence which specifies the sequence of the
polypeptide such that a number of possible amino acids (e.g., all
20 or a subset thereof) can be incorporated at that position. Using
the IUPAC nomenclature, the most versatile codon is NNK, which
encodes all amino acids as well as the TAG stop codon. The NNK
codon may be used in order to introduce the required diversity.
Other codons which achieve the same ends are also of use, including
the NNN codon, which leads to the production of the additional stop
codons TGA and TAA. Such a targeted approach can allow the full
sequence space in a target area to be explored.
[0237] The libraries can comprise protease resistant antibody
polypeptides that have a known main-chain conformation. (See, e.g.,
Tomlinson et al., WO 99/20749.) Libraries can be prepared in a
suitable plasmid or vector. As used herein, vector refers to a
discrete element that is used to introduce heterologous DNA into
cells for the expression and/or replication thereof. Any suitable
vector can be used, including plasmids (e.g., bacterial plasmids),
viral or bacteriophage vectors, artificial chromosomes and episomal
vectors. Such vectors may be used for simple cloning and
mutagenesis, or an expression vector can be used to drive
expression of the library. Vectors and plasmids usually contain one
or more cloning sites (e.g., a polylinker), an origin of
replication and at least one selectable marker gene. Expression
vectors can further contain elements to drive transcription and
translation of a polypeptide, such as an enhancer element,
promoter, transcription termination signal, signal sequences, and
the like. These elements can be arranged in such a way as to be
operably linked to a cloned insert encoding a polypeptide, such
that the polypeptide is expressed and produced when such an
expression vector is maintained under conditions suitable for
expression (e.g., in a suitable host cell).
[0238] Cloning and expression vectors generally contain nucleic
acid sequences that enable the vector to replicate in one or more
selected host cells. Typically in cloning vectors, this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety
of bacteria, yeast and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the 2
micron plasmid origin is suitable for yeast, and various viral
origins (e.g. SV40, adenovirus) are useful for cloning vectors in
mammalian cells. Generally, the origin of replication is not needed
for mammalian expression vectors, unless these are used in
mammalian cells able to replicate high levels of DNA, such as COS
cells.
[0239] Cloning or expression vectors can contain a selection gene
also referred to as selectable marker. Such marker genes encode a
protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will
therefore not survive in the culture medium. Typical selection
genes encode proteins that confer resistance to antibiotics and
other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, complement auxotrophic deficiencies, or supply
critical nutrients not available in the growth media.
[0240] Suitable expression vectors can contain a number of
components, for example, an origin of replication, a selectable
marker gene, one or more expression control elements, such as a
transcription control element (e.g., promoter, enhancer,
terminator) and/or one or more translation signals, a signal
sequence or leader sequence, and the like. Expression control
elements and a signal or leader sequence, if present, can be
provided by the vector or other source. For example, the
transcriptional and/or translational control sequences of a cloned
nucleic acid encoding an antibody chain can be used to direct
expression.
[0241] A promoter can be provided for expression in a desired host
cell. Promoters can be constitutive or inducible. For example, a
promoter can be operably linked to a nucleic acid encoding an
antibody, antibody chain or portion thereof, such that it directs
transcription of the nucleic acid. A variety of suitable promoters
for procaryotic (e.g., the .beta.-lactamase and lactose promoter
systems, alkaline phosphatase, the tryptophan (trp) promoter
system, lac, tac, T3, T7 promoters for E. coli) and eucaryotic
(e.g., simian virus 40 early or late promoter, Rous sarcoma virus
long terminal repeat promoter, cytomegalovirus promoter, adenovirus
late promoter, EG-1a promoter) hosts are available.
[0242] In addition, expression vectors typically comprise a
selectable marker for selection of host cells carrying the vector,
and, in the case of a replicable expression vector, an origin of
replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and may be used in
procaryotic (e.g., .beta.-lactamase gene (ampicillin resistance),
Tet gene for tetracycline resistance) and eucaryotic cells (e.g.,
neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin,
or hygromycin resistance genes). Dihydrofolate reductase marker
genes permit selection with methotrexate in a variety of hosts.
Genes encoding the gene product of auxotrophic markers of the host
(e.g., LEU2, URA3, HIS3) are often used as selectable markers in
yeast. Use of viral (e.g., baculovirus) or phage vectors, and
vectors which are capable of integrating into the genome of the
host cell, such as retroviral vectors, are also contemplated.
[0243] Suitable expression vectors for expression in prokaryotic
(e.g., bacterial cells such as E. coli) or mammalian cells include,
for example, a pET vector (e.g., pET-12a, pET-36, pET-37, pET-39,
pET-40, Novagen and others), a phage vector (e.g., pCANTAB 5 E,
Pharmacia), pRIT2T (Protein A fusion vector, Pharmacia), pCDM8,
pCDNA1.1/amp, pcDNA3.1, pRc/RSV, pEF-1 (Invitrogen, Carlsbad,
Calif.), pCMV-SCRIPT, pFB, pSG5, pXT1 (Stratagene, La Jolla,
Calif.), pCDEF3 (Goldman, L. A., et al., Biotechniques,
21:1013-1015 (1996)), pSVSPORT (GibcoBRL, Rockville, Md.), pEF-Bos
(Mizushima, S., et al., Nucleic Acids Res., 18:5322 (1990)) and the
like. Expression vectors which are suitable for use in various
expression hosts, such as prokaryotic cells (E. coli), insect cells
(Drosophila Schnieder S2 cells, Sf9), yeast (P. methanolica, P.
pastoris, S. cerevisiae) and mammalian cells (eg, COS cells) are
available.
[0244] Examples of vectors are expression vectors that enable the
expression of a nucleotide sequence corresponding to a polypeptide
library member. Thus, selection with generic and/or target ligands
can be performed by separate propagation and expression of a single
clone expressing the polypeptide library member. As described
above, the selection display system may be bacteriophage display.
Thus, phage or phagemid vectors may be used. Example vectors are
phagemid vectors which have an E. coli. origin of replication (for
double stranded replication) and also a phage origin of replication
(for production of single-stranded DNA). The manipulation and
expression of such vectors is well known in the art (Hoogenboom and
Winter (1992) supra; Nissim et al. (1994) supra). Briefly, the
vector can contain a .beta.-lactamase gene to confer selectivity on
the phagemid and a lac promoter upstream of an expression cassette
that can contain a suitable leader sequence, a multiple cloning
site, one or more peptide tags, one or more TAG stop codons and the
phage protein pIII. Thus, using various suppressor and
non-suppressor strains of E. coli and with the addition of glucose,
iso-propyl thio-.beta.-D-galactoside (IPTG) or a helper phage, such
as VCS M13, the vector is able to replicate as a plasmid with no
expression, produce large quantities of the polypeptide library
member only or product phage, some of which contain at least one
copy of the polypeptide-pIII fusion on their surface.
[0245] The libraries and repertoires described herein can contain
antibody formats. For example, the polypeptide contained within the
libraries and repertoires can be whole separate V.sub.H or V.sub.L
domains, any of which are either modified or unmodified. scFv
fragments, as well as other antibody polypeptides, can be readily
produced using any suitable method. A number of suitable antibody
engineering methods are well known in the art. For example, a scFv
can be formed by linking nucleic acids encoding two variable
domains with a suitable oligonucleotide that encodes an appropriate
linker peptide, such as (Gly-Gly-Gly-Gly-Ser).sub.3 or other
suitable linker peptides. The linker bridges the C-terminal end of
the first V region and the N-terminal end of the second V region.
Similar techniques for the construction of other antibody formats,
such as Fv, Fab and F(ab').sub.2 fragments can be used. To format
Fab and F(ab').sub.2 fragments, V.sub.H and V.sub.L polypeptides
can be combined with constant region segments, which may be
isolated from rearranged genes, germline C genes or synthesized
from antibody sequence data. A library or repertoire described
herein can be a V.sub.H or V.sub.L library or repertoire.
[0246] The polypeptides comprising a protease resistant variable
domain may comprise a target ligand (e.g. VEGF) binding site and a
generic ligand binding site. In certain embodiments, the generic
ligand binding site is a binding site for a superantigen, such as
protein A, protein L or protein G. The variable domains can be
based on any desired variable domain, for example a human VH (e.g.,
V.sub.H 1a, V.sub.H 1b, V.sub.H 2, V.sub.H 3, V.sub.H 4, V.sub.H 5,
V.sub.H 6), a human V.lamda. (e.g., V.lamda.I, V.lamda.II,
V.lamda.III, V.lamda.IV, V.lamda.V, V.lamda.VI or V.kappa.1) or a
human V.kappa. (e.g., V.kappa.2, V.kappa.3, V.kappa.4, V.kappa.5,
V.kappa.6, V.kappa.7, V.kappa.8, V.kappa.9 or V.kappa.10) or a
Camelid V.sub.HH, optionally that has been humanized.
Nucleic Acids, Host Cells and Methods for Producing Protease
Resistant Polypeptides
[0247] The invention relates to isolated and/or recombinant nucleic
acids encoding protease resistant peptides or polypeptides e.g.,
that are selectable or selected by the methods described
herein.
[0248] Nucleic acids referred to herein as "isolated" are nucleic
acids which have been separated away from other material (e.g.,
other nucleic acids such as genomic DNA, cDNA and/or RNA) in its
original environment (e.g., in cells or in a mixture of nucleic
acids such as a library). An isolated nucleic acid can be isolated
as part of a vector (e.g., a plasmid).
[0249] Nucleic acids referred to herein as "recombinant" are
nucleic acids which have been produced by recombinant DNA
methodology, including methods which rely upon artificial
recombination, such as cloning into a vector or chromosome using,
for example, restriction enzymes, homologous recombination, viruses
and the like, and nucleic acids prepared using the polymerase chain
reaction (PCR).
[0250] The invention also relates to a recombinant host cell which
comprises a (one or more) recombinant nucleic acid or expression
construct comprising a nucleic acid encoding a protease resistant
peptide or polypeptide, e.g., a peptide or polypeptide selectable
or selected by the methods described herein. There is also provided
a method of preparing a protease resistant peptide or polypeptide,
comprising maintaining a recombinant host cell of the invention
under conditions appropriate for expression of a protease resistant
peptide or polypeptide. The method can further comprise the step of
isolating or recovering the protease resistant peptide or
polypeptide, if desired.
[0251] For example, a nucleic acid molecule (i.e., one or more
nucleic acid molecules) encoding a protease resistant peptide or
polypeptide, or an expression construct (i.e., one or more
constructs) comprising such nucleic acid molecule(s), can be
introduced into a suitable host cell to create a recombinant host
cell using any method appropriate to the host cell selected (e.g.,
transformation, transfection, electroporation, infection), such
that the nucleic acid molecule(s) are operably linked to one or
more expression control elements (e.g., in a vector, in a construct
created by processes in the cell, integrated into the host cell
genome). The resulting recombinant host cell can be maintained
under conditions suitable for expression (e.g., in the presence of
an inducer, in a suitable animal, in suitable culture media
supplemented with appropriate salts, growth factors, antibiotics,
nutritional supplements, etc.), whereby the encoded peptide or
polypeptide is produced. If desired, the encoded peptide or
polypeptide can be isolated or recovered (e.g., from the animal,
the host cell, medium, milk). This process encompasses expression
in a host cell of a transgenic animal (see, e.g., WO 92/03918,
GenPharm International).
[0252] The protease resistant peptide or polypeptide selected by
the method described herein can also be produced in a suitable in
vitro expression system, by chemical synthesis or by any other
suitable method.
Polypeptides, dAbs & Antagonists
[0253] As described and exemplified herein, protease resistant dAbs
of the invention generally bind their target ligand with high
affinity. Thus, in another aspect, there is provided a method for
selecting, isolating and/or recovering a polypeptide or dAb of the
invention that binds VEGF with high affinity. Generally, the method
comprises providing a library or repertoire of peptides or
polypeptides (eg dAbs), combining the library or repertoire with a
protease (e.g., trypsin, elastase, leucozyme, pancreatin, sputum)
under conditions suitable for protease activity, and selecting,
isolating and/or recovering a peptide or polypeptide that binds a
ligand (e.g., target ligand). Because the library or repertoire has
been exposed to protease under conditions where protease sensitive
peptides or polypeptides will be digested, the activity of protease
can eliminate the less stable polypeptides that have low binding
affinity, and thereby produce a collection of high affinity binding
peptides or polypeptides.
[0254] For example, the polypeptide or dAb of the invention can
bind VEGF with an affinity (KD; KD=K.sub.off(kd)/K.sub.on(ka) as
determined by surface plasmon resonance) of 300 nM to 1 pM (i.e.,
3.times.10.sup.-7 to 5.times.10.sup.-12M), e.g. 50 nM to 1 pM, e.g.
5 nM to 1 pM and e.g. 1 nM to 1 pM; for example K.sub.D of
1.times.10.sup.-7 M or less, e.g. 1.times.10.sup.-8 M or less, e.g.
1.times.10.sup.-9 M or less, e.g. 1.times.10.sup.-10 M or less and
e.g. 1.times.10.sup.-11 M or less; and/or a K.sub.off rate constant
of 5.times.10.sup.-1 s.sup.-1 to 1.times.10.sup.-7 s.sup.-1, e.g.
1.times.10.sup.-2 s.sup.-1 to 1.times.10.sup.-6 s.sup.-1, e.g.
5.times.10.sup.-3 s.sup.-1 to 1.times.10.sup.-5 s.sup.-1, for
example 5.times.10.sup.-1 s.sup.-1 or less, e.g. 1.times.10.sup.-2
s.sup.-1 or less, e.g. 1.times.10.sup.-3 s.sup.-1 or less, e.g.
1.times.10.sup.-4 s.sup.-1 or less, e.g. 1.times.10.sup.-5 s.sup.-1
or less, and e.g. 1.times.10.sup.-6 s.sup.-1 or less as determined
by surface plasmon resonance.
[0255] Although we are not bound by any particular theory, peptides
and polypeptides that are resistant to proteases are believed to
have a lower entropy and/or a higher stabilization energy. Thus,
the correlation between protease resistance and high affinity
binding may be related to the compactness and stability of the
surfaces of the peptides and polypeptides and dAbs selected by the
method described herein.
[0256] In one embodiment, the polypeptide, dAb or antagonist of the
invention inhibits binding of VEGF at a concentration 50 (IC50) of
IC50 of about 1 .mu.M or less, about 500 nM or less, about 100 nM
or less, about 75 nM or less, about 50 nM or less, about 10 nM or
less or about 1 nM or less.
[0257] In certain embodiments, the polypeptide, dAb or antagonist
specifically binds VEGF, eg, human VEGF, and dissociates from human
VEGF with a dissociation constant (K.sub.D) of 300 nM to 1 pM or
300 nM to 5 pM or 50 nM to 1 pM or 50 nM to 5 pM or 50 nM to 20 pM
or about 10 pM or about 15 pM or about 20 pM as determined by
surface plasmon resonance In certain embodiments, the polypeptide,
dAb or antagonist specifically binds VEGF, eg, human VEGF, and
dissociates from human VEGF with a K.sub.off rate constant of
5.times.10.sup.-1 s.sup.-1 to 1.times.10.sup.-7s.sup.-1, e.g.
1.times.10.sup.-2 s.sup.-1 to 1.times.10.sup.-6 s.sup.-1, e.g.
5.times.10.sup.-3 s.sup.-1 to 1.times.10.sup.-5 s.sup.-1, for
example 5.times.10.sup.-1 s.sup.-1 or less, e.g. 1.times.10.sup.-2
s.sup.-1 or less, e.g. 1.times.10.sup.-3 s.sup.-1 or less, e.g.
1.times.10.sup.-4 s.sup.-1 or less, e.g. 1.times.10.sup.-5 s.sup.-1
or less, and e.g. 1.times.10.sup.-6 s.sup.-1 or less as determined
by surface plasmon resonance
[0258] In certain embodiments, the polypeptide, dAb or antagonist
specifically binds VEGF, eg, human VEGF, with a K.sub.on of
1.times.10.sup.-3 M.sup.-1s.sup.-1 to 1.times.10.sup.-7
M.sup.-1s.sup.-1 or 1.times.10.sup.-3 M.sup.-1s.sup.-1 to
1.times.10.sup.-6 M.sup.-1s.sup.-1 or about 1.times.10.sup.-4
M.sup.-1s.sup.-1 or about 1.times.10.sup.-5 M.sup.-1s.sup.-1. In
one embodiment, the polypeptide, dAb or antagonist specifically
binds VEGF, eg, human VEGF, and dissociates from human VEGF with a
dissociation constant (K.sub.D) and a K.sub.off as defined in this
paragraph. In one embodiment, the polypeptide, dAb or antagonist
specifically binds VEGF, eg, human VEGF, and dissociates from human
VEGF with a dissociation constant (K.sub.D) and a K.sub.on as
defined in this paragraph. In some embodiments, the polypeptide or
dAb specifically binds VEGF (eg, human VEGF) with a K.sub.D and/or
K.sub.off and/or K.sub.on as recited in this paragraph and
comprises an amino acid sequence that is at least or at least about
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to the amino acid sequence of a dAb with the amino acid
sequence selected from that of : DOM15-26-595, DOM15-26-591,
DOM15-26-589, DOM15-26-588, DOM15-26-555 and DOM15-10-11.
[0259] The polypeptide, dAb or antagonist can be expressed in E.
coli or in Pichia species (e.g., P. pastoris). In one embodiment,
the ligand or dAb monomer is secreted in a quantity of at least
about 0.5 mg/L when expressed in E. coli or in Pichia species
(e.g., P. pastoris). Although, the ligands and dAb monomers
described herein can be secretable when expressed in E. coli or in
Pichia species (e.g., P. pastoris), they can be produced using any
suitable method, such as synthetic chemical methods or biological
production methods that do not employ E. coli or Pichia
species.
[0260] In some embodiments, the polypeptide, dAb or antagonist does
not comprise a Camelid immunoglobulin variable domain, or one or
more framework amino acids that are unique to immunoglobulin
variable domains encoded by Camelid germline antibody gene segments
, eg at position 108, 37, 44, 45 and/or 47.
[0261] Antagonists of VEGF according to the invention can be
monovalent or multivalent. In some embodiments, the antagonist is
monovalent and contains one binding site that interacts with VEGF,
the binding site provided by a polypeptide or dAb of the invention.
Monovalent antagonists bind one VEGF and may not induce
cross-linking or clustering of VEGF on the surface of cells which
can lead to activation of the receptor and signal transduction.
[0262] In other embodiments, the antagonist of VEGF is multivalent.
Multivalent antagonists of VEGF can contain two or more copies of a
particular binding site for VEGF or contain two or more different
binding sites that bind VEGF, at least one of the binding sites
being provided by a polypeptide or dAb of the invention. For
example, as described herein the antagonist of VEGF can be a dimer,
trimer or multimer comprising two or more copies of a particular
polypeptide or dAb of the invention that binds VEGF, or two or more
different polypeptides or dAbs of the invention that bind VEGF. In
certain embodiments, the multivalent antagonist of VEGF contains
two or more binding sites for a desired epitope or domain of
VEGF.
[0263] Some ligands (and antagonists) may have utility as
diagnostic agents, because they can be used to bind and detect,
quantify or measure VEGF in a sample. Accordingly, an accurate
determination of whether or how much VEGF is in the sample can be
made.
[0264] In other embodiments, the polypeptide, dAb or antagonist
specifically binds VEGF with a K.sub.D described herein and
inhibits tumour growth in a standard murine xenograft model (e g ,
inhibits tumour growth by at least about 10%, as compared with a
suitable control). In one embodiment, the polypeptide, dAb or
antagonist inhibits tumour growth by at least about 10% or by at
least about 25%, or by at least about 50%, as compared to a
suitable control in a standard murine xenograft model when
administered at about 1 mg/kg or more, for example about 5 or 10
mg/kg.
[0265] In other embodiments, the polypeptide, dAb or antagonist
binds VEGF and antagonizes the activity of the VEGF in a standard
cell assay with an ND.sub.50 of .ltoreq.100 nM.
[0266] In certain embodiments, the polypeptide, dAb or antagonist
of the invention are efficacious in animal models of inflammatory
diseases such as those described in WO 2006038027 and WO 2006059108
and WO 2007049017 when an effective amount is administered.
Generally an effective amount is about 1 mg/kg to about 10 mg/kg
(e.g., about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,
about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9
mg/kg, or about 10 mg/kg). The models of chronic inflammatory
disease are recognized by those skilled in the art as being
predictive of therapeutic efficacy in humans.
[0267] Generally, the present ligands (e.g., antagonists) will be
utilised in purified form together with pharmacologically
appropriate carriers. Typically, these carriers include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, any
including saline and/or buffered media. Parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride and lactated Ringer's. Suitable physiologically-acceptable
adjuvants, if necessary to keep a polypeptide complex in
suspension, may be chosen from thickeners such as
carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
[0268] Intravenous vehicles include fluid and nutrient replenishers
and electrolyte replenishers, such as those based on Ringer's
dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition). A variety of suitable formulations can be used,
including extended release formulations.
[0269] The ligands (e.g., antagonists) of the present invention may
be used as separately administered compositions or in conjunction
with other agents. These can include various immunotherapeutic
drugs, such as cylcosporine, methotrexate, adriamycin or
cisplatinum, and immunotoxins. Pharmaceutical compositions can
include "cocktails" of various cytotoxic or other agents in
conjunction with the ligands of the present invention, or even
combinations of ligands according to the present invention having
different specificities, such as ligands selected using different
target antigens or epitopes, whether or not they are pooled prior
to administration.
[0270] The route of administration of pharmaceutical compositions
according to the invention may be any of those commonly known to
those of ordinary skill in the art. For therapy, including without
limitation immunotherapy, the selected ligands thereof of the
invention can be administered to any patient in accordance with
standard techniques.
[0271] The administration can be by any appropriate mode, including
parenterally, intravenously, intramuscularly, intraperitoneally,
transdermally, via the pulmonary route, or also, appropriately, by
direct infusion with a catheter. The dosage and frequency of
administration will depend on the age, sex and condition of the
patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician. Administration can be local (e.g., local delivery to
the lung by pulmonary administration, e.g., intranasal
administration) or systemic as indicated.
[0272] The ligands of this invention can be lyophilised for storage
and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilisation and reconstitution can lead to
varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss
than IgG antibodies) and that use levels may have to be adjusted
upward to compensate.
[0273] The compositions containing the present ligands (e.g.,
antagonists) or a cocktail thereof can be administered for
prophylactic and/or therapeutic treatments. In certain therapeutic
applications, an adequate amount to accomplish at least partial
inhibition, suppression, modulation, killing, or some other
measurable parameter, of a population of selected cells is defined
as a "therapeutically-effective dose". Amounts needed to achieve
this dosage will depend upon the severity of the disease and the
general state of the patient's own immune system, but generally
range from 0.005 to 5.0 mg of ligand, e.g. dAb or antagonist per
kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being
more commonly used. For prophylactic applications, compositions
containing the present ligands or cocktails thereof may also be
administered in similar or slightly lower dosages, to prevent,
inhibit or delay onset of disease (e.g., to sustain remission or
quiescence, or to prevent acute phase). The skilled clinician will
be able to determine the appropriate dosing interval to treat,
suppress or prevent disease. When an ligand of VEGF (e.g.,
antagonist) is administered to treat, suppress or prevent disease,
it can be administered up to four times per day, twice weekly, once
weekly, once every two weeks, once a month, or once every two
months, at a dose off, for example, about 10 .mu.g/kg to about 80
mg/kg, about 100 .mu.g/kg to about 80 mg/kg, about 1 mg/kg to about
80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kg to about
60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about
40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about
20 mg/kg , about 1 mg/kg to about 10 mg/kg, about 10 .mu.g/kg to
about 10 mg/kg, about 10 .mu.g/kg to about 5 mg/kg, about 10
.mu.g/kg to about 2.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3
mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,
about 8 mg/kg, about 9 mg/kg or about 10 mg/kg. In particular
embodiments, the ligand of VEGF (e.g., antagonist) is administered
to treat, suppress or prevent disease once every two weeks or once
a month at a dose of about 10 .mu.g/kg to about 10 mg/kg (e.g.,
about 10 .mu.g/kg, about 100 .mu.g/kg, about 1 mg/kg, about 2
mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,
about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg.)
[0274] Treatment or therapy performed using the compositions
described herein is considered "effective" if one or more symptoms
are reduced (e.g., by at least 10% or at least one point on a
clinical assessment scale), relative to such symptoms present
before treatment, or relative to such symptoms in an individual
(human or model animal) not treated with such composition or other
suitable control. Symptoms will obviously vary depending upon the
disease or disorder targeted, but can be measured by an ordinarily
skilled clinician or technician. Such symptoms can be measured, for
example, by monitoring the level of one or more biochemical
indicators of the disease or disorder (e.g., levels of an enzyme or
metabolite correlated with the disease, affected cell numbers,
etc.), by monitoring physical manifestations (e.g., inflammation,
tumor size, etc.), or by an accepted clinical assessment scale.
[0275] Similarly, prophylaxis performed using a composition as
described herein is "effective" if the onset or severity of one or
more symptoms is delayed, reduced or abolished relative to such
symptoms in a similar individual (human or animal model) not
treated with the composition.
[0276] A composition containing a ligand (e.g., antagonist) or
cocktail thereof according to the present invention may be utilised
in prophylactic and therapeutic settings to aid in the alteration,
inactivation, killing or removal of a select target cell population
in a mammal. In addition, the selected repertoires of polypeptides
described herein may be used extracorporeally or in vitro
selectively to kill, deplete or otherwise effectively remove a
target cell population from a heterogeneous collection of cells.
Blood from a mammal may be combined extracorporeally with the
ligands whereby the undesired cells are killed or otherwise removed
from the blood for return to the mammal in accordance with standard
techniques.
[0277] A composition containing an ligand (e.g., antagonist)
according to the present invention may be utilised in prophylactic
and therapeutic settings to aid in the alteration, inactivation,
killing or removal of a select target cell population in a
mammal.
[0278] The ligands (e.g., anti-VEGF antagonists, dAb monomers) can
be administered and or formulated together with one or more
additional therapeutic or active agents. When a ligand (eg, a dAb)
is administered with an additional therapeutic agent, the ligand
can be administered before, simultaneously with or subsequent to
administration of the additional agent. Generally, the ligand and
additional agent are administered in a manner that provides an
overlap of therapeutic effect.
[0279] In one embodiment, the invention is a method for treating,
suppressing or preventing disease, selected from for example Cancer
(e.g. a solid tumour), inflammatory disease, autoimmune disease,
vascular proliferative disease (e.g. AMD (age related macular
degeneration)) comprising administering to a mammal in need thereof
a therapeutically-effective dose or amount of a polypeptide, dAb
which binds to VEGF or antagonist of VEGF according to the
invention.
[0280] The invention provides a method for treating, suppressing or
preventing pulmonary diseases. Thus, in another embodiment, the
invention is a method for treating, suppressing or preventing a
pulmonary disease (e.g., lung cancer) comprising administering to a
mammal in need thereof a therapeutically-effective dose or amount
of a polypeptide, dAb or antagonist of VEGF according to the
invention.
[0281] In particular embodiments, an antagonist of VEGF is
administered via pulmonary delivery, such as by inhalation (e.g.,
intrabronchial, intranasal or oral inhalation, intranasal drops) or
by systemic delivery (e.g., parenteral, intravenous, intramuscular,
intraperitoneal, subcutaneous).
[0282] In a further aspect of the invention, there is provided a
composition comprising a a polypeptide, dAb or antagonist of VEGF
according to the invention and a pharmaceutically acceptable
carrier, diluent or excipient.
