U.S. patent application number 13/193167 was filed with the patent office on 2012-05-03 for targeted binding agents directed to upar and uses thereof.
This patent application is currently assigned to MedImmune Limited. Invention is credited to Paul Elvin, Stephen Charles Emery, Qing Zhou.
Application Number | 20120108797 13/193167 |
Document ID | / |
Family ID | 38610146 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120108797 |
Kind Code |
A1 |
Elvin; Paul ; et
al. |
May 3, 2012 |
TARGETED BINDING AGENTS DIRECTED TO UPAR AND USES THEREOF
Abstract
Targeted binding agents directed to the antigen uPAR and uses of
such antibodies are described. In particular, fully human
monoclonal antibodies directed to the antigen uPAR. Nucleotide
sequences encoding, and amino acid sequences comprising, heavy and
light chain immunoglobulin molecules, particularly sequences
corresponding to contiguous heavy and light chain sequences
spanning the framework regions and/or complementary determine
regions (CDR's), specifically from FR1 through FR4 or CDR1 through
CDR3. Hybridomas or other cell lines expressing such immunoglobulin
molecules and monoclonal antibodies.
Inventors: |
Elvin; Paul; (Northwich
Cheshire, GB) ; Emery; Stephen Charles; (Winterly
Cheshire, GB) ; Zhou; Qing; (Fremont, CA) |
Assignee: |
MedImmune Limited
Cambridge
GB
|
Family ID: |
38610146 |
Appl. No.: |
13/193167 |
Filed: |
July 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12296593 |
Oct 9, 2008 |
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PCT/US2007/008913 |
Apr 9, 2007 |
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13193167 |
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60790642 |
Apr 10, 2006 |
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Current U.S.
Class: |
530/388.22 |
Current CPC
Class: |
A61P 19/02 20180101;
C07K 16/2896 20130101; C07K 2317/76 20130101; C07K 2317/21
20130101; A61P 35/04 20180101; A61P 9/10 20180101; A61P 19/00
20180101; A61K 2039/505 20130101; A61P 25/00 20180101; A61P 27/00
20180101; C07K 2317/73 20130101; A61P 27/02 20180101; A61P 35/00
20180101; A61P 37/08 20180101; C07K 2317/92 20130101 |
Class at
Publication: |
530/388.22 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. An isolated targeted binding agent that specifically binds to
urokinase-type plasminogen activator receptor (uPAR) and inhibits
binding of urokinase-type plasminogen activator (uPA) to uPAR.
2-63. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.119
to U.S. Provisional Application Ser. No. 60/790,642, filed Apr. 10,
2006, the entirety of which is hereby incorporated by
reference.
FIELD
[0002] The invention relates to targeted binding agents against
urokinase-type plasminogen activator receptor (uPAR) and uses of
such agents. More specifically, the invention relates to fully
human monoclonal antibodies directed to uPAR. The described binding
agents are useful as diagnostics and for the treatment of diseases
associated with the activity and/or overproduction of uPAR.
BACKGROUND
[0003] The urokinase-type plasminogen activator receptor (uPAR,
CD87), GenBank Accession Number NP.sub.--002650, is a 55-60 kDa
highly glycosylated 313 residue polypeptide that is linked to the
outer leaflet of the cell membrane by a glycophosphatidylinositol
(GPI)-anchor. The protein is folded into three highly homologous
domains, DI, DII and DIII (Huai et al, Science 311: 656-659 2006).
The N-terminal end of uPAR provides a urokinase plasminogen
activator (uPA)-binding site (DI) (Gardsvoll et al, J Biol Chem
274: 37995-38003 1999). Evidence suggests that residues in domain 3
participate in the assembly of the ligand-binding site, and that
domains 2 and 3 increase the affinity of uPA binding to domain 1
(Liang et al, J Biol Chem 276: 28946-28953 2001). The C-terminal
domain (DIII) is processed to add a glycophosphatidylinositol
(GPI)-anchor involving residues ser282, gly283, ala284. Proteolytic
cleavage in the linker region between DI and DII (Hoyer-Hansen et
al, Eur J Biochem 243: 21-26 1997; Andolfo et al, Thromb Hemost 88:
298-306 2002) and at residues within the C-terminal domain
(Beaufort et al, J Immunol 172: 5450-549 2004) result in the
presence of both a membrane anchored form comprising DII and DIII
and a soluble forms of uPAR (suPAR) that comprise DI-DIII or
DII-DIII. Cleavage of the glycolipid anchor may also release uPAR
from the cell surface (Wilhelm et al, J Cell Physiol 180: 225-235
1999). Soluble uPAR incorporating DI retains the ability to bind
urokinase (Higazi et al, J Biol Chem 270: 17375-17380 1995).
[0004] uPAR is expressed on the surface of many types of cells,
including circulating leukocytes, vascular smooth muscle cells,
angiogenic endothelial cells, bone marrow cells, and fibroblasts.
Upregulation of uPAR expression at the cell surface, and
plasminogen activation, has been linked to a number of conditions
including inflammation, wound repair, arthritis (Szekanecz et al, J
Clin Pathol 50: 314-319 1997), atherosclerosis (Carmeliet et al,
Nature Genetics, 1-7: 439-444 1997); angiogenesis and tumor
invasion and metastasis. The presence of elevated levels of soluble
uPAR in plasma has been shown to be a prognostic marker in
colorectal cancer (Stephens et al, J Natl Cancer Inst 91: 869-874
1999).
[0005] Activation of the inactive zymogen plasminogen to the serine
protease plasmin is a key event regulating fibrinolysis. There are
two plasminogen activators, tissue plasminogen activator (tPA) and
urokinase plasminogen activator (uPA). Whereas tPA appears to be
the key plasminogen activator in plasma, uPA is associated with
cell surface plasminogen activation as a result of binding to uPAR.
Urokinase is secreted as a single chain zymogen (scuPA) that
exhibits very low or no intrinsic enzymatic activity. After
enzymatic cleavage by plasmin, the scuPA is converted into an
active, disulfide bond-linked, two-chain high molecular weight
(HMW)-uPA. This HMW-uPA, is comprised of an A-chain (aa 1-158) and
a B-chain, or LMW-uPA (aa 159-411). The primary interaction of uPA
with uPAR is mediated through the growth factor domain (GFD) as
1-48 of uPA in the A-chain, located in the amino-terminal fragment
(ATF). A second site in uPA that interacts with uPAR (connecting
peptide, as 136-143) has been identified.
[0006] Binding affinity of uPA for uPAR is about 1 nM (Kd).
Comparing the primary structure of uPAR among different species,
human uPAR is 95% identical to that of Cynomolgus monkey (Macaca
fascicularis), and only 60% identical to that of mouse (Mus
musculus). Furthermore, the high affinity binding of uPA and uPAR
is species specific. The affinities of the uPA for uPAR across
species (e.g. human uPA to rat uPAR) differ by at least two orders
of magnitude.
[0007] Cell surface recruitment of uPA to uPAR regulates cell
surface plasminogen activation: scuPA is activated more efficiently
when receptor bound (Ellis et al, J Biol Chem 264: 2185-2188 1989);
the catalytic efficiency of receptor bound uPA is increased
relative to uPA in solution phase (Higazi et al, J Biol Chem 270:
17375-17380 1995); PAI-1 is less efficient as an inhibitor of
receptor bound uPA (Ellis et al, J Biol Chem 265: 9904-9908 1990).
Plasminogen in turn is recruited to the cell surface through low
and high affinity interactions and plasmin generation proceeds more
effectively at the cell surface and is also less susceptible to its
physiological inhibitors. At the tumor cell surface, plasminogen
activation facilitates a number of processes that contribute to
tumor progression including, activation of matrix-metalloproteases,
degradation of extracellular matrix proteins, release and
activation of growth factors that drive tumor growth and invasion
(Andreasen et al, Int J Cancer 72: 1-22 1997).
[0008] In addition to the well established role in plasminogen
activation, more recently evidence has established the role of uPAR
as an adhesion receptor for vitronectin, a complex interaction that
involves uPAR, uPA and PAI-1 (Waltz and Chapman, J Biol Chem-269:
14746-14750 1994; Czekay et al, J Cell Biol 160: 781-791 2003; Li
et al, J Biol Chem 278: 29925-29932 2003). Cleavage of cell surface
uPAR exposes an epitope on the DII domain that has been shown to be
chemotactic in vitro (Degryse et al, J Biol Chem 280: 24792-24803
2005). Interactions with integrin receptors have been reported
(e.g. Wei et al, J Cell Biol 168: 501-511 2005; Wei et al, Mol Biol
Cell 12: 2975-2986 2001), and activation of cellular signaling
pathways as a result of uPA binding have been demonstrated
(reviewed in Blasi and Carmeliet, Nature Rev Mol Cell Biol 3:
932-943 2002)
[0009] Increased expression of uPAR and urokinase have been
correlated with tumor progression in a range of human cancers
including, breast, urinary bladder, gastric cancer, endometrial
cancer, colorectal cancer (Edo de Bock and Wang, Med Res Reviews
24: 13-39 2004). uPAR gene expression is increased by tumor
promoters, growth factors, cytokines and hormones as well as by
atherogenic lipoproteins or hypoxia. Immunohistochemical and in
situ hybridization studies have shown the uPA/uPAR complex to be
localized to the invasive edge of a tumor, (Pyke et al, Am J Pathol
138: 1059-1067 1991) and correlated with tumor invasion. High
expression levels of each uPA, uPAR or PAI-1 is linked to poor
prognosis in different types of tumors (Duffy and Duggan, Clin
Biochem 37: 541-548 2004).
[0010] Others have shown that a polyclonal urokinase receptor
antibody can reduce tumor volume and detect the presence of occult
tumor metastases in vivo (Rabbani and Gladu, Cancer Res. 62:2390-7
2002). Adenovirus mediated delivery of the uPA-ATF (Li et al, Gene
Therapy 5 1105-1113 1998; Human Gene Therapy 16: 1157-1167 2005),
stable transfection of uPA-ATF (Zhu et al, DNA Cell Biol 20:
297-305 2001), anti-sense uPAR (Mohan et al, Cancer Res 59:
3369-3373 1999) or combined antisense uPAR/uPA (Gondi et al,
Oncogene 22: 5967-5975 2003) resulted in blockade or loss of uPAR
activity and inhibition of invasion in vitro and tumor growth and
invasion in vivo. In addition, a peptide derived from the
non-receptor binding region of urokinase plasminogen activator
(uPA) inhibited tumor progression and angiogenesis and induced
tumor cell death in vivo (Guo et al, FASEB J. 14:1400-1410
2000).
SUMMARY
[0011] Embodiments of the invention relate to fully human targeted
binding agents that specifically bind to uPAR and thereby inhibit
plasminogen activation and activation of certain
matrix-metalloproteases. The targeted binding agents also inhibit
tumor cell adhesion and/or invasion, and/or cellular metastasis. In
addition, the targeted binding agents are useful for reducing tumor
growth. Mechanisms by which this can be achieved can include and
are not limited to either inhibiting binding of uPA to its receptor
uPAR, inhibiting uPAR/uPA localized uPA enzymatic activity,
abrogation of intereactions with integrins or extracellular matrix
proteins such as vitronectin, thereby reducing the effective
concentration of uPAR.
[0012] Thus one embodiment of the invention is a targeted binding
agent that specifically binds to uPAR and inhibits binding of uPA
to uPAR. The targeted binding agent may bind uPAR with a K.sub.d of
less than 100 .mu.M, 10 .mu.M, 1 .mu.M, 100 nM, 10 nM, 1 nM, 500
pM, 400 pM, 300 pM, 200 pM, 100 pM or 50 pM.
[0013] Yet another embodiment is a targeted binding agent that
binds to uPAR and inhibits greater than 90% of plasminogen
activation on U937 cells at antibody concentrations as low as 100
.mu.g/ml, 10 .mu.g/ml, 8 .mu.g/ml, 6 .mu.g/ml, 4 .mu.g/ml, 2
.mu.g/ml, 1 .mu.g/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, or 100 pg/ml or
less.