[0283] Moreover, the present invention provides a method for the
treatment of disease using a polypeptide, dAb or antagonist of VEGF
or a composition according to the present invention. In an
embodiment the disease is Cancer (e.g. a solid tumour), or an
inflammatory disease, eg rheumatoid arthritis, or an autoimmune
disease, or a vascular proliferative disease such as AMD (Age
Related Macular Degeneration).
Formats
[0284] Increased half-life is useful in in vivo applications of
immunoglobulins, especially antibodies and most especially antibody
fragments of small size. Such fragments (Fvs, disulphide bonded
Fvs, Fabs, scFvs, dAbs) suffer from rapid clearance from the body;
thus, whilst they are able to reach most parts of the body rapidly,
and are quick to produce and easier to handle, their in vivo
applications have been limited by their only brief persistence in
vivo. One embodiment of the invention solves this problem by
providing increased half-life of the ligands in vivo and
consequently longer persistence times in the body of the functional
activity of the ligand.
[0285] Methods for pharmacokinetic analysis and determination of
ligand half-life will be familiar to those skilled in the art.
Details may be found in Kenneth, A et al: Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists and in Peters et al,
Pharmacokinetc analysis: A Practical Approach (1996). Reference is
also made to "Pharmacokinetics", M Gibaldi & D Perron,
published by Marcel Dekker, 2.sup.nd Rev. ex edition (1982), which
describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
[0286] Half lives (t1/2 alpha and t1/2 beta) and AUC can be
determined from a curve of serum concentration of ligand against
time. The WinNonlin analysis package (available from Pharsight
Corp., Mountain View, Calif. 94040, USA) can be used, for example,
to model the curve. In a first phase (the alpha phase) the ligand
is undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the terminal phase when
the ligand has been distributed and the serum concentration is
decreasing as the ligand is cleared from the patient. The t alpha
half life is the half life of the first phase and the t beta half
life is the half life of the second phase. Thus, in one embodiment,
the present invention provides a ligand or a composition comprising
a ligand according to the invention having a t.alpha. half-life in
the range of 15 minutes or more. In one embodiment, the lower end
of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.
In addition, or alternatively, a ligand or composition according to
the invention will have a t.alpha. half life in the range of up to
and including 12 hours. In one embodiment, the upper end of the
range is 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable
range is 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
[0287] In one embodiment, the present invention provides a ligand
(polypeptide, dAb or antagonist) or a composition comprising a
ligand according to the invention having a t.beta. half-life in the
range of 30 minutes or more. In one embodiment, the lower end of
the range is 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5
hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In
addition, or alternatively, a ligand or composition according to
the invention has a t.beta. half-life in the range of up to and
including 21 days. In one embodiment, the upper end of the range is
12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20
days. In one embodiment a ligand or composition according to the
invention will have a t.beta. half life in the range 12 to 60
hours. In a further embodiment, it will be in the range 12 to 48
hours. In a further embodiment still, it will be in the range 12 to
26 hours.
[0288] In addition, or alternatively to the above criteria, the
present invention provides a ligand or a composition comprising a
ligand according to the invention having an AUC value (area under
the curve) in the range of 1 mg.min/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300
mg.min/ml. In addition, or alternatively, a ligand or composition
according to the invention has an AUC in the range of up to 600
mg.min/ml. In one embodiment, the upper end of the range is 500,
400, 300, 200, 150, 100, 75 or 50 mg.min/ml. In one embodiment a
ligand according to the invention will have a AUC in the range
selected from the group consisting of the following: 15 to 150
mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50
mg.min/ml.
[0289] Polypeptides and dAbs of the invention and antagonists
comprising these can be formatted to have a larger hydrodynamic
size, for example, by attachment of a PEG group, serum albumin,
transferrin, transferrin receptor or at least the
transferrin-binding portion thereof, an antibody Fc region, or by
conjugation to an antibody domain. For example, polypeptides dAbs
and antagonists formatted as a larger antigen-binding fragment of
an antibody or as an antibody (e.g., formatted as a Fab, Fab',
F(ab).sub.2, F(ab').sub.2, IgG, scFv).
[0290] Hydrodynamic size of the ligands (e.g., dAb monomers and
multimers) of the invention may be determined using methods which
are well known in the art. For example, gel filtration
chromatography may be used to determine the hydrodynamic size of a
ligand. Suitable gel filtration matrices for determining the
hydrodynamic sizes of ligands, such as cross-linked agarose
matrices, are well known and readily available.
[0291] The size of a ligand format (e.g., the size of a PEG moiety
attached to a dAb monomer), can be varied depending on the desired
application. For example, where ligand is intended to leave the
circulation and enter into peripheral tissues, it is desirable to
keep the hydrodynamic size of the ligand low to facilitate
extravazation from the blood stream. Alternatively, where it is
desired to have the ligand remain in the systemic circulation for a
longer period of time the size of the ligand can be increased, for
example by formatting as an Ig like protein.
Half-Life Extension by Targeting an Antigen or Epitope that
Increases Half-Live in vivo
[0292] The hydrodynaminc size of a ligand and its serum half-life
can also be increased by conjugating or associating a VEGF binding
polypeptide, dAb or antagonist of the invention to a binding domain
(e.g., antibody or antibody fragment) that binds an antigen or
epitope that increases half-live in vivo, as described herein. For
example, the VEGF binding agent (e.g., polypeptide) can be
conjugated or linked to an anti-serum albumin or anti-neonatal Fc
receptor antibody or antibody fragment, eg an anti-SA or
anti-neonatal Fc receptor dAb, Fab, Fab' or scFv, or to an anti-SA
affibody or anti-neonatal Fc receptor Affibody or an anti-SA
avimer, or an anti-SA binding domain which comprises a scaffold
selected from, but preferably not limited to, the group consisting
of CTLA-4, lipocallin, SpA, an affibody, an avimer, GroEl and
fibronectin (see PCT/GB2008/000453 filed 8 Feb. 2008 for disclosure
of these binding domain, which domains and their sequences are
incorporated herein by reference and form part of the disclosure of
the present text). Conjugating refers to a composition comprising
polypeptide, dAb or antagonist of the invention that is bonded
(covalently or noncovalently) to a binding domain that binds serum
albumin.
[0293] Suitable polypeptides that enhance serum half-life in vivo
include, for example, transferrin receptor specific
ligand-neuropharmaceutical agent fusion proteins (see U.S. Pat. No.
5,977,307, the teachings of which are incorporated herein by
reference), brain capillary endothelial cell receptor, transferrin,
transferrin receptor (e.g., soluble transferrin receptor), insulin,
insulin-like growth factor 1 (IGF 1) receptor, insulin-like growth
factor 2 (IGF 2) receptor, insulin receptor, blood coagulation
factor X, .alpha.1-antitrypsin and HNF 1.alpha.. Suitable
polypeptides that enhance serum half-life also include alpha-1
glycoprotein (orosomucoid; AAG), alpha-1 antichymotrypsin (ACT),
alpha-1 microglobulin (protein HC; AIM), antithrombin III (AT III),
apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B),
ceruloplasmin (Cp), complement component C3 (C3), complement
component C4 (C4), C1 esterase inhibitor (C1 INH), C-reactive
protein (CRP), ferritin (FER), hemopexin (HPX), lipoprotein(a)
(Lp(a)), mannose-binding protein (MBP), myoglobin (Myo), prealbumin
(transthyretin; PAL), retinol-binding protein (RBP), and rheumatoid
factor (RF).
[0294] Suitable proteins from the extracellular matrix include, for
example, collagens, laminins, integrins and fibronectin. Collagens
are the major proteins of the extracellular matrix. About 15 types
of collagen molecules are currently known, found in different parts
of the body, e.g. type I collagen (accounting for 90% of body
collagen) found in bone, skin, tendon, ligaments, cornea, internal
organs or type II collagen found in cartilage, vertebral disc,
notochord, and vitreous humor of the eye.
[0295] Suitable proteins from the blood include, for example,
plasma proteins (e.g., fibrin, .alpha.-2 macroglobulin, serum
albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum
amyloid protein A, haptoglobin, profilin, ubiquitin, uteroglobulin
and .beta.-2-microglobulin), enzymes and enzyme inhibitors (e.g.,
plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin and
pancreatic trypsin inhibitor), proteins of the immune system, such
as immunoglobulin proteins (e.g., IgA, IgD, IgE, IgG, IgM,
immunoglobulin light chains (kappa/lambda)), transport proteins
(e.g., retinol binding protein, .alpha.-1 microglobulin), defensins
(e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin
2 and neutrophil defensin 3) and the like.
[0296] Suitable proteins found at the blood brain barrier or in
neural tissue include, for example, melanocortin receptor, myelin,
ascorbate transporter and the like.
[0297] Suitable polypeptides that enhance serum half-life in vivo
also include proteins localized to the kidney (e.g., polycystin,
type IV collagen, organic anion transporter Kl, Heymann's antigen),
proteins localized to the liver (e.g., alcohol dehydrogenase,
G250), proteins localized to the lung (e.g., secretory component,
which binds IgA), proteins localized to the heart (e.g., HSP 27,
which is associated with dilated cardiomyopathy), proteins
localized to the skin (e.g., keratin), bone specific proteins such
as morphogenic proteins (BMPs), which are a subset of the
transforming growth factor .beta. superfamily of proteins that
demonstrate osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6,
BMP-7, BMP-8), tumor specific proteins (e.g., trophoblast antigen,
herceptin receptor, oestrogen receptor, cathepsins (e.g., cathepsin
B, which can be found in liver and spleen)).
[0298] Suitable disease-specific proteins include, for example,
antigens expressed only on activated T-cells, including LAG-3
(lymphocyte activation gene), osteoprotegerin ligand (OPGL; see
Nature 402, 304-309 (1999)), OX40 (a member of the TNF receptor
family, expressed on activated T cells and specifically
up-regulated in human T cell leukemia virus type-I
(HTLV-I)-producing cells; see Immunol. 165 (1):263-70 (2000)).
Suitable disease-specific proteins also include, for example,
metalloproteases (associated with arthritis/cancers) including
CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2,
murine ftsH; and angiogenic growth factors, including acidic
fibroblast growth factor (FGF-1), basic fibroblast growth factor
(FGF-2), vascular endothelial growth factor/vascular permeability
factor (VEGF/VPF), transforming growth factor-.alpha. (TGF
.alpha.), tumor necrosis factor-alpha (TNF-.alpha.), angiogenin,
interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived
endothelial growth factor (PD-ECGF), placental growth factor
(P1GF), midkine platelet-derived growth factor-BB (PDGF), and
fractalkine
[0299] Suitable polypeptides that enhance serum half-life in vivo
also include stress proteins such as heat shock proteins (HSPs).
HSPs are normally found intracellularly. When they are found
extracellularly, it is an indicator that a cell has died and
spilled out its contents. This unprogrammed cell death (necrosis)
occurs when as a result of trauma, disease or injury, extracellular
HSPs trigger a response from the immune system. Binding to
extracellular HSP can result in localizing the compositions of the
invention to a disease site.
[0300] Suitable proteins involved in Fc transport include, for
example, Brambell receptor (also known as FcRB). This Fc receptor
has two functions, both of which are potentially useful for
delivery. The functions are (1) transport of IgG from mother to
child across the placenta (2) protection of IgG from degradation
thereby prolonging its serum half-life. It is thought that the
receptor recycles IgG from endosomes. (See, Holliger et al, Nat
Biotechnol 15(7):632-6 (1997).)
dAbs that Bind Serum Albumin
[0301] The invention in one embodiment provides a polypeptide or
antagonist (e.g., dual specific ligand comprising an anti-TNFR1 dAb
(a first dAb) that binds to TNFR1 and a second dAb that binds serum
albumin (SA), the second dAb binding SA with a K.sub.D as
determined by surface plasmon resonance of 1 nM to 1, 2, 3, 4, 5,
10, 20, 30, 40, 50, 60, 70, 100, 200, 300, 400 or 500 .mu.M (i.e.,
.times.10.sup.-9 to 5.times.10.sup.-4), or 100 nM to 10 .mu.M, or 1
to 5 .mu.M or 3 to 70 nM or 10 nM to 1, 2, 3, 4 or 5 .mu.M. For
example 30 to 70 nM as determined by surface plasmon resonance In
one embodiment, the first dAb (or a dAb monomer) binds SA (e.g.,
HSA) with a K.sub.D as determined by surface plasmon resonance of
approximately 1, 50, 70, 100, 150, 200, 300 nM or 1, 2 or 3 .mu.M.
In one embodiment, for a dual specific ligand comprising a first
anti-SA dAb and a second dAb to VEGF, the affinity (eg K.sub.D
and/or K.sub.off as measured by surface plasmon resonance, eg using
BiaCore) of the second dAb for its target is from 1 to 100000 times
(eg, 100 to 100000, or 1000 to 100000, or 10000 to 100000 times)
the affinity of the first dAb for SA. In one embodiment, the serum
albumin is human serum albumin (HSA). For example, the first dAb
binds SA with an affinity of approximately 10 .mu.M, while the
second dAb binds its target with an affinity of 100 pM. In one
embodiment, the serum albumin is human serum albumin (HSA). In one
embodiment, the first dAb binds SA (eg, HSA) with a K.sub.D of
approximately 50, for example 70, 100, 150 or 200 nM. Details of
dual specific ligands are found in WO03002609, WO04003019 and
WO04058821.
[0302] The ligands of the invention can in one embodiment comprise
a dAb that binds serum albumin (SA) with a K.sub.D as determined by
surface plasmon resonance of 1 nM to 1, 2, 3, 4, 5, 10, 20, 30, 40,
50, 60, 70, 100, 200, 300, 400 or 500 .mu.M (i.e., .times.10-9 to
5.times.10-4), or 100 nM to 10 .mu.M, or 1 to 5 .mu.M or 3 to 70 nM
or 10 nM to 1, 2, 3, 4 or 5 .mu.M.
[0303] For example 30 to 70 nM as determined by surface plasmon
resonance In one embodiment, the first dAb (or a dAb monomer) binds
SA (e.g., HSA) with a K.sub.D as determined by surface plasmon
resonance of approximately 1, 50, 70, 100, 150, 200, 300 nM or 1, 2
or 3 .mu.M. In one embodiment, the first and second dAbs are linked
by a linker, for example a linker of from 1 to 4 amino acids or
from 1 to 3 amino acids, or greater than 3 amino acids or greater
than 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids. In one embodiment,
a longer linker (greater than 3 amino acids) is used to enhance
potency (K.sub.D of one or both dAbs in the antagonist).
[0304] In particular embodiments of the ligands and antagonists,
the dAb binds human serum albumin and competes for binding to
albumin with a dAb selected from the group consisting of
[0305] MSA-16, MSA-26 (See WO04003019 for disclosure of these
sequences, which sequences and their nucleic acid counterpart are
incorporated herein by reference and form part of the disclosure of
the present text),
[0306] DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474),
DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ
ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479),
DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID
NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484),
DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID
NO: 487), DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490),
DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ
ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27 (SEQ ID NO: 495),
DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ
ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500),
DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19 (SEQ
ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),
DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ
ID NO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510),
DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ
ID NO: 513), DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515),
DOM7r-32 (SEQ ID NO: 516), DOM7r-33 (SEQ ID NO: 517) (See
WO2007080392 for disclosure of these sequences, which sequences and
their nucleic acid counterpart are incorporated herein by reference
and form part of the disclosure of the present text; the SEQ ID
No's in this paragraph are those that appear in WO2007080392),
[0307] dAb8 (dAb10), dAb 10, dAb36, dAb7r20 (DOM7r20), dAb7r2l
(DOM7r21), dAb7r22 (DOM7r22), dAb7r23 (DOM7r23), dAb7r24 (DOM7r24),
dAb7r25 (DOM7r25), dAb7r26 (DOM7r26), dAb7r27 (DOM7r27), dAb7r28
(DOM7r28), dAb7r29 (DOM7r29), dAb7r29 (DOM7r29), dAb7r3l (DOM7r31),
dAb7r32 (DOM7r32), dAb7r33 (DOM7r33), dAb7r33 (DOM7r33), dAb7h22
(DOM7h22), dAb7h23 (DOM7h23), dAb7h24 (DOM7h24), dAb7h25 (DOM7h25),
dAb7h26 (DOM7h26), dAb7h27 (DOM7h27), dAb7h30 (DOM7h30), dAb7h3l
(DOM7h31), dAb2 (dAbs 4,7,41), dAb4, dAb7, dAb11, dAb12 (dAb7m12),
dAb13 (dAb 15), dAb15, dAb16 (dAb21, dAb7m16), dAb17, dAb18, dAb19,
dAb21, dAb22, dAb23, dAb24, dAb25 (dAb26, dAb7m26), dAb27, dAb30
(dAb35), dAb31, dAb33, dAb34, dAb35, dAb38 (dAb54), dAb41, dAb46
(dAbs 47, 52 and 56), dAb47, dAb52, dAb53, dAb54, dAb55, dAb56,
dAb7m12, dAb7m16, dAb7m26, dAb7r1 (DOM 7r1), dAb7r3 (DOM7r3),
dAb7r4 (DOM7r4), dAb7r5 (DOM7r5), dAb7r7 (DOM7r7), dAb7r8 (DOM7r8),
dAb7r13 (DOM7r13), dAb7r14 (DOM7r14), dAb7r15 (DOM7r15), dAb7r16
(DOM7r16), dAb7r17 (DOM7r17), dAb7r18 (DOM7r18), dAb7r19 (DOM7r19),
dAb7h1 (DOM7h1), dAb7h2 (DOM7h2), dAb7h6 (DOM7h6), dAb7h7 (DOM7h7),
dAb7h8 (DOM7h8), dAb7h9 (DOM7h9), dAb7h10 (DOM7h10), dAb7h11
(DOM7h11), dAb7h12 (DOM7h12), dAb7h13 (DOM7h13), dAb7h14 (DOM7h14),
dAb7p1 (DOM7p1), and dAb7p2 (DOM7p2) (see PCT/GB2008/000453 filed
8.sup.th Feb. 2008 for disclosure of these sequences, which
sequences and their nucleic acid counterpart are incorporated
herein by reference and form part of the disclosure of the present
text). Alternative names are shown in brackets after the dAb, e.g.
dAb8 has an alternative name which is dAb10 i.e. dAb8 (dAb10).
These sequences are also set out in FIGS. 51a and b.
[0308] In certain embodiments, the dAb binds human serum albumin
and comprises an amino acid sequence that has at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99% amino acid sequence identity with
the amino acid sequence of a dAb selected from the group consisting
of MSA-16, MSA-26,
[0309] DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474),
DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ
ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479),
DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID
NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484),
DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID
NO: 487), DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490),
DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ
ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27 (SEQ ID NO: 495),
DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ
ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500),
DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19 (SEQ
ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),
DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ
ID NO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510),
DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ
ID NO: 513), DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515),
DOM7r-32 (SEQ ID NO: 516), DOM7r-33 (SEQ ID NO: 517) (the SEQ ID
No's in this paragraph are those that appear in WO2007080392),
[0310] dAb8, dAb 10, dAb36, dAb7r20, dAb7r2l, dAb7r22, dAb7r23,
dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,
dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24,
Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11,
dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,
dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,
dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,
dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,
dAb7r18, dAb7r.sup.19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,
dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and
dAb7p2.
[0311] For example, the dAb that binds human serum albumin can
comprise an amino acid sequence that has at least about 90%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99% amino acid sequence
identity with DOM7h-2 (SEQ ID NO:482), DOM7h-3 (SEQ ID NO:483),
DOM7h-4 (SEQ ID NO:484), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ ID
NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496), DOM7r-13
(SEQ ID NO:497), DOM7r-14 (SEQ ID NO:498), DOM7h-22 (SEQ ID
NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ ID NO:491),
DOM7h-25 (SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493), DOM7h-21 (SEQ
ID NO:494), DOM7h-27 (SEQ ID NO:495) (the SEQ ID No's in this
paragraph are those that appear in WO2007080392),
[0312] dAb8, dAb 10, dAb36, dAb7h21, dAb7h22, dAb7h23, Ab7h24,
Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11,
dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,
dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,
dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,
dAb7h11, dAb7h12, dAb7h13 and dAb7h14
[0313] In certain embodiments, the dAb binds human serum albumin
and comprises an amino acid sequence that has at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99% amino acid sequence identity with
the amino acid sequence of a dAb selected from the group consisting
of
[0314] DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1
(SEQ ID NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496),
DOM7h-22 (SEQ ID NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ
ID NO:491), DOM7h-25 (SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493),
DOM7h-21 (SEQ ID NO:494), DOM7h-27 (SEQ ID NO:495) (the SEQ ID No's
in this paragraph are those that appear in WO2007080392),
[0315] dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,
dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb38, dAb41, dAb7h1, dAb7h2,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13
and dAb7h14.
[0316] In more particular embodiments, the dAb is a V.sub..kappa.
dAb that binds human serum albumin and has an amino acid sequence
selected from the group consisting of
[0317] DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1
(SEQ ID NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496)
(the SEQ ID No's in this paragraph are those that appear in
WO2007080392),
[0318] dAb2, dAb4, dAb7, dAb38, dAb41, dAb54, dAb7h1, dAb7h2,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13
and dAb7h14.
[0319] In more particular embodiments, the dAb is a V.sub.H dAb
that binds human serum albumin and has an amino acid sequence
selected from dAb7h30 and dAb7h31.
[0320] In more particular embodiments, the dAb is dAb7h11 or
dAb7h14.
[0321] In other embodiments, the dAb, ligand or antagonist binds
human serum albumin and comprises one, two or three of the CDRs of
any of the foregoing amino acid sequences, eg one, two or three of
the CDRs of dAb7h11 or dAb7h14.
[0322] Suitable Camelid V.sub.HH that bind serum albumin include
those disclosed in WO 2004/041862 (Ablynx N.V.) and in WO2007080392
(which V.sub.HH sequences and their nucleic acid counterpart are
incorporated herein by reference and form part of the disclosure of
the present text), such as Sequence A (SEQ ID NO:518), Sequence B
(SEQ ID NO:519), Sequence C (SEQ ID NO:520), Sequence D (SEQ ID
NO:521), Sequence E (SEQ ID NO:522), Sequence F (SEQ ID NO:523),
Sequence G (SEQ ID NO:524), Sequence H (SEQ ID NO:525), Sequence I
(SEQ ID NO:526), Sequence J (SEQ ID NO:527), Sequence K (SEQ ID
NO:528), Sequence L (SEQ ID NO:529), Sequence M (SEQ ID NO:530),
Sequence N (SEQ ID NO:531), Sequence O (SEQ ID NO:532), Sequence P
(SEQ ID NO:533), Sequence Q (SEQ ID NO:534), these sequence numbers
corresponding to those cited in WO2007080392 or WO 2004/041862
(Ablynx N.V.). In certain embodiments, the Camelid V.sub.HH binds
human serum albumin and comprises an amino acid sequence that has
at least about 80%, or at least about 85%, or at least about 90%,
or at least about 95%, or at least about 96%, or at least about
97%, or at least about 98%, or at least about 99% amino acid
sequence identity with ALB1 disclosed in WO2007080392 or with any
one of SEQ ID NOS:518-534, these sequence numbers corresponding to
those cited in WO2007080392 or WO 2004/041862.
[0323] In some embodiments, the ligand or antagonist comprises an
anti-serum albumin dAb that competes with any anti-serum albumin
dAb disclosed herein for binding to serum albumin (e.g., human
serum albumin).
[0324] In an alternative embodiment, the antagonist or ligand
comprises a binding moiety specific for VEGF (eg, human VEGF),
wherein the moiety comprises non-immunoglobulin sequences as
described in co-pending application PCT/GB2008/000453 filed 8 Feb.
2008, the disclosure of these binding moieties, their methods of
production and selection (eg, from diverse libraries) and their
sequences are incorporated herein by reference as part of the
disclosure of the present text)
Conjugation to a Half-Life Extending Moiety (eg, Albumin)
[0325] In one embodiment, a (one or more) half-life extending
moiety (eg, albumin, transferrin and fragments and analogues
thereof) is conjugated or associated with the VEGF-binding
polypeptide, dAb or antagonist of the invention. Examples of
suitable albumin, albumin fragments or albumin variants for use in
a VEGF-binding format are described in WO 2005077042, which
disclosure is incorporated herein by reference and forms part of
the disclosure of the present text. In particular, the following
albumin, albumin fragments or albumin variants can be used in the
present invention: [0326] SEQ ID NO:1 (as disclosed in WO
2005077042, this sequence being explicitly incorporated into the
present disclosure by reference); [0327] Albumin fragment or
variant comprising or consisting of amino acids 1-387 of SEQ ID
NO:1 in WO 2005077042; [0328] Albumin, or fragment or variant
thereof, comprising an amino acid sequence selected from the group
consisting of: (a) amino acids 54 to 61 of SEQ ID NO:1 in WO
2005077042; (b) amino acids 76 to 89 of SEQ ID NO:1 in WO
2005077042; (c) amino acids 92 to 100 of SEQ ID NO:1 in WO
2005077042; (d) amino acids 170 to 176 of SEQ ID NO:1 in WO
2005077042; (e) amino acids 247 to 252 of SEQ ID NO:1 in WO
2005077042; (f) amino acids 266 to 277 of SEQ ID NO:1 in WO
2005077042; (g) amino acids 280 to 288 of SEQ ID NO:1 in WO
2005077042; (h) amino acids 362 to 368 of SEQ ID NO:1 in WO
2005077042; (i) amino acids 439 to 447 of SEQ ID NO:1 in WO
2005077042 (j) amino acids 462 to 475 of SEQ ID NO:1 in WO
2005077042; (k) amino acids 478 to 486 of SEQ ID NO:1 in WO
2005077042; and (1) amino acids 560 to 566 of SEQ ID NO:1 in WO
2005077042.
[0329] Further examples of suitable albumin, fragments and analogs
for use in a VEGF binding format are described in WO 03076567,
which disclosure is incorporated herein by reference and which
forms part of the disclosure of the present text. In particular,
the following albumin, fragments or variants can be used in the
present invention: [0330] Human serum albumin as described in WO
03076567, eg, in FIG. 3 (this sequence information being explicitly
incorporated into the present disclosure by reference); [0331]
Human serum albumin (HA) consisting of a single non-glycosylated
polypeptide chain of 585 amino acids with a formula molecular
weight of 66,500 (See, Meloun, et al., FEBS Letters 58:136 (1975);
Behrens, et al., Fed. Proc. 34:591 (1975); Lawn, et al., Nucleic
Acids Research 9:6102-6114 (1981); Minghetti, et al., J. Biol.
Chem. 261:6747 (1986)); [0332] A polymorphic variant or analog or
fragment of albumin as described in Weitkamp, et al., Ann. Hum.
Genet. 37:219 (1973); [0333] An albumin fragment or variant as
described in EP 322094, eg, HA(1-373, HA(1-388), HA(1-389),
HA(1-369), and HA(1-419) and fragments between 1-369 and 1-419;
[0334] An albumin fragment or variant as described in EP 399666,
eg, HA(1-177) and HA(1-200) and fragments between HA(1-X), where X
is any number from 178 to 199.
[0335] Where a (one or more) half-life extending moiety (eg,
albumin, transferrin and fragments and analogues thereof) is used
to format the VEGF-binding polypeptides, dAbs and antagonists of
the invention, it can be conjugated using any suitable method, such
as, by direct fusion to the VEGF-binding moiety (eg, anti-VEGF
dAb), for example by using a single nucleotide construct that
encodes a fusion protein, wherein the fusion protein is encoded as
a single polypeptide chain with the half-life extending moiety
located N- or C-terminally to the VEGF binding moiety.
Alternatively, conjugation can be achieved by using a peptide
linker between moieties, eg, a peptide linker as described in WO
03076567 or WO 2004003019 (these linker disclosures being
incorporated by reference in the present disclosure to provide
examples for use in the present invention). Typically, a
polypeptide that enhances serum half-life in vivo is a polypeptide
which occurs naturally in vivo and which resists degradation or
removal by endogenous mechanisms which remove unwanted material
from the organism (e.g., human). For example, a polypeptide that
enhances serum half-life in vivo can be selected from proteins from
the extracellular matrix, proteins found in blood, proteins found
at the blood brain barrier or in neural tissue, proteins localized
to the kidney, liver, lung, heart, skin or bone, stress proteins,
disease-specific proteins, or proteins involved in Fc
transport.
[0336] In embodiments of the invention described throughout this
disclosure, instead of the use of an anti-VEGF "dAb" in an
antagonist or ligand of the invention, it is contemplated that the
skilled addressee can use a polypeptide or domain that comprises
one or more or all 3 of the CDRs of a dAb of the invention that
binds VEGF (e.g., CDRs grafted onto a suitable protein scaffold or
skeleton, eg an affibody, an SpA scaffold, an LDL receptor class A
domain or an EGF domain) The disclosure as a whole is to be
construed accordingly to provide disclosure of antagonists using
such domains in place of a dAb. In this respect, see
PCT/GB2008/000453 filed 8 Feb. 2008, the disclosure of which is
incorporated by reference).
[0337] In one embodiment, therefore, an antagonist of the invention
comprises an immunoglobulin single variable domain or domain
antibody (dAb) that has binding specificity for VEGF or the
complementarity determining regions of such a dAb in a suitable
format. The antagonist can be a polypeptide that consists of such a
dAb, or consists essentially of such a dAb. The antagonist can be a
polypeptide that comprises a dAb (or the CDRs of a dAb) in a
suitable format, such as an antibody format (e.g., IgG-like format,
scFv, Fab, Fab', F(ab').sub.2), or a dual specific ligand that
comprises a dAb that binds VEGF and a second dAb that binds another
target protein, antigen or epitope (e.g., serum albumin).
[0338] Polypeptides, dAbs and antagonists according to the
invention can be formatted as a variety of suitable antibody
formats that are known in the art, such as, IgG-like formats,
chimeric antibodies, humanized antibodies, human antibodies, single
chain antibodies, bispecific antibodies, antibody heavy chains,
antibody light chains, homodimers and heterodimers of antibody
heavy chains and/or light chains, antigen-binding fragments of any
of the foregoing (e.g., a Fv fragment (e.g., single chain Fv
(scFv), a disulfide bonded Fv), a Fab fragment, a Fab' fragment, a
F(ab').sub.2 fragment), a single variable domain (e.g., V.sub.H,
V.sub.L), a dAb, and modified versions of any of the foregoing
(e.g., modified by the covalent attachment of polyalkylene glycol
(e.g., polyethylene glycol, polypropylene glycol, polybutylene
glycol) or other suitable polymer).
[0339] In some embodiments, the invention provides a ligand (eg, an
anti-VEGF antagonist) that is an IgG-like format. Such formats have
the conventional four chain structure of an IgG molecule (2 heavy
chains and two light chains), in which one or more of the variable
regions (V.sub.H and or V.sub.L) have been replaced with a dAb of
the invention. In one embodiment, each of the variable regions (2
V.sub.H regions and 2 V.sub.L regions) is replaced with a dAb or
single variable domain, at least one of which is an anti-VEGF dAb
according to the invention. The dAb(s) or single variable domain(s)
that are included in an IgG-like format can have the same
specificity or different specificities. In some embodiments, the
IgG-like format is tetravalent and can have one (anti-VEGF only),
two (eg, anti-VEGF and anti-SA), three or four specificities. For
example, the IgG-like format can be monospecific and comprises 4
dAbs that have the same specificity; bispecific and comprises 3
dAbs that have the same specificity and another dAb that has a
different specificity; bispecific and comprise two dAbs that have
the same specificity and two dAbs that have a common but different
specificity; trispecific and comprises first and second dAbs that
have the same specificity, a third dAb with a different specificity
and a fourth dAb with a different specificity from the first,
second and third dAbs; or tetraspecific and comprise four dAbs that
each have a different specificity. Antigen-binding fragments of
IgG-like formats (e.g., Fab, F(ab').sub.2, Fab', Fv, scF.sub.v) can
be prepared. In one embodiment, the IgG-like formats or
antigen-binding fragments thereof do not crosslink VEGF, for
example, the format may be monovalent for VEGF. If complement
activation and/or antibody dependent cellular cytotoxicity (ADCC)
function is desired, the ligand can be an IgG1-like format. If
desired, the IgG-like format can comprise a mutated constant region
(variant IgG heavy chain constant region) to minimize binding to Fc
receptors and/or ability to fix complement (see e.g. Winter et al.,
GB 2,209,757 B; Morrison et al., WO 89/07142; Morgan et al., WO
94/29351, Dec. 22, 1994).
[0340] The ligands of the invention (polypeptides, dAbs and
antagonists) can be formatted as a fusion protein that contains a
first immunoglobulin single variable domain that is fused directly
to a second immunoglobulin single variable domain. If desired such
a format can further comprise a half-life extending moiety. For
example, the ligand can comprise a first immunoglobulin single
variable domain that is fused directly to a second immunoglobulin
single variable domain that is fused directly to an immunoglobulin
single variable domain that binds serum albumin.
[0341] Generally the orientation of the polypeptide domains that
have a binding site with binding specificity for a target, and
whether the ligand comprises a linker, is a matter of design
choice. However, some orientations, with or without linkers, may
provide better binding characteristics than other orientations. All
orientations (e.g., dAb1-linker-dAb2; dAb2-linker-dAb1) are
encompassed by the invention are ligands that contain an
orientation that provides desired binding characteristics can be
easily identified by screening.
[0342] Polypeptides and dAbs according to the invention, including
dAb monomers, dimers and trimers, can be linked to an antibody Fc
region, comprising one or both of C.sub.H2 and C.sub.H3 domains,
and optionally a hinge region. For example, vectors encoding
ligands linked as a single nucleotide sequence to an Fc region may
be used to prepare such polypeptides.
[0343] The invention moreover provides dimers, trimers and polymers
of the aforementioned dAb monomers e.g. of anti-VEGF dAb
monomers.
Codon Optimised Sequences
[0344] As described above, embodiments of the invention provide
codon optimized nucleotide sequences encoding polypeptides and
variable domains of the invention. As shown in the following
illustration, codon optimized sequences of about 70% identity can
be produced that encode for the same variable domain (in this case
the variable domain amino acid sequence is identical to
DOM1h-131-206). In this instance, the sequences were optimized for
expression by Pichia pastoris (codon optimized sequences 1-3) or E.
coli (codon optimized sequences 4 and 5).
[0345] We performed a calculation taking into account the
degeneracy in the genetic code and maximised the number of
nucleotide changes within each degenerate codon encoded by the
nucleotide sequence of DOM1h-131-206 (as shown below as
DOM1h-131-206 WT) and a theoretical nucleotide sequence which still
encodes a variable domain that is identical to DOM1h-131-206. The
calculation revealed that the theoretical sequence would have only
57% identity to the nucleotide sequence of DOM1h-131-206.
Codon Optimised Sequence 1
DNA Sequence
TABLE-US-00002 [0346]
gaggttcaattgttggaatccggtggtggattggttcaacctggtggttc
tttgagattgtcctgtgctgcttccggttttactttcgctcacgagacta
tggtttgggttagacaggctccaggtaaaggattggaatgggtttcccac
attccaccagatggtcaagatccattctacgctgactccgttaagggaag
attcactatctccagagacaactccaagaacactttgtacttgcagatga
actccttgagagctgaggatactgctgtttaccactgtgctttgttgcca
aagagaggaccttggtttgattactggggacagggaactttggttactgt ttcttcc
Corresponding AA Sequence
TABLE-US-00003 [0347]
evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvsh
ippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp
krgpwfdywgqgtlvtvss
[0348] 74.1% nucleotide sequence identity to WT sequence
##STR00001##
Codon Optimised Sequence 2
DNA Sequence
TABLE-US-00004 [0349]
gagaaaagagaggttcaattgcttgaatctggaggaggtttggtccagcc
aggagggtcccttcgactaagttgtgctgccagtgggtttacgtttgctc
atgaaactatggtatgggtccgacaggcacctggtaaaggtcttgaatgg
gtttcacatatccctccagacggtcaagacccattttacgctgattccgt
gaaaggcagatttacaatttcacgagataattctaaaaacaccttgtact
tacaaatgaactcattgagagctgaggacactgcagtttatcactgcgct
ttactaccaaaacgtggaccttggtttgattattggggccaaggtacgtt
agtgactgttagttct
Corresponding AA Sequence
TABLE-US-00005 [0350]
ekrevqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglew
vshippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhca
llpkrgpwfdywgqgtlvtvss
[0351] 71.1% nucleotide sequence identity to WT sequence
##STR00002##
Codon Optimised Sequence 3
DNA Sequence
TABLE-US-00006 [0352]
gaagtgcagcttcttgaaagtggtggagggctagtgcagccagggggatc
tttaagattatcatgcgctgccagtggatttacttttgctcacgagacga
tggtctgggtgagacaagctcctggaaaaggtttagagtgggtttctcac
attccacctgatggtcaagatcctttctacgcagattccgtcaaaggaag
atttactatctccagagataatagtaaaaacactttgtacctacagatga
actcacttagagccgaagataccgctgtgtaccactgcgccttgttgcca
aagagaggtccttggttcgattactggggtcagggtactctggttacagt ctcatct
Corresponding AA Sequence
TABLE-US-00007 [0353]
evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvsh
ippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp
krgpwfdywgqgtlvtvss
[0354] 72.6% nucleotide sequence identity to WT sequence
##STR00003##
Codon Optimised Sequence 4
DNA Sequence
TABLE-US-00008 [0355]
gaagtacaactgctggagagcggtggcggcctggttcaaccgggtggttc
cctgcgcctgtcctgtgcggcatctggtttcaccttcgcacacgaaacca
tggtgtgggttcgccaagctccgggcaaaggcctggaatgggtaagccac
attcctccagatggccaggacccattctatgcggattccgttaagggtcg
ctttaccatttctcgtgataactccaaaaacaccctgtacctgcagatga
actccctgcgcgccgaggatactgcggtgtaccattgtgcgctgctgcct
aaacgtggcccgtggttcgattactggggtcagggtactctggtcaccgt aagcagc
Corresponding AA Sequence
TABLE-US-00009 [0356]
evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvsh
ippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp
krgpwfdywgqgtlvtvss
[0357] 76.5% nucleotide sequence identity to WT sequence
##STR00004##
Codon Optimised Sequence 5
DNA Sequence
TABLE-US-00010 [0358]
gaggttcaactgctggaatctggtggtggtctggtacaaccgggtggttc
cctgcgtctgagctgtgcagcctctggtttcaccttcgctcatgagacca
tggtttgggtacgccaggctccgggtaaaggcctggagtgggtaagccat
atccctcctgatggtcaggacccgttctatgctgattccgtcaaaggccg
ttttaccatttctcgtgacaacagcaaaaacactctgtacctgcaaatga
actccctgcgtgcagaagacacggcggtttatcactgtgcactgctgcca
aaacgcggcccttggttcgactactggggccagggtactctggtcactgt atcttct
Corresponding AA Sequence
TABLE-US-00011 [0359]
evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvsh
ippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp
krgpwfdywgqgtlvtvss
[0360] 78.4% nucleotide sequence identity to WT sequence
##STR00005##
Exemplification
Example A
Lead Selection & Characterisation of Domain Antibodies to Human
TNFR1
[0361] Domain antibodies generated were derived from phage
libraries. Both soluble selections and panning to passively
absorbed human TNFR1 were performed according to the relevant
standard methods. Human TNFR1 was purchased as a soluble
recombinant protein either from R&D systems (Cat No
636-R1-025/CF) or Peprotech (Cat no. 310-07) and either used
directly (in the case of passive selections) or after biotinylation
using coupling via primary amines followed by quality control of
its activity in a biological assay and analysis of its MW and
extent of biotinylation by mass spectrometry. Typically 3 rounds of
selection were performed utilising decreasing levels of antigen in
every next round.
[0362] Outputs from selections were screened by phage ELISA for the
presence of anti-TNFR1 binding clones. DNA was isolated from these
phage selections and subcloned into a expression vector for
expression of soluble dAb fragments. Soluble dAb fragments were
expressed in 96-well plates and the supernantants were used to
screen for the presence of anti-TNFR1 binding dAbs, either using a
direct binding ELISA with anti-c-myc detection or BIAcore.TM. using
a streptavidin/biotinylated TNFR1 BIAcore.TM. chip and ranked
according to off-rates.
[0363] The lead molecules, described below, were derived from the
parental dAb, designated DOM1h-131 (disclosed in WO2006038027).
This molecule was selected from the phage display library after 3
rounds of selections using 60 nM of biotinylated antigen.
Streptavidin or neutravidin coated Dyna beads were alternated as
capture reagents in each round of selection to prevent selection of
binders against either streptavidin or neutravidin. The potency of
the lead DOM1h-131 at this stage was in the low micromolar range as
determined in the MRC-5 fibroblast/IL-8 release cell assay. The
binding kinetics as determined by BIAcore.TM. typically displayed
fast-on/fast-off rates. E. coli expression levels of this DOM1h-131
lead molecule, as a C-terminally myc tagged monomer were in the
region of 8 mg/l.
Affinity Maturation of Leads:
[0364] DOM1h-131 was taken forward into affinity maturation to
generate mutants with higher potency and improved biophysical
characteristics (see FIG. 3 for amino acid sequences of DOM1h-131
derived leads). After generation of an error-prone library (average
number of 1 amino acid change per dAb sequence, library size
8.times.10.sup.7) using an error-prone PCR polymerase (Genemorph
II, Stratagene), seven rounds of selection utilising these
error-prone libraries were performed. This strategy led to the
isolation of clone DOM1h-131-8, a molecule where 4 amino acid
changes (one in framework 1 (FR1), one in CDR1, one in CDR3 and one
in FR4) gave an approximate 100-fold improvement in potency as
measured by the MRC-5 cell assay (.about.4 nM). In this assay MRC-5
cells were incubated with the test samples for one hour then
TNF-.alpha. (200 pg/ml) was added. After an overnight incubation
IL-8 release was determined using an IL-8 ABI 8200 cellular
detection assay (FMAT). A TNF-.alpha. dose curve was included in
each experiment. The concentration of TNF-.alpha. used to compete
with dAb binding to TNFR1 (200 pg/ml) was approximately 70% of the
maximum TNF-.alpha. response in this assay.
[0365] In order to further improve potency, single amino acid
positions were diversified by oligo-directed mutagenesis at key
positions suggested by the error-prone lead consensus information.
During this process an improved version of the DOM1h-131-8 clone,
DOM1h-131-24 (originally named DOM1h-131-8-2 prior to correction)
was isolated through BIAcore.TM. screening that had a single K94R
amino acid mutation (amino acid numbering according to Kabat) and
an RBA potency of 200-300 pM.
[0366] Further error-prone libraries based on this lead and the NNS
library from which it was derived were generated and subjected to
three rounds of phage selections using heat treatment (for method
see Jespers L, et al., Aggregation-resistant domain antibodies
selected on phage by heat denaturation. Nat Biotechnol. 2004
September; 22(9):1161-5). During this selection, libraries were
pooled and clones derived from round two of the selection yielded
dAbs such as DOM1h-131-53 which were considered to be more heat
stable. It was hypothesised that these clones would possess better
biophysical characteristics. Some framework mutations in clone
DOM1h-131-53 were germlined to generate clone DOM1h-131-83. This
clone formed the basis for further diversification via
oligo-directed individual CDR mutagenesis either using phage
display selection as described above or using the in-vitro
compartmentalization technology using emulsions. The phage display
strategy generated leads DOM1h-131-117 and DOM1h-131-151. The
in-vitro compartmentalization technology generated
DOM1h-131-511.
[0367] At this stage these three leads were compared in biophysical
and biological assays and DOM1h-131-511 was the molecule with the
best properties. Furthermore these molecules were tested for their
resistance to proteolytic cleavage in the presence of trypsin or
leucozyme. Leucozyme consists of pooled sputum from patients with
cystic fibrosis and contains high levels of elastase and other
proteases and was used as a surrogate for in vivo conditions in
lung diseases. This data indicated that all three leads
DOM1h-131-117, DOM1h-131-151 and DOM1h-131-511 were rapidly
degraded in presence of trypsin or leucozyme. This finding raised
concerns about the in vivo persistence of DOM1h-131-511 when in the
patient and a strategy was developed to select for improved
resistance to trypsin. It was hypothesised that such improved
trypsin resistance could have a beneficial effect on other
biophysical properties of the molecule. Essentially the standard
phage selection method was modified to allow for selection in the
presence of proteases prior to selection on antigen. To this end a
new phage vector was engineered in which the c-myc tag was deleted
to allow selections in the presence of trypsin without cleaving the
displayed dAb off the phage. DOM1h-131-511 based error-prone
libraries were generated and cloned in the pDOM33 vector (see FIG.
50 for pDOM33 vector map). Phage stocks generated from this library
were pre-treated with either 1 mg/ml or 100 .mu.g/ml trypsin at
37.degree. C. for 24 hours, subsequently protease inhibitor which
was Roche Complete Protease Inhibitors (2.times.) was added to
block the trypsin activity prior to selection on the relevant
antigen. Four rounds of selection were performed. Soluble expressed
TNFR1 binding dAbs were assessed using the BIAcore.TM. for their
ability to bind TNFR1 with or without the presence of proteases
during one hour or overnight incubations at 37.degree. C. in the
presence or absence of trypsin (at 100 .mu.g/ml or 1000 .mu.g/ml
final trypsin concentration). This led to the isolation of two lead
molecules DOM1h-131-202 and DOM1h-131-206 which demonstrated
improved protease resistance as shown by BIAcore.TM. antigen
binding experiments. It is interesting to note that DOM1h-131-202
contained only one mutation in CDR2 (V53D), all amino acid
numbering according to Kabat) in comparison to DOM1h-131-511,
whereas DOM1h-131-206 contained only two mutations: the first
mutation is the same as in DOM1h-131-202 (V53D mutation in CDR2)
and the second is a Y91H mutation in FR3 (see FIG. 3). This Y91H
mutation in FR3 does occur in the 3-20 human germline gene
indicating that this residue occurs in human antibodies. The three
clones DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206 have amino
acid sequences as shown in FIG. 3.
[0368] Activity of the Molecules was determined as below:
[0369] BIAcore.TM. binding affinity assessment of DOM1H-131-202,
DOM1H-131-511 and DOM1H-131-206 for binding to human TNFR1.
[0370] The binding affinities of DOM1H-131-202, DOM1H-131-511 and
DOM1H-131-206 for binding to human recombinant E. coli-expressed
human TNFR1 were assessed by BIAcore.TM. analysis. Analysis was
carried out using biotinylated human TNFR1. 1400 RU of biotinylated
TNFR1 was coated to a streptavidin (SA) chip. The surface was
regenerated back to baseline using mild acid elution conditions.
DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 were passed over
this surface at defined concentrations using a flow rate of 50
.mu.l/min. The work was carried out on a BIAcore.TM. 3000 machine
and data were analysed and fitted to the 1:1 model of binding. The
binding data fitted well to the 1:1 model for all tested molecules.
All K.sub.D values were calculated from k.sub.on and k.sub.off
rates. BIAcore.TM. runs were carried out at 25.degree. C.
[0371] The data below were produced from three independent
experiments. In each experiment the results were calculated by
averaging a number of fits using highest dAb concentrations for kd
and lower concentrations for ka. The data are presented as the mean
and standard deviation (in brackets) of the results (Table 1).
TABLE-US-00012 TABLE 1 BIAcore .TM. data for DOM1H-131-202,
DOM1H-131-511 and DOM1H-131-206 binding to human TNFR1 k.sub.on
k.sub.off K.sub.D (nM) DOM1H-131-511 5.03E+05 5.06E-04 1.07 (511)
(1.07E+05) (1.01E-04) (0.44) DOM1H-131-202 1.02E+06 5.42E-04 0.55
(202) (2.69E+05) (3.69E-05) (0.11) DOM1H-131-206 1.55E+06 7.25E-04
0.47 (206) (3.57E+05) (1.95E-04) (0.06) DOM1H-131-202,
DOM1H-131-511 and DOM1H-131-206 bound similarly and with high
affinity to human TNFR1. DOM1H-131-202 and DOM1H-131-206 bind with
average affinities of 0.55 nM and 0.47 nM respectively. Both
DOM1H-131-202 and DOM1H-131-206 have a slightly better affinity in
comparison to DOM1H-131-511 which has an average affinity of 1.07
nM.
Receptor Binding Assay:
[0372] The potency of the dAbs was determined against human TNFR1
in a receptor binding assay. This assay measures the binding of
TNF-alpha to TNFR1 and the ability of soluble dAb to block this
interaction. The TNFR1-FC fusion is captured on a bead pre-coated
with goat anti-human IgG (H&L). The receptor coated beads are
incubated with TNF-alpha (10 ng/ml), dAb, biotin conjugated
anti-TNF-alpha and streptavidin alexa fluor 647 in a black sided
clear bottomed 384 well plate. After 6 hours the plate is read on
the ABI 8200 Cellular Detection system and bead associated
fluorescence determined. If the dAb blocks TNF-alpha binding to
TNFR1 the fluorescent intensity will be reduced.
[0373] Data was analysed using the ABI 8200 analysis software.
Concentration effect curves and potency (EC.sub.50) values were
determined using GraphPad Prism and a sigmoidal dose response curve
with variable slope. The assay was repeated on three separate
occasions. A TNF-alpha dose curve was included in each experiment
(FIGS. 38 and 39). The concentration of TNF-alpha used to compete
with dAb binding to TNFR1 (10 ng/ml) is approximately 90% of the
maximum TNF-alpha response in this assay.
[0374] A representative graph is shown in FIG. 39 showing the
ability of dAbs to inhibit the binding of TNF-alpha to TNFR1. In
all three experiments the negative control samples (HEL4, an
anti-hen egg white lysozyme dAb and V.sub.H dummy) weakly inhibit
the interaction between TNF-alpha and TNFR1 at high concentrations.
The average potency (EC.sub.50) values for the test samples and
positive controls (anti-TNFR1 mAb obtained from R&D Systems,
mAb225) and Enbrel.TM. (etanercept; a dimeric fusion consisting of
TNFR2 linked to the Fc portion of IgG1; licensed for the treatment
of rheumatoid arthritis) are shown in Table 2.
TABLE-US-00013 TABLE 2 Potency (EC.sub.50) values for
DOM1H-131-202, DOM1H-131-206 and DOM1H-131-511 in a TNFR1 receptor
binding assay for three repeat experiments. Average Sample
EC.sub.50 (nM) SEM DOM1H-131-202 0.11 0.008 DOM1H-131-206 0.07 0.01
DOM1H-131-511 0.19 0.01 Enbrel .TM. (Etanercept) 0.20 0.07
Anti-TNFR1 mAb # mAb225 0.08 0.003
[0375] In this assay DOM1H-131-206 appears more potent than the
other two dAbs being tested and has a similar potency to the
commercially available anti-TNFR1 mAb, MAB225 (R and D
Systems).
[0376] Expression of lead clones from Pichia pastoris was carried
out as described below: The primary amino acid sequence of the
three lead molecules was used to produce codon optimised genes for
secreted expression in Pichia pastoris. There is 75% sequence
identity between the codon optimized and the non-codon optimized
DOM1H-131-206. The three synthetic genes were cloned into the
expression vector pPIC-Z.alpha. (from Invitrogen) and then
transformed into two Pichia strains, X33 and KM71H. The transformed
cells were plated out onto increasing concentrations of Zeocin
(100, 300, 600 and 900 .mu.g/ml) to select for clones with multiple
integrants. Approximately 15 clones for each cell line and
construct were selected for expression screening. As the
correlation between high/low gene copy number and expression level
is not fully understood in Pichia pastoris, several clones were
picked from across the Zeocin concentration range. 5 L fermenter
runs were carried out using clones that had not been extensively
screened for high productivity. This allowed the production of
significant amounts of material for further studies.
Material Production for Protein Characterisation:
[0377] Protein A based chromatography resins have been extensively
used to purify V.sub.H dAbs from microbial culture supernatants.
Although this allows a single step purification method for
producing high purity material, usually >90% in most cases, for
some molecules the low pH elution conditions can result in the
formation of aggregates. There is also the issue of the limited
capacity of affinity resins for dAbs; this would mean the use of
significant quantities of resin to process from fermenters. In
order to produce high quality material for characterisation and
further stability and nebuliser studies, a downstream purification
process was devised using a mixed modal charge induction resin as
the primary capture step followed by anion exchange. Without
significant optimisation, this allowed the recovery of .about.70%
of the expressed dAb at a purity of .about.95%.
[0378] For the capture step on the mixed modal charge induction
resin, Capto MMC from GE Healthcare, column equilibration is
performed using 50 mM sodium phosphate pH6.0 and the supernatant is
loaded without any need for dilution or pH adjustment. After
washing the column, the protein is eluted by pH gradient using an
elution buffer which is 50 mM Tris pH 9.0. The specific wash and
gradient conditions will vary slightly depending on the pI of the
protein being eluted
[0379] The eluate peak is then further purified with a flow through
step using anion exchange chromatography. This removes residual HMW
contamination such as alcohol oxidase and reduces endotoxin. The
resin is equilibrated with either PBS or phosphate buffer pH 7.4
without salt. Upon loading the eluate from Capto MMC onto the anion
exchange resin the dAb does not bind and is recovered from the flow
through. Endotoxin and other contaminants bind to the resin. The
presence of salt if using PBS buffer improves protein recovery to
91% for this step rather than 86% recovery achieved without salt.
However the presence of salt reduces the effectiveness of endotoxin
removal such that a typical endotoxin level of dAb following this
step with the inclusion of salt was measured as 58 EU/ml compared
with a level of <1.0 EU/ml obtained when no salt was
present.
Protein Characterisation:
[0380] The material produced from the 5 L fermenter runs was
characterised for identity using electrospray mass spectrometry,
amino terminal sequencing and isoelectric focusing and for purity
using SDS-PAGE, SEC and Gelcode glycoprotein staining kit
(Pierce).
Identity:
[0381] The amino terminal sequence analysis of the first five
residues of each protein, was as expected (EVQLL . . . ). Mass
spectrometry was performed on samples of the proteins which had
been buffer exchanged into 50:50 H.sub.2O:acetonitrile containing
0.1% glacial acetic acid using C4 Zip-tips (Millipore). The
measured mass for each of the three proteins was within 0.5 Da of
the theoretical mass based on the primary amino acid sequence
(calculated using average masses) when allowing for a mass
difference of -2 from the formation of the internal disulphide
bond. IEF was used to identify the proteins based on their pI which
was different for each protein.
Purity:
[0382] The three proteins were loaded onto non-reducing SDS-PAGE
gels in 1 .mu.g and 10 .mu.g amounts in duplicate. A single band
was observed in all instances. Size exclusion chromatography was
also performed to demonstrate levels of purity. For size exclusion
chromatography (SEC) 100 .mu.g of each protein were loaded onto a
TOSOH G2000 SWXL column flowing at 0.5 ml/min. Mobile phase was
PBS/10% ethanol.
Investigation of dAb Stability for Candidate Selection:
[0383] For the indication of COPD, it would be necessary to deliver
the dAb into the lung, eg using a nebuliser device. This would mean
the protein could potentially experience a range of shear and
thermal stresses depending on the type of nebuliser used and could
be subjected to enzymatic degradation by proteases in the lung
environment. It was determined if the protein could be delivered
using this type of device, form the correct particle size
distribution and remain functional following nebuliser delivery.