[0014] Yet another embodiment is a targeted binding agent that
binds to uPAR and inhibits plasminogen activation on U937 cells
with an IC.sub.50 of less than 10 .mu.g/ml, 1 .mu.g/ml, 0.1
.mu.g/ml, 0.08 .mu.g/ml, 0.06 .mu.g/ml, 0.04 .mu.g/ml, 0.02
.mu.g/ml or 0.01 .mu.g/ml.
[0015] Yet another embodiment is a targeted binding agent that
binds to uPAR and inhibits greater than 90% of uPAR-mediated cell
adhesion of U937 cells to vitronectin at targeted binding agent
concentrations as low as 100 .mu.g/ml, 10 .mu.g/ml, 8 .mu.g/ml, 6
.mu.g/ml, 4 .mu.g/ml, 2 .mu.g/ml, 1 .mu.g/ml, 100 ng/ml, 10 ng/ml,
1 ng/ml, or 100 pg/ml or less. Yet another embodiment is a targeted
binding agent that binds to uPAR and inhibits greater than 80% of
uPAR-mediated cell adhesion of U937 cells to vitronectin at
targeted binding agent concentrations as low as 100 .mu.g/ml, 10
.mu.g/ml, 8 .mu.g/ml, 6 .mu.g/ml, 4 .mu.g/ml, 2 .mu.g/ml, 1
.mu.g/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, or 100 pg/ml or less.
[0016] Yet another embodiment is a targeted binding-agent that
binds to uPAR and inhibits greater than 90% of invasion of HT-1080
cells through Matrigel.TM. at targeted binding agent concentrations
as low as 1000 .mu.g/ml, 100 .mu.g/ml, 10 .mu.g/ml, 8 .mu.g/ml, 6
.mu.g/ml, 4 .mu.g/ml, 2 .mu.g/ml, 1 .mu.g/ml, 100 ng/ml, 10 ng/ml,
1 ng/ml or 100 pg/ml or less.
[0017] In another embodiment the targeted binding agent may
comprise a sequence comprising any one of the complementarity
determining regions (CDR), CDR1, CDR2 or CDR3 sequences as shown in
Table 18. In another embodiment the targeted binding agent may
comprise a sequence comprising any two of a CDR1, CDR2 or CDR3
sequence as shown in Table 18 (that is either a CDR1 and CDR2, a
CDR1 and CDR3, or a CDR2 and CDR3). In another embodiment the
targeted binding agent may comprise a sequence comprising a CDR1,
CDR2 and CDR3 sequence as shown in Table 18. It is noted that those
of ordinary skill in the art can readily accomplish CDR
determinations. See for example, Kabat et al., Sequences of
Proteins of Immunological Interest, Fifth Edition, NIH Publication
91-3242, Bethesda Md. (1991), vols. 1-3.
[0018] In another embodiment the targeted binding agent may
comprise a sequence comprising any one of a CDR1, CDR2 or CDR3
sequence as shown in Table 18. In another embodiment the targeted
binding agent may comprise a sequence comprising any two of a CDR1,
CDR2 or CDR3 sequence as shown in Table 18 (that is either a CDR1
and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another
embodiment the targeted binding agent may comprise a sequence
comprising a CDR1, CDR2 and CDR3 sequence as shown in Table 18.
[0019] In another embodiment the targeted binding agent may
comprise a sequence comprising any one of a CDR1, CDR2 or CDR3
sequence as shown in Table 19. In another embodiment the targeted
binding agent may comprise a sequence comprising any two of a CDR1,
CDR2 or CDR3 sequence as shown in Table 19 (that is either a CDR1
and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another
embodiment the targeted binding agent may comprise a sequence
comprising a CDR1, CDR2 and CDR3 sequence as shown in Table 19.
[0020] In another embodiment the targeted binding agent may
comprise a sequence comprising any one of a CDR1, CDR2 or CDR3
sequence of any one of either fully human monoclonal antibody
2.19.2 (ATCC Accession Number PTA-7474), 2.6.1 (ATCC Accession
Number PTA-7475) or 4.18.1 (ATCC Accession Number PTA-7476), as
shown in Table 18. In another embodiment the targeted binding agent
may comprise a sequence comprising any two of a CDR1, CDR2 or CDR3
sequence of either fully human monoclonal antibody 2.19.2, 2.6.1 or
4.18.1 as shown in Table 18 (that is either a CDR1 and CDR2, a CDR1
and CDR3 or a CDR2 and CDR3). In another embodiment the targeted
binding agent may comprise a sequence comprising a CDR1, CDR2 and
CDR3 sequence of either fully human monoclonal antibody 2.19.2,
2.6.1 or 4.18.1 as shown in Table 18.
[0021] In another embodiment the targeted binding agent may
comprise a sequence comprising any one of a CDR1, CDR2 or CDR3
sequence of either fully human monoclonal antibody 2.19.2, 2.6.1 or
4.18.1 as shown in Table 19. In another embodiment the targeted
binding agent may comprise a sequence comprising any two of a CDR1,
CDR2 or CDR3 sequence of either fully human monoclonal antibody
2.19.2, 2.6.1 or 4.18.1 as shown in Table 19 (that is either a CDR1
and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another
embodiment the targeted binding agent may comprise a sequence
comprising a CDR1, CDR2 and CDR3 sequence of either fully human
monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table
19.
[0022] In another embodiment the targeted binding agent comprises a
sequence comprising any one of a CDR1, CDR2 or CDR3 sequence as
shown in Table 18 and any one of a CDR1, CDR2 or CDR3 sequence as
shown in Table 19. In another embodiment the targeted binding agent
comprises any two of a CDR1, CDR2 or CDR3 sequence shown in Table
18 and any two of a CDR1, CDR2 or CDR3 sequence as shown in Table
19 (that is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and
CDR3). In another embodiment the targeted binding agent comprises a
CDR1, CDR2 and CDR3 sequence as shown in Table 18 and a CDR1, CDR2
and CDR3 sequence as shown in Table 19.
[0023] In another embodiment the targeted binding agent comprises a
sequence comprising any one of a CDR1, CDR2 or CDR3 sequence of
either fully human monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as
shown in Table 18, and any one of a CDR1, CDR2 or CDR3 sequence of
either fully human monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as
shown in Table 19. In another embodiment the targeted binding agent
comprises any two of a CDR1, CDR2 or CDR3 sequence of either fully
human monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table
18, and any two of a CDR1, CDR2 or CDR3 sequence of either fully
human monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table
19 (that is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and
CDR3). In another embodiment the targeted binding agent comprises a
CDR1, CDR2 and CDR3 sequence of either fully human monoclonal
antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table 18, and a CDR1,
CDR2 and CDR3 sequence of either fully human monoclonal antibody
2.19.2, 2.6.1 or 4.18.1 as shown in Table 19.
[0024] In another embodiment the targeted binding agent comprises a
sequence comprising a CDR1, CDR2 and CDR3 sequence of fully human
monoclonal antibody 2.19.2 as shown in Table 18, and a CDR1, CDR2
and CDR3 sequence of fully human monoclonal antibody 2.19.2 as
shown in Table 19. In another embodiment the targeted binding agent
comprises a sequence comprising a CDR1, CDR2 and CDR3 sequence of
fully human monoclonal antibody 2.6.1 as shown in Table 18, and a
CDR1, CDR2 and CDR3 sequence of fully human monoclonal antibody
2.6.1 as shown in Table 19. In another embodiment the targeted
binding agent comprises a sequence comprising a CDR1, CDR2 and CDR3
sequence of fully human monoclonal antibody 4.18.1 as shown in
Table 18, and a CDR1, CDR2 and CDR3 sequence of fully human
monoclonal antibody 4.18.1 as shown in Table 19.
[0025] In one embodiment of the invention, the targeted binding
agent is an antibody. In one embodiment of the invention, the
targeted binding agent is a monoclonal antibody. In one embodiment
of the invention, the targeted binding agent is a fully human
monoclonal antibody.
[0026] In another embodiment there is an antibody that binds to
uPAR and comprises a light chain amino acid sequence comprising any
one of a CDR1, CDR2 or CDR3 sequence as shown in Table 19. In
another embodiment there is an antibody that binds to uPAR and
comprises a light chain amino acid sequence comprising any two of a
CDR1, CDR2 or CDR3 sequence as shown in Table 19 (that is either a
CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another
embodiment there is an antibody that binds to uPAR and comprises a
light chain amino acid sequence comprising a CDR1, a CDR2 and a
CDR3 sequence as shown in Table 19. In certain embodiments the
antibody is a fully human monoclonal antibody.
[0027] Yet another embodiment is an antibody that binds to uPAR and
comprises a heavy chain amino acid sequence comprising any one of a
CDR1, CDR2 or CDR3 sequence as shown in Table 18. In another
embodiment there is an antibody that binds to uPAR and comprises a
heavy chain amino acid sequence comprising any two of a CDR1, CDR2
or CDR3 sequence as shown in Table 18 (that is either a CDR1 and
CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another embodiment
there is an antibody that binds to uPAR and comprises a heavy chain
amino acid sequence comprising a CDR1, a CDR2 and a CDR3 sequence
as shown in Table 18. In certain embodiments the antibody is a
fully human monoclonal antibody.
[0028] In another embodiment the antibody that binds to uPAR may
comprise an amino acid sequence comprising any one of a CDR1, CDR2
or CDR3 sequence of either fully human monoclonal antibody 2.19.2,
2.6.1 or 4.18.1 as shown in Table 18. In another embodiment the
antibody may comprise an amino acid sequence comprising any two of
a CDR1, CDR2 or CDR3 sequence of either fully human monoclonal
antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table 18 (that is
either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In
another embodiment the antibody may comprise an amino acid sequence
comprising a CDR1, CDR2 and CDR3 sequence of either fully human
monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table
18.
[0029] In another embodiment the antibody that binds to uPAR may
comprise an amino acid sequence comprising any one of a CDR1, CDR2
or CDR3 sequence of either fully human monoclonal antibody 2.19.2,
2.6.1 or 4.18.1 as shown in Table 19. In another embodiment the
antibody may comprise an amino acid sequence comprising any two of
a CDR1, CDR2 or CDR3 sequence of either fully human monoclonal
antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table 19 (that is
either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In
another embodiment the antibody may comprise an amino acid sequence
comprising a CDR1, CDR2 and CDR3 sequence of either fully human
monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table
19.
[0030] In another embodiment the antibody that binds to uPAR
comprises a heavy chain amino acid sequence comprising any one of a
CDR1, CDR2 or CDR3 sequence as shown in Table 18 and a light chain
amino acid sequence comprising one of a CDR1, CDR2 or CDR3 sequence
as shown in Table 19. In another embodiment the antibody comprises
a heavy chain amino acid sequence comprising any two of a CDR1,
CDR2 or CDR3 sequence as shown in Table 18 and a light chain amino
acid sequence comprising any two of a CDR1, CDR2 or CDR3 sequence
as shown in Table 19 (that is either a CDR1 and CDR2, a CDR1 and
CDR3 or a CDR2 and CDR3). In another embodiment the antibody
comprises a heavy chain amino acid sequence comprising a CDR1, CDR2
and CDR3 sequence as shown in Table 18 and a light chain amino acid
sequence comprising a CDR1, CDR2 and CDR3 sequence as shown in
Table 19.
[0031] In another embodiment the antibody that binds to uPAR
comprises a heavy chain amino acid sequence comprising any one of a
CDR1, CDR2 or CDR3 sequence of either fully human monoclonal
antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table 18 and a light
chain amino acid sequence comprising any one of a CDR1, CDR2 or
CDR3 sequence of either fully human monoclonal antibody 2.19.2,
2.6.1 or 4.18.1 as shown in Table 19. In another embodiment the
antibody comprises a heavy chain amino acid sequence comprising any
two of a CDR1, CDR2 or CDR3 sequence of either fully human
monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table 18
and a light chain amino acid sequence comprising any two of a CDR1,
CDR2 or CDR3 sequence of either fully human monoclonal antibody
2.19.2, 2.6.1 or 4.18.1 as shown in Table 19 (that is either a CDR1
and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another
embodiment the antibody comprises a heavy chain amino acid sequence
comprising a CDR1, CDR2 and CDR3 sequence of either fully human
monoclonal antibody 2.19.2, 2.6.1 or 4.18.1 as shown in Table 18
and a light chain amino acid sequence comprising a CDR1, CDR2 and
CDR3 sequence of either fully human monoclonal antibody 2.19.2,
2.6.1 or 4.18.1 as shown in Table 19.