Therefore the intrinsic stability of each molecule to a range of
physical stresses was investigated to determine the baseline
stability and the most sensitive stability indicating assays. As
the stability of each protein will be dependent on the buffer
solution it is solubilised in, some pre-formulation work was
necessary. This information, such as buffer, pH, would also be
useful for understanding the stability of the protein during the
downstream purification process and subsequent storage. In order to
characterise the changes in the molecules during exposure to a
range of physical stresses, a range of analytical techniques were
used such as size exclusion chromatography (SEC), SDS-PAGE and
isoelectric focusing (IEF).
[0384] Assessment of protease stability of DOM1H-131-202,
DOM1H-131-511 and DOM1H-131-206:
[0385] The protease stability of DOM1H-131-202, DOM1H-131-511 and
DOM1H-131-206 was assessed by BIAcore.TM. analysis of the residual
binding activity after pre-incubation for defined timepoints in
excess of proteases. Approximately 1400RU of biotinylated TNFR1 was
coated to a streptavidin (SA) chip. 250 nM of DOM1H-131-202,
DOM1H-131-511 and DOM1H-131-206 was incubated with PBS only or with
100 .mu.g/ml of trypsin, elastase or leucozyme for 1, 3, and 24
hours at 30.degree. C. The reaction was stopped by the addition of
a cocktail of protease inhibitors. The dAb/protease mixtures were
then passed over the TNFR1 coated chip using reference cell
subtraction. The chip surface was regenerated with 10 ul 0.1M
glycine pH 2.2 between each injection cycle. The fraction of
DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 bound to human TNFR1
(at 10 secs) pre-incubated with proteases was determined relative
to dAb binding without proteases. BIAcore.TM. runs were carried out
at 25.degree. C.
[0386] The data was produced from three independent experiments.
The bar graph indicates mean values and the error bars indicate
standard deviation of the results (for results see FIG. 24).
[0387] It was found that DOM1H-131-202 and DOM1H-131-206 were shown
to have greater resistance to proteolytic degradation by trypsin,
elastase or leucozyme in comparison to DOM1H-131-511. The
difference between DOM1H-131-202 and DOM1H-131-206 in comparison to
DOM1H-131-511 is most pronounced after 1 hr with trypsin and after
3 hrs with elastase or leucozyme.
Thermal Stability as Determined Using DSC:
[0388] In order to determine at which pH the molecules had the
greatest stability, differential scanning calorimeter (DSC) was
used to measure the melting temperatures (T.sub.m) of each dAb in
Britton-Robinson buffer. As Britton-Robinson is made up of three
component buffer systems (acetate, phosphate and borate), it is
possible to produce a pH range from 3-10 in the same solution. The
theoretical pI was determined from the proteins primary amino acid
sequence. From the DSC, the pH at which the dAbs had their greatest
intrinsic thermal stability was found to be pH 7 for DOM1H-131-202
(202), pH 7-7.5 for DOM1H-131-206 (206) and pH 7.5 for
DOM1H-131-511 (511). For all subsequent stress and stability work
the following pHs were used for each dAb; for DOM1H-131-202 (202)
and DOM1H-131-206 (206) pH 7.0 and for DOM1H-131-511 (511) pH 7.5
in Britton-Robinson buffer. The results are summarised in Table 3
below:
TABLE-US-00014 TABLE 3 Summary of the pH and T.sub.ms of
DOM1H-131-202 (202), DOM1H-131-206 (206) and DOM1H-131-511 (511) as
determined by DSC in Britton-Robinson buffer at 1 mg/ml pH that
gives greatest Tm (.degree. C.) of the dAb intrinsic thermal
stability dAb at the given pH DOM1H-131-202 (202) 7.0 68.6
DOM1H-131-206 (206) 7.0-7.5 65.8 DOM1H-131-511 (511) 7.5 58.0
Intrinsic Solubility Testing:
[0389] All the lead dAbs were concentrated in centrifugal Vivaspin
concentrators (5K cut-off), to determine their maximum solubility
and the levels of recovery upon concentration. Experiments were
performed in Britton-Robinson buffer at the most stable pH. Sample
volumes and concentrations were measured over a time course and
deviation from expected concentration recorded as well as percent
recovery of the sample.
[0390] It was found that all proteins could be concentrated to over
100 mg/ml in Britton-Robinson buffer. Both DOM1H-131-202 (202) and
DOM1H-131-206 (206) showed lower recoveries than expected compared
to DOM1H-131-511 (511), but still within acceptable levels.
Nebuliser Delivery of the Lead dAbs:
[0391] By testing different nebulisers and formulation buffers it
was demonstrated that the dAb could effectively be delivered using
a wide range of nebulising devices. More importantly, it was shown
for the first time that nebulisation of the dAb in the formulation
buffer produced the desired particle size distribution (compared
using the percentage of droplets <5 .mu.m) for effective lung
delivery whilst maintaining protein functionality. This is further
described below.
Comparison of Performance in Various Devices:
[0392] DOM1H-131-511 (511) was tested in six nebuliser devices
comprising two devices from each of the three main groups of
nebulisers for liquid formulations i.e. ultrasonic nebulisers, jet
nebulisers and vibrating mesh nebulisers. In each device the dAb
was tested at 5 mg/ml with a range of PEG concentrations. For each
sample the percentage of droplet size <5 .mu.m was measured
using a Malvern Spraytek Device (Malvern Instruments Limited, UK)
and the results are shown in FIG. 35. The stability of each sample
after being nebulised was assessed using SEC to analyse the amount
of sample which had dimerised both in the material remaining in the
cup and in collected aerosol. The results may be seen in FIG. 36.
The less the extent of dimer formation the greater the
stability.
[0393] Most devices can deliver 40% or more of the liquid
formulation in the correct size range but the eFlow (a vibrating
mesh nebuliser device) and PARI LC (a jet nebuliser) devices
perform better, with the PARI LC* (star) device delivering more
than 80% when PEG is included in the buffer. This increase in
delivery with PEG is also observed with the eFlow and, to a lesser
extent, with the PARI LC+.
[0394] Importantly activity of the dAb was also found to be
retained after nebulisation (see results in FIG. 8)
Effect of Buffer Additives:
[0395] Due to the lower stability of DOM1H-131-511 (511), the 50 mM
phosphate formulation buffer contained both PEG 1000 and sucrose
(and has a viscosity which is within the range which is defined as
about equal to the viscosity of a solution of about 2% to about 10%
PEG 1000 in 50 mM phosphate buffer containing 1.2%(w/v sucrose) to
help protect the dAb from both shear and thermal stress. As both
DOM1H-131-202 (202) and DOM1H-131-206 (206) have higher T.sub.m's
and showed considerably improved stability to thermal stress, all
the molecules were tested in both the original formulation buffer
and in Britton-Robinson buffer (which has a lower viscosity than
the formulation buffer). The dAbs were tested in both the E-flow
and Pari LC+ devices with run time of 3.5 minutes at a protein
concentration of 5 mg/ml and the particle size distribution
determined using a Malvern Spraytek Device. As a comparison, a
marketed drug for cystic fiborosis (designated standard protein X)
that is delivered using a nebuliser device, was tested in its own
formulation buffer. The results are shown in FIG. 37. For good
delivery and distribution into the deep lung, the ideal particle
size is less than 6 microns, e.g. <5 .mu.m. All the dAbs give
comparable levels of particle sizes that were less than 5 .mu.m in
both the Britton-Robinson buffer and formulation buffer (as
described earlier). However, the higher viscosity of the
formulation buffer could be particularly beneficial for producing
particles within the correct size range, e.g. particles <5
.mu.m. The concentration of the dAb in the cup of the device was
determined by A.sub.280 measurements before and after nebulisation.
It was found that the protein concentration did not change
significantly indicating that neither the protein nor vehicle is
preferentially nebulised during delivery.
Conclusion:
[0396] It has been demonstrated as described above that
polypeptides such as dAbs can be nebulised in a range of
commercially available nebuliser devices and importantly that they
retain stability and biological activity after nebulisation and
there is no significant aggregation observed following
nebulisation. When viscosity enhancing excipients, such as PEG are
added to the buffer formulation, particle size distribution and
percentage droplet size less than 5 .mu.m can be improved, thus
potentially improving dAb delivery to the deep lung.
[0397] Delivery of dAb to the lung can also be improved by
increasing the dAb concentration for example a concentration of up
to about 40 mg/ml and delivery time without any reduction in dAb
stability or activity.
Example 1
[0398] Phage Vector pDOM13
[0399] A filamentous phage (fd) display vector, pDOM13 was used.
This vector produces fusion proteins with phage coat protein III.
The multiple cloning site of pDOM13 is illustrated in FIG. 1. The
genes encoding dAbs were cloned as SalI/NotI fragments.
Example 2
[0400] Test Protease Selections on Phage-Displayed Domain
Antibodies (dAbs) with a Range of Resistance to Trypsin
[0401] The genes encoding dAbs DOM4-130-54 which binds IL-1R1,
DOM1h-131-511 which binds TNFR1, and DOM15-10, DOM15-26 and
DOM15-26-501, which bind VEGFA, were cloned in pDOM13 and phages
displaying these dAbs were produced according to standard
techniques. Phages were purified by PEG precipitation, resuspended
in PBS and titered.
[0402] The above dAbs displayed a range of ability to resist
degradation by trypsin when tested as isolated proteins. Resistance
to degradation was assessed as follows: dAb (1 mg/ml) in PBS was
incubated with trypsin at 40 .mu.g/ml at 30.degree. C., resulting
in a molecular ratio of 25:1 dAb: trypsin. Samples (30 .mu.l) were
taken immediately before addition of trypsin, and then at T=1 hour,
3 hours, and 24 hours. Protease activity was neutralized by
addition of Roche Complete Protease Inhibitors (2.times.) followed
by immersion in liquid nitrogen and storage on dry ice. 15 .mu.g of
each dAb sample was subsequently analyzed by electrophoresis on a
Novex 10-20% Tricine gel and proteins were stained with SureBlue
(1.times.).
[0403] Both DOM15-10 and DOM15-26-501 were significantly digested
during the first three hours. DOM15-26, DOM4-130-54 and
DOM1h-131-511 were more stable, with digestion of the dAbs only
becoming apparent after 24 hours (FIG. 2).
[0404] The phage-displayed dAbs were also incubated in the presence
of trypsin to evaluate if trypsin resistance of phage-displayed
dAbs correlated with the results obtained with the isolated soluble
dAbs. Various concentrations of trypsin and incubation times were
tested. In all cases, after neutralization of trypsin with Roche
Complete Protease Inhibitors, the phages were tested for their
ability to bind a generic ligand: protein A, which binds all
V.sub.H domain antibodies (e.g., DOM1h-131, DOM15-26, DOM15-26-501)
or protein L, which binds all V.sub.K domain antibodies (e.g.,
DOM4-130-54, DOM15-10). Phage were also tested for binding to
target antigens. In both cases, binding was assumed to correlate
with retention of the dAb structural integrity through resistance
to proteolysis. The binding activity was measured either by ELISA
(using conjugated antibodies against phage) or by elution of bound
phages and titre analysis following infection of exponentially
growing E. coli TG1 cells.
[0405] Tests with DOM15-10, DOM15-26 and DOM15-26-501 on Phage
[0406] Each dAb was treated for one hour at room temperature with a
range of trypsin concentrations (100 .mu.g/ml, 10 .mu.g/ml and 0
.mu.g/ml). Trypsin activity was blocked with Roche Complete
Protease Inhibitor (1.times.) and then the phages were diluted in
2% Marvell in PBS, incubated with 50 nM of biotinylated antigen
(recombinant human VEGF (R&D systems)) for one hour at room
temperature. Strepavidin-coated beads (Dynabeads M-280
(Invitrogen)) that were pre-blocked for one hour at room
temperature with 2% Marvell in PBS were added, and the mixture was
then incubated for five minutes at room temperature. All of the
incubation steps with Dynabeads were carried out on a rotating
wheel. Unbound phages were washed away by washing the beads eight
times with 1 ml of 0.1% Tween-20 in PBS. Bound phages were eluted
with 0.5 ml of 0.1M Glycine pH2.2 and neutralized with 100 .mu.l of
1M Tris-HCL pH 8.0. Eluted phage were used to infect exponentially
growing TG1 cells (one hour at 37.degree. C.) and plated on
Tetracycline plates. Plates were incubated overnight at 37.degree.
C. and colony counts were made (see Table 4). The best results were
observed from selection with incubation with 100 .mu.g/ml trypsin.
There was about a 10-fold increase in the yield of DOM15-26 in
comparison to DOM15-10 and DOM15-26-501.
[0407] A second experiment was done to further confirm these
results under more severe incubation conditions. Phage displayed
dAbs were treated for 1 hour or 2 hours at 37.degree. C. with
agitation (250 rpm). The best results were observed from selections
with 2 hour incubation with 100 ug/ml trypsin. The yield of
DOM15-26 was 200-fold higher than the yield of DOM15-26-501 and
1000-fold higher than the yield of DOM15-10.
[0408] In a third experiment, phages displaying DOM15-26 and
DOM15-26-501 were mixed 1:1 at the start. They were then either
incubated with trypsin (1000 .mu.g/ml) or without trypsin for two
hours at 37.degree. C. with agitation (250 rpm), and then selected
for antigen binding as described above. Sequencing of ten colonies
from each selection revealed a mixed population of clones for
selection without trypsin pre-treatment (DOM15-26: 4/10;
DOM15-26-501: 6/10), whereas all clones from the selection with
trypsin encoded for DOM15-26 as expected.
[0409] These experiments indicate that a selection pressure can be
obtained by adding a protease to phages displaying dAbs, such that
phages displaying the most proteolytically stable dAbs are
preferentially selected (following panning on a generic ligand or
the antigen).
TABLE-US-00015 TABLE 4 1:1 Length of Trypsin DOM15-26 DOM15-26-501
mixed DOM15-10 Experiment incubation Temp. concentration titre
titre titre titre 1 1 hr Room 100 .mu.g/ml 1.6 .times. 10.sup.8 6.3
.times. 10.sup.7 1.1 .times. 10.sup.7 input 10.sup.10 temp 1 hr
Room 10 .mu.g/ml 3 .times. 10.sup.8 4.4 .times. 10.sup.8 2.4
.times. 10.sup.8 temp 1 hr Room 0 .mu.g/ml 0.9 .times. 10.sup.8 2
.times. 10.sup.8 0.7 .times. 10.sup.8 temp 2 1 hr, 250 rpm
37.degree. C. 100 .mu.g/ml 2 .times. 10.sup.7 1 .times. 10.sup.6 1
.times. 10.sup.5 input 10.sup.9 2 hr, 250 rpm 37.degree. C. 100
.mu.g/ml 1 .times. 10.sup.7 6 .times. 10.sup.4 1 .times. 10.sup.4 2
hr, 250 rpm 37.degree. C. 0 .mu.g/ml 5.4 .times. 10.sup.7 4.1
.times. 10.sup.7 3 .times. 10.sup.8 3 2 h, 250 rpm 37.degree. C.
100 .mu.g/ml 2.3 .times. 10.sup.8 8 .times. 10.sup.5 6.8 .times.
10.sup.7 input 10.sup.10 2 h, 250 rpm 37.degree. C. 0 .mu.g/ml 3.9
.times. 10.sup.8 4.4 .times. 10.sup.8 4.8 .times. 10.sup.8
[0410] Tests with DOM4-130-54 on Phage
[0411] DOM4-130-54 was tested in a similar protocol as described
above. The parameters that were varied were: concentration of
trypsin, temperature and length of incubation. Biopanning was done
against IL-RI-Fc (a fusion of IL-1RI and Fc) at 1 nM concentration
in PBS. Significant reductions in phage titre were only observed
after incubation of the phage with 100 .mu.g/ml trypsin overnight
at 37.degree. C. (see Table 5).
TABLE-US-00016 TABLE 5 Trypsin Length of incubation Temperature
concentration Titre 1 hr Room temp 100 .mu.g/ml .sup. 1.8 .times.
10.sup.10 1 hr Room temp 10 .mu.g/ml 7.2 .times. 10.sup.9 1 hr Room
temp 0 .mu.g/ml 6.6 .times. 10.sup.9 Overnight Room temp 100
.mu.g/ml 2.16 .times. 10.sup.9 Overnight Room temp 10 .mu.g/ml 7.2
.times. 10.sup.9 Overnight Room temp 0 .mu.g/ml 7.8 .times.
10.sup.9 Overnight 37.degree. C. 100 .mu.g/ml 2.04 .times. 10.sup.6
Overnight 37.degree. C. 10 .mu.g/ml 3.84 .times. 10.sup.8 Overnight
37.degree. C. 0 .mu.g/ml 7.2 .times. 10.sup.9
[0412] Tests with DOM1h-131 Phage
[0413] DOM1h-131 phage (closely related to DOM1h-131-511 by amino
acid sequence) were treated with 0 .mu.g/ml, 10 .mu.g/ml, 100
.mu.g/ml and 1000 .mu.g/ml trypsin for one hour at room
temperature. Digestion was inhibited by the addition of 25.times.
Complete Protease Inhibitors (Roche). Serial 2-fold dilutions of
the phage were carried out down an ELISA plate coated with 1 nM
TNFRI, and binding phage were detected with anti-M13-HRP. The
results are shown below in Table 6.
TABLE-US-00017 TABLE 6 DOM1h-131 Trypsin concentration 1 100 10 0
Phage mg/ml .mu.g/ml .mu.g/ml .mu.g/ml input 0.284 0.418 0.784
0.916 4.51E+10 0.229 0.377 0.802 0.944 2.26E+10 0.183 0.284 0.860
0.949 1.13E+10 0.133 0.196 0.695 0.962 5.64E+09 0.114 0.141 0.573
0.946 2.82E+09 0.089 0.115 0.409 0.850 1.41E+09 0.084 0.084 0.286
0.705 7.05E+08 0.080 0.084 0.213 0.577 3.52E+08
[0414] These test experiments clearly show that 100 .mu.g/ml of
trypsin and a temperature of 37.degree. C. are appropriate to apply
a selection pressure on phages displaying dAbs of various degrees
of resistance to proteolysis by trypsin. Incubation time with the
protease can be optimized for each phage-displayed dAb, if
desired.
Example 3
[0415] Protease Selection of Phage-Displayed Repertoires of Domain
Antibodies
[0416] Four repertoires were created using the following dAbs as
parent molecules: DOM4-130-54, DOM1h-131-511, DOM15-10 and
DOM15-26-555. Random mutations were introduced in the genes by PCR
using the Stratagene Mutazyme II kit, biotinylated primers and 5-50
pg of template for a 50 .mu.l reaction. After digestion with SalI
and NotI, the inserts were purified from undigested products with
streptavidin-coated beads and ligated into pDOM13 at the
corresponding sites. E. coli TB1 cells were transformed with the
purified ligation mix resulting in large repertoires of
tetracycline-resistant clones: 8.5.times.10.sup.8 (DOM4-130-54),
1.5.times.10.sup.9 (DOM1h-131-511), 6.times.10.sup.8 (DOM15-10) and
3.times.10.sup.9 (DOM15-26-555).
[0417] Phage libraries were prepared by double precipitation with
PEG and resuspended in PBS.
[0418] The rates of amino acid mutations were 2.3 and 4.4 for the
DOM1h-131-511 and DOM4-130-54 repertoires, respectively. The
functionality was assessed by testing 96 clones in phage ELISA
using wells coated with protein A or protein L (at 1 .mu.g/ml).
62.5% and 27% of the clones exhibited functional display of dAbs in
the DOM1h-131-511 and DOM4-130-54 repertoires, respectively.
[0419] The rates of amino acid mutations were 2.5 and 4.6 for the
DOM15-10 and DOM15-26-555 repertoires, respectively. The
functionality was assessed by testing 96 clones in phage ELISA
using wells coated with protein A or protein L (at 1 .mu.g/ml).
31.3% and 10.4% of the clones exhibited functional display of dAbs
in the DOM15-10 and DOM15-26-555 repertoires, respectively.
[0420] DOM4-130-54 and DOM1h-131-511 Repertoires
[0421] Four rounds of selection were carried out with these
libraries to select for dAbs with improved protease resistance.
[0422] The first round of selection was by antigen binding (1 nM or
10 nM antigen) without protease treatment to clean-up the library
to remove any clones that no longer bound antigen with high
affinity. The outputs from round 1 were in the 10.sup.8-10.sup.10
range (compared to an input of 10.sup.11 phage) indicating that the
majority of the library bound antigen with high affinity.
[0423] In round 2, protease treatment with 100 .mu.g/ml trypsin was
introduced, and the outputs are as shown below in Table 7:
TABLE-US-00018 TABLE 7 Trypsin incubation DOM1h-131-511 DOM4-130-54
conditions library library 37.degree. C. overnight 1.86 .times.
10.sup.6 2.1 .times. 10.sup.6 37.degree. C. 2 hrs 4.8 .times.
10.sup.8 5.1 .times. 10.sup.8 Room temperature 2 hrs 1.2 .times.
10.sup.9 4.62 .times. 10.sup.9 No trypsin ~1 .times. 10.sup.9 ~4
.times. 10.sup.9 No antigen 1.8 .times. 10.sup.4 <6 .times.
10.sup.3
[0424] There was significant selection when the dAbs were treated
with trypsin at 37.degree. C. overnight. This output was taken
forward to round 3, where the phage were treated with either 1
mg/ml or 100 .mu.g/ml trypsin at 37.degree. C. for 24 hours. The
titres of the trypsin treated phage from round 3 were
10.sup.5-10.sup.6 for the DOM1h-131-511 repertoire and
10.sup.7-10.sup.8 for the DOM4-130-154 repertoire.
[0425] All outputs from round 3 (DOM1h-131-511 and DOM4-130-154
with 1 mg/ml and 100 .mu.g/ml) underwent a fourth round of
selection against 1 nM antigen with 100 .mu.g/ml trypsin. The
titres were in the range of 10.sup.6-10.sup.8, similar to that seen
in round 3. Some enrichment was seen for the DOM1h-131-511
repertoire, but no enrichment was seen for the DOM4-130-54
repertoire.
[0426] DOM15-10 and DOM15-26-555 Repertoires
[0427] The first round of selection was carried out with 2 nM
biotinylated hVEGF (human vascular endothelial growth factor)
concentration and without protease treatment to clean-up the
library to remove any clones that no longer bound antigen with high
affinity. The outputs from round 1 were about 10.sup.8 (compared to
an input of 10.sup.10 phage for DOM15-10 and 10.sup.11 phage for
DOM15-26-555) indicating that the majority of the library bound
antigen with high affinity.
[0428] The second and third rounds of selection were performed with
2 nM biotinylated hVEGF. Prior to panning on hVEGF, the phages were
incubated in the presence of trypsin (100 .mu.g/ml) at 37.degree.
C. in a shaker (250 rpm). Incubation time was one hour for the
DOM15-10 repertoire and two hours for the DOM15-26-555
repertoire.
[0429] The outputs were as follows: 1.5.times.10.sup.6 and
9.times.10.sup.5 for the second and third rounds of selection with
the DOM15-10 repertoire; 2.2.times.10.sup.8 and 3.9.times.10.sup.9
for the second and third rounds of selection with the
DOM15-26-555.
Example 4
[0430] Analysis of Selection Outputs: DOM4-130-54 and DOM1h-131-511
Repertoires
[0431] All outputs from round 3 and round 4 were subcloned into the
pDOM5 vector and transformed into JM83 cells. The pDOM5 vector is a
pUC119-based vector. Expression of proteins is driven by the Plac
promoter. A GAS 1 leader sequence (see WO 2005/093074) ensured
secretion of isolated, soluble dAbs into the periplasm and culture
supernatant of E. coli JM83. 96 and 72 individual colonies from
round 3 and round 4 were randomly picked for expression
[0432] 12-24 clones were sequenced from each round 3 and round 4
output. Consensus mutations were observed in both selections and
approximately 25 clones harboring consensus motifs were chosen for
further characterization. The amino acid sequences of these clones
are shown in FIG. 3 (DOM1h-131-511 selected variants) and FIG. 4
(DOM4-130-54 selected variants) and listed as DNA sequences in
FIGS. 19A-19L. The amino acids that differ from the parent sequence
in selected clones are highlighted (those that are identical are
marked by dots). The loops corresponding to CDR1, CDR2 and CDR3 are
outlined with boxes.
[0433] These clones were expressed in a larger amount, purified on
protein L (for DOM4-130-54 variants) and protein A (for
DOM1h-131-511 variants) and tested for antigen binding on BIAcore
after one hour or overnight incubation at 37.degree. C. in the
presence or absence of trypsin (100 .mu.g/ml or 1000 .mu.g/ml final
concentration).
[0434] Generally, the outputs from the DOM4-130-54 selections were
more stable with most clones remaining resistant to trypsin for one
hour and the best clones resistant overnight. In comparison, a
small number of clones from the DOM1h-131-511 selections were
resistant to trypsin for one hour, whilst none of the clones were
resistant overnight.
Example 5
[0435] Analysis of Selection Outputs: DOM15-10 and DOM15-26-555
Repertoires
[0436] The effectiveness of selection with trypsin pre-treatment
was first tested on monoclonal phage ELISA with and without trypsin
digestion. Eighteen colonies from the second round of selection and
24 colonies from the third round of selection of each library were
picked. Clones DOM15-10, DOM15-26-501 and DOM15-26 were used as
controls. Additional controls included amplified and purified phage
solution from each library after second and third rounds of trypsin
selection.
[0437] Each phage sample was divided into two fractions, the first
was treated with 100 ug/ml trypsin, the second was not treated with
trypsin. Incubation of both fractions was carried out for one hour
at 37.degree. C. with agitation (250 rpm) and blocked by adding
Roche Complete Protease Inhibitor (1.times.).
[0438] Phage ELISA was performed using the trypsin-digested and
undigested samples. ELISA wells were coated with neutravidin in
0.1M bicarbonate buffer at a concentration of 1 .mu.g/ml. After the
washing steps with PBS and blocking of the antigen-coated wells
with 1% Tween-20 in PBS for one hour at room temperature, the wells
were coated with biotinylated hVEGF diluted in 1% Tween-20 in PBS
at a concentration of 100 ng/ml. Next, the wells were washed with
PBS and treated or untreated phage supernatants diluted 1:1 with 1%
Tween-20/PBS, were added. After 30 minutes of incubation at
37.degree. C., the wells were washed with 1% Tween-20/PBS, followed
by a 30 minute incubation at 37.degree. C. with anti-M13 phage-HRP
conjugate (diluted 1/5000 in 1% Tween-20/PBS). The wells were then
washed with PBS and peroxidase. Reaction was initiated by adding
SureBlue reagent. After about ten minutes, the reaction was stopped
with an equivalent volume of 1M HCl and the wells were read at
OD.sub.450 nm.
[0439] ELISA read-outs of unstable controls DOM15-10 and
DOM15-26-501 treated with trypsin gave an OD.sub.450 lower than
0.404 and this value was assumed as a border value of an unstable
clone. All samples that gave an OD lower than 0.404 were considered
to be unstable. All samples above that value were considered to be
stable.
TABLE-US-00019 TABLE 8 Trypsin No trypsin 2nd 3rd 2nd 3rd Library
selection selection selection selection DOM15-10 33% 89% 100% 100%
DOM15-26-555 94.4% 100% 100% 100%
[0440] Table 8 shows the percentage of stable clones after the
second and third rounds of trypsin selection of each library. The
enrichment of trypsin resistant clones is visible in both libraries
after the third round of selection. The values of control ELISA
wells containing amplified purified phage mix after each selection
were much higher than 0.404 in each case after trypsin digestion.
Moreover, a small increase in signal was observed when comparing
trypsin-treated phage from the third round of selection with
trypsin-treated phage from the second round of selection. The
DOM15-10 phage library showed an increase of about 14% of the
starting value. DOM15-26-555 phage library showed an increase that
represents about 2% of the starting value.
[0441] Overall these results show that selection with trypsin
pre-treatment was effective to select trypsin-resistant phage
clones from the DOM15-10 and DOM15-26-555 repertoires.
[0442] All outputs from the second and third rounds of selection
(DOM15-26-555) and from the third round of selection only
(DOM15-10) were subcloned into the pDOM5 vector and transformed
into HB2151 electrocompetent cells. The pDOM5 vector is a
pUC119-based vector. Expression of proteins is driven by the Plac
promoter. A GAS1 leader sequence ensured secretion of isolated,
soluble dAbs into the periplasm and culture supernatant of E. coli
HB2151. 184 individual colonies from each round of selection (3 and
4) were randomly picked for expression in 1 ml culture volumes.
[0443] Bacterial supernatants were diluted in HBS-EP BIAcore buffer
(1:1 volume ratio) and split to duplicates. Trypsin was added to
only one vial at a final concentration of 20 .mu.g/ml. Incubation
was carried out for 40 minutes at 37.degree. C. with agitation (250
rpm). After blocking the reaction with Roche Complete Protease
Inhibitor (1.times.), both trypsin treated and untreated phage
supernatants were tested on BIAcore 3000 for antigen binding (2,000
RU of biotinylated hVEGF on a SA sensorchip).
[0444] The criteria for picking clones were: a decrease in antigen
binding of <15% of dAbs treated with trypsin relative to
untreated dAbs (based on max RU reached on selected time point),
which would reflect dAbs stability to protease treatment in
general; and off-rate decrease of <40% between two time points
during dissociation of a dAb from the antigen. Based on these
values, 60 clones from both the second and third rounds of
selection of the DOM15-26-555 library and 17 clones from the third
round of selection of the DOM15-10 library were sequenced.
Consensus mutations were observed in both libraries' outputs and 17
clones from each library harboring consensus motifs were chosen for
further characterization. The amino acid sequences of these clones
are shown in FIG. 5 (DOM15-26-555 selected variants) and FIG. 6
(DOM15-10 selected variants) and listed as DNA sequences in FIGS.
20A-20E. The amino acids that differ from the parent sequence in
selected clones are highlighted (those that are identical are
marked by dots). The loops corresponding to CDR1, CDR2 and CDR3 are
outlined by boxes.
[0445] These clones were expressed in 50 ml expression cultures,
purified on protein A (for DOM15-26-555 variants) or protein L (for
DOM15-10 variants) diluted to 100 nM concentration in HBS-EP buffer
and tested for antigen binding on BIAcore after 1.5 hours of
incubation at 37.degree. C. with agitation (250 rpm) in the
presence or absence of trypsin (20 .mu.g/ml final
concentration).
[0446] These clones were also tested for trypsin resistance using
the method described in Example 2. Proteins were buffer exchanged
to PBS and concentrated to 1 mg/ml. 25 .mu.g of protein was mixed
with 1 .mu.g of trypsin (Promega) and incubated for 0 hours and 24
hours at 30.degree. C. After this time, the reaction was blocked
with Roche Complete Protease Inhibitor (1.times.) and DTT, as well
as loading agent, was added Samples were denatured for five minutes
at 100.degree. C. Then 15 .mu.g of each sample was analyzed by
electrophoresis on Novex 10-20% Tricine gels and proteins were
stained with SureBlue (1.times.).
[0447] Generally, the outputs from the DOM15-26-555 selections were
more stable, with most clones remaining resistant to trypsin for
1.5 hours when tested on BIAcore and overnight when run on
SDS-PAGE. In comparison, only a small number of clones from the
DOM15-10 selections were resistant to trypsin for overnight
treatment when run on SDS-PAGE.
Example 6
[0448] Identification of DOM1h-131-511 Variants
[0449] DOM1h-131-203, DOM1h-131-204 and DOM1h-131-206 were analyzed
in further detail. They were compared on the BIAcore at a dAb
concentration of 500 nM after incubation with different
concentrations of trypsin (ranging from 0 to 100 .mu.g/ml)
overnight at 37.degree. C. The BIAcore traces are shown in FIG. 7.
The results clearly show that both variants are more resistant than
their parent to proteolysis at high concentration of trypsin (100
.mu.g/ml). Two of the dAbs, DOM1h-131-202 and DOM1h-131-206, were
also compared along with their parent against a range of other
proteases including leucozyme, elastase and pancreatin under the
conditions described above, with a protease concentration of 100
.mu.g/ml. The dAbs showed increased resistance to proteolysis
compared to the parent against all proteases tested. The BIAcore
traces for elastase and leucozyme are shown in FIG. 8.
[0450] 5 .mu.M of each dAb was treated with 100 .mu.g/ml sequencing
grade trypsin for 0, 1, 3 and 24 hours. The reaction was inhibited
with 25.times. Roche Complete Protease Inhibitor and the reactions
were run on a 4-12% Novex Bis-Tris gel. The gels are shown in FIG.
9.
Example 7
[0451] Identification of DOM4-130-54 Variants
[0452] DOM4-130-201 and DOM4-130-202 were analyzed in further
detail. They were compared on the BIAcore at a dAb concentration of
500 nM after incubation with different concentrations of trypsin
(ranging from 0 to 100 .mu.g/ml) overnight at 37.degree. C. The
BIAcore traces are shown in FIG. 10. The results clearly show that
all three variants are more resistant than their parent to
proteolysis at high concentrations of trypsin (100 .mu.g/ml).
DOM4-130-201 and DOM4-130-202 were also compared with the parent
against a range of other proteases including leucozyme, elastase
and pancreatin under the conditions described above with a protease
concentration of 100 .mu.g/ml. Although the results were less
apparent than with trypsin, the lead dAbs showed increased
resistance to proteolysis compared to parent against all proteases
tested. The BIAcore traces for elastase and leucozyme are shown in
FIG. 11.
[0453] 5 .mu.M of each dAb was treated with 100 ug/ml sequencing
grade trypsin for 0, 1, 3 and 24 hours. The reaction was inhibited
with 25.times. Roche Complete Protease Inhibitor and the reactions
were run on a 4-12% Novex Bis-Tris gel. The gels are shown in FIG.
9.
Example 8
[0454] Further Characterization of DOM1h-131-511 and DOM4-130-54
Variants
[0455] The dAbs were first analyzed using Differential Scanning
Calorimetry (DSC) to determine whether the increase in trypsin
resistance correlated with an increase in melting temperature (Tm).
An increase in trypsin stability does correlate with an increase in
Tm (see Table 9)
TABLE-US-00020 TABLE 9 Name Tm, .degree. C. DOM1h-131-511 57.9
DOM1h-131-202 67.5 DOM1h-131-203 65.7 DOM1h-131-204 62.3
DOM1h-131-206 64.9 DOM4-130-54 54.1 DOM4-130-201 64.7 DOM4-130-202
64.5
[0456] The DOM1h-131-511 derived dAbs were also compared in a MRC-5
cell-based assay (see Table 10). In this assay, the ability of the
dAbs to neutralize TNF.alpha. stimulated IL-8 release was measured
to determine whether the increase in trypsin stability had led to a
decrease in efficacy. However, the activity of the
trypsin-resistant dAbs in the assay was substantially
unaffected.
TABLE-US-00021 TABLE 10 Sample ND50 nM DOM1h-131-511 1.98
DOM1h-131-511 1.71 DOM1h-131-511 (230307CE) 1.89 DOM1h-131-203
(230307CE) 2.28 DOM1h-131-204 (230307CE) 1.89 DOM1h-131-511 1.46
DOM1h-131-206 (230307CE) 0.71
[0457] The DOM4-130-54 derived dAbs were tested in a Receptor
Binding Assay to see if they still had the same ability to inhibit
the binding of IL-1 to IL-RI (see Table 11). The activity of the
trypsin resistant dAbs was unaffected in this assay.
TABLE-US-00022 TABLE 11 dAb IC50 (nM) DOM4-130-54 280 pM
DOM4-130-201 257 pM DOM4-130-202 254 pM
Example 9
[0458] Identification of DOM15-26-555 Variants
[0459] DOM15-26-588, DOM15-26-589, DOM15-26-591, and DOM15-26-593
were analyzed in further detail together with their parent and two
additional dAbs, DOM15-26-594 and DOM15-26-595, which were created
by mutagenesis to combine mutations that would have the greatest
impact on potency and stability (E6V and F100S/I). Sequences are
shown in FIG. 12. Clones were compared on the BIAcore for hVEGF
binding at the dAb concentration of 100 nM after incubation with
trypsin at a concentration of 200 .mu.g/ml. The reaction was
carried out for three hours and 24 hours at 37.degree. C. with
agitation (250 rpm). The BIAcore traces of the best clone,
DOM15-26-593, and the parent are shown in FIG. 13. Other results
are presented as a chart in FIG. 14. The results clearly show that
all variants are more resistant than the parent to proteolysis
after 24 hours of trypsin treatment.
[0460] Trypsin resistance of DOM15-26-593 and the parent was also
examined by running treated and un-treated samples on SDS-PAGE.
Briefly, proteins were buffer exchanged to PBS and concentrated to
1 mg/ml. 25 ug of protein was mixed with 1 .mu.g of sequencing
grade trypsin (Promega) and incubated for 0 hours, 1 hour, 3 hours
and 24 hours at 30.degree. C. After this time, the reaction was
blocked with Roche Complete Protease Inhibitor (1.times.) and DTT,
as well as loading agent, was added. Samples were denatured for
five minutes at 100.degree. C. 15 ug of each sample was loaded on
Novex 10-20% Tricine gels and proteins were stained with SureBlue
(1.times.). The results are shown in FIG. 15. The trypsin
resistance profile of DOM15-26-593 in this experiment varied from
the profile shown by the BIAcore experiment, suggesting that
differences in reaction conditions may influence the final result
of trypsin cleavage. Nonetheless, DOM15-26-593 has better
biophysical properties, as well as affinity, than other selected
clones, as shown below. A summary of the properties of the
DOM15-26-555 variants is also shown in the table 12 below.
TABLE-US-00023 TABLE 12 Attribute Trypsin Stability SEC-MALLS DSC %
binding % Est. Tm RBA BIAcore @ +24 dAb monomer mw .degree. C. nM
KD nM hrs 15-26 0 37-136 64 10 28.2 30 15-26- 0-40 18-290 51 1.14
9.1 5 501 15-26- 0 28-78 63 11.7 26.1 10 555 15-26- 10 33 70 27
59.1 15 588 15-26- 90 17 63 1.94 9.6 65 589 15-26- 20 21-234 63 16
38 35 591 15-26- 80 17 65 0.323 3.2 80 593 15-26- 60 17 65 0.828 5
70 595
Example 10
[0461] Identification of DOM15-10 Variants
[0462] DOM15-10-11 was analyzed in further detail, together with
its parent, DOM15-10. Sequences are shown in FIG. 16. The dAbs were
compared on the BIAcore for hVEGF binding at the dAb concentration
of 100 nM after incubation with trypsin at a concentration of 200
.mu.g/ml. The reaction was carried out for 1 hour, 3 hours and 24
hours at 37.degree. C. with agitation (250 rpm). The BIAcore traces
of these dAbs are shown in FIG. 17. The result clearly shows that
the selected variant is more resistant than the parent to
proteolysis after 24 hours of trypsin treatment.
[0463] Trypsin resistance of the lead and the parent was also
examined by running treated and un-treated samples of SDS-PAGE.
Briefly, proteins were buffer exchanged to PBS and concentrated to
1 mg/ml. 25 .mu.g of protein was mixed with 1 .mu.g of sequencing
grade trypsin (Promega) and incubated for 0 hours, 1 hour, 3 hours
and 24 hours at 30.degree. C. After this time, the reaction was
blocked with Roche Complete Protease Inhibitor (1.times.) and DTT,
as well as loading agent, was added. Samples were denatured for
five minutes at 100.degree. C. 15 .mu.g of each sample was loaded
on Novex 10-20% Tricene gels and proteins were stained with
SureBlue (1.times.). The results are presented in FIG. 18. In this
case, the trypsin resistant profile correlates well with the
BIAcore trypsin test, showing that the binding activity directly
reflects the protein's integrity.
Example 11
[0464] Further Characterization of DOM15-26-555 and DOM15-10
Variants
[0465] The dAbs were analyzed using Differential Scanning
Calorimetry (DSC) to determine whether the increase in trypsin
resistance correlated with an increase in Tm. The results are shown
in Table 13. There is a correlation between the trypsin resistance
of DOM15-26-555 variants and melting temperature. The lead
DOM15-26-588 and DOM15-26-593 showed improved Tm, but the other
clones did not. It is worth noting that both DOM15-26-555 and
DOM15-10 parent molecules have much higher Tm at the start
(63.3-63.7.degree. C.) than the DOM4-130-54 and DOM1h-131-511
parent molecules (Tm at start: 57.9-54.1.degree. C.), but overall
the protease resistant clones reach a Tm in a similar range
(average Tm of 65.1.degree. C. for the DOM1h-131-511/DOM4-130-54
variants and average Tm of 64.9.degree. C. for the
DOM15-26-55/DOM15-10 variants).
TABLE-US-00024 TABLE 13 Name Tm .degree. C. DOM15-26-555 63.3
DOM15-26-588 70.1 DOM15-26-589 63 DOM15-26-591 63 DOM15-26-593 65
DOM15-10 63.7 DOM15-10-11 63.3
[0466] The dAbs were also compared in a receptor binding assay and
BIAcore kinetics were measured to determine whether the increase in
trypsin stability had led to a decrease in efficacy. However, the
activity of the dAbs in the assay was substantially unaffected or
even improved. The results are presented in Table 14.
TABLE-US-00025 TABLE 14 Clone ID EC.sub.50 (nM) K.sub.D (nM)
DOM15-26-555 11.7 26.1 DOM15-26-588 27 59.1 DOM15-26-589 1.94 9.6
DOM15-26-591 16 38 DOM15-26-593 0.323 3.2 DOM15-26-594 4.09 15.1
DOM15-26-595 0.828 5 DOM15-10 10.23 23.6 DOM15-10-11 3.58 14.6
Advantages of an Enhanced Tm
[0467] Most proteins--including domain antibodies--exist in two
states: a folded state (which leads to a biologically active
molecule) and an unfolded state (which does not bear functional
activity). These two states co-exist at all temperatures and the
relative proportion of each state is usually determined by a
constant K that is a function of the kinetic constants of folding
and unfolding. The melting temperature is usually defined as the
temperature at which K=1, i.e. the temperature at which the
fraction of folded protein is equal to be fraction of unfolded
protein. The constant K is determined by the stabilizing and
destabilizing intramolecular interactions of a protein and
therefore is primarily determined by the amino acid sequence of the
protein. Extrinsic parameters such as temperature, pH, buffer
composition, pressure influence K and therefore the melting
temperature.
[0468] Unfolded proteins are easy targets for degradation
mechanisms: (i) exposure of disulfide bonds increase risks of
oxidation or reduction depending on the circumstances, (ii)
enhanced backbone flexibility favours auto-proteolytic reactions,
(iii) exposure of peptide segments offers targets to proteases in
vivo, to proteases during production processes and to carry-over
proteases during downstream processing and long-term storage, and
(iv) exposure of aggregation-prone segments leads to
inter-molecular aggregation and protein precipitation. In all
cases, a loss of protein integrity, protein content and protein
activity happens, thereby compromising efforts to (i) ensure batch
reproducibility, (ii) ensure long-term stability on shelf, and
(iii) in vivo efficacy.
[0469] In nature proteins have been designed by evolution to
adequately perform at body temperature and to be readily replaced
via homeostatic mechanisms. Therapeutic proteins manufactured
through biotechnogical processes face a different environment: they
are frequently produced by recombinant DNA technology in a foreign
host, are expressed at higher amount in large vessels, undergo very
important changes in pH or buffer composition throughout downstream
processes and finally are stored at high concentrations in
non-physiological buffers for prolonged period of time. New
delivery techniques (e.g. inhalation, sc patch, slow delivery
nanoparticles) are also adding on the stress undergone by
therapeutic proteins. Finally the advent of protein engineering
techniques has resulted in the production of enhanced or totally
novel therapeutic proteins. Because most engineering techniques are
in-vitro based techniques aimed at altering or creating new amino
acid sequences, evolution processes that have gradually improved
biological proteins do not take place, hence resulting in proteins
of sub-optimal performances with regards to stress resistance.
[0470] The technique of the present invention aims at reproducing
one of the conditions faced by proteins throughout Darwinian
evolution. Peptides or polypeptides, eg immunoglobulin single
variable domains are infused with proteases that play a major role
in tissue remodelling and protein homeostasis. Any particular
mutation that may result in a protein with an improved fit to its
function is also tested for its ability to fit within the
environment it is performing in. This process is reproduced in one
embodiment of the present invention: a repertoire of peptide or
polypeptide variants is created and exposed to a protease. In a
second step, the repertoire of variants is contacted with a
specific target. Only those protein variants that have sustained
degradation by the protease are able to engage with the target and
therefore recovered, eg, by a simple affinity purification process
named `biopanning`. The system offers a number of advantages in
comparison to in vivo processes: the protein repertoire can be
faced with a wider range of conditions, eg a range of proteases, at
higher concentrations, for longer times, in different buffers or
pHs and at different temperatures. Thus this in vitro technology
offers a means to design proteins that may perform and remain
stable in a wider range of environments than those they originate
from. Clearly this offers multiple advantages for the
biotechnological industry and for the area of therapeutic proteins
in particular.
Example 12
PK Correlation Data for Protease Resistant Leads
[0471] The parent dAb and a protease-resistant dAb in each of the
four dAb lineages, were further evaluated in vivo (see Table15
below for list and details)
TABLE-US-00026 TABLE 15 Resistance Tm Activity ID as Fc Lineage dAb
ID to trypsin (.degree. C.) (nM) fusion DOM4-130 DOM4-130-54 Good
54 0.128(*) DMS1541 DOM4-130-202 Very high 64 0.160(*) DMS1542
DOM1h-131 DOM1h-131-511 Good 57 0.048.dagger. DMS1543 DOM1h-131-206
Very high 64 0.047.dagger. DMS1544 DOM15-10 DOM15-10 Low 64
0.913.dagger. DMS1546 DOM15-10-11 High 63 0.577.dagger. DMS1531
DOM15-26 DOM15-26-501(*) Low 52 0.330.dagger. DMS1545 DOM15-26-593
High 65 0.033.dagger. DMS1529 (*)as determined by MRC5/IL-a
bioassay; .dagger.as determined by RBA assay Note: DOM15-26-501 is
a parent version of DOM15-26-555 exemplified above in this patent
application. DOM15-26-555 has one germline amino acid mutation in
CDR1 (I34M). DOM15-26-501 has a lower melting temperature than
DOM15-26-555 (52 C. v 63.3 C.) and an increased susceptibility to
digestion by trypsin. DOM15-26-501 was chosen over DOM15-26-555 for
the PK study as it is a better representative for poor stability in
comparison to DOM15-26-593.
[0472] We can translate the resistance as follows:
[0473] 1 is low
[0474] 2 is moderate
[0475] 3 is good
[0476] 4 is high
[0477] 5 is very high
[0478] Then this means that the trypsin resistance of the parent
molecules is:
[0479] DOM4-130-54 is Good
[0480] DOM1h-131-511 is Good
[0481] DOM15-10 is Low
[0482] DOM15-26-501 is Low
[0483] As for the selected leads:
[0484] DOM4-130-202 is Very high
[0485] DOM1h-131-206 is Very high
[0486] DOM15-10-11 is High
[0487] DOM15-26-593 is High
[0488] Because domain antibodies are small in size (12-15 kDa) they
are rapidly cleared from the circulation upon iv or sc injection.
Indeed the renal glomerular filtration cut-off is above 50 kDa and
therefore small proteins such as dAbs are not retained in the
circulation as they pass through the kidneys. Therefore, in order
to evaluate the long term effects of resistance to proteases in
vivo, we tag domain antibodies with a moiety that increases
systemic residence. Several approaches (e.g.
[0489] PEG, Fc fusions, albumin fusion, etc) aiming at extending
half-life have been reported in the literature. In this application
the domain antibodies have been tagged (or formatted) with the Fc
portion of the human IgG1 antibody. This format offers two
advantages: (i) the molecular size of the resulting dAb-Fc is
.about.75 kDa which is large enough to ensure retention in
circulation, (ii) the antibody Fc moiety binds to the FcRn receptor
(also know as "Brambell" receptor). This receptor is localized in
epithelial cells, endothelial cells and hepatocytes and is involved
in prolonging the life-span of antibodies and albumin: indeed upon
pinocytosis of antibodies and other serum proteins, the proteins
are directed to the acidified endosome where the FcRn receptor
intercepts antibodies (through binding to the Fc portion) before
transit to the endosome and return these to the circulation. Thus
by tagging the Fc portion to the dAb, it is ensured that the dAbs
will exposed for long period to two at least compartments--the
serum and the pre-endosomal compartments, each of which containing
a specific set of proteolytic enzymes. In addition, the FcRn
receptor mediates transcytosis whereby Fc-bearing proteins migrate
to and from the extravascular space.
[0490] Formatting with Fc was accomplished by fusing the gene
encoding the VH and VK dAbs to the gene encoding the human IgG1 Fc,
through a short intervening peptide linker (in bold):
[0491] For a VH dAb (underlined):
TABLE-US-00027 EVQ......GQGTLVTVSSASTHTCPPCPAPELLGGP . . .
(hIgGlFc) . . . PGK*
[0492] For a VK dAb (underlined):
TABLE-US-00028 DIQ.........GQGTKVEIKRTVAAPSTHTCPPCPAPELLGGP . . .
(hIgGlFc) . . . PGK*
[0493] Material was produced by transient transfection of HEK293/6E
cells using 293-fectin (Invitrogen) according to standard
protocols. These cells are designed for high-level transient
expression when used in conjunction with the pTT series of vectors
(Durocher et al 2002). Thus the dAb genes were cloned into a
modified pTT5 vector (pDOM38) to generate the Fc fusion expression
vector (see FIG. 21). The supernatant from the transfected cells
was harvested at 5 days post-transfection, clarified by
centrifugation and filtered through a 0.2 .mu.m filter. The dAb-Fc
fusion proteins were purified by capture onto Protein-A streamline
resin (GE Healthcare). Protein was eluted from the column in 10 mM
sodium citrate pH3, followed by the addition of and 1M sodium
citrate pH6, to achieve a final composition of 100 mM sodium
citrate pH6.
[0494] The dAb-Fc molecules were tested for in vivo half life in
the rat at a target dose of 5 mg/kg into female Sprague-Dawley rats
(n=3 per group). It should be noted that the target dose vastly
exceeds target concentration in rats, so it is expected that
differences in affinities between parent dAbs and trypsin-resistant
dAbs (see example 11) will not impact on the fate of the molecules
in vivo. Hence differences in PK profiles between dAbs are expected
to reflect on an antigen-independent elimination process.
[0495] Blood samples were taken after 0.03, 1, 4, 8, 24, 48, 72,
96, 120 and 168 hours post administration. After clot formation,
serum was withdrawn and then tested in hIL-1R1, TNFR1 or VEGF
antigen capture assays:
[0496] hIL-1R1 Antigen Capture Assays:
[0497] Coat with 4 ug/mL anti-hIL-1R1
[0498] Block
[0499] Add 500 ng/mL shIL-1R1
[0500] Add samples
[0501] Detect with anti-human Fc HRP @1:10,000
[0502] TNFR1 Antigen Capture Assays:
[0503] Coat with 0.1 ug/mL sTNFR1
[0504] Block
[0505] Add samples
[0506] Detect with anti-human Fc HRP @1:10,000
[0507] VEGF Antigen Capture Assays:
[0508] Coat with 0.25 ug/mL VEGF
[0509] Block
[0510] Add samples
[0511] Detect with anti-human Fc HRP @1:10,000
[0512] Raw data from the assays were converted into concentrations
of drug in each serum sample. The mean .mu.g/mL values at each time
point were then analysed in WinNonLin using non-compartmental
analysis (NCA). The PK profiles of each dAb-Fc pair are shown in
Table 16 which summarises the determined PK parameters.
TABLE-US-00029 TABLE 16 AUC/D (0-inf) Half Life (hr * .mu.g/mL)/ %
AUC ID dAb (hr) (mg/kg) Extrapolated DMS1541 4-130-54 93.2 691.5
22.7 DMS1542 4-130-202 176.8 710.1 49 DMS1543 1h-131-511 140.8
1807.5 40 DMS1544 1h-131-206 158.6 2173.0 43.6 DMS1546 15-10 43.2
324.6 3.8 DMS1531 15-10-11 56.6 770.5 n.d. DMS1545 15-26-501 12.9
89 5.1 DMS1529 15-26-593 86.2 804.7 21.0
[0513] The results clearly indicate that--whilst the PK profiles of
the dAb-Fc pairs 4-130-54 to 1h-131-206 are almost
superimposable--the profiles vary widely with the other pairs. The
effects are mostly visible when AUC/D is considered: the AUC/D of
15-10 is only 42% of that of 15-10-11. The AUC/D of 15-26-501 is
only 11% of that of 15-26-593. These important differences also
impact (to a lesser extent) half-lives: 43.2 h versus 56.6 h for
15-10 and 15-10-11, respectively. A greater difference is seen with
the DOM15-26 lineage: 12.9 h versus 86.2 h for 15-26-501 and
15-26-593, respectively. Indeed for a good PK analysis using
non-compartmental analysis, there should be at least 4 data points
used to fit the linear regression slope and the period of time over
which the half life is estimated should be at least 3 times that of
the calculated half life.
[0514] In light of the biophysical properties described in the
examples herein, it appears that the ability of any given dAb to
resist degradation by trypsin is correlated with the ability of the
dAb-Fc fusion to circulate for longer period in the rat serum.
Indeed as shown in the examples, such as Example 10, DOM15-10 and
DOM15-26-501 are the most degradable dAbs: incubation of 25 ug dAb
in the presence of 1 ug of trypsin at 30.degree. C. for .about.3 h
resulted in complete degradation. All other dAbs in this study
(whether they had been selected with trypsin (ie. DOM15-10-11,
DOM15-26-593, DOM4-130-202 and DOM1h-131-206) or whether they
already had some trypsin resistance as parent molecules
(DOM4-130-54 and DOM1h-131-511)) have comparable PK profile in rats
when re-formatted into dAb-Fc molecules. Thus, the present PK study
suggests that susceptibility to proteolysis has its biggest impact
on the in vivo stability of dAbs when those dAbs have very low
resistance to proteolysis. It also shows that--beyond a certain
level--further increments in resistance to degradation by trypsin
(e.g. DOM4-130-206 v DOM4-130-54) do not significantly add up to
the ability of the dAb-Fc molecule to further slow down elimination
in vivo.
[0515] In three cases, selection in the presence of trypsin
resulted in new molecules with increased thermal stability (defined
by the melting temperature): DOM4-130-202, DOM1h-131-206 and
DOM15-26-593. The PK study indicates that--in the present
dataset--melting temperature is not an adequate parameter to
rationalize the observed PK profiles: indeed DOM15-10 has a higher
Tm than DOM15-10-11 and yet is more rapidly cleared than
DOM15-10-11 from the circulation. Elsewhere, the two dAbs of the
DOM4-130 lineage have markedly different Tm (by 10.degree. C.) and
yet show almost identical stability in vivo when formatted into
dAb-Fc molecules. It should be noted that melting temperature is
not per se excluded as key parameter to predict in vivo stability.
It just happens that with the present dataset, large Tm differences
(from 54.degree. C. and above) have not a significant impact on the
fate of dAbs in vivo. This doesn't exclude the possibility that at
melting temperature lower than 54.degree. C., the in vivo stability
of dAbs may correlate with thermal stability, or perhaps even with
thermal stability and resistance to proteases altogether.