[0032] In another embodiment the antibody comprises a heavy chain
amino acid sequence comprising a CDR1, CDR2 and CDR3 sequence of
fully human monoclonal antibody 2.19.2 as shown in Table 18 and a
light chain amino acid sequence comprising a CDR1, CDR2 and CDR3
sequence of fully human monoclonal antibody 2.19.2 as shown in
Table 19. In another embodiment the antibody comprises a heavy
chain amino acid sequence comprising a CDR1, CDR2 and CDR3 sequence
of fully human monoclonal antibody 2.6.1 as shown in Table 18 and a
light chain amino acid sequence comprising a CDR1, CDR2 and CDR3
sequence of fully human monoclonal antibody 2.6.1 as shown in Table
19. In another embodiment the antibody comprises a heavy chain
amino acid sequence comprising a CDR1, CDR2 and CDR3 sequence of
fully human monoclonal antibody 4.18.1 as shown in Table 18 and a
light chain amino acid sequence comprising a CDR1, CDR2 and CDR3
sequence of fully human monoclonal antibody 4.18.1 as shown in
Table 19.
[0033] A further embodiment of the invention is a targeted binding
agent which cross-competes for binding to uPAR with the targeted
binding agent or antibodies of the invention. In another embodiment
of the invention there is an antibody which cross-competes for
binding to uPAR with the targeted binding agent or antibodies of
the invention.
[0034] In another embodiment the targeted binding agent or antibody
cross-competes for binding to uPAR with any one of fully human
monoclonal antibodies 2.19.2, 2.6.1 and 4.18.1. In one embodiment
of the invention is an antibody which cross-competes with any one
of fully human monoclonal antibodies 2.19.2, 2.6.1 and 4.18.1 for
binding to uPAR.
[0035] A further embodiment of the invention is a targeted binding
agent that binds to the same epitope on uPAR as the targeted
binding agent or antibodies of the invention. In another embodiment
of the invention there is an antibody that binds to the same
epitope on uPAR as the targeted binding agent or antibodies of the
invention.
[0036] In one embodiment of the invention is a targeted binding
agent that binds to the same epitope on uPAR as any one of fully
human monoclonal antibodies 2.19.2, 2.6.1 and 4.18.1. In one
embodiment of the invention is an antibody that binds to the same
epitope on uPAR as any one of fully human monoclonal antibodies
2.19.2, 2.6.1 and 4.18.1.
[0037] In one embodiment, the targeted binding agent comprises one
or more of fully human monoclonal antibodies 2.19.2 (ATCC Accession
Number PTA-7474), 2.6.1 (ATCC Accession Number PTA-7475) and/or
4.18.1 (ATCC Accession Number PTA-7476) which specifically bind to
uPAR, as discussed in more detail below.
[0038] In one embodiment there is provided a targeted binding agent
or antibody produced by the hybridoma selected from ATCC Accession
Number PTA-7474, ATCC Accession Number PTA-7475 and ATCC Accession
Number PTA-7476. In one embodiment there is provided a targeted
binding agent or an antibody derivable from the antibody produced
by the hybridoma selected from ATCC Accession Number PTA-7474, ATCC
Accession Number PTA-7475 and ATCC Accession Number PTA-7476.
[0039] In one embodiment, the targeted binding agent comprises an
antibody derivable from one or more of fully human monoclonal
antibodies 2.19.2 (ATCC Accession Number PTA-7474), 2.6.1 (ATCC
Accession Number PTA-7475) and/or 4.18.1 (ATCC Accession Number
PTA-7476).
[0040] In one embodiment, the targeted binding agent comprises a
polypeptide having the sequence of SEQ ID NO.: 26, 30 or 50. In one
embodiment, the targeted binding agent comprises a polypeptide
having the sequence of SEQ ID NO.: 28, 32 or 52. In one embodiment,
the antibody of the invention comprises a heavy chain polypeptide
having the sequence of SEQ ID NO.: 26, 30 or 50. In one embodiment,
the antibody of the invention comprises a light chain polypeptide
having the sequence of SEQ ID NO.: 28, 32 or 52.
[0041] In one embodiment, the targeted binding agent of the
invention comprises a polypeptide comprising the sequence of SEQ ID
NO.: 26, wherein the sequence has any one of the unique combination
of germline and non-germline residues indicated by each row of
Table 15. In one embodiment, the targeted binding agent of the
invention comprises a polypeptide comprising the sequence of SEQ ID
NO.: 30, wherein the sequence has any one of the unique combination
of germline and non-germline residues indicated by each row of
Table 13. In one embodiment, the targeted binding agent of the
invention comprises a polypeptide comprising the sequence of SEQ ID
NO.: 50, wherein the sequence has any one of the unique combination
of germline and non-germline residues indicated by each row of
Table 17. In one embodiment, the targeted binding agent of the
invention comprises a polypeptide comprising the sequence of SEQ ID
NO.: 28, wherein the sequence has any one of the unique combination
of germline and non-germline residues indicated by each row of
Table 14. In one embodiment, the targeted binding agent of the
invention comprises a polypeptide comprising the sequence of SEQ ID
NO.: 32, wherein the sequence has any one of the unique combination
of germline and non-germline residues indicated by each row of
Table 12. In one embodiment, the targeted binding agent of the
invention comprises a polypeptide comprising the sequence of SEQ ID
NO.: 52, wherein the sequence has any one of the unique combination
of germline and non-germline residues indicated by each row of
Table 16.
[0042] In one embodiment, the antibody of the invention comprises a
heavy chain polypeptide comprising the sequence of SEQ ID NO.: 26,
wherein the sequence has any one of the unique combination of
germline and non-germline residues indicated by each row of Table
15. In one embodiment, the antibody of the invention comprises a
heavy chain polypeptide comprising the sequence of SEQ ID NO.: 30,
wherein the sequence has any one of the unique combination of
germline and non-germline residues indicated by each row of Table
13. In one embodiment, the antibody of the invention comprises a
heavy chain polypeptide comprising the sequence of SEQ ID NO.: 50,
wherein the sequence has any one of the unique combination of
germline and non-germline residues indicated by each row of Table
17. In one embodiment, the antibody of the invention comprises a
light chain polypeptide comprising the sequence of SEQ ID NO.: 28,
wherein the sequence has any one of the unique combination of
germline and non-germline residues indicated by each row of Table
14. In one embodiment, the antibody of the invention comprises a
light chain polypeptide comprising the sequence of SEQ ID NO.: 32,
wherein the sequence has any one of the unique combination of
germline and non-germline residues indicated by each row of Table
12. In one embodiment, the antibody of the invention comprises a
light chain polypeptide comprising the sequence of SEQ ID NO.: 52,
wherein the sequence has any one of the unique combination of
germline and non-germline residues indicated by each row of Table
16.
[0043] The invention further provides methods for assaying the
level of uPAR in a patient sample, comprising contacting a targeted
binding agent or an anti-uPAR antibody of the invention with a
biological sample from a patient, and detecting the level of
binding between said antibody and uPAR in said sample. In more
specific embodiments, the biological sample is blood, plasma,
saliva, cerebrospinal fluid, bone marrow or urine.
[0044] In other embodiments the invention provides compositions,
including a targeted binding agent of the invention or binding
fragment thereof, and a pharmaceutically acceptable carrier. In
other embodiments the invention provides compositions, including an
antibody of the invention or binding fragment thereof, and a
pharmaceutically acceptable carrier.
[0045] The invention provides methods of treating a malignant tumor
in an animal by selecting an animal in need of treatment for a
malignant tumor and administering to the animal a therapeutically
effective dose of the targeted binding agent of the invention that
binds to uPAR. In certain embodiments, the animal is human. In
certain embodiments, the targeted binding agent is an antibody of
the invention and may be selected from the group consisting of
2.19.2 (ATCC Accession Number PTA-7474), 2.6.1 (ATCC Accession
Number PTA-7475) and 4.18.1 (ATCC Accession Number PTA-7476).
[0046] In certain embodiments, the malignant tumor can be selected
from the group consisting of: melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, gastric (stomach) cancer, prostate
cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer,
glioblastoma, endometrial cancer, kidney cancer, colon cancer,
pancreatic cancer, esophageal carcinoma, head and neck cancers,
mesothelioma, sarcomas, biliary (cholangiocarcinoma), small bowel
adenocarcinoma, pediatric malignancies and epidermoid
carcinoma.
[0047] Still further embodiments of the invention include methods
of effectively treating an animal suffering from a cell adhesion-
and/or invasion-related diseases, including selecting an animal in
need of treatment for a cell adhesion- and/or invasion-related
disease, and administering to the animal a therapeutically
effective dose of a targeted binding agent of the invention that
specifically binds to uPAR. In certain embodiments, the animal is
human. In certain embodiments, the targeted binding agent is a
fully human monoclonal antibody. In certain embodiments, the cell
adhesion- or cell invasion-related disease is tumor metastasis. In
certain embodiments, the targeted binding agent is an antibody of
the invention and may be selected from the group consisting of
2.19.2 (ATCC Accession Number PTA-7474), 2.6.1 (ATCC Accession
Number PTA-7475) and 4.18.1 (ATCC Accession Number PTA-7476).
[0048] Treatable cell adhesion- and invasion-related diseases
include neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, gastric (stomach) cancer, prostate
cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer,
glioblastoma, endometrial cancer, kidney cancer, colon cancer,
pancreatic cancer, esophageal carcinoma, head and neck cancers,
mesothelioma, sarcomas, biliary (cholangiocarcinoma), small bowel
adenocarcinoma, pediatric malignancies and epidermoid
carcinoma.
[0049] Treatable cell adhesion- and invasion-related diseases
include non-neoplastic diseases, such as, inflammatory, or
hyperproliferative diseases including but not limited to arthritis,
atherosclerosis, allergic conjunctivitis.
[0050] Additional embodiments of the invention include methods of
inhibiting uPAR induced cell adhesion and/or invasion in an animal.
These methods include selecting an animal in need of treatment for
uPAR induced cell adhesion and invasion, and administering to said
animal a therapeutically effective dose of a targeted binding agent
or fully human monoclonal antibody of the invention wherein said
antibody specifically binds to uPAR.
[0051] Further embodiments of the invention include the use of a
targeted binding agent or an antibody of the invention in the
preparation of medicament for the treatment of cell adhesion- or
invasion-related diseases in an animal, wherein said monoclonal
antibody specifically binds to uPAR.
[0052] In still further embodiments, the targeted binding agents
described herein can be used for the preparation of a medicament
for the effective treatment of uPAR induced cell adhesion, and/or
invasion in an animal, wherein said targeted binding agent
specifically binds to uPAR. In still further embodiments, the
antibodies described herein can be used for the preparation of a
medicament for the effective treatment of uPAR induced cell
adhesion, and/or invasion in an animal, wherein said antibody
specifically binds to uPAR.
[0053] Embodiments of the invention described herein relate to
targeted binding agents that bind uPAR and affect uPAR function.
Other embodiments of the invention described herein relate to
monoclonal antibodies that bind uPAR and affect uPAR function.
Other embodiments relate to anti-uPAR antibodies of the invention
with desirable properties from a therapeutic perspective, including
high binding affinity for uPAR, the ability to neutralize uPAR in
vitro and in vivo, and the ability to inhibit uPAR induced cell
adhesion and/or invasion and/or tumor growth. Other embodiments
relate to preparations of an anti-uPAR antibody of the invention
with desirable properties from a therapeutic perspective, including
high binding affinity for uPAR, the ability to neutralize uPAR in
vitro and in vivo, and the ability to inhibit uPAR induced cell
adhesion and/or invasion and/or tumor growth. Other embodiments
relate to fully human anti-uPAR antibodies of the invention with
desirable properties from a therapeutic perspective, including high
binding affinity for uPAR, the ability to neutralize uPAR in vitro
and in vivo, and the ability to inhibit uPAR induced cell adhesion
and/or invasion and/or tumor growth. Other embodiments relate to
preparations of a fully human anti-uPAR antibody of the invention
with desirable properties from a therapeutic perspective, including
high binding affinity for uPAR, the ability to neutralize uPAR in
vitro and in vivo, and the ability to inhibit uPAR induced cell
adhesion and/or invasion and/or tumor growth.