Example 13
Trypsin Selections on DOM10-53-474
Trypsin Stability of Purified DOM10-53-474:
[0516] DOM10-53-474 is a domain antibody which binds to IL-13 with
a high potency. To assess the stability of this dAb in the presence
of trypsin, purified dAb was digested with trypsin for increased
time points and run on a gel to examine any possible protein
degradation. 25 .mu.l of purified DOM10-53-474 at 1 mg/ml was
incubated with 1 .mu.l of sequencing grade trypsin at 1 mg/ml at
30.degree. C., resulting in molecular ratio of 25:1 dAb:trypsin.
dAb was incubated with trypsin for 1 h, 4 h and 24 h and the
protease activity was neutralised by addition of 4 .mu.l of Roche
complete protease inhibitors followed by incubation on ice. Time 0
sample was made by adding protease inhibitors to dAb without adding
trypsin. 2 .mu.l of sample was subsequently analysed by
electrophoresis using Labchip according to manufacturers
instructions.
[0517] FIG. 22 shows a gel run with DOM10-53-474 incubated with
typsin for increased time points. For comparison one of the trypsin
stable dAbs, DOM15-26-593 was also treated with trypsin as
explained above and was run alongside. As shown in the figure,
DOM15-26-593 looks stable even after 24 h incubation with trypsin.
However, DOM10-53-474 is degraded to a certain extent after 24 h,
but looking stable at 1 h and 4 h time points. These data suggests
that DOM10-53-474 is resistant to degradation by trypsin to a
certain extent, but is not as stable as one of the most trypsin
stable dAbs DOM15-26-593.
Trypsin Stability of Phage--Displayed DOM10-53-474:
[0518] To assess the trypsin stability of phage displayed
DOM10-53-474, the gene encoding DOM10-53-474 was cloned into
Sal/Not sites of pDOM33 (FIG. 50) and phage produced according to
standard techniques. Phage was purified by PEG precipitation,
re-suspended in PBS and titered.
[0519] Phage displayed dAbs were incubated with trypsin for
different time points to evaluate trypsin resistance. Following
incubation with trypsin, stability was measured by titre analysis
following infection of exponentially growing E. coli TG1 cells.
[0520] 100 .mu.l of phage was incubated in 100 .mu.g/ml trypsin for
1 h, 2 h, 4 h and overnight at 37 C, in a shaking incubator.
Trypsin activity was blocked with Roche complete protease inhibitor
(.times.2) and then phage was diluted in 2% marvel in PBS,
incubated with 10 nM biotinylated IL-13 for one hour at room
temperature. Streptavidin-coated beads (Dynabeads M-280
(Invitrogen) that were pre-blocked for one hour at room temperature
with 2% marvel in PBS was added, and the mixture was then incubated
for 5 minutes at room temperature. All of the incubation steps with
Dynabeads were carried out on a rotating wheel. Unbound phage was
washed away by washing the beads eight times with 1 ml of 0.1%
Tween-20 in PBS. Bound phage was eluted with 0.5 ml of 0.1M Glycine
pH 2.2 and neutralized with 100 .mu.l of 1M Tris-HCL pH 8.0. Eluted
phage was used to infect exponentially growing TG1 (1 h at
37.degree. C.) and plated on tetracycline plates. Plates were
incubated at 37.degree. C. overnight and colony counts were made.
Phage output titres following digestion with trypsin is summarised
in Table 17. Phage titres decreased when incubated with trypsin for
increased time points. After 24 h incubation all phage was
digested.
TABLE-US-00030 TABLE 17 Output titres of trypsin selections
performed on phage displayed DOM-10-53-474 parent: Length of
Trypsin trypsin incubation concentration Titre No trypsin control
-- 3 .times. 10.sup.7 1 h 100 .mu.g/ml 1 .times. 10.sup.7 2 h 100
.mu.g/ml 7 .times. 10.sup.6 4 h 100 .mu.g/ml 5 .times. 10.sup.6
overnight 100 .mu.g/ml 0
Selection of dAbs More Resistant to Trypsin:
[0521] In order to select for dAbs which are more resistant to
degradation by trypsin, random mutations were introduced to gene
encoding DOM10-53-474 by PCR using Stratergene Mutazyme 11 kit,
biotinylated primers and 5-50 pg of template for 50 .mu.l reaction.
After digestion with SalI and NotI, inserts were purified from
undigested products with streptavidin coated beads and ligated into
pDOM33 at the corresponding sites. E. Coli TB 1 cells were
transformed with purified ligation mix resulting in an error prone
library of DOM10-53-474. The size of the library was
1.9.times.10.sup.9 and the rate of amino acid mutation was 1.3.
[0522] Three rounds of selections were performed with this library
to select for dAbs with improved protease resistance. First round
of selection was performed only with antigen without trypsin
treatment to clean up the library to remove any clones that no
longer bound antigen with high affinity. Selection was carried out
at 10 nM IL-13. The outputs from round one were 2.times.10.sup.9
compared to input phage of 6.times.10.sup.10 indicating that
majority of library bound antigen with high affinity.
[0523] The second and third rounds of selections were performed
with 1 nM biotinylated IL-13. Prior to panning on IL-13, phage was
incubated with 100 .mu.g/ml of trypsin at 37.degree. C. in a shaker
(250 rpm). For second round selection, trypsin incubation was
carried out for 1 h either at room temperature or at 37.degree. C.
The outputs from round 2 selection is shown in Table 18:
TABLE-US-00031 TABLE 18 Output phage titres following second round
selection. Trypsin treatment Titre No treatment 1 .times. 10.sup.8
1 h room temperature 5 .times. 10.sup.7 1 h 37.degree. C. 2 .times.
10.sup.7
[0524] Phage outputs from round 2 selection with 1 h trypsin
treatment at 37.degree. C. was used as the input for 3.sup.rd round
selection. For 3.sup.rd round selection, phage was treated with 100
.mu.g/ml trypsin but for longer time points: 2 h at 37.degree. C.,
4 h at 37.degree. C., overnight at room temperature or overnight at
37.degree. C. The output titres for 3.sup.rd round selection are
summarised in Table 19:
TABLE-US-00032 TABLE 19 Output phage titres following third round
selection Trypsin treatment Titre No trypsin 1.3 .times. 10.sup.8 2
h at 37.degree. C. 1.9 .times. 10.sup.7 4 h at 37.degree. C. 2
.times. 10.sup.6 Overnight at room temperature 4 .times. 10.sup.7
Overnight at 37.degree. C. 2.1 .times. 10.sup.6
[0525] Several clones from each selection outputs from round 1, 2
and 3 were sequenced to assess the sequence diversity. Following
first round of selection without trypsin treatment, 50% of the
selection outputs had parent DOM10-53-474 sequence. After 2.sup.nd
round of selection, percentage of parent increased to 75%. After
3.sup.rd round of selection, percentage of parent increased to
80%.
[0526] This data indicate that DOM10-53-474 is already resistant to
degradation by trypsin and not many new clones can be selected from
these trypsin selections. FIG. 22 showed that when purified protein
was digested with trypsin, DOM10-53-474 was not completely digested
even after overnight trypsin treatment. However to see whether
there are any new clones that are more trypsin resistant than
DOM10-53-474 in selection outputs, selection 3 output where phage
was treated overnight with trypsin at 37.degree. C. was sub-cloned
into pDOM5. Hundred clones were then sequenced to look for any
trypsin resistant clones. Out of hundred clones analysed, only 26
clones had new sequences, however none of these clones had
mutations at trypsin cleavage sites (Lysine or Arginine) suggesting
that these clones are not more resistant to trypsin than
DOM10-53-474.
Example 14
[0527] Storage and Biophysical Improvements Introduced into the
Lead DOM0101 (Anti-TNFR1) dAbs by Phage Selections in the Presence
of Trypsin:
[0528] To improve the protease resistance of the lead molecule
DOM1h-131-511, phage selections in the presence of trypsin were
carried out as described earlier. The method produced a range of
clones with improved trypsin stability compared to the parental
DOM1h-131-511 molecule. Two clones, DOM1h-131-202 and DOM1h-131-206
were selected for further characterisation as they showed the most
significant improvement to the action of trypsin. Further work as
outlined below shows that with the improved resistance to the
action of trypsin there are other beneficial effects, primarily on
the stability of the molecules to shear and thermal stress. These
two parameters are central to increasing the storage and shelf life
stability of biopharmaceutical products.
Production of Lead DOM0101 dAbs in Pichia pastoris:
[0529] The genes encoding the primary amino acid sequence of the
three lead molecules was used to produce secreted protein in Pichia
pastoris. The three synthetic genes (DOM1h-131-511, DOM1h-131-202
and DOM1h-131-206) were cloned into the expression vector
pPIC-Z.alpha. and then transformed into two Pichia strain, X33 and
KM71H. The transformed cells were plated out onto increasing
concentrations of Zeocin (100, 300, 600 and 900 .mu.g/ml) to select
for clones with multiple integrants. Several clones were then
screened in 2 L flasks to identify high expressing cell lines. The
best expressing clones were then used to produce material at 5 L
scale in fermenters.
Protein Purification and Material Characterization:
[0530] In order to produce high quality material for
characterisation and further stability studies, a downstream
purification process was devised using a mixed modal charge
induction resin (Capto MMC) as the primary capture step followed by
anion exchange (Q Sepharose). Without significant optimisation,
this allowed the recovery of .about.70% of the expressed dAb at a
purity of .about.95%. The material was characterised for identity
using electrospray mass spectrometry, amino terminal sequencing and
isoelectric focusing and for purity using SDS-PAGE and SEC (size
exclusion chromatography).
Protein Identity:
[0531] The amino terminal sequence analysis of the first five
residues of each protein, was as expected (EVQLL . . . ). Mass
spectrometry was performed on samples of the proteins which had
been buffer exchanged into 50:50 H.sub.2O:acetonitrile containing
0.1% glacial acetic acid using C4 Zip-tips (Millipore). The
measured mass for each of the three proteins was within 0.5 Da of
the theoretical mass based on the primary amino acid sequence
(calculated using average masses) when allowing for a mass
difference of -2 from the formation of the internal disulphide
bond. IEF was used to identify the proteins based on their pI which
was different for each protein.
Protein Purity:
[0532] The three proteins were loaded onto non-reducing SDS-PAGE
gels in 1 .mu.g and 10 .mu.g amounts in duplicate. A single band
was observed in all instance.
[0533] Size exclusion chromatography was also performed to
demonstrate levels of purity. For size exclusion chromatography
(SEC) 100 .mu.g of each protein were loaded onto a TOSOH G2000 SWXL
column flowing at 0.5 ml/min. Mobile phase was PBS/10% ethanol. The
percentage of monomer was measured based on the area under the
curve (see FIG. 23).
Comparison of Stability of DOM1h-131-511, -202 and -206
Assessment of Protease Stability:
[0534] The protease stability of DOM1h-131-511, DOM1h-131-202 and
DOM1h-131-206 was assessed by BIAcore.TM. analysis of the residual
binding activity after pre-incubation for defined timepoints in
excess of proteases. Approximately 1400RU of biotinylated TNFR1 was
coated to a streptavidin (SA) chip. 250 nM of DOM1h-131-511,
DOM1h-131-202 and DOM1h-131-206 was incubated with PBS only or with
100 ug/ml of trypsin, elastase or leucozyme for 1, 3, and 24 hour
at 30.degree. C. The reaction was stopped by the addition of a
cocktail of protease inhibitors. The dAb/protease mixtures were
then passed over the TNFR1 coated chip using reference cell
subtraction. The chip surface was regenerated with 10 ul 0.1M
glycine pH 2.2 between each injection cycle. The fraction of
DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206 bound to human TNFR1
(at 10 secs) pre-incubated with proteases was determined relative
to dAb binding without proteases. BIAcore.TM. runs were carried out
at 25.degree. C. The data below was produced from three independent
experiments. The bar graph indicates mean values and the error bars
indicate standard deviation of the results (FIG. 24).
[0535] It was found that DOM1h-131-202 and DOM1h-131-206 were shown
to have greater resistance to proteolytic degradation by trypsin,
elastase or leucozyme in comparison to DOM1h-131-511. The
difference between DOM1h-131-202 and DOM1h-131-206 in comparison to
DOM1h-131-511 is most pronounced after 1 hr with trypsin and after
3 hrs with elastase or leucozyme. There is a trend that
DOM1h-131-206 is slightly more stable compared to DOM1h-131-202 in
most of the conditions tested.
Thermal Stability of the dAbs as Determined Using DSC:
[0536] In order to determine at which pH the lead molecules had the
greatest stability, differential scanning calorimeter (DSC) was
used to measure the melting temperatures (T.sub.m) of each dAb in
Britton-Robinson buffer. As Britton-Robinson is made up of three
component buffer systems (40 mM of each of acetic, phosphoric and
boric acid), it is possible to produce a pH range from 3-10 in the
same solution. The theoretical pI was determined from the proteins
primary amino acid sequence. From the DSC, the pH at which the dAbs
had their greatest intrinsic thermal stability was found to be pH 7
for DOM1h-131-202, pH 7-7.5 for DOM1h-131-206 and pH 7.5 for
DOM1h-131-511. For all subsequent stress and stability work the
following pHs were used for each dAb; for DOM1h-131-202 and
GSK1995057A DOM1h-131-206 pH 7.0 and for DOM1h-131-511 pH 7.5 in
Britton-Robinson buffer. The results are summarised in Table
20.
TABLE-US-00033 TABLE 20 Summary of the pH and T.sub.ms of
DOM1h-131-202, DOM1h-131-206 and DOM1h-131-511 as determined by DSC
in Britton-Robinson buffer at 1 mg/ml. The temperature was ramped
at 180.degree. C./hour. pH that gives Tm (.degree. C.) of greatest
intrinsic the dAb at dAb thermal stability the given pH
DOM1h-131-202 7.0 68.6 DOM1h-131-206 7.0-7.5 65.8 DOM1h-131-511 7.5
58.0
[0537] Two Week Thermal Stability Testing
[0538] The ability of a protein to endure prolonged periods of time
at elevated temperatures is usually a good indication of its
stability. Under these conditions, protein may undergo several
physical processes such as aggregation or chemical modification.
The dAbs (at 1 mg/ml) were incubated at 37 and 50.degree. C. for 14
days in Britton-Robinson buffer. SEC was used to determine how much
monomer was left in solution over the 14 day period (FIG. 25).
[0539] From FIG. 25 it can be seen that both DOM1h-131-202 and
DOM1h-131-206 are significantly more stable than DOM1h-131-511 to
thermal stress. Exposing proteins to elevated temperatures, such as
37 and 50.degree. C., are routinely used to give an indication on a
drug's long term shelf-life. These higher temperatures are used to
accelerate the normal process associated with long term storage at
room temperature such as deamidation, oxidation or aggregation. The
level of aggregation formation in solution can also be monitored
using SEC (FIG. 26A to I). After 14 days at 37.degree. C., the loss
of DOM1h-131-511 from solution can be attributed to both
precipitation and the formation of higher ordered aggregates as
determined by SEC (FIG. 26B). A significantly lower loss in protein
is also seen with both DOM1h-131-202 and DOM1h-131-206 at
37.degree. C. after 14 days with very little or no substantial
increase in aggregate formation, especially in the case of
DOM1h-131-206 (FIG. 26H). At 50.degree. C., the difference between
the molecules is even more pronounced, with DOM1h-131-206 showing
better stability at the higher temperature than DOM1h-131-202 after
14 days, showing significantly reduced formation of higher
molecular weight aggregates (FIG. 26). Relative to the t=0,
DOM1h-131-206 shows only a small increased in aggregate formation
after 14 days (FIG. 26I), whereas DOM1h-131-511 has all but
precipitated out of solution (FIG. 26C).
[0540] This shows that the changes introduced into the dAb by the
trypsin selections, e.g. the improved thermal stability, has
significantly improved the protein storage stability at 37 and
50.degree. C. Both DOM1h-131-202 and more significantly
DOM1h-131-206, clearly have improved solution stability and lower
tendency to form aggregates at elevated temperatures which can
directly be translated to improved long term storage stability at
more relevant temperatures such +4.degree. C. and room
temperature.
[0541] Samples from 24 hr, 48 hr, 7 days and 14 days time points
from the thermal stress experiment were then analysed by IEF to see
if the proteins had undergone any biophysical changes which would
affect the overall charge of the protein (FIG. 27).
[0542] Again both DOM1h-131-202 and DOM1h-131-206 show no
significant changes at 37.degree. C. compared to DOM1h-131-511.
With DOM1h-131-511 a faint second band appears at 37.degree. C.
after 24 hrs. It is believed this extra banding is due to
dimerisation of the protein, thus masking charge and producing two
populations of molecules. At 50.degree. C. the difference between
the molecules is more pronounced, DOM1h-131-206 clearly shows no
significant changes at the elevated temperature whereas
DOM1h-131-202 is showing some sign of modification after 24 hr. The
majority of DOM1h-131-511 is lost by precipitation after 48 hr in
Britton-Robinson.
[0543] The T=0, 7 and 14 day time points at 50.degree. C. were
analysed by the TNFR-1 RBA to determine the functionality of the
protein after exposure to high temperatures (FIG. 28). The assay is
currently not as sensitive as SEC or IEF at detecting subtle
changes to the molecule due to stress, but it can be used show that
the dAb can still bind to the antigen.
[0544] The shift in the curve to the left for DOM1h-131-511
reflects the fact that the majority of the dAb has been lost due to
precipitation. The material that is left in solution is still able
to bind antigen. As shown in FIG. 25, the majority of both
DOM1h-131-202 and DOM1h-131-206 are able to be maintained in
solution even after 14 days. The RBA shows that all the soluble
protein is still functional and able to bind to TNFR1.
Storage Stability Testing at High Protein Concentrations:
[0545] Experiments were carried out to investigate the storage
stability at +4.degree. C. at very high protein concentrations to
see how each molecule performed under these conditions. All the
lead dAbs were concentrated in centrifugal Vivaspin concentrators
(5K cut-off) in Britton-Robinson buffer at their most stable pH, to
.about.100 mg/ml. The samples at .about.100 mg/ml were then left at
+4.degree. C. for 7 days and then analysed by SEC to see if any
other physical changes had occurred to the samples during storage
at high concentrations (FIG. 29). The samples were diluted to
.about.1 mg/ml before being run on the SEC column in 1.times.PBS
10% ethanol (v/v).
[0546] From the SEC traces it can be seen that neither
DOM1h-131-202 nor DOM1h-131-206 show any significant increase in
the formation of aggregates after 7 days, where as there is
.about.2% reduction in the monomer concentration for
DOM1h-131-511.
Nebuliser Delivery of the Lead dAbs:
[0547] For early stage toxicology and clinical work, the dAbs will
be formulated as a liquid and delivered via a nebulising device.
Depending on the device (eg, ultrasonic, jet or vibrating mesh),
the dAb will experience a degree of shear and thermal stress as it
was nebulised to form a aerosol of defined particle size. As both
DOM1h-131-202 and -206 have higher T.sub.m's and showed
considerably improved stability to thermal stress compared to
DOM1h-131-511, all the dAbs were tested in two nebuliser devices to
see how they responded to shear/thermal stress induced during
nebulisation. Both the protein from the nebulised aerosol and the
remaining dAb in the device (i.e. in the cup) were then analysed by
SEC to determine the amount of aggregation generated during the
process.
[0548] All the molecules were tested in Britton-Robinson buffer at
their most stable pH. The dAbs were tested in both the E-flow Rapid
(vibrating mesh) and Pari LC+ (jet nebuliser) with run time of 3.5
minutes at a protein concentration of 5 mg/ml and the particle size
distribution determined using a Malvern Spraytek. The results are
shown in FIG. 30. For good delivery and distribution into the deep
lung, the ideal particle size is <5 .mu.m. All the dAbs give
comparable levels of particle sizes that were less than 5 .mu.m in
Britton-Robinson buffer. The concentration of the dAb in the cup of
the device was determined by A.sub.280 measurements before and
after nebulisation (data not shown). It was found that the protein
concentration did not change significantly indicating that neither
the protein nor vehicle are preferentially nebulised during
delivery.
[0549] Samples of the dAbs nebulised in Britton-Robinson buffer
were run on SEC to determine if during delivery the protein had
undergone any physical changes. FIG. 31 shows the relative
percentage change in either the cup or the aerosol as determined by
SEC. It can be seen that both DOM1h-131-202 and DOM1h-131-206
undergo relative small changes in the concentration of monomer
relative to DOM1h-131-511. This demonstrates that both
DOM1h-131-202 and DOM1h-131-206 with their improved T.sub.m's have
less propensity to aggregate during nebulisation.
[0550] FIG. 32 shows the actual SEC traces for DOM1h-131-206 and
DOM1h-131-511 in Britton-Robinson buffer post nebulisation and
demonstrates that the relative loss in monomer (FIG. 31) is due to
dimer formation. This again provides further supporting evidence to
the theory that the greater thermal stability shown by
DOM1h-131-202 and DOM1h-131-206 can prevent significant aggregation
even in an unoptimised formulation buffer.
[0551] For toxicology and safety assessment work, it is necessary
to delivery the dAb at significantly higher levels into the animal
than the therapeutic doses given to patients. This can only be
achieved by using significantly higher protein concentrations
and/or delivering the dAb over a prolonged period of time. As it
had already been shown that DOM1h-131-511 forms aggregates on
nebulisation at 5 mg/ml over 3.5 mins, DOM1h-131-206 was tested at
40 mg/ml in PBS and nebulised using the Pari LC+ for up to 1 hour.
Samples from the cup and aerosol were taken at the time points to
throughout the run to see if the prolong nebulisation caused the
dAbs to aggregate due to shear or thermal stress as determined by
SEC and the protein concentration (A280 nm measurements). Table 21
shows the protein concentration of the dAb both in the cup and
aerosol as determined by A280.
TABLE-US-00034 TABLE 21 Measured protein concentration of
DOM1h-131-206 as determined by A280 absorbance readings for both
the cup and aerosol during nebulisation of the dAb at ~40 mg/ml
using the Pari LC+. Allowing for dilution errors and instrumental
error the sample concentration does not change after nebulising the
dAb over 1 hr. Time Cup Sample Aerosol Sample (Mins) (mg/ml)
(mg/ml) 1 43.8 43.4 29 44.5 43.5 59 44.6 44.1
[0552] From Table 21 it can be seen that the concentration of the
protein did not significantly change during the run, demonstrating
that there was no significant loss of the protein due to
aggregation. FIG. 33 shows that over the period of 1 hour of
nebulisation, DOM1h-131-206 does not form any higher ordered
aggregates such as dimers as determined by SEC. This clearly
demonstrates that the improved biophysical properties, as
introduced into the molecule by trypsin selections, significantly
increases the dAbs resistance to shear and thermal stress and that
this can be directly correlated to improved storage shelf-life and
the ability to nebulise the protein so that higher ordered
aggregates do not form.
Solution State of the Lead dAbs:
[0553] Since the major route of degradation for all the three lead
dAbs appears to be self-association leading initially to
dimerisation followed by further aggregation and ultimately
precipitation, the three lead molecules were investigated by
Analytical Ultra-Centrifugation (AUC) to determine the degree of
self--association. The proteins were investigated by two methods,
sedimentation equilibrium and sedimentation velocity.
[0554] For the sedimentation equilibrium method the three samples
were run at three different concentrations ranging from 0.5 mg/ml
to 5 mg/ml with centrifugation effects using three different rotor
speeds. By this method it was determined that DOM1h-131-511 is a
stable dimer (26.1-34.4 kDa), DOM1h-131-202 is monomer/dimer
equilibrium (22.7-27.8 kDa) with a relatively stable dimeric state
at the concentrations measured with K.sub.d=1.3 .mu.M and
DOM1h-131-206 is predominantly monomeric (15.4-17.9 kDa) with a
K.sub.d for the monomer to dimer association of 360 .mu.M.
[0555] By the sedimentation velocity method all samples showed some
degree of dissociation upon dilution. From the results obtained,
shown in FIG. 34, the sedimentation coefficient observed for
DOM1h-131-511 is indicative of higher order aggregates and the peak
shift upon dilution is an indication of dissociation of these
aggregates. The protein aggregation and dissociation cancel each
other out which can give the impression of being a stable dimer as
observed by sedimentation equilibrium. The sedimentation
coefficients observed for DOM1h-131-202 are indicative of a rapid
dynamic equilibrium and therefore the monomer and dimer peaks could
not be separated from each other, giving the single peak with a
higher sedimentation coefficient than is appropriate for the mass
of the sample. This result agrees with the result obtained by the
sedimentation equilibrium method and the dissociation constant was
measured as being 1 .mu.M. DOM1h-131-206 was determined to be more
monomeric than the other two samples, having a sedimentation
coefficient of 1.9 s as compared to 2.5 s for the other two
samples. This data agrees well with the sedimentation equilibrium
data. At the concentrations measured, .about.10-fold below the
K.sub.d of 360 .mu.M, the sample is predominantly monomeric.
Example 15
Potency Enhancement of the DOM15-26-593 dAb:
[0556] An example of the enhancement of potency in VEGFR2 Receptor
Binding Assay of the DOM15-26-593 dAb over DOM15-26 parent is shown
in FIG. 40. In this assay, the ability of a potential inhibitor to
prevent binding of VEGF to VEGFR2 is measured in a plate-based
assay. In this assay a VEGFR2-Fc chimera is coated on a 96-well
ELISA plate, and to this is added a predetermined amount of VEGF
that has been pre-incubated with a dilution series of the test dAb.
Following the washing-off of unbound protein, the amount of VEGF
bound to the receptor is detected with an anti-VEGF antibody, the
level of which is determined colorimetrically. A dose-response
effect is plotted as percentage inhibition of VEGF binding as a
function of test substance concentration. An effective inhibitor is
therefore one that demonstrates substantial blocking of ligand
binding at low concentrations.
FC Fusions Potency and Half Life:
[0557] The therapeutic potential of VEGF blockade in the treatment
of tumours has been realised for over 30 years. The chronic nature
of cancer dictates that biopharmaceuticals require a long serum
half life to mediate their effects, and this is not consistent with
the rapid clearance of free dAbs from the circulation by renal
filtration. To assess the utility of the VEGF dAbs as
anti-angiogenics for the treatment of cancer, the lead domain
antibodies were formatted as fusions with wild type human IgG1 Fc
via a hybrid linker so as to form a bivalent molecule with a serum
half life extended by the use of FcRn-mediated antibody salvage
pathways.
[0558] In this Fc fusion format, the potency of the lead trypsin
selected dAb, DOM15-26-593 was compared with the initial parent dAb
(DOM15-26) & the trypsin labile dAb (DOM15-26-501) using the
assay described previously. The results are shown in the Table 22
below:
TABLE-US-00035 TABLE 22 Potency (RBA) & half life
characteristics of DOM15-26 leads in the Fc fusion format Potency
T1/2b dAb Fc (nM) (hrs) DOM15-26 hIgG1 0.506 ND DOM15-26-501 hIgG1
0.323 12.9 DOM15-26-593 hIgG1 0.033 84.6
[0559] It can be seen from these results that in the dimeric Fc
fusion format, affinity & potency are enhanced in relation to
the free dAbs due to the effect of avidity. It is clear that the
potency enhancement obtained in DOM15-26-593 by virtue of trypsin
selection is maintained and is even more pronounced in this Fc
format. Furthermore, the improvements in thermal and protease
stability translate into profound changes in the in vivo
pharmacokinetic behaviour of the molecules. The improvement in the
elimination half life (see FIG. 41) of DOM15-26-593 compared with
DOM15-26-501 is likely to be a direct consequence of the increased
stability of the dAb, rendering it more resistant to the
degradative processes that occur within the endosomal compartment.