[0054] In one embodiment, the invention includes antibodies that
bind to uPAR with very high affinities (Kd). For example a human,
rabbit, mouse, chimeric or humanized antibody that is capable of
binding uPAR with a Kd less than, but not limited to, 10.sup.-5,
10.sup.-6, 10.sup.-7, 10.sup.-9, 10.sup.-9, 10.sup.-10 or
10.sup.-11M, or any range or value therein. Affinity and/or avidity
measurements can be measured by KinExA.RTM. and/or BIACORE.RTM., as
described herein.
[0055] One embodiment of the invention includes isolated
antibodies, or fragments of those antibodies, that bind to uPAR. As
known in the art, the antibodies can be, for example, polyclonal,
oligoclonal, monoclonal, chimeric, humanized, and/or fully human
antibodies. Embodiments of the invention described herein also
provide cells for producing these antibodies.
[0056] It will be appreciated that embodiments of the invention are
not limited to any particular form of an antibody or method of
generation or production. For example, the anti-uPAR antibody of
the invention may be a full-length antibody (e.g., having an intact
human Fc region) or an antibody fragment (e.g., a Fab, Fab',
F(ab').sub.2, Fv or Dab (Dabs are the smallest functional binding
units of human antibodies). In addition, the antibody may be
manufactured from a hybridoma that secretes the antibody, or from a
recombinantly produced cell that has been transformed or
transfected with a gene or genes encoding the antibody.
[0057] Other embodiments of the invention include isolated nucleic
acid molecules encoding any of the targeted binding agents,
antibodies or fragments thereof as described herein, vectors having
isolated nucleic acid molecules encoding anti-uPAR antibodies or a
host cell transformed with any of such nucleic acid molecules. In
addition, one embodiment of the invention is a method of producing
an anti-uPAR antibody of the invention by culturing host cells
under conditions wherein a nucleic acid molecule is expressed to
produce the antibody followed by recovering the antibody. It should
be realized that embodiments of the invention also include any
nucleic acid molecule which encodes an antibody or fragment of an
antibody of the invention including nucleic acid sequences
optimized for increasing yields of antibodies or fragments thereof
when transfected into host cells for antibody production.
[0058] A further embodiment includes a method of producing high
affinity antibodies to uPAR by immunizing a mammal with human uPAR,
or a fragment thereof, and one or more orthologous sequences or
fragments thereof.
[0059] Other embodiments are based upon the generation and
identification of isolated antibodies that bind specifically to
uPAR. uPAR is expressed at elevated levels in cell adhesion- and/or
invasion-related diseases, such as neoplastic diseases. Inhibition
of the biological activity of uPAR can prevent uPAR induced cell
adhesion and invasion and other desired effects.
[0060] Another embodiment of the invention includes a method of
diagnosing diseases or conditions in which an antibody prepared as
described herein is utilized to detect the level of uPAR in a
patient sample. In one embodiment, the patient sample is blood or
blood serum. In further embodiments, methods for the identification
of risk factors, diagnosis of disease, and staging of disease is
presented which involves the identification of the overexpression
of uPAR using anti-uPAR antibodies.
[0061] Another embodiment of the invention includes a method for
diagnosing a condition associated with the expression of uPAR in a
cell by contacting the serum or a cell with a targeted binding
agent or an anti-uPAR antibody of the invention, and thereafter
detecting the presence of uPAR. Preferred conditions include cell
adhesion- and/or invasion-related diseases including, but not
limited to, neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0062] In another embodiment, the invention includes an assay kit
for detecting uPAR in mammalian tissues, cells, or body fluids to
screen for uPAR-related diseases. The kit includes a targeted
binding agent or an antibody of the invention that binds to uPAR
and a means for indicating the reaction of the antibody with uPAR,
if present. In one embodiment, the antibody is a monoclonal
antibody. In another embodiment, the antibody that binds uPAR is
labeled. In still another embodiment the antibody is an unlabeled
primary antibody and the kit further includes a means for detecting
the primary antibody. In one embodiment, the means for detecting
includes a labeled second antibody that is an anti-immunoglobulin.
The antibody may be labeled with a marker selected from the group
consisting of a fluorochrome, an enzyme, a radionuclide and a
radiopaque material.
[0063] Other embodiments of the invention include pharmaceutical
compositions having an effective amount of a targeted binding agent
or an anti-uPAR antibody of the invention in admixture with a
pharmaceutically acceptable carrier or diluent. In yet other
embodiments, the targeted binding agent or anti-uPAR antibody of
the invention, or a fragment thereof, is conjugated to a
therapeutic agent. The therapeutic agent can be, for example, a
toxin or a radioisotope.
[0064] Yet another embodiment includes methods for treating
diseases or conditions associated with the expression of uPAR in a
patient, by administering to the patient an effective amount of a
targeted binding agent or an anti-uPAR antibody of the invention.
The targeted binding agent or anti-uPAR antibody of the invention
can be administered alone, or can be administered in combination
with additional antibodies or chemotherapeutic drug or radiation
therapy. For example, a monoclonal, oligoclonal or polyclonal
mixture of uPAR antibodies that block cell adhesion and/or invasion
can be administered in combination with a drug shown to inhibit
tumor cell proliferation directly. The method can be performed in
vivo and the patient is preferably a human patient. In a preferred
embodiment, the method concerns the treatment of cell adhesion-
and/or invasion-related diseases including, but not limited to,
neoplastic diseases, such as, melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0065] In another embodiment, the invention provides an article of
manufacture including a container. The container includes a
composition containing a targeted binding agent or an anti-uPAR
antibody of the invention, and a package insert or label indicating
that the composition can be used to treat cell adhesion- and/or
invasion-related diseases characterized by the overexpression of
uPAR.
[0066] In some embodiments, the targeted binding agent or anti-uPAR
antibody of the invention is administered to a patient, followed by
administration of a clearing agent to remove excess circulating
antibody from the blood.
[0067] Yet another embodiment is the use of a targeted binding
agent or an anti-uPAR antibody of the invention in the preparation
of a medicament for the treatment of diseases such as cell
adhesion- and/or invasion-related diseases. In one embodiment, the
cell adhesion- and/or invasion-related diseases include carcinoma,
such as breast, ovarian, stomach, endometrial, salivary gland,
lung, kidney, colon, colorectum, esophageal, thyroid, pancreatic,
prostate and bladder cancer. In another embodiment, the cell
adhesion- and/or invasion-related diseases include, but are not
limited to, neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, sarcoma, head and neck cancers, mesothelioma, biliary
(cholangiocarcinoma), small bowel adenocarcinoma, pediatric
malignancies and glioblastoma.
[0068] Yet another embodiment is the use of a targeted binding
agent or an anti-uPAR antibody of the invention in the preparation
of a medicament for the treatment of a malignant tumour in an
animal.
[0069] Yet another embodiment of the invention is the use of a
targeted binding agent or an anti-uPAR antibody of the invention in
the preparation of a medicament for the treatment of a
non-neoplastic disease. Yet another embodiment of the invention is
the use of a targeted binding agent or an anti-uPAR antibody of the
invention in the preparation of a medicament for the treatment of
inflammatory, or hyperproliferative diseases including but not
limited to arthritis, atherosclerosis, allergic conjunctivitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1 is a line graph showing the inhibitory effect of
fifteen anti-uPAR monoclonal antibodies in a plasminogen activation
inhibition assay. The dose-response relationship curve for each mAb
is illustrated. The eight antibodies which exhibited the highest
efficacy and potency are indicated by the vertical bracket. Bold
face legends indicate antibodies which exhibited the strongest
inhibition. Percentage of inhibition ("% Inhibition") is shown as a
function of antibody concentration with units of Log
[mAb](.mu.g/ml).
[0071] FIG. 2 is a line graph showing the ability of uPAR mAbs to
inhibit adhesion of U937 cells to vitronectin. Shown are
dose-response relationship curves for eight mAbs, as well as human
and goat isotype negative controls and goat anti-uPAR positive
control. Percent inhibition ("% Inhibition") is shown as a function
of antibody concentration with units of Log [mAb](ng/ml).
[0072] FIG. 3 shows the inhibitory effect of uPAR mAbs on HT-1080
invasion through Matrigel.TM. membrane preparation. The antibodies
at concentrations of 0.1 .mu.g/ml (top graph), 1.0 .mu.g/ml (middle
graph), and 10 .mu.g/ml (bottom graph) were tested. The ratios of
cell numbers that invade into the Matrigel.TM. beyond 60 micrometer
(.mu.m) with treatment versus isotype control are plotted.
[0073] FIG. 4 is a bar graph showing binding of anti-uPAR
monoclonal antibodies to monkey (cynomolgus) uPAR expressed on the
surface of 293T cells. The GeoMean values derived from the FACS
analysis of the histograph peak shift are plotted.
[0074] FIG. 5 is a bar graph showing the effect of anti-uPAR mAbs
on tumor cell invasion. The percentage of HT-1080 cells invading
through Matrigel.TM. beyond 80 micrometer (.mu.M) is shown for each
antibody at concentrations ranging from 0.1 to 100 ng/ml is
shown.
[0075] FIG. 6 shows the results of epitope binding analysis of
anti-human uPAR mAbs by Western Blotting. Human-murine uPAR
chimeras (His-tagged) were expressed in HEK293T cells and uPAR
expression was detected using the anti-human uPAR mAbs as
indicated. Lane 1--murine DI/DIII--human DII; lane 2--murine
DI/DII--human DIII; lane 3--murine DI--human DI/DIII; lane 4--human
DI/DII--murine DIII; lane 5--human uPAR.
[0076] FIG. 7 is a line graph showing the effect of anti-uPAR mAbs
on solid tumor growth of MDA-MB231-GFP orthotopic xenografts. Mean
tumor volume (in cm.sup.3) at time points post transplantation
(weeks post implant) are shown for each antibody tested, and for
IgG4 isotype and doxorubicin controls. Untreated control tumor
growth (not shown) was not significantly different to IgG4 isotype
controls.
[0077] FIGS. 8A-F are bar graphs showing the activity of anti-uPAR
monoclonal antibodies on tumour biomarker expression, measured in
primary tumors of MDA-MB231-GFP orthotopic xenografts. The data
represents the mean+/-standard deviation of staining intensity
(density) measured from tumors of six animals per treatment group;
ki-67 (FIG. 8a), CD31 (FIG. 8b), MAPK (FIG. 8c), pMAPK (FIG. 8d),
FAK (FIG. 8e), pFAK (FIG. 5f), including untreated, doxorubicin and
IgG4 controls.
[0078] FIGS. 9A-B are bar graphs representing the activity of
anti-uPAR mAbs to inhibit metastasis in mice with MDA-MB231-GFP
orthotopic xenografts. The data represents the mean+/-standard
deviation of the number of tumor foci in 10 fields of view in
tissue sections from lung (FIG. 9A) or liver (FIG. 9B) from six
animals per treatment group as indicated.
DETAILED DESCRIPTION
[0079] Embodiments of the invention relate to targeted binding
agents that bind to urokinase-type plasminogen activator receptor
(uPAR). In some embodiments, the binding agents bind to uPAR and
inhibit the binding of urokinase-type plasminogen activator (uPA)
to its receptor, uPAR. In one embodiment, the targeted binding
agents are monoclonal antibodies, or binding fragments thereof.
[0080] Other embodiments of the invention include fully human
anti-uPAR antibodies, and antibody preparations that are
therapeutically useful. In one embodiment, preparations of the
anti-uPAR antibody of the invention have desirable therapeutic
properties, including strong binding affinity for uPAR, the ability
to inhibit plasminogen activation in vitro, and the ability to
inhibit uPAR-induced cell adhesion and invasion in vitro and in
vivo.
[0081] Embodiments of the invention also include isolated binding
fragments of anti-uPAR antibodies. In one embodiment the binding
fragments are derived from fully human anti-uPAR antibodies.