It is also to be expected, therefore, that dAbs with enhanced
protease stability are able to persist for longer in other
biological compartments such as the serum, mucosal surfaces and
various tissue compartments where proteolysis is an active process
involved in the turnover of biological molecules.
[0560] Pharmacokinetic Clearance Profiles:
[0561] Pharmacokinetic clearance profiles of DOM15-26-593 and
DOM15-26-501 were measured after i.v. administration DOM15-26-593
and DOM15-26-501 to 3 rats at concentrations of 5 mg/kg. Levels of
DOM15-26-593 and DOM15-26-501 in the serum were then measured using
a direct VEGF binding standard ELISA assay and an anti-human Fc
antibody, therefore only intact drug in the serum samples were
detected. The full pharmacokinetic profile is shown in the Table 23
below:
TABLE-US-00036 TABLE 23 Summary Pharmacokinetic parameters of the
DOM15-26 & DOM15- 26-593 Fc fusions in rat Half Life Cmax AUC
(0-inf) Clearance dAb (hr) (.mu.g/ml) (hr * .mu.g/ml) (ml/hr/kg)
DOM15-26-501 12.9 91.4 445.1 11.8 DOM15-26-593 84.6 101.8 3810
1.3
[0562] It can be seen from these results that DOM15-26-593 has a
significantly improved pharmacokinetic profile with e.g. an
extended half life and reduce clearance rate.
[0563] The significantly improved potency and pharmacokinetic
properties of the
[0564] DOM15-26-593 resulted in analysis of the compound for a
range of other biophysical attributes.
Solution State Properties: Analysis by SEC-MALLs & AUC:
[0565] The in-solution state of DOM15-26-593 was assessed by both
size exclusion chromatography-multi-angle laser light scattering
(SEC-MALLS) and analytical ultracentrifugation (AUC). SEC-MALLS was
run on a Superdex 200 GF column (agarose matrix) at a flow rate of
0.3 ml.min.sup.-1 in Dulbecco's PBS (Sigma) with refractive index
(RI detection on a Wyatt Optilab rEX) and MALLS detection on a
Wyatt TREOS MALLS detector. Data were analysed using ASTRA software
Two separate batches of DOM15-26-593 were analysed and both were
shown to behave as monomers in solution at concentrations of up to
2.5 mg/ml with a calculated molecular mass of 78-81 KDa, consistent
with the calculated intact molecular mass of approx 76 kDa.
[0566] For the AUC analysis, DOM15-26-593 was diluted to
concentrations of 0.2, 0.5 and 1.0 mg/ml in PBS & sedimentation
velocity runs carried out at 40000 rpm in a Beckman XL-A analytical
ultracentrifuge. Data was acquired at 5 minute intervals at a set
temperature of 20.degree. C. Data was analysed using SEDFIT
software and sedimentation coefficient distributions were generated
using either c(S) or ls-g(s*) routines.
[0567] The results of this analysis show that DOM15-26-593 behaves
as a monomer in solution at concentrations of up to 2.5 mg/ml with
a calculated molecular mass of 78-81 KDa, consistent with the
calculated intact molecular mass of approx 76 kDa (FIGS. 42a &
42b).
Thermal Melting Properties: Analysis by DSC
[0568] Experiments were Done with DOM15-26-593 (and Fc Fusion) as
Follows:
[0569] Differential scanning calorimetry was used to analyse the
thermal stability of the dAbs and Fc fusions. Briefly the proteins
were placed in a Microcal calorimeter at a concentration of 2
mg.ml.sup.-1 with a buffer reference. The samples were heated from
20.degree. C. to 100.degree. C. at a rate of 180.degree.
C.hr.sup.-1 in an appropriate buffer, and the thermal denaturation
data analysed using "Origin" software using fitting models
appropriate to the nature of the protein under analysis.
[0570] The increased thermal stability of the trypsin selected dAb
(65.degree. C., FIG. 43 middle panel) is maintained in the Fc
fusion (64.5.degree. C., FIG. 43 upper panel). The Tm curve of the
DOM15-26-501 dAb (52.degree. C., FIG. 43 lower panel) is shown for
comparison.
Stability to Freeze-Thaw, Temperature Stress and Serum
Components
[0571] Experiments were Done with DOM15-26-593 (and Fc Fusion) as
Follows:
[0572] The stability properties of the DOM15-26-593 dAb mean that
it can be subjected to physical and biological stress with minimal
effects on its ability to bind VEGF (see FIGS. 44-47 (a and b)).
The binding of the VEGF dAb-Fc fusions to VEGF was in all cases
determined by ELISA. Briefly, a 96-well ELISA plate was coated with
250 mg/ml VEGF.sub.165 in carbonate buffer overnight. The plate was
then blocked with 1% BSA in PBS prior to addition of the test
substances diluted in the same buffer. The unbound material was
washed away after 60 minutes incubation, and the bound material
detected with a 1:10,000 dilution of HRP-conjugated anti-human IgG
followed by "SureBlue" chromogenic substrate and stopping with 1M
HCl.
[0573] For example, the molecule can be repeatedly freeze thawed
from liquid nitrogen (-196.degree. C.) to body temperature
(37.degree. C.) for 10 cycles without loss of binding activity as
determined by ELISA (FIG. 44). This treatment also resulted in no
obvious alterations in the molecule's aggregation state, as
assessed by conventional size exclusion chromatography (FIG. 45).
Further tests demonstrated that the molecule can be placed at a
range of different temperatures from -80.degree. C. to 55.degree.
C. with only a minor drop in antigen binding activity after 168
hours at only the highest incubation temperature (FIG. 46).
Furthermore, incubation with serum from human or cynomolgus monkeys
at 37.degree. C. for 14 days caused no loss of antigen binding
ability (FIGS. 47a and 47b), as determined by the VEGF binding
ELISA
Potency in VEGFR2 Receptor Binding Assay & HUVEC Cell
Assay:
[0574] The receptor binding assay described above was used to
assess the potency of the DOM15-26-593 dAb-Fc fusion (FIG. 48). It
was found that the DOM15-26-593 dAb has enhanced potency in this
assay, which establishes the ability of the dAb to block the
binding of VEGF to VEGFR2 in vitro. The potency of the DMS1529 was
also demonstrated in a HUVEC (Human Umbilical Vein Endothelial
Cell) assay, where the ability of VEGF antagonists to block the
VEGF stimulated proliferation of HUVE cells is measured. Briefly,
approximately 4e3 HUVE cells are dispensed into the wells of a
96-well plate to which is added a mixture of VEGF and a dilution
series of the test substance, such that the final concentration of
VEGF is 5 ng/ml, or as otherwise determined by a dose-response
titration. The cells are incubated at 37.degree. C. for a further 4
days, at which point the cell number is determined by the use of a
cell quantification reagent such as "CellTiter". This allows the
colorimetric determination of cell proliferation in comparison with
standards over the 4 days of the experiment. Cell numbers are
determined at the end of a fixed incubation period with a
pre-determined amount of VEGF and a varying amount of test article.
The more potent the antagonist, the lower the cell proliferation
observed (FIG. 49).
Sequence CWU 1
1
2381109DNAArtificial SequencepDOM13 1taatgttatt taaatcatta
tcaaaattag caaccgcagc agcatttttt gcaggcgtgg 60caacagcgtc gacacactgc
aggaggcggc cgcagaaact gttgaacgt 1092119PRTHomo sapiens 2Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30Thr
Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser His Ile Pro Pro Val Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1153108PRTHomo sapiens 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Tyr Leu Asn 20 25 30Leu Asp Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Asn Phe Gly Ser Glu Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Pro Ser Phe Tyr Phe Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 1054116PRTHomo sapiens 4Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Ala Tyr 20 25
30Pro Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Glu Ile Ser Pro Ser Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Arg Lys Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser 1155108PRTHomo
sapiens 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile
Gly Pro Glu 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Ile Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Met Phe Gln Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Arg Arg 100 1056358DNAHomo sapiens 6gaggtgcagc
tgttggagtc tgggggaggc atggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 3587358DNAHomo sapiens
7gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cgcctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg agccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 3588358DNAHomo sapiens
8gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt caactttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 3589358DNAHomo sapiens
9gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgacg
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35810358DNAHomo sapiens
10gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctat 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acaacctgcg cgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35811358DNAHomo sapiens
11gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggcc
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ctggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagag cacgctatat 240ctgcaaatga acggcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35812358DNAHomo sapiens
12gaggtgcagc tgttggagtc tgggggaggc ttggtaaagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120cctgggaagg gtctagagtg ggtctcacat attcccccgg ctggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35813358DNAHomo sapiens
13gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggcc
120ccagggaagg gtctagagtg ggtctcacat attcccccgg acggtcaaga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35814358DNAHomo sapiens
14gaggtgcagc tgtgggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggattga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35815358DNAHomo sapiens
15gaggtgcagc tgtcggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccag atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aatagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagcg 35816358DNAHomo sapiens
16gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc aggtcaccgt ctcgagcg 35817358DNAHomo sapiens
17gaggtgcggc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtacag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagagccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcagatga acagcctgcg tgccgaggac
acagcggtgt attactgtgc gctgcttcct 300aagagagggc cttggtttga
ctactggggt cagggaaccc aggtcaccgt ctcgagcg 35818358DNAHomo sapiens
18gaggtgcggc tgttggagtc tgggggaggc ttggtacagc ctgaggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atagccagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgg gctgcttcct 300aagagggggc cttggtttga
ctacaggggt cagggaaccc tggtcaccgt ctcgagcg 35819358DNAHomo sapiens
19gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt caccattgcg catgaaacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctaccggggt cagggaaccc tggtcaccgt ctcgagcg 35820358DNAHomo sapiens
20gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcctccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gcggcttcct 300aagagggggc cttggtttga
ctactggggt cagggaacct tggtcaccgt ctcgagcg 35821357DNAHomo sapiens
21gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggca
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat atcactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35722357DNAHomo sapiens
22gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccgggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35723357DNAHomo sapiens
23gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag ccaccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaggg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35724357DNAHomo sapiens
24gaggtgcagc tgttggagtc tgggggaggc ctggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaggg gtctagagtg ggtctcacat attccctcgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggttcga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35725357DNAHomo sapiens
25gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg atggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagaaggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35726357DNAHomo sapiens
26gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg cgtgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35727357DNAHomo sapiens
27gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg caagagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaagac
acagcggtat attactgtgc gcggcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35728357DNAHomo sapiens
28gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cttccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ttggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gctgcttcct 300aagaaggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35729357DNAHomo sapiens
29gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttgcg catgagacga tggtgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcacat attcccccgg ctggtcagga
tcccttctac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctatat 240ctgcaaatga acagcctgcg tgccgaggac
acagcggtat attactgtgc gcggcttcct 300aagagggggc cttggtttga
ctactggggt cagggaaccc tggtcaccgt ctcgagc 35730325DNAHomo sapiens
30gacatcctga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaggccc ctaagctcct gatcaatctt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatgtcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acggg
32531325DNAHomo sapiens 31gacatcctga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggctccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagcggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatttcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccag 300gggaccaagg tggaaatcaa acggg 32532325DNAHomo sapiens
32gacatccaga cgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac cagaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatct 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acggg
32533325DNAHomo sapiens 33gacatccagg tgacccagtc tccatccacc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaacttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatttcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccga 300gggaccaagg tggaaatcaa acggg 32534325DNAHomo sapiens
34gacatccaga tgacccagtc tccatccttc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac cagaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata cgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatatcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gtttggccaa 300gggaccaagg tggaaatcaa agggg
32535325DNAHomo sapiens 35gacatccaga tgacccagtc tccatcctcc
ctgtcggcat ctgaaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
cagaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagaa ttcactctca ccatcagcag tctgcaacct
240gaagacttcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acggg 32536325DNAHomo sapiens
36gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60gtcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcagcct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acggg
32537325DNAHomo sapiens 37gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctggatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcgg tctgcaacct
240gaagatttcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccga 300gggaccaggg tggaaatcaa acggg
32538325DNAHomo sapiens 38gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcagtttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatttcg ctacgtacta ctgtcaaccg tctttttact tcccttacac
gttcggccaa 300gggaccaagg tggaaatcaa acggg 32539325DNAHomo sapiens
39gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaaattt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcaa tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa atggg
32540325DNAHomo sapiens 40gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcatttat ggttccgagt tgcaaagtgg tgtcccacca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattccg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acggg 32541325DNAHomo sapiens
41gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatatac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccacca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acagg
32542325DNAHomo sapiens 42gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcgctctca ccatcagcag tctgcaacct
240gaagattccg ctacgtacta ctgtcaaccg tctttttact tcccatatac
gttcggccaa 300gggaccaagg tggaaatcaa acagg 32543325DNAHomo sapiens
43gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcact
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatgtcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acggg
32544325DNAHomo sapiens 44gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagacttcg ctacgtacta ctgtcaaccg tctttttact tcccatatac
gtttggccaa 300gggaccaagg tggaaatcaa acagg 32545325DNAHomo sapiens
45gacatccaaa tggcccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttgg actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcagtttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattccg ctacgtacta ctgtcagccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acagg
32546325DNAHomo sapiens 46gacatccaga tgacccagtc accatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggacatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatttcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acggg 32547324DNAHomo sapiens
47gacatccaga tgacccagtc tccatcctcc ctgtctgcgt ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcacactca
ccatcagcag tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa actg
32448324DNAHomo sapiens 48gacatccaga taacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatttcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa accg 32449324DNAHomo sapiens
49gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact acccttatac gttcggccaa 300gggaccaagg tggaaatcaa acag
32450324DNAHomo sapiens 50gacatccaga tgacacagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atctcttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcagtttt ggttccgagt tgcaaagtgg tgttccttca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcagcct
240gaagattccg ctacgtacta ctgtcaaccg tctttttact acccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 32451324DNAHomo sapiens
51gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaatttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact acccttatac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32452324DNAHomo sapiens 52gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg agtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattccg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa tcgg 32453324DNAHomo sapiens
53gacatccaga tgacccagtc tccatcctcc ctgtctgcat atgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcagtttt ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatttcg gtacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acgg
32454324DNAHomo sapiens 54gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgtc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatcaatttt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatttcg gtacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 32455324DNAHomo sapiens
55gacatccaga tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga ccttgtcacc
60atcacttgcc gggcaagtca ggatatttac ctgaatttag actggtatca gcagaaacca
120gggaaagccc ctaagctcct gatcaattta ggttccgagt tgcaaagtgg
tgtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagatttcg ctacgtacta ctgtcaaccg
tctttttact tcccttatac gttcggccaa 300gggaccaagg tggaaatcaa acgt
32456324DNAHomo sapiens 56gacatcctga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggatatttac
ctgaatttag actggtatca gcagaaacca 120gggaaggccc ctaagctcct
gatcaatctt ggttccgagt tgcaaagtgg tgtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagatgtcg ctacgtacta ctgtcaaccg tctttttact tcccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 32457348DNAHomo sapiens
57gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34858348DNAHomo sapiens 58gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtccagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgtgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34859348DNAHomo sapiens 59gaggtgcagc
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtccagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cgcgctgtat 240ctgcaaatga acagcctgcg tgcagaggac
accgcggtat attactgtgc gaaagatcct 300cggaagtttg actactgggg
tcagggagcc ctggtcaccg tctcgagc 34860348DNAHomo sapiens 60gaggtgcagc
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg gatctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc aaaagatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34861348DNAHomo sapiens 61gaggtgcagc
tgctggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttcata
tacatactat 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagtttg aatactgggg
tcagggaacc ctggtcaccg tctcgagc 34862348DNAHomo sapiens 62gaggtgcagc
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcggactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaatgatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34863348DNAHomo sapiens 63gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacatttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acggcctgcg tgccgaggac
accgcggtat attactgtgc gaacgatcct 300cggaagattg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34864348DNAHomo sapiens 64gaggtgcagc
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcatactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgca tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagttag actactgggg
tcagggaacc ctggtcaccg tctcgagc 34865348DNAHomo sapiens 65gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc caggggggtc cctgcgtctc 60tcctgtgctg
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaaga gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcacaatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagattg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34866348DNAHomo sapiens 66gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagagtccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtac gaaagatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34867348DNAHomo sapiens 67gaggtgcagt
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatt tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34868348DNAHomo sapiens 68gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcaaactccg tgaagggtcg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagattg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34869348DNAHomo sapiens 69gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagtctg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34870348DNAHomo sapiens 70gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atctcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagattg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34871348DNAHomo sapiens 71gaggtgcatc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcaaactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagattg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34872348DNAHomo sapiens 72gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagttag actactgggg
tcagggaacc ctggtcaccg tctcgagc 34873348DNAHomo sapiens 73gaggtgcagc
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtac gaaagatcct 300cggaagtttg actactgggg
tcagggaacc ctggtcaccg tctcgagc 34874348DNAHomo sapiens 74gaggtgcagc
tgttggtgtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt cacctttaag gcttatccga tgatgtgggt ccgccaggct
120ccagggaagg gtctagagtg ggtttcagag atttcgcctt cgggttctta
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaagatcct 300cggaagttag actactgggg
tcagggaacc ctggtcaccg tctcgagc 34875324DNAHomo sapiens 75gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tatatgtttc agcctaggac gttcggccaa 300gggaccaagg tggaaatcag acgg
32476324DNAHomo sapiens 76gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttaa gttggtacca gcagaaacca 120gggaaagccc ctaagcgcct
gatctatcat tcgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tatatgtttg agcctaggac
gttcggccaa 300gggaccaagg tggaaatcag acag 32477324DNAHomo sapiens
77gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgtc gggcaagtca gtggattggt ccggagttaa gatggtacca gaagaaacca
120gggaaagccc ctaagctcct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tatatgtttc agcctaggac
gttcggccaa 300gggaccaagg tggaaatcag acgg 32478324DNAHomo sapiens
78gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120gggaaagccc ctaagcacct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtagatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tatatgtttc agcctaagac gttcggccaa 300gggaccaagg tggaaatcag atgg
32479324DNAHomo sapiens 79gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
cctgagttaa gatggtacca gaagaaacca 120gggaaagccc ctaagctcct
gatctatcat acgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactttca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tatatgtttc agcctaggac
gttcggccca 300gggaccaagg tggaaattag acgg 32480324DNAHomo sapiens
80gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcat acgtccattt tgcgaagtgg
ggtcccatct 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tatatgtttc agcctaggac gttcggccaa 300gggaccaagg tggaaatcag atgg
32481324DNAHomo sapiens 81gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttaa gttggtacca gcagaaacca 120gggaaagccc ctaagcgcct
gatctatcat acgtccattt tacagagtgg ggtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gcagattttg caacgtacta ctgtcaacag tatatgtttc agcctaggac
gttcggccaa 300gggaccaagg tggaaatcag acag 32482324DNAHomo sapiens
82gacatccaga tgacccagtc cccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tatatgtttt ggcctaggac gttcggccaa 300gggaccaagg tggaaatcag acaa
32483324DNAHomo sapiens 83gacatccaga tgacccagtc tccatcctcc
ctgtccgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttaa gttggtacca gcagaaacca 120gggaaagctc ctaagctcct
gatctatcat acgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaactt
240gaagattttg ctacgtacta ctgtcaacag tatatgtttc tgcctaggac
gttcggccaa 300gggaccaagg tggaaatcag aggg 32484324DNAHomo sapiens
84gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120gggaaagccc ctaagttcct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggata tgggacagat ttcactctca
ccatcaacag tctgcaacct 240ggagattttg ctacgtacta ctgtcaacag
tatatgtttc agcctaggac gttcggccaa 300gggaccaagg tggaaatcag acgg
32485324DNAHomo sapiens 85gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttaa gttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcat acgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tatatgtatc agcctaggac
gttcggccaa 300gggaccaagg tggaaatcag acag 32486324DNAHomo sapiens
86gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gatggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tatatgtttc agcctatgac gttcggccaa 300gggaccaagg tggaaatcag aggg
32487324DNAHomo sapiens 87gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttaa gatggtacca gaagaaacca 120gggaaagccc ctaagctcct
gatctatcat acgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaagct
240gaagattctg ctacgtacta ctgtcaacag tatatgtttc agcctaggac
gttcggccaa 300gggaccaagg tggaaaccag acgg 32488324DNAHomo sapiens
88gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgaaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120cggaaagccc ctaagctcct gatctatcat acgtccattt tgcaaagtgg
ggtcccatca 180cgtttcactg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
tatatgtttc agcctatgac gttcggccaa 300gggaccaagg tggaaatcag acgg
32489324DNAHomo sapiens 89gacatcctga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttac gttggtacca gcataaacca 120gggaaagccc ctaagctcct
gatctatcat acgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggata tgggacagat ttcactctct ccatcagcag tctgcaacct
240gaagatttcg ctacgtacta ctgtcaacag tatatgtttc agcctaggac
gttcggccaa 300gggaccaagg tggaaatcag atgg 32490324DNAHomo sapiens
90gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggt ccggagttaa gttggtacca gcagaaacca
120gggaaagccc caaagctcct gatctatcat acgtccattt tgcaaggtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaggattttg ctacgtacta ctgtcaacag
tatatgtttt ggcctaggac gttcggccaa 300gggaccaagg tggaaatcag acag
32491300DNAHomo sapiens 91gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggt
ccggagttac gttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcat acgtccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag tatatgtttc agcctatgac
gttcggccaa 3009222PRTArtificial SequenceGas leader sequence 92Met
Leu Phe Lys Ser Leu Ser Lys Leu Ala Thr Ala Ala Ala Phe Phe1 5 10
15Ala Gly Val Ala Thr Ala 20938PRTArtificial SequenceGene III
Sequence 93Ala Ala Ala Glu Thr Val Glu Ser1 594108PRTHomo sapiens
94Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Asn Ser Phe Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Thr Val Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Gln 100 10595108PRTHomo sapiens 95Asp Ile Gln Met Thr Gln
Ser Pro Pro Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Asn
Ser Pro Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Ile Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10596108PRTHomo sapiens 96Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Arg Pro Ile Gly Thr Thr 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Trp Phe Gly Ser Arg Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10597108PRTHomo sapiens
97Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Tyr Ile Gly Ser
Gln 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Ala Trp Ala Ser Val Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Arg Gln
Gly Ala Ala Ser Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10598108PRTHomo sapiens 98Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Phe Ile Tyr Arg Tyr 20 25 30Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala
Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Ala His Leu Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10599108PRTHomo sapiens 99Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Trp Ile Gly Ser Gln 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Met Trp Arg Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Ala Gln Gly Ala Ala Leu Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105100108PRTHomo sapiens
100Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Lys Ile Ala Thr
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Ser Ser Ser Leu Gln Ser Ala Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Ala Val Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105101108PRTHomo sapiens 101Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Trp Ile Asp Thr Gly 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asn Val
Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Gly Ser Pro Thr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105102108PRTHomo sapiens 102Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Glu Ile Tyr Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala Ser His Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Val Ile Gly Asp Pro Val 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105103108PRTHomo sapiens
103Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Thr Leu
Leu Ile 35 40 45Tyr Arg Leu Ser Val Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Asn Val Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105104108PRTHomo sapiens 104Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Asn
Ser Gln Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Phe Ala Val Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105105108PRTHomo sapiens 105Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Asn Ser Pro Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Thr Tyr Arg Val Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105106108PRTHomo sapiens
106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Gly Leu
Trp 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Ser Ser Leu Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Lys Tyr Asn Leu Pro Tyr 85 90 95Thr Ser Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105107108PRTHomo sapiens 107Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Phe Arg His 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Ala Leu Tyr Pro Lys
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105108108PRTHomo sapiens 108Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ile Lys His 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Arg Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly Ala Arg Trp Pro Gln 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105109108PRTHomo sapiens
109Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Tyr
His 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Arg Lys Val Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105110108PRTHomo sapiens 110Asp Ile Gln Thr Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Tyr Ile Gly Arg Tyr 20 25 30Leu Arg Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ser
Ser Val Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Arg Met Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys Arg 100
105111108PRTHomo sapiens 111Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln His Ile His Arg Glu 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ala Ser Arg Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Lys Tyr Leu Pro Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105112108PRTHomo sapiens
112Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile His Arg
Glu 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gln Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Arg Tyr Arg Val Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105113108PRTHomo sapiens 113Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Arg 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Ala Ala Pro Arg Leu Leu Ile 35 40 45Tyr Arg Thr
Ser Trp Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Ser Gln Trp Pro His
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105114108PRTHomo sapiens 114Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Lys Ile Tyr Lys Asn 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ser Ser Ile Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Arg Tyr Leu Ser Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105115108PRTHomo sapiens
115Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Lys Ile Tyr Asn
Asn 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asn Thr Ser Ile Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Arg Trp Arg Ala Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105116108PRTHomo sapiens 116Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Trp Ile Tyr Lys Ser 20 25 30Leu Gly Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gln Ser
Ser Leu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Gln Met Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105117108PRTHomo sapiens 117Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Trp Ile Tyr Arg His 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Arg Leu Gln
Ser Gly Val Pro Thr Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Thr His Asn Pro Pro Lys 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105118108PRTHomo sapiens
118Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile Gly Arg
Tyr 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Ser Ser Val Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Arg Tyr Met Gln Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105119108PRTHomo sapiens 119Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Trp Ile Gly Arg Tyr 20 25 30Leu Arg Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Gly
Ser Gln Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Leu Gln Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105120108PRTHomo sapiens 120Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Tyr Ile Gly Arg Tyr 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ser Ser Val Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Ser Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105121108PRTHomo sapiens
121Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile Ser Arg
Gln 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Val Leu Gln Ser Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Arg Tyr Ile Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Val Lys Arg 100 105122108PRTHomo sapiens 122Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Trp Ile His Arg Gln 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ala
Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Phe Ser Lys Pro Ser
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105123118PRTHomo sapiens 123Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Tyr Asp Tyr 20 25 30Asn Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile Thr His Thr Gly
Gly Val Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gln
Asn Pro Ser Tyr Gln Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 115124120PRTHomo sapiens 124Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Leu Tyr 20 25 30Asp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser
Ile Val Asn Ser Gly Val Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Leu Asn Gln Ser Tyr His Trp Asp Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120125123PRTHomo
sapiens 125Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Lys Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ser Ile Asp Phe Met Gly Pro His Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Arg Thr Ser Met
Leu Pro Met Lys Gly Lys Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120126118PRTHomo sapiens 126Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Arg Tyr 20 25 30Ser
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Thr Ile Leu Pro Gly Gly Asp Val Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Gln Thr Pro Asp Tyr Met Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115127117PRTHomo
sapiens 127Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Trp Lys Tyr 20 25 30Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Thr Ile Leu Gly Glu Gly Asn Asn Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Thr Met Asp Tyr Lys
Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115128118PRTHomo sapiens 128Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser
Gly Phe Thr Phe Asp Glu Tyr 20 25 30Asn Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile Leu Pro His Gly
Asp Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gln
Asp Pro Leu Tyr Arg Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 115129118PRTHomo sapiens 129Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Arg Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr
Ile Ile Ser Asn Gly Lys Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys
85 90 95Ala Lys Gln Asp Trp Met Tyr Met Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115130118PRTHomo sapiens
130Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr
Tyr 20 25 30Thr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Thr Ser Ser Gly Ser Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Asn Ser Leu Tyr Lys Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115131119PRTHomo sapiensVARIANT119Xaa = Any Amino Acid 131Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Pro Thr 20 25
30Asn Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Thr Gly Thr Gly Ala Ala Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Gln Asn Ser Arg Tyr Arg Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Xaa
115132117PRTHomo sapiensVARIANT117Xaa = Any Amino Acid 132Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Gly Gly Lys Asp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Xaa 115133123PRTHomo
sapiensVARIANT123Xaa = Any Amino Acid 133Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25 30Thr Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile
Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ser Asp Val Leu Lys Thr Gly Leu Asp Gly Phe Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Xaa 115
120134121PRTHomo sapiensVARIANT121Xaa = Any Amino Acid 134Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Met Ala Tyr 20 25
30Gln Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile His Gln Thr Gly Phe Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Val Arg Ser Met Arg Pro Tyr Lys Phe
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Xaa
115 120135118PRTHomo sapiensVARIANT118Xaa = Any Amino Acid 135Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr
20 25 30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Asn Leu Glu Pro Phe Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Xaa
115136119PRTHomo sapiensVARIANT119Xaa = Any Amino Acid 136Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Lys Thr Gly Pro Ser Ser Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Xaa
115137118PRTHomo sapiensVARIANT118Xaa = Any Amino Acid 137Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Lys Leu Ser Asn Gly Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Xaa
115138119PRTHomo sapiensVARIANT119Xaa = Any Amino Acid 138Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Val Val Lys Asp Asn Thr Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Xaa
115139119PRTHomo sapiensVARIANT119Xaa = Any Amino Acid 139Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Asn Thr Gly Gly Lys Gln Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Xaa
115140121PRTHomo sapiensVARIANT121Xaa = Any Amino Acid 140Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Pro Tyr 20 25
30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Arg Thr Glu Asn Arg Gly Val Ser Phe
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Xaa
115 120141121PRTHomo sapiensVARIANT121Xaa = Any Amino Acid 141Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Asp Tyr
20 25 30Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Met Ile Ser Ser Ser Gly Leu Trp Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Phe Arg Leu Phe Pro Arg Thr
Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Xaa 115 120142108PRTHomo sapiens 142Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Pro
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Val Pro Pro 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105143108PRTHomo
sapiens 143Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser Pro Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Thr Tyr Arg Ile Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105144108PRTHomo sapiens 144Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Phe Lys Ser 20 25 30Leu Lys
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Asn Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Tyr Trp Pro
Val 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105145107PRTHomo sapiens 145Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Tyr Tyr His 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Val Arg Lys Val Pro Arg Thr 85 90 95Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105146108PRTHomo sapiens 146Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Arg Tyr
20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Gln Ala Ser Val Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
Leu Tyr Pro Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105147108PRTHomo sapiens 147Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Tyr Asn Trp 20 25 30Leu Lys Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Val Val Ile Pro Arg 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105148108PRTHomo sapiens 148Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Leu Trp His 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr His Ala Ser Leu Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Ala Val Tyr Pro Lys 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105149108PRTHomo sapiens
149Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Phe Arg
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr His Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Arg Leu Leu Tyr Pro Lys 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105150108PRTHomo sapiens 150Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Phe Tyr His 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Pro Ala
Ser Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ala Arg Trp Pro Arg 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105151108PRTHomo sapiens 151Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ile Trp His 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser Arg Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Val Ala Arg Val Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105152108PRTHomo sapiens
152Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Arg
Tyr 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Val Gly Tyr Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105153108PRTHomo sapiens 153Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Leu Lys Tyr 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala
Ser His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Thr Tyr Tyr Pro Ile
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105154108PRTHomo sapiens 154Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Leu Arg Tyr 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Trp Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Val Leu Tyr Tyr Pro Gln 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105155108PRTHomo sapiens
155Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Leu Arg
Ser 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Val Tyr Trp Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105156108PRTHomo sapiens 156Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Phe Arg His 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Ala Leu Tyr Pro Lys
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105157108PRTHomo sapiens 157Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Arg Lys Tyr 20 25 30Leu Arg Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Thr Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Asn Leu Phe Trp Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105158108PRTHomo sapiens
158Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Arg
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Met Leu Phe Tyr Pro Lys 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105159108PRTHomo sapiens 159Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ile Lys His 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala
Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Arg Trp Pro Gln
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105160108PRTHomo sapiens 160Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Tyr Tyr His 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Val Arg Lys Val Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105161108PRTHomo sapiens
161Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Lys
His 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asn Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Gly Arg Tyr Pro Lys 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105162108PRTHomo sapiens 162Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Phe Lys Ser 20 25 30Leu Lys Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala
Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Tyr Trp Pro Val
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105163116PRTHomo sapiens 163Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Trp Val Tyr 20 25 30Gln Met Asp Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Ala Phe Gly
Ala Lys Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Leu
Ser Gly Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 115164120PRTHomo sapiens 164Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Trp Ser Tyr 20 25 30Gln Met Thr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser
Ser Phe Gly Ser Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Lys Gly Arg Asp His Asn Tyr Ser Leu Phe Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120165324DNAHomo
sapiens 165gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcgg aattcctttt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaaccc 240gaagattttg ctacgtacta
ctgtcaacag acgtatactg ttcctcctac gtttggccaa 300gggaccaagg
tggaaatcaa acag 324166324DNAHomo sapiens 166gacatccaga tgacccagtc
tccaccctcc ctgtccgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatcgg aattcccctt tgcaaagtgg ggtcccatca
180cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag acttattcga
ttcctcctac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324167324DNAHomo sapiens 167gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtcg tccgattggg
acgacgttaa gttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctggttt ggttcccggt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtgcgcag gctgggacgc atcctacgac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324168324DNAHomo sapiens
168gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtaa gtatattggt tcgcagttaa attggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcgcttgg gcgtccgttt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcgtcag ggtgctgcgt cgcctcggac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324169324DNAHomo sapiens 169gacattcaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtttatttat cggtatttat cgtggtatca gcagaaacca 120gggaaagtcc
ctaagctcct gatctataat gcgtcctatt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag catgctcatt
tgcctcgtac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324170324DNAHomo sapiens 170gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg
tctcagttat cttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatcatgtgg cgttcctcgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtgctcag ggtgcggcgt tgcctaggac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324171324DNAHomo sapiens
171gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gaagattgct acttatttaa attggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagg tcttcctctt
tgcaaagcgc ggtcccatca 180cgtttcagtg gcagtggatc tgggacagtt
ttcacactca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag acgtatgctg ttcctcctac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324172324DNAHomo sapiens 172gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtggattgat actgggttag cgtggtacca gcagaaacca 120gggaaagccc
ctaggctcct gatctataat gtgtccaggt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag tattggggta
gtcctacgac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324173324DNAHomo sapiens 173gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca ggagatttat
tcgtggttag cgtggtacca gcagagacca 120gggaaagccc ctaagctcct
gatctataat gcttcccatt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gtgattggtg atcctgttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324174324DNAHomo sapiens
174gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtacca
gcagaaacca 120gggaaagccc ctacgctcct gatctatcgg ttgtccgtgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag acttataatg ttcctcctac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324175324DNAHomo sapiens 175gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcattagc agctatctaa attggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatagg aattcccagt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag acttttgcgg
ttcctcctac gttcggccaa 300gggaccaagg tggagatcaa acgg
324176324DNAHomo sapiens 176gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattagc
agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcgg aattcccctt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag acgtataggg tgcctcctac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324177324DNAHomo sapiens
177gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gcatattggg ttgtggttac attggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagg tcttccttgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag aagtataatt tgccttatac gtccggccaa 300gggaccaagg
tggaaatcaa acgg 324178324DNAHomo sapiens 178gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcattttt cggcatttaa agtggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgcg gcatcccgtt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag gttgcgctgt
atcctaagac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324179324DNAHomo sapiens 179gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattatt
aagcatttaa agtggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatggt gcatcccggt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag ggggctcggt ggcctcagac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324180324DNAHomo sapiens
180gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gagcatttat tatcatttaa agtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctataag gcatccacgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag gttcggaagg tgcctcggac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324181324DNAHomo sapiens 181gacatccaga cgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtatattggt aggtatttac gttggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgat tcttccgtgt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag cgttatcgta
tgccttatac gttcggccaa 300gggaccaggg tagaaatcaa acgg
324182324DNAHomo sapiens 182gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gcatattcat
agggagttaa ggtggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatcag gcgtcccgtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag aagtatctgc ctccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324183324DNAHomo sapiens
183gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gcatattcat agggagttaa ggtggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatcag gcgtcccgtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag cgttataggg tgccttatac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324184324DNAHomo sapiens 184gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagtattggg cggaggttaa agtggtacca gcagaaacca 120ggggcagccc
ctaggctcct gatctatcgt acgtcctggt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag acgtcgcagt
ggcctcatac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324185324DNAHomo sapiens 185gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gaagatttat
aagaatttac gttggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctataat tcttccattt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag aggtatctgt cgccttatac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324186324DNAHomo sapiens
186gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gaagatttat aataatttaa ggtggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatctataat acttccattt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag cgatggcgtg cgccttatac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324187324DNAHomo sapiens 187gacattcaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtggatttat aagtcgttag ggtggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatcag tcttctttgt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag tatcatcaga
tgcctcggac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324188324DNAHomo sapiens 188gacatccaga tgacccagtc tccatcctcc
ctatctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggatttat
aggcatttaa ggtggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgat gcgtccaggt tgcaaagtgg ggtcccaaca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag actcataatc ctcctaagac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 324189324DNAHomo sapiens
189gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtatattggt aggtatttac gttggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgat tcttccgtgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag aggtatatgc agccttttac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324190324DNAHomo sapiens 190gacatccaga tgacccagtc
tccatcctcc ctgtccgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtggattggt cggtatttac ggtggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctataat gggtcccagt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag cggtatcttc
agccttatac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324191324DNAHomo sapiens 191gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtatattggt
aggtatttac gttggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgat tcttccgtgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag cgttattctt cgccttatac
gttcggccaa 300gggaccaagg tggaaatcaa gcgg 324192324DNAHomo sapiens
192gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtatatttcg cgtcagttaa ggtggtacca
gcagaaacca 120gggaaagccc ctaggctcct gatctatggg gcgtccgttt
tgcaaagcgg gatcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtcaacag aggtatatta ctccttatac gttcggccaa 300gggaccaagg
tggaagtcaa acgg 324193324DNAHomo sapiens 193gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gtggattcat aggcagttaa agtggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctattat gcttccattt tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag acgttttcta
agccttctac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324194354DNAHomo sapiens 194gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt caccttttat
gattataata tgtcttgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcaact attacgcata cgggtggggt tacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggat accgcggtat attactgtgc
gaaacagaat 300ccttcttatc agtttgacta ctggggtcag ggaaccctgg
tcaccgtctc gagc 354195360DNAHomo sapiens 195gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt
cacctttgat ctttatgata tgtcgtgggt ccgccaggct 120ccagggaagg
gtctagagtg ggtctcatcg attgttaatt cgggtgttag gacatactac
180gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac accgcggtat
attactgtgc gaaacttaat 300cagagttatc attgggattt tgactactgg
ggtcagggaa ccctggtcac cgtctcgagc 360196369DNAHomo sapiens
196gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttcg aagtattgga tgtcgtgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcatct attgatttta
tgggtccgca tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggat accgcggtat attactgtgc gaaagggagg 300acgtcgatgt
tgccgatgaa ggggaagttt gactactggg gtcagggaac cctggtcacc 360gtctcgagc
369197354DNAHomo sapiens 197gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttcat
cgttattcga tgtcttgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcaacg attttgcctg gtggtgatgt tacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggat accgcggtat attactgtgc
gaaacagacg 300cctgattata tgtttgacta ctggggtcag ggaaccctgg
tcaccgtctc gagc 354198351DNAHomo sapiens 198gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt
caccttttgg aagtataata tggcgtgggt ccgccaggct 120ccagggaagg
gtctagagtg ggtctcaact attcttggtg agggtaataa tacatactac
180gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggat accgcggtat
attactgtgc gaaaacgatg 300gattataagt ttgactactg gggtcaggga
accctggtca ccgtctcgag c 351199354DNAHomo sapiens 199gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtacag
cctccggatt cacctttgat gagtataata tgtcttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcaacg attctgccgc atggtgatcg
gacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggat
accgcggtat attactgtgc gaaacaggat 300cctttgtata ggtttgacta
ctggggtcag ggaaccctgg tcaccgtctc gagc 354200354DNAHomo sapiens
200gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttcg gattatcgga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg attatttcga
atggtaagtt tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaacaggat 300tggatgtata
tgtttgacta ctggggtcag ggaaccctgg tcaccgtctc gagc 354201354DNAHomo
sapiens 201gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttcgg acgtatacta tggcttgggt
ccgccaggcc 120ccagggaagg gtctagagtg ggtctcatcg attactagta
gtggttcttc tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggat accgcggtat attactgtgc gaaagtgaat 300tctttgtata
agtttgacta ctggggtcag ggaaccctgg tcaccgtctc gagc 354202355DNAHomo
sapiens 202gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttcgg ccgactaata tgtcgtgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaact attactggta
ctggtgctgc gacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaacagaat 300tctcgttata
ggtttgacta ctggggtcag ggaaccctgg tcaccgtctc gagcg 355203349DNAHomo
sapiens 203gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaagggggg 300aaggattttg
actactgggg tcagggaacc ctggtcaccg tctcgagcg 349204367DNAHomo sapiens
204gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaagtgat 300gttcttaaga
cgggtctgga tggttttgac tactggggtc agggaaccct ggtcaccgtc 360tcgagcg
367205361DNAHomo sapiens 205gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttatg
gcgtatcaga tggcttgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcaact attcatcaga cgggtttttc tacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggat accgcggtat attactgtgc
gaaagtgcgt 300tctatgcgtc cttataagtt tgactactgg ggtcagggaa
ccctggtcac cgtctcgagc 360g 361206352DNAHomo sapiens 206gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag
cctccggatt caccttttgg ccgtatacga tgagttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt ttggttcgac
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaaggtaat 300cttgagccgt ttgactactg
gggtcaggga accctggtca ccgtctcgag cg 352207355DNAHomo sapiens
207gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaaagacg 300ggtccgtcgt
cgtttgacta ctggggtcag ggaaccctgg tcaccgtctc gagcg 355208352DNAHomo
sapiens 208gaggtgcagc tgttggagtc tgggggaggt ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggat accgcggtat attactgtgc gaaaaagctt 300agtaatggtt
ttgactactg gggtcaggga accctggtca ccgtctcgag cg 352209355DNAHomo
sapiens 209gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaagtggtt 300aaggataata
cgtttgacta ctggggtcag ggaaccctgg tcaccgtctc gagcg 355210355DNAHomo
sapiens 210gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatt
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaaatact 300gggggtaagc
agtttgacta ctggggtcag ggaaccctgg tcaccgtctc gagcg 355211361DNAHomo
sapiens 211gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt caccttttgg ccgtatacga tgagttgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcaacg atttcgccgt
ttggttcgac tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaaggact 300gagaataggg
gggtttcttt tgactactgg ggtcagggaa ccctggtcac cgtctcgagc 360g
361212361DNAHomo sapiens 212gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttaag
gattatgata tgacttgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcaatg atttcttcgt cgggtctttg gacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaaggtttt 300aggctgtttc ctcggacttt tgactactgg ggtcagggaa
ccctggtcac cgtctcgagc 360g 361213343PRTHomo sapiens 213Glu Val Gln
Leu Leu Val Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Ala Tyr 20 25 30Pro
Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Glu Ile Ser Pro Ser Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ile Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Arg Lys Leu Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr His Thr Cys
Pro Pro Cys Pro Ala Pro 115 120 125Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys 130 135 140Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val145 150 155 160Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 165 170 175Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 180 185
190Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
195 200 205Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu 210 215 220Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg225 230 235 240Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys 245 250 255Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 260 265
270Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
275 280 285Thr Ile Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 290 295 300Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser305 310 315 320Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 325 330 335Leu Ser Leu Ser Pro Gly Lys
340214225PRTHomo sapiens 214Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro1 5 10 15Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 20 25 30Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 35 40 45Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 50 55 60Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val65 70 75 80Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 85 90 95Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 100 105 110Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120
125Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
130 135 140Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu145 150 155 160Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 165 170 175Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 180 185 190Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 195 200 205Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215
220Lys2252151029DNAHomo sapiens 215gaggtgcagc tgttggtgtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttaag
gcttatccga tgatgtgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtttcagag atttcgcctt cgggttctta tacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaagatcct 300cggaagttag actactgggg tcagggaacc ctggtcaccg
tctcgagcgc tagcacccac 360acctgccccc cctgccctgc ccccgagctg
ctgggcggac ctagcgtgtt cctgttcccc 420cccaagccta aggacaccct
gatgatcagc aggacccccg aagtgacctg cgtggtggtg 480gatgtgagcc
acgaggaccc tgaagtgaag ttcaactggt acgtggacgg cgtggaagtg
540cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctaccg
cgtggtgtct 600gtgctgaccg tgctgcacca ggattggctg aacggcaagg
agtacaagtg caaagtgagc 660aacaaggccc tgcctgcccc tatcgagaaa
accatcagca aggccaaggg ccagcctaga 720gagccccagg tctacaccct
gcctccctcc agagatgagc tgaccaagaa ccaggtgtcc 780ctgacctgtc
tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac
840ggccagcccg agaacaacta caagaccacc ccccctgtgc tggacagcga
tggcagcttc 900ttcctgtact ccaagctgac cgtggacaag agcagatggc
agcagggcaa cgtgttcagc 960tgcagcgtga tgcacgaggc cctgcacaat
cactacaccc agaagagtct gagcctgtcc 1020cctggcaag 1029216357DNAHomo
sapiens 216gaggttcaat tgttggaatc cggtggtgga ttggttcaac ctggtggttc
tttgagattg 60tcctgtgctg cttccggttt tactttcgct cacgagacta tggtttgggt
tagacaggct 120ccaggtaaag gattggaatg ggtttcccac attccaccag
atggtcaaga tccattctac 180gctgactccg ttaagggaag attcactatc
tccagagaca actccaagaa cactttgtac 240ttgcagatga actccttgag
agctgaggat actgctgttt accactgtgc tttgttgcca 300aagagaggac
cttggtttga ttactgggga cagggaactt tggttactgt ttcttcc
357217119PRTHomo sapiens 217Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ala His Glu 20 25 30Thr Met Val Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser His Ile Pro Pro Asp Gly
Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Leu Leu
Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 115218357DNAArtificial SequenceConsensus
218gaggtncann tgttggantc nggnggnggn ttggtncanc ctggnggntc
nntgngnntn 60tcctgtgcng cntccggntt nacnttngcn cangagacna tggtntgggt
nngncaggcn 120ccaggnaang gnntngantg ggtntcncan attccnccng
atggtcanga tccnttctac 180gcngactccg tnaagggnng nttcacnatc
tccngngaca antccaagaa cacnntntan 240ntgcanatga acnncntgng
ngcngaggan acngcngtnt ancactgtgc nntgntnccn 300aagagnggnc
cttggtttga ntactggggn cagggaacnn tggtnacngt ntcnnnc
357219366DNAHomo sapiens 219gagaaaagag aggttcaatt gcttgaatct
ggaggaggtt tggtccagcc aggagggtcc 60cttcgactaa gttgtgctgc cagtgggttt
acgtttgctc atgaaactat ggtatgggtc 120cgacaggcac ctggtaaagg
tcttgaatgg gtttcacata tccctccaga cggtcaagac 180ccattttacg
ctgattccgt gaaaggcaga tttacaattt cacgagataa ttctaaaaac
240accttgtact tacaaatgaa ctcattgaga gctgaggaca ctgcagttta
tcactgcgct 300ttactaccaa aacgtggacc ttggtttgat tattggggcc
aaggtacgtt agtgactgtt 360agttct 366220122PRTHomo sapiens 220Glu Lys
Arg Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln1 5 10 15Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25
30Ala His Glu Thr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45Glu Trp Val Ser His Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr
Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn65 70 75 80Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 85 90 95Tyr His Cys Ala Leu Leu Pro Lys Arg Gly Pro
Trp Phe Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120221366DNAPichia pastoris 221gagaaaagag aggttcaatt
gcttgaatct ggaggaggtt tggtccagcc aggagggtcc 60cttcgactaa gttgtgctgc
cagtgggttt acgtttgctc atgaaactat ggtatgggtc 120cgacaggcac
ctggtaaagg tcttgaatgg gtttcacata tccctccaga cggtcaagac
180ccattttacg ctgattccgt gaaaggcaga tttacaattt cacgagataa
ttctaaaaac 240accttgtact tacaaatgaa ctcattgaga gctgaggaca
ctgcagttta tcactgcgct 300ttactaccaa aacgtggacc ttggtttgat
tattggggcc aaggtacgtt agtgactgtt 360agttct 366222358DNAArtificial
SequenceConsensus 222gaggtncann tgntngantc tggnggaggn ttggtncagc
cnggngggtc cctncgnctn 60nnntgtgcng ccnnnggntt nacntttgcn catganacna
tggtntgggt ccgncaggca 120ccnggnaang gtctngantg ggtntcacat
atnccnccng anggtcanga nccnttntac 180gcngantccg tgaanggcng
nttnacnatn tcncgngana attcnaanaa cacnntntan 240ntncaaatga
acnnnntgng ngcngaggac acngcngtnt atcactgngc nntnctnccn
300aanngnggnc cttggtttga ntantggggn canggnacnn tngtnacngt ntngnncn
358223357DNAHomo sapiens 223gaagtgcagc ttcttgaaag tggtggaggg
ctagtgcagc cagggggatc tttaagatta 60tcatgcgctg ccagtggatt tacttttgct
cacgagacga tggtctgggt gagacaagct 120cctggaaaag gtttagagtg
ggtttctcac attccacctg atggtcaaga tcctttctac 180gcagattccg
tcaaaggaag atttactatc tccagagata atagtaaaaa cactttgtac
240ctacagatga actcacttag agccgaagat accgctgtgt accactgcgc
cttgttgcca 300aagagaggtc cttggttcga ttactggggt cagggtactc
tggttacagt ctcatct 357224119PRTHomo sapiens 224Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30Thr Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser His
Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115225357DNAPichia
pastoris 225gaagtgcagc ttcttgaaag tggtggaggg ctagtgcagc cagggggatc
tttaagatta 60tcatgcgctg ccagtggatt tacttttgct cacgagacga tggtctgggt
gagacaagct 120cctggaaaag gtttagagtg ggtttctcac attccacctg
atggtcaaga tcctttctac 180gcagattccg tcaaaggaag atttactatc
tccagagata atagtaaaaa cactttgtac 240ctacagatga actcacttag
agccgaagat accgctgtgt accactgcgc cttgttgcca 300aagagaggtc
cttggttcga ttactggggt cagggtactc tggttacagt ctcatct
357226358DNAArtificial SequenceConsensus 226gangtgcagc tnntngannn
tggnggaggn ntngtncagc cngggggntc nntnngnntn 60tcntgngcng ccnnnggatt
nacntttgcn cangagacga tggtntgggt nngncangcn 120ccnggnaang
gtntagagtg ggtntcncan attccnccng atggtcanga tccnttctac
180gcagantccg tnaanggnng nttnacnatc tccngngana atnnnaanaa
cacnntntan 240ctncanatga acnnnctnng ngccgangan acngcngtnt
ancactgngc nntgntnccn 300aagagnggnc cttggttnga ntactggggt
cagggnacnc tggtnacngt ctcnancn 358227357DNAHomo sapiens
227gaagtacaac tgctggagag cggtggcggc ctggttcaac cgggtggttc
cctgcgcctg 60tcctgtgcgg catctggttt caccttcgca cacgaaacca tggtgtgggt
tcgccaagct 120ccgggcaaag gcctggaatg ggtaagccac attcctccag
atggccagga cccattctat 180gcggattccg ttaagggtcg ctttaccatt
tctcgtgata actccaaaaa caccctgtac 240ctgcagatga actccctgcg
cgccgaggat actgcggtgt accattgtgc gctgctgcct 300aaacgtggcc
cgtggttcga ttactggggt cagggtactc tggtcaccgt aagcagc
357228119PRTHomo sapiens 228Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ala His Glu 20 25 30Thr Met Val Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser His Ile Pro Pro Asp Gly
Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Leu Leu
Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 115229357DNAEscherichia coli 229gaagtacaac
tgctggagag cggtggcggc ctggttcaac cgggtggttc cctgcgcctg 60tcctgtgcgg
catctggttt caccttcgca cacgaaacca tggtgtgggt tcgccaagct
120ccgggcaaag gcctggaatg ggtaagccac attcctccag atggccagga
cccattctat 180gcggattccg ttaagggtcg ctttaccatt tctcgtgata
actccaaaaa caccctgtac 240ctgcagatga actccctgcg cgccgaggat
actgcggtgt accattgtgc gctgctgcct 300aaacgtggcc cgtggttcga
ttactggggt cagggtactc tggtcaccgt aagcagc 357230357DNAArtificial
SequenceConsensus 230gangtncanc tgntggagnn nggnggnggc ntggtncanc
cnggnggntc cctgcgnctn 60tcctgtgcng cntcnggntt caccttngcn canganacna
tggtgtgggt ncgccangcn 120ccnggnaang gnctngantg ggtnnnncan
attccnccng atggncagga nccnttctan 180gcngantccg tnaagggncg
nttnaccatn tcncgngana antccaanaa cacnctntan 240ctgcanatga
acnncctgcg ngccgaggan acngcggtnt ancantgtgc gctgctncct
300aanngnggnc cntggttnga ntactggggt cagggnacnc tggtcaccgt nnnnagc
357231357DNAHomo sapiens 231gaggttcaac tgctggaatc tggtggtggt
ctggtacaac cgggtggttc cctgcgtctg 60agctgtgcag cctctggttt caccttcgct
catgagacca tggtttgggt acgccaggct 120ccgggtaaag gcctggagtg
ggtaagccat atccctcctg atggtcagga cccgttctat 180gctgattccg
tcaaaggccg ttttaccatt tctcgtgaca acagcaaaaa cactctgtac
240ctgcaaatga actccctgcg tgcagaagac acggcggttt atcactgtgc
actgctgcca 300aaacgcggcc cttggttcga ctactggggc cagggtactc
tggtcactgt atcttct 357232119PRTHomo sapiens 232Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Glu 20 25 30Thr Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser His
Ile Pro Pro Asp Gly Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Leu Leu Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115233357DNAEscherichia
coli 233gaggttcaac tgctggaatc tggtggtggt ctggtacaac cgggtggttc
cctgcgtctg 60agctgtgcag cctctggttt caccttcgct catgagacca tggtttgggt
acgccaggct 120ccgggtaaag gcctggagtg ggtaagccat atccctcctg
atggtcagga cccgttctat 180gctgattccg tcaaaggccg ttttaccatt
tctcgtgaca acagcaaaaa cactctgtac 240ctgcaaatga actccctgcg
tgcagaagac acggcggttt atcactgtgc actgctgcca 300aaacgcggcc
cttggttcga ctactggggc cagggtactc tggtcactgt atcttct
357234357DNAArtificial SequenceConsensus 234gaggtncanc tgntggantc
tggnggnggn ntggtacanc cnggnggntc cctgcgtctn 60nnctgtgcag cctcnggntt
caccttngcn catgagacna tggtntgggt ncgccaggcn 120ccnggnaang
gnctngagtg ggtnnnncat atnccnccng atggtcagga nccnttctan
180gcngantccg tnaanggccg nttnaccatn tcncgngaca annncaanaa
cacnctntan 240ctgcaaatga acnncctgcg tgcngangac acngcggtnt
atcactgtgc nctgctnccn 300aanngnggnc cttggttnga ctactggggn
cagggnacnc tggtcacngt ntcnnnn 3572355PRTHomo sapiens 235Glu Val Gln
Leu Leu1 523628PRTArtificial Sequencepeptide linker 236Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr His Thr Cys1 5 10 15Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 20 2523732PRTArtificial
Sequencepeptide linker 237Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala Pro Ser1 5 10 15Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro 20 25 30238348DNAHomo sapiens
238gaggtgcagc tgttggtttc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60tcctgtgcag cctccggatt cacctttaag gcttatccga tgatgtgggt
ccgccaggct 120ccagggaagg gtctagagtg ggtctcagag atctcgcctt
cgggttctta tacatactac 180gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaagatcct 300cggaagattg
actactgggg tcagggaacc ctggtcaccg tctcgagc 348
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