Exemplary fragments include Fv, Fab', Dab or other well-known
antibody fragments, as described in more detail below. Embodiments
of the invention also include cells that express fully human
antibodies against uPAR. Examples of cells include hybridomas, or
recombinantly created cells, such as Chinese hamster ovary (CHO)
cells, variants of CHO cells (for example DG44) and NS0 cells that
produce antibodies against uPAR. Additional information about
variants of CHO cells can be found in Andersen and Reilly (2004)
Current Opinion in Biotechnology 15, 456-462 which is incorporated
herein in its entirety by reference.
[0082] In addition, embodiments of the invention include methods of
using these antibodies for treating diseases. Anti-uPAR antibodies
of the invention are useful for preventing uPAR-mediated
plasminogen activation, thereby inhibiting cell adhesion and/or
invasion. While not being limited to any particular theory, the
mechanism of action of this inhibition may include inhibition of
uPA from binding to its receptor, uPAR. Anti-uPAR antibodies of the
invention may also inhibit uPAR/uPA localized uPA enzymatic
activity, thereby reducing the effective concentration of uPAR.
Anti-uPAR antibodies of the invention may inhibit uPAR dependent
adhesion of cells to vitronectin and may also disrupt uPAR
modification of integrin mediated cell adhesion. Anti-uPAR
antibodies of the invention may also modify intracellular
signalling as a result of inhibiting uPA binding to uPAR or
disruption of uPAR interactions with integrins and growth factor
receptors. Diseases that are treatable through this inhibition
mechanism include, but are not limited to, neoplastic diseases,
such as melanoma, small cell lung cancer, non-small cell lung
cancer, glioma, hepatocellular (liver) carcinoma, glioblastoma, and
cancers and tumors of the thyroid, stomach, prostate, breast,
ovary, bladder, lung, uterus, kidney, colon, and pancreas, salivary
gland, and colorectum.
[0083] Other embodiments of the invention include diagnostic assays
for specifically determining the quantity of uPAR in a biological
sample. The assay kit can include anti-uPAR antibodies of the
invention along with the necessary labels for detecting such
antibodies. These diagnostic assays are useful to screen for cell
adhesion- and invasion-related diseases including, but not limited
to, neoplastic diseases, such as, melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0084] Another aspect of the invention is an antagonist of the
biological activity of uPAR wherein the antagonist binds to uPAR.
In one embodiment, the antagonist is a targeted binding agent, such
as an antibody. The antagonist may bind to:
[0085] i) uPAR; or
[0086] ii) the uPA/uPAR complex,
or a combination of these. In one embodiment the antagonist is able
to antagonize the biological activity of uPAR in vitro and in vivo.
The antagonist may be selected from an antibody described herein,
for example fully human monoclonal antibody 2.19.2 (ATCC Accession
Number PTA-7474), 2.6.1 (ATCC Accession Number PTA-7475) and 4.18.1
(ATCC Accession Number PTA-7476).
[0087] In one embodiment the antagonist of the biological activity
of uPAR may bind to uPAR and thereby prevent uPAR mediated
plasminogen activation, thereby inhibiting cell adhesion and/or
invasion. The mechanism of action of this inhibition may include
binding of the antagonist to uPAR and inhibiting the binding of uPA
to its receptor, uPAR. Without wishing to be bound by any
particular theoretical considerations, mechanisms by which
antagonism of the biological activity of uPAR can be achieved
include, but are not limited to, inhibition of binding of uPA to
the receptor uPAR, or inhibition of uPAR/uPA localized uPA
enzymatic activity.
[0088] One embodiment is a targeted binding agent which binds to
the same epitope or epitopes as fully human monoclonal antibody
2.19.2, 2.6.1 or 4.18.1.
[0089] One embodiment is an antibody which binds to the same
epitope or epitopes as fully human monoclonal antibody 2.19.2,
2.6.1 or 4:18.1.
[0090] In one embodiment, the targeted binding agent binds uPAR
with a K.sub.d of less than 1 nanomolar (nM). The targeted binding
agent may bind with a K.sub.d less than 500 picomolar (pM). The
targeted binding agent may bind with a K.sub.d less than 400
picomolar (pM). The targeted binding agent may bind with a K.sub.d
less than 300 picomolar (pM). In another embodiment, the targeted
binding agent binds with a K.sub.d of less than 200 .mu.M.
[0091] One embodiment is a hybridoma that produces the targeted
binding agent as described hereinabove. In one embodiment is a
hybridoma that produces the light chain and/or the heavy chain of
the antibodies as described hereinabove. In one embodiment the
hybridoma produces the light chain and/or the heavy chain of a
fully human monoclonal antibody. In another embodiment the
hybridoma produces the light chain and/or the heavy chain of the
fully human monoclonal antibody 2.19.2, 2.6.1 or 4.18.1.
Alternatively the hybridoma may produce an antibody which binds to
the same epitope or epitopes as fully human monoclonal antibody
2.19.2, 2.6.1 or 4.18.1.
[0092] Another embodiment is a nucleic acid molecule encoding the
targeted binding agent as described hereinabove. In one embodiment
is a nucleic acid molecule encoding the light chain or the heavy
chain of an antibody as described hereinabove. In one embodiment
the nucleic acid molecule encodes the light chain or the heavy
chain of a fully human monoclonal antibody. Still another
embodiment is a nucleic acid molecule encoding the light chain or
the heavy chain of a fully human monoclonal antibody selected from
antibodies 2.19.2, 2.6.1 or 4.18.1.
[0093] Another embodiment of the invention is a vector comprising a
nucleic acid molecule or molecules as described hereinabove,
wherein the vector encodes a targeted binding agent as defined
hereinabove. In one embodiment of the invention is a vector
comprising a nucleic acid molecule or molecules; as described
hereinabove, wherein the vector encodes a light chain and/or a
heavy chain of an antibody as defined hereinabove.
[0094] Yet another embodiment of the invention is a host cell
comprising a vector as described hereinabove. Alternatively the
host cell may comprise more than one vector.
[0095] In addition, one embodiment of the invention is a method of
producing a targeted binding, agent or of the invention by
culturing host cells under conditions wherein a nucleic acid
molecule is expressed to produce the targeted binding agent,
followed by recovery of the targeted binding agent. In one
embodiment of the invention is a method of producing an antibody of
the invention by culturing host cells under conditions wherein a
nucleic acid molecule is expressed to produce the antibody,
followed by recovery of the antibody.
[0096] In one embodiment the invention includes a method of making
an targeted binding agent by transfecting at least one host cell
with at least one nucleic acid molecule encoding the targeted
binding agent as described hereinabove, expressing the nucleic acid
molecule in the host cell and isolating the targeted binding agent.
In one embodiment the invention includes a method of making an
antibody by transfecting at least one host cell with at least one
nucleic acid molecule encoding the antibody as described
hereinabove, expressing the nucleic acid molecule in the host cell
and isolating the antibody.
[0097] According to another aspect, the invention includes a method
of antagonising the biological activity of uPAR by administering an
antagonist as described herein. The method may include selecting an
animal in need of treatment for disease-related cell adhesion
and/or invasion, and administering to the animal a therapeutically
effective dose of an antagonist of the biological activity of
uPAR.
[0098] Another aspect of the invention includes a method of
antagonising the biological activity of uPAR by administering a
targeted binding agent as described hereinabove. The method may
include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion, and administering to
the animal a therapeutically effective dose of a targeted binding
agent which antagonises the biological activity of uPAR.
[0099] Another aspect of the invention includes a method of
antagonising the biological activity of uPAR by administering an
antibody as described hereinabove. The method may include selecting
an animal in need of treatment for disease-related cell adhesion
and/or invasion, and administering to the animal a therapeutically
effective dose of an antibody which antagonises the biological
activity of uPAR.
[0100] According to another aspect there is provided a method of
treating disease-related cell adhesion and/or invasion in a mammal
by administering a therapeutically effective amount of an
antagonist of the biological activity of uPAR. The method may
include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion, and administering to
the animal a therapeutically effective dose of an antagonist of the
biological activity of uPAR.
[0101] According to another aspect there is provided a method of
treating disease-related cell adhesion and/or invasion in a mammal
by administering a therapeutically effective amount of a targeted
binding agent which antagonizes the biological activity of uPAR.
The method may include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion, and administering to
the animal a therapeutically effective dose of a targeted binding
agent which antagonises the biological activity of uPAR. The
targeted binding agent can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drugs or radiation therapy.
[0102] According to another aspect there is provided a method of
treating disease-related cell adhesion and/or invasion in a mammal
by administering a therapeutically effective amount of an antibody
which antagonizes the biological activity of uPAR. The method may
include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion, and administering to
the animal a therapeutically effective dose of an antibody which
antagonises the biological activity of uPAR. The antibody can be
administered alone, or can be administered in combination with
additional antibodies or chemotherapeutic drugs or radiation
therapy.
[0103] According to another aspect there is provided a method of
treating cancer in a mammal by administering a therapeutically
effective amount of an antagonist of the biological activity of
uPAR. The method may include selecting an animal in need of
treatment for cancer, and administering to the animal a
therapeutically effective dose of an antagonist which antagonises
the biological activity of uPAR. The antagonist can be administered
alone, or can be administered in combination with additional
antibodies or chemotherapeutic drugs or radiation therapy.
[0104] According to another aspect there is provided a method of
treating cancer in a mammal by administering a therapeutically
effective amount of a targeted binding agent which antagonizes the
biological activity of uPAR. The method may include selecting an
animal in need of treatment for cancer, and administering to the
animal a therapeutically effective dose of a targeted binding agent
which antagonises the biological activity of uPAR. The targeted
binding agent can be administered alone, or can be administered in
combination with additional antibodies or chemotherapeutic drugs or
radiation therapy.
[0105] According to another aspect there is provided a method of
treating cancer in a mammal by administering a therapeutically
effective amount of an antibody which antagonizes the biological
activity of uPAR. The method may include selecting an animal in
need of treatment for cancer, and administering to the animal a
therapeutically effective dose of an antibody which antagonises the
biological activity of uPAR. The antibody can be administered
alone, or can be administered in combination with additional
antibodies or chemotherapeutic drugs or radiation therapy.
[0106] According to another aspect of the invention there is
provided the use of an antagonist of the biological activity of
uPAR for the manufacture of a medicament for the treatment of
disease-related cell adhesion and/or invasion.
[0107] According to another aspect of the invention there is
provided the use of a targeted binding agent which antagonizes the
biological activity of uPAR for the manufacture of a medicament for
the treatment of disease-related cell adhesion and/or invasion.
[0108] According to another aspect of the invention there is
provided the use of an antibody which antagonizes the biological
activity of uPAR for the manufacture of a medicament for the
treatment of disease-related cell adhesion and/or invasion.
[0109] In one embodiment the present invention is particularly
suitable for use in antagonizing uPAR, in patients with a tumor
which is dependent alone, or in part, on a uPAR receptor.
[0110] Another embodiment of the invention includes an assay kit
for detecting uPAR in mammalian tissues, cells, or body fluids to
screen for cell adhesion- and invasion-related diseases. The kit
includes a targeted binding agent that binds to uPAR and a means
for indicating the reaction of the targeted binding agent with
uPAR, if present. In one embodiment, the targeted binding agent
that binds uPAR is labeled. In another embodiment the targeted
binding agent is an unlabeled and the kit further includes a means
for detecting the targeted binding agent. Preferably the targeted
binding agent is labeled with a marker selected from the group
consisting of a fluorochrome, an enzyme, a radionuclide and a
radio-opaque material.
[0111] Another embodiment of the invention includes an assay kit
for detecting uPAR in mammalian tissues, cells, or body fluids to
screen for cell adhesion- and invasion-related diseases. The kit
includes an antibody that binds to uPAR and a means for indicating
the reaction of the antibody with uPAR, if present. The antibody
may be a monoclonal antibody. In one embodiment, the antibody that
binds uPAR is labeled. In another embodiment the antibody is an
unlabeled primary antibody and the kit further includes a means for
detecting the primary antibody. In one embodiment, the means
includes a labeled second antibody that is an anti-immunoglobulin.
Preferably the antibody is labeled with a marker selected from the
group consisting of a fluorochrome, an enzyme, a radionuclide and a
radio-opaque material.
[0112] Further embodiments, features, and the like regarding
anti-uPAR antibodies are provided in additional detail below.
Sequence Listing
[0113] Embodiments of the invention include the specific anti-uPAR
antibodies listed below in Table 1. This table reports the
identification number of each anti-uPAR antibody, along with the
SEQ ID number of the variable domain of the corresponding heavy
chain and light chain genes. Each antibody has been given an
identification number that includes three numbers separated by two
decimal points.
TABLE-US-00001 TABLE 1 mAb SEQ ID ID No.: Sequence NO: 1.4.2
Nucleotide sequence encoding the variable region of the heavy chain
1 Amino acid sequence encoding the variable region of the heavy
chain 2 Nucleotide sequence encoding the variable region of the
light chain 3 Amino acid sequence encoding the variable region of
the light chain 4 1.41.1 Nucleotide sequence encoding the variable
region of the heavy chain 5 Amino acid sequence encoding the
variable region of the heavy chain 6 Nucleotide sequence encoding
the variable region of the light chain 7 Amino acid sequence
encoding the variable region of the light chain 8 1.61.1 Nucleotide
sequence encoding the variable region of the heavy chain 9 Amino
acid sequence encoding the variable region of the heavy chain 10
Nucleotide sequence encoding the variable region of the light chain
11 Amino acid sequence encoding the variable region of the light
chain 12 1.99.1 Nucleotide sequence encoding the variable region of
the heavy chain 13 Amino acid sequence encoding the variable region
of the heavy chain 14 Nucleotide sequence encoding the variable
region of the light chain 15 Amino acid sequence encoding the
variable region of the light chain 16 1.100.1 Nucleotide sequence
encoding the variable region of the heavy chain 17 Amino acid
sequence encoding the variable region of the heavy chain 18
Nucleotide sequence encoding the variable region of the light chain
19 Amino acid sequence encoding the variable region of the light
chain 20 1.113.2 Nucleotide sequence encoding the variable region
of the heavy chain 21 Amino acid sequence encoding the variable
region of the heavy chain 22 Nucleotide sequence encoding the
variable region of the light chain 23 Amino acid sequence encoding
the variable region of the light chain 24 2.6.1 Nucleotide sequence
encoding the variable region of the heavy chain 25 Amino acid
sequence encoding the variable region of the heavy chain 26
Nucleotide sequence encoding the variable region of the light chain
27 Amino acid sequence encoding the variable region of the light
chain 28 2.19.2 Nucleotide sequence encoding the variable region of
the heavy chain 29 Amino acid sequence encoding the variable region
of the heavy chain 30 Nucleotide sequence encoding the variable
region of the light chain 31 Amino acid sequence encoding the
variable region of the light chain 32 3.83 Nucleotide sequence
encoding the variable region of the heavy chain 33 Amino acid
sequence encoding the variable region of the heavy chain 34
Nucleotide sequence encoding the variable region of the light chain
35 Amino acid sequence encoding the variable region of the light
chain 36 3.176.2 Nucleotide sequence encoding the variable region
of the heavy chain 37 Amino acid sequence encoding the variable
region of the heavy chain 38 Nucleotide sequence encoding the
variable region of the light chain 39 Amino acid sequence encoding
the variable region of the light chain 40 4.12.1 Nucleotide
sequence encoding the variable region of the heavy chain 41 Amino
acid sequence encoding the variable region of the heavy chain 42
Nucleotide sequence encoding the variable region of the light chain
43 Amino acid sequence encoding the variable region of the light
chain 44 4.13.1 Nucleotide sequence encoding the variable region of
the heavy chain 45 Amino acid sequence encoding the variable region
of the heavy chain 46 Nucleotide sequence encoding the variable
region of the light chain 47 Amino acid sequence encoding the
variable region of the light chain 48 4.18.1 Nucleotide sequence
encoding the variable region of the heavy chain 49 Amino acid
sequence encoding the variable region of the heavy chain 50
Nucleotide sequence encoding the variable region of the light chain
51 Amino acid sequence encoding the variable region of the light
chain 52 4.19.1 Nucleotide sequence encoding the variable region of
the heavy chain 53 Amino acid sequence encoding the variable region
of the heavy chain 54 Nucleotide sequence encoding the variable
region of the light chain 55 Amino acid sequence encoding the
variable region of the light chain 56 4.50.1 Nucleotide sequence
encoding the variable region of the heavy chain 57 Amino acid
sequence encoding the variable region of the heavy chain 58
Nucleotide sequence encoding the variable region of the light chain
59 Amino acid sequence encoding the variable region of the light
chain 60
DEFINITIONS
[0114] Unless otherwise defined, scientific and technical terms
used herein shall have the meanings that are commonly understood by
those of ordinary skill in the art. Further, unless otherwise
required by context, singular terms shall include pluralities and
plural terms shall include the singular. Generally, nomenclatures
utilized in connection with, and techniques of, cell and tissue
culture, molecular biology, and protein and oligo- or
polynucleotide chemistry and hybridization described herein are
those well known and commonly used in the art.
[0115] Standard techniques are used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques are performed according to manufacturer's
specifications or as commonly accomplished in the art or as
described herein. The foregoing techniques and procedures are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. See e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by
reference. The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0116] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0117] An antagonist may be a polypeptide, nucleic acid,
carbohydrate, lipid, small molecular weight compound, an
oligonucleotide, an oligopeptide, RNA interference (RNAi),
antisense, a recombinant protein, an antibody, or fragments thereof
or conjugates or fusion proteins thereof. For a review of RNAi see
Milhavet O, Gary D S, Mattson M P. (Pharmacol Rev. 2003 December;
55(4); 629-48. Review.) and antisense see Opalinska J B, Gewirtz A
M. (Sci STKE. 2003 Oct. 28; 2003(206):pe 47.)
[0118] Disease-related cell adhesion and/or invasion may be any
abnormal, undesirable or pathological cell adhesion and/or
invasion, for example tumor-related cell adhesion and/or invasion.
Cell adhesion- and/or invasion-related diseases include, but are
not limited to, non-solid tumors such as leukemia, multiple myeloma
or lymphoma, and also solid tumors such as melanoma, small cell
lung cancer, non-small cell lung cancer, glioma, hepatocellular
(liver) carcinoma, glioblastoma, carcinoma of the thyroid, bile
duct, bone, gastric, brain/CNS, head and neck, hepatic system,
stomach, prostate, breast, renal, testicle, ovary, skin, cervix,
lung, muscle, neuron, oesophageal, bladder, lung, uterus, vulva,
endometrium, kidney, colorectum, pancreas, pleural/peritoneal
membranes, salivary gland, and epidermis.
[0119] A compound refers to any small molecular weight compound
with a molecular weight of less than about 2000 Daltons.
[0120] The term "uPAR" refers to the molecule urokinase-type
plasminogen activator receptor.
[0121] The term "neutralizing" when referring to an targeted
binding agent such as an antibody relates to the ability of an
antibody to eliminate, or significantly reduce, the activity of a
target antigen. Accordingly, a "neutralizing" anti-uPAR antibody of
the invention is capable of eliminating or significantly reducing
the activity of uPAR. A neutralizing uPAR antibody may, for
example, act by blocking the binding of uPA to its receptor uPAR.
By blocking this binding, the uPA mediated plasminogen activation
is significantly, or completely, eliminated. Ideally, a
neutralizing antibody against uPAR inhibits cell adhesion and/or
invasion.
[0122] The term "polypeptide" is used herein as a generic term to
refer to native protein, fragments, or analogs of a polypeptide
sequence. Hence, native protein, fragments, and analogs are species
of the polypeptide genus. Preferred polypeptides in accordance with
the invention comprise the human heavy chain immunoglobulin
molecules and the human kappa light chain immunoglobulin molecules,
as well as antibody molecules formed by combinations comprising the
heavy chain immunoglobulin molecules with light chain
immunoglobulin molecules, such as the kappa or lambda light chain
immunoglobulin molecules, and vice versa, as well as fragments and
analogs thereof. Preferred polypeptides in accordance with the
invention may also comprise solely the human heavy chain
immunoglobulin molecules or fragments thereof.
[0123] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory or otherwise is
naturally-occurring.
[0124] The term "operably linked" as used herein refers to
positions of components so described that are in a relationship
permitting them to function in their intended manner. For example,
a control sequence "operably linked" to a coding sequence is
connected in such a way that expression of the coding sequence is
achieved under conditions compatible with the control
sequences.
[0125] The term "polynucleotide" as referred to herein means a
polymeric form of nucleotides of at least 10 bases in length,
either ribonucleotides or deoxynucleotides or a modified form of
either type of nucleotide, or RNA-DNA hetero-duplexes. The term
includes single and double stranded forms of DNA.
[0126] The term "oligonucleotide" referred to herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and non-naturally occurring linkages.
Oligonucleotides are a polynucleotide subset generally comprising a
length of 200 bases or fewer. Preferably, oligonucleotides are 10
to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17,
18, 19, or 20 to 40 bases in length. Oligonucleotides are usually
single stranded, e.g. for probes; although oligonucleotides may be
double stranded, e.g. for use in the construction of a gene mutant.
Oligonucleotides can be either sense or antisense
oligonucleotides.
[0127] The term "naturally occurring nucleotides" referred to
herein includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" referred to herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotides linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoroamidate, and the
like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl.
Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures
of which are hereby incorporated by reference. An oligonucleotide
can include a label for detection, if desired.
[0128] Two amino acid sequences are "homologous" if there is a
partial or complete identity between their sequences. For example,
85% homology means that 85% of the amino acids are identical when
the two sequences are aligned for maximum matching. Gaps (in either
of the two sequences being matched) are allowed in maximizing
matching; gap lengths of 5 or less are preferred with 2 or less
being more preferred. Alternatively and preferably, two protein
sequences (or polypeptide sequences derived from them of at least
about 30 amino acids in length) are homologous, as this term is
used herein, if they have an alignment score of at more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O.,
in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5,
National Biomedical Research Foundation (1972)) and Supplement 2 to
this volume, pp. 1-10. The two sequences or parts thereof are more
preferably homologous if their amino acids are greater than or
equal to 50% identical when optimally aligned using the ALIGN
program. It should be appreciated that there can be differing
regions of homology within two orthologous sequences. For example,
the functional sites of mouse and human orthologues may have a
higher degree of homology than non-functional regions.
[0129] The term "corresponds to" is used herein to mean that a
polynucleotide sequence is homologous (i.e., is identical, not
strictly evolutionarily related) to all or a portion of a reference
polynucleotide sequence, or that a polypeptide sequence is
identical to a reference polypeptide sequence.
[0130] In contradistinction, the term "complementary to" is used
herein to mean that the complementary sequence is homologous to all
or a portion of a reference polynucleotide sequence. For
illustration, the nucleotide sequence "TATAC" corresponds to a
reference sequence "TATAC" and is complementary to a reference
sequence "GTATA".
[0131] The term "sequence identity" means that two polynucleotide
or amino acid sequences are identical (i.e., on a
nucleotide-by-nucleotide or residue-by-residue basis) over the
comparison window. The term "percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the comparison window (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The terms "substantial identity"
as used herein denotes a characteristic of a polynucleotide or
amino acid sequence, wherein the polynucleotide or amino acid
comprises a sequence that has at least 85 percent sequence
identity, preferably at least 90 to 95 percent sequence identity,
more preferably at least 99 percent sequence identity, as compared
to a reference sequence over a comparison window of at least 18
nucleotide (6 amino acid) positions, frequently over a window of at
least 24-48 nucleotide (8-16 amino acid) positions, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the sequence which may include deletions or
additions which total 20 percent or less of the reference sequence
over the comparison window. The reference sequence may be a subset
of a larger sequence.
[0132] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2.sup.nd Edition, E. S. Golub and D. R. Gren, Eds.,
Sinauer Associates, Sunderland, Mass. (1991)), which is
incorporated herein by reference. Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl
amino acids, lactic acid, and other unconventional amino acids may
also be suitable components for polypeptides of the present
invention. Examples of unconventional amino acids include:
4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxy-terminal direction, in accordance with standard usage and
convention.
[0133] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end; the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction; sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences"; sequence
regions on the DNA strand having the same sequence as the RNA and
which are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences".
[0134] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity, and most preferably at least 99 percent sequence
identity. Preferably, residue positions that are not identical
differ by conservative amino acid substitutions. Conservative amino
acid substitutions refer to the interchangeability of residues
having similar side chains. For example, a group of amino acids
having aliphatic side chains is glycine, alanine, valine, leucine,
and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, glutamic-aspartic, and
asparagine-glutamine.
[0135] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75%, more preferably at least 80%, 90%, 95%, and most
preferably 99% sequence identity to the antibodies or
immunoglobulin molecules described herein. In particular,
conservative amino acid replacements are contemplated. Conservative
replacements are those that take place within a family of amino
acids that have related side chains. Genetically encoded amino
acids are generally divided into families: (1) acidic=aspartate,
glutamate; (2) basic=lysine, arginine, histidine; (3)
non-polar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. More preferred families are: serine and threonine are an
aliphatic-hydroxy family; asparagine and glutamine are an
amide-containing family; alanine, valine, leucine and isoleucine
are an aliphatic family; and phenylalanine, tryptophan, and
tyrosine are an aromatic family. For example, it is reasonable to
expect that an isolated replacement of a leucine with an isoleucine
or valine, an aspartate with a glutamate, a threonine with a
serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
binding function or properties of the resulting molecule,
especially if the replacement does not involve an amino acid within
a framework site. Whether an amino acid change results in a
functional peptide can readily be determined by assaying the
specific activity of the polypeptide derivative. Assays are
described in detail herein. Fragments or analogs of antibodies or
immunoglobulin molecules can be readily prepared by those of
ordinary skill in the art. Preferred amino- and carboxy-termini of
fragments or analogs occur near boundaries of functional domains.
Structural and functional domains can be identified by comparison
of the nucleotide and/or amino acid sequence data to public or
proprietary sequence databases. Preferably, computerized comparison
methods are used to identify sequence motifs or predicted protein
conformation domains that occur in other proteins of known
structure and/or function. Methods to identify protein sequences
that fold into a known three-dimensional structure are known. Bowie
et al. Science 253:164 (1991). Thus, the foregoing examples
demonstrate that those of skill in the art can recognize sequence
motifs and structural conformations that may be used to define
structural and functional domains in accordance with the antibodies
described herein.
[0136] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various muteins of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain(s)
forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991),
which are each incorporated herein by reference.
[0137] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion, but where the remaining amino acid sequence is identical
to the corresponding positions in the naturally-occurring sequence
deduced, for example, from a full-length cDNA sequence. Fragments
typically are at least 5, 6, 8 or 10 amino acids long, preferably
at least 14 amino acids long, more preferably at least 20 amino
acids long, usually at least 50 amino acids long, and even more
preferably at least 70 amino acids long. The term "analog" as used
herein refers to polypeptides which are comprised of a segment of
at least 25 amino acids that has substantial identity to a portion
of a deduced amino acid sequence and which has at least one of the
following properties: (1) specific binding to a uPAR, under
suitable binding conditions, (2) ability to block appropriate
uPA/uPAR binding, or (3) ability to inhibit uPA activity.
Typically, polypeptide analogs comprise a conservative amino acid
substitution (or addition or deletion) with respect to the
naturally-occurring sequence. Analogs typically are at least 20
amino acids long, preferably at least 50 amino acids long or
longer, and can often be as long as a full-length
naturally-occurring polypeptide.
[0138] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv.
Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);
and Evans et al. J. Med. Chem. 30:1229 (1987), which are
incorporated herein by reference. Such compounds are often
developed with the aid of computerized molecular modeling. Peptide
mimetics that are structurally similar to therapeutically useful
peptides may be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biochemical property or pharmacological activity), such as human
antibody, but have one or more peptide linkages optionally replaced
by a linkage selected from the group consisting of: --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-(cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods
well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) may be used to generate more
stable peptides. In addition, constrained peptides comprising a
consensus sequence or a substantially identical consensus sequence
variation may be generated by methods known in the art (Rizo and
Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by
reference); for example, by adding internal cysteine residues
capable of forming intramolecular disulfide bridges which cyclize
the peptide.
[0139] As used herein, the term "antibody" refers to a polypeptide
or group of polypeptides that are comprised of at least one binding
domain that is formed from the folding of polypeptide chains having
three-dimensional binding spaces with internal surface shapes and
charge distributions complementary to the features of an antigenic
determinant of an antigen. An antibody typically has a tetrameric
form, comprising two identical pairs of polypeptide chains, each
pair having one "light" and one "heavy" chain. The variable regions
of each light/heavy chain pair form an antibody binding site.
[0140] As used herein, targeted binding agent, targeted binding
protein, specific binding protein and like terms refer to an
antibody, or binding fragment thereof, that preferentially binds to
a target site. In one embodiment, the targeted binding agent is
specific for only one target site. In other embodiments, the
targeted binding agent is specific for more than one target site.
In one embodiment, the targeted binding agent may be a monoclonal
antibody and the target site may be an epitope.
[0141] "Binding fragments" of an antibody are produced by
recombinant DNA techniques, or by enzymatic or chemical cleavage of
intact antibodies. Binding fragments include Fab, Fab',
F(ab').sub.2, Fv, Dab and single-chain antibodies. An antibody
other than a "bispecific" or "bifunctional" antibody is understood
to have each of its binding sites identical. An antibody
substantially inhibits adhesion of a receptor to a counter-receptor
when an excess of antibody reduces the quantity of receptor bound
to counter-receptor by at least about 20%, 40%, 60% or 80%, and
more usually greater than about 85% (as measured in an in vitro
competitive binding assay).
[0142] An antibody may be oligoclonal, a polyclonal antibody, a
monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a
multi-specific antibody, a bi-specific antibody, a catalytic
antibody, a chimeric antibody, a humanized antibody, a fully human
antibody, an anti-idiotypic antibody and antibodies that can be
labeled in soluble or bound form as well as fragments, variants or
derivatives thereof, either alone or in combination with other
amino acid sequences provided by known techniques. An antibody may
be from any species. The term antibody also includes binding
fragments of the antibodies of the invention; exemplary fragments
include Fv, Fab, Fab', single stranded antibody (svFC), dimeric
variable region (Diabody) and disulphide stabilized variable region
(dsFv).
[0143] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
may, but not always, have specific three-dimensional structural
characteristics, as well as specific charge characteristics. An
antibody is said to specifically bind an antigen when the
dissociation constant is .ltoreq.1 .mu.M, preferably .ltoreq.100 nM
and most preferably .ltoreq.10 nM.
[0144] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0145] "Active" or "activity" in regard to a uPAR polypeptide
refers to a portion of an uPAR polypeptide that has a biological or
an immunological activity of a native uPAR polypeptide.
"Biological" when used herein refers to a biological function that
results from the activity of the native uPAR polypeptide. A
preferred uPAR biological activity includes, for example, uPAR
induced cell adhesion and invasion.
[0146] "Mammal" when used herein refers to any animal that is
considered a mammal. Preferably, the mammal is human.
[0147] Digestion of antibodies with the enzyme, papain, results in
two identical antigen-binding fragments, known also as "Fab"
fragments, and a "Fc" fragment, having no antigen-binding activity
but having the ability to crystallize. Digestion of antibodies with
the enzyme, pepsin, results in the a F(ab').sub.2 fragment in which
the two arms of the antibody molecule remain linked and comprise
two-antigen binding sites. The F(ab').sub.2 fragment has the
ability to crosslink antigen.
[0148] "Fv" when used herein refers to the minimum fragment of an
antibody that retains both antigen-recognition and antigen-binding
sites.
[0149] "Fab" when used herein refers to a fragment of an antibody
that comprises the constant domain of the light chain and the CH1
domain of the heavy chain.
[0150] "Dab" when used herein refers to a fragment of an antibody
that is the smallest functional binding unit of a human
antibodies.
[0151] The term "mAb" refers to monoclonal antibody.
[0152] "Liposome" when used herein refers to a small vesicle that
may be useful for delivery of drugs that may include the uPAR
polypeptide of the invention or antibodies to such an uPAR
polypeptide to a mammal.
[0153] "Label" or "labeled" as used herein refers to the addition
of a detectable moiety to a polypeptide, for example, a radiolabel,
fluorescent label, enzymatic label chemiluminescent labeled or a
biotinyl group. Radioisotopes or radionuclides may include .sup.3H,
.sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.125I,
.sup.131I, fluorescent labels may include rhodamine, lanthanide
phosphors or FITC and enzymatic labels may include horseradish
peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase.
[0154] The term "pharmaceutical agent or drug" as used herein
refers to a chemical compound or composition capable of inducing a
desired therapeutic effect when properly administered to a patient.
Other chemistry terms herein are used according to conventional
usage in the art, as exemplified by The McGraw-Hill Dictionary of
Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco
(1985)), (incorporated herein by reference).
[0155] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than
about 80 percent of all macromolecular species present in the
composition, more preferably more than about 85%, 90%, 95%, and
99%. Most preferably, the object species is purified to essential
homogeneity (contaminant species cannot be detected in the
composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0156] The term "patient" includes human and veterinary
subjects.
Human Antibodies and Humanization of Antibodies
[0157] Human antibodies avoid some of the problems associated with
antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a patient.
In order to avoid the utilization of murine or rat derived
antibodies, fully human antibodies can be generated through the
introduction of functional human antibody loci into a rodent, other
mammal or animal so that the rodent, other mammal or animal
produces fully human antibodies.
[0158] One method for generating fully human antibodies is through
the use of XenoMouse.RTM. strains of mice that have been engineered
to contain up to but less than 1000 kb-sized germline configured
fragments of the human heavy chain locus and kappa light chain
locus. See Mendez et al. Nature Genetics 15:146-156 (1997) and
Green and Jakobovits J. Exp. Med. 188:483-495 (1998). The
XenoMouse.RTM. strains are available from Abgenix, Inc. (Fremont,
Calif.).
[0159] The production of the XenoMouse.RTM. strains of mice is
further discussed and delineated in U.S. patent application Ser.
No. 07/466,008, filed Jan. 12, 1990, Ser. No. 07/610,515, filed
Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24, 1992, Ser. No.
07/922,649, filed Jul. 30, 1992, Ser. No. 08/031,801, filed Mar.
15, 1993, Ser. No. 08/112,848, filed Aug. 27, 1993, Ser. No.
08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279, filed Jan.
20, 1995, Ser. No. 08/430,938, filed Apr. 27, 1995, Ser. No.
08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5,
1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837,
filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser.
No. 08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun.
5, 1995, Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No.
08/724,752, filed Oct. 2, 1996, Ser. No. 08/759,620, filed Dec. 3,
1996, U.S. Publication 2003/0093820, filed Nov. 30, 2001 and U.S.
Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598
and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507
B2. See also European Patent No., EP 0 463 151 B1, grant published
Jun. 12, 1996, International Patent Application No., WO 94/02602,
published Feb. 3, 1994, International Patent Application No., WO
96/34096, published Oct. 31, 1996, WO 98/24893, published Jun. 11,
1998, WO 00/76310, published Dec. 21, 2000. The disclosures of each
of the above-cited patents, applications, and references are hereby
incorporated by reference in their entirety.
[0160] In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more D.sub.H genes, one or more
J.sub.H genes, a mu constant region, and usually a second constant
region (preferably a gamma constant region) are formed into a
construct for insertion into an animal. This approach is described
in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.
5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,
5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to
Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to
Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and
5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and
Dunn, and GenPharm International U.S. patent application Ser. No.
07/574,748, filed Aug. 29, 1990, Ser. No. 07/575,962, filed Aug.
31, 1990, Ser. No. 07/810,279, filed Dec. 17, 1991, Ser. No.
07/853,408, filed Mar. 18, 1992, Ser. No. 07/904,068, filed Jun.
23, 1992, Ser. No. 07/990,860, filed Dec. 16, 1992, Ser. No.
08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762, filed Jul.
22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.
08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,
1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of
which are hereby incorporated by reference. See also European
Patent No. 0 546 073 B1, International Patent Application Nos. WO
92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO
94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884
and U.S. Pat. No. 5,981,175, the disclosures of which are hereby
incorporated by reference in their entirety. See further Taylor et
al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al.,
1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon
et al., (1995), Fishwild et al., (1996), the disclosures of which
are hereby incorporated by reference in their entirety.
[0161] Kirin has also demonstrated the generation of human
antibodies from mice in which, through microcell fusion, large
pieces of chromosomes, or entire chromosomes, have been introduced.
See European Patent Application Nos. 773 288 and 843 961, the
disclosures of which are hereby incorporated by reference.
Additionally, KM.TM.-mice, which are the result of cross-breeding
of Kirin's Tc mice with Medarex's minilocus (Humab) mice have been
generated., These mice possess the human IgH transchromosome of the
Kirin mice and the kappa chain transgene of the Genpharm mice
(Ishida et al., Cloning Stem Cells, (2002) 4:91-102).
[0162] Human antibodies can also be derived by in vitro methods.
Suitable examples include but are not limited to phage display
(CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion
(formerly, Proliferon), Affimed) ribosome display (CAT), yeast
display, and the like.
Preparation of Antibodies
[0163] Antibodies, as described herein, were prepared through the
utilization of the XenoMouse.RTM. technology, as described below.
Such mice, then, are capable of producing human immunoglobulin
molecules and antibodies and are deficient in the production of
murine immunoglobulin molecules and antibodies. Technologies
utilized for achieving the same are disclosed in the patents,
applications, and references disclosed in the background section
herein. In particular, however, a preferred embodiment of
transgenic production of mice and antibodies therefrom is disclosed
in U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996
and International Patent Application Nos. WO 98/24893, published
Jun. 11, 1998 and WO 00/76310, published Dec. 21, 2000, the
disclosures of which are hereby incorporated by reference. See also
Mendez et al. Nature Genetics 15:146-156 (1997), the disclosure of
which is hereby incorporated by reference.
[0164] Through the use of such technology, fully human monoclonal
antibodies to a variety of antigens have been produced.
Essentially, XenoMouse.RTM. lines of mice are immunized with an
antigen of interest (e.g. uPAR), lymphatic cells (such as B-cells)
are recovered from the hyper-immunized mice, and the recovered
lymphocytes are fused with a myeloid-type cell line to prepare
immortal hybridoma cell lines. These hybridoma cell lines are
screened and selected to identify hybridoma cell lines that
produced antibodies specific to the antigen of interest. Provided
herein are methods for the production of multiple hybridoma cell
lines that produce antibodies specific to uPAR. Further, provided
herein are characterization of the antibodies produced by such cell
lines; including nucleotide and amino acid sequence analyses of the
heavy and light chains of such antibodies.
[0165] Alternatively, instead of being fused to myeloma cells to
generate hybridomas, B cells can be directly assayed. For example,
CD19+ B-cells can be isolated from hyperimmune XenoMouse.RTM. mice
and allowed to proliferate and differentiate into
antibody-secreting plasma cells. Antibodies from the cell
supernatants are then screened by ELISA for reactivity against the
uPAR immunogen. The supernatants might also be screened for
immunoreactivity against fragments of uPAR to further map the
different antibodies for binding to domains of functional interest
on uPAR. The antibodies may also be screened other related human
receptors and against the rat, the mouse, and non-human primate,
such as Cynomolgus monkey, orthologues of uPAR, the last to
determine species cross-reactivity. B cells from wells containing,
antibodies of interest may be immortalized by various methods
including fusion to make hybridomas either from individual or from
pooled wells, or by infection with EBV or transfection by known
immortalizing genes and then plating in suitable medium.
Alternatively, single plasma cells secreting antibodies with the
desired specificities are then isolated using a uPAR-specific
hemolytic plaque assay (see for example Babcook et al., Proc. Natl.
Acad. Sci. USA 93:7843-48 (1996)). Cells targeted for lysis are
preferably sheep red blood cells (SRBCs) coated with the uPAR
antigen.
[0166] In the presence of a B-cell culture containing plasma cells
secreting the immunoglobulin of interest and complement, the
formation of a plaque indicates specific uPAR-mediated lysis of the
sheep red blood cells surrounding the plasma cell of interest. The
single antigen-specific plasma cell in the center of the plaque can
be isolated and the genetic information that encodes the
specificity of the antibody is isolated from the single plasma
cell. Using reverse-transcription followed by PCR (RT-PCR), the DNA
encoding the heavy and light chain variable regions of the antibody
can be cloned. Such cloned DNA can then be further inserted into a
suitable expression vector, preferably a vector cassette such as a
pcDNA, more preferably such a pcDNA vector containing the constant
domains of immunoglobulin heavy and light chain. The generated
vector can then be transfected into host cells, e.g., HEK293 cells,
CHO cells, and cultured in conventional nutrient media modified as
appropriate for inducing transcription, selecting transformants, or
amplifying the genes encoding the desired sequences.
[0167] In general, antibodies produced by the fused hybridomas were
human IgG2 heavy chains with fully human kappa or lambda light
chains. Antibodies described herein possess human IgG4 heavy chains
as well as IgG2 heavy chains. Antibodies can also be of other human
isotypes, including IgG1. The antibodies possessed high affinities,
typically possessing a Kd of from about 10.sup.-6 through about
10.sup.-12 M or below, when measured by solid phase and solution
phase techniques. Antibodies possessing a KD of at least 10.sup.-11
M are preferred to inhibit the activity of uPAR.
[0168] As will be appreciated, antibodies can be expressed in cell
lines other than hybridoma cell lines. Sequences encoding
particular antibodies can be used to transform a suitable mammalian
host cell. Transformation can be by any known method for
introducing polynucleotides into a host cell, including, for
example packaging the polynucleotide in a virus (or into a viral
vector) and transducing a host cell with the virus (or vector) or
by transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which
patents are hereby incorporated herein by reference). The
transformation procedure used depends upon the host to be
transformed. Methods for introducing heterologous polynucleotides
into mammalian cells are well known in the art and include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0169] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), human
epithelial kidney 293 cells, and a number of other cell lines. Cell
lines of particular preference are selected through determining
which cell lines have high expression levels and produce antibodies
with constitutive uPAR binding properties.
[0170] Based on the ability of mAbs to significantly neutralize
uPAR activity (as demonstrated in the Examples below), these
antibodies will have therapeutic effects in treating symptoms and
conditions resulting from uPAR expression. In specific embodiments,
the antibodies and methods herein relate to the treatment of
symptoms resulting from uPAR induced cell adhesion, invasion and
intracellular signalling.
[0171] According to another aspect of the invention there is
provided a pharmaceutical composition comprising an antagonist of
the biological activity of uPAR, and a pharmaceutically acceptable
carrier. In one embodiment the antagonist comprises an antibody.
According to another aspect of the invention there is provided a
pharmaceutical composition comprising an antagonist of the
biological activity of uPAR, and a pharmaceutically acceptable
carrier. In one embodiment the antagonist comprises an
antibody.
[0172] Anti-uPAR antibodies are useful in the detection of uPAR in
patient samples and accordingly are useful as diagnostics for
disease states as described herein. In addition, based on their
ability to significantly inhibit uPA activity (as demonstrated in
the Examples below), anti-uPAR antibodies have therapeutic effects
in treating symptoms and conditions resulting from uPAR expression.
In specific embodiments, the antibodies and methods herein relate
to the treatment of symptoms resulting from uPAR induced cell
adhesion, invasion and intracellular signalling. Further
embodiments involve using the antibodies and methods described
herein to treat cell adhesion- and invasion-related diseases
including neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, gastric (stomach) cancer, prostate
cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer,
glioblastoma, endometrial cancer, kidney cancer, colon cancer, and
pancreatic cancer. The antibodies may also be useful in treating
cell adhesion and/or invasion in arthritis, atherosclerosis and
diseases involving angiogenesis.
Therapeutic Administration and Formulations
[0173] Embodiments of the invention include sterile pharmaceutical
formulations of anti-uPAR antibodies that are useful as treatments
for diseases. Such formulations would inhibit the binding of uPA to
its receptor uPAR, thereby effectively treating pathological
conditions where, for example, serum or tissue uPAR is abnormally
elevated. Anti-uPAR antibodies preferably possess adequate affinity
to potently inhibit uPA, and preferably have an adequate duration
of action to allow for infrequent dosing in humans. A prolonged
duration of action will allow for less frequent and more convenient
dosing schedules by alternate parenteral routes such as
subcutaneous or intramuscular injection.
[0174] Sterile formulations can be created, for example, by
filtration through sterile filtration membranes, prior to or
following lyophilization and reconstitution of the antibody. The
antibody ordinarily will be stored in lyophilized form or in
solution. Therapeutic antibody compositions generally are placed
into a container having a sterile access port, for example, an
intravenous solution bag or vial having an adapter that allows
retrieval of the formulation, such as a stopper pierceable by a
hypodermic injection needle.
[0175] The route of antibody administration is in accord with known
methods, e.g., injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intrathecal, inhalation or intralesional routes,
direct injection to a tumor site, or by sustained release systems
as noted below. The antibody is preferably administered
continuously by infusion or by bolus injection.
[0176] An effective amount of antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it is preferred that the therapist titer the
dosage and modify the route of administration as required to obtain
the optimal therapeutic effect. Typically, the clinician will
administer antibody until a dosage is reached that achieves the
desired effect. The progress of this therapy is easily monitored by
conventional assays or by the assays described herein.
[0177] Antibodies, as described herein, can be prepared in a
mixture with a pharmaceutically acceptable carrier. This
therapeutic composition can be administered intravenously or
through the nose or lung, preferably as a liquid or powder aerosol
(lyophilized). The composition may also be administered
parenterally or subcutaneously as desired. When administered
systemically, the therapeutic composition should be sterile,
pyrogen-free and in a parenterally acceptable solution having due
regard for pH, isotonicity, and stability. These conditions are
known to those skilled in the art. Briefly, dosage formulations of
the compounds described herein are prepared for storage or
administration by mixing the compound having the desired degree of
purity with pharmaceutically acceptable carriers, excipients, or
stabilizers. Such materials are non-toxic to the recipients at the
dosages and concentrations employed, and include buffers such as
TRIS HCl, phosphate, citrate, acetate and other organic acid salts;
antioxidants such as ascorbic acid; low molecular weight (less than
about ten residues) peptides such as polyarginine; proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidinone; amino acids such as glycine,
glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as mannitol or sorbitol; counterions
such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS
or polyethyleneglycol.
[0178] Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice as described in
Remington: The Science and Practice of Pharmacy (20.sup.th ed,
Lippincott Williams & Wilkens Publishers (2003)). For example,
dissolution or suspension of the active compound in a
pharmaceutically acceptable carrier such as water or naturally
occurring vegetable oil like sesame, peanut, or cottonseed oil or a
synthetic fatty vehicle like ethyl oleate or the like may be
desired. Buffers, preservatives, antioxidants and the like can be
incorporated according to accepted pharmaceutical practice.
[0179] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
films or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.
Biomed Mater. Res., (1981) 15:167-277 and Langer, Chem. Tech.,
(1982) 12:98-105, or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22:547-556),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the LUPRON
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0180] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated proteins remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for protein stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered
to be intermolecular S--S bond formation through disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0181] Sustained-released compositions also include preparations of
crystals of the antibody suspended in suitable formulations capable
of maintaining crystals in suspension. These preparations when
injected subcutaneously or intraperitonealy can produce a sustained
release effect. Other compositions also include liposomally
entrapped antibodies. Liposomes containing such antibodies are
prepared by methods known per se: U.S. Pat. No. DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692;
Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP
52,322; EP 36,676; EP 88,046; EP 143,949; 142,641; Japanese patent
application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324.
[0182] The dosage of the antibody formulation for a given patient
will be determined by the attending physician taking into
consideration various factors known to modify the action of drugs
including severity and type of disease, body weight, sex, diet,
time and route of administration, other medications and other
relevant clinical factors. Therapeutically effective dosages may be
determined by either in vitro or in viva methods.
[0183] An effective amount of the antibodies, described herein, to
be employed therapeutically will depend, for example, upon the
therapeutic objectives, the route of administration, and the
condition of the patient. Accordingly, it is preferred for the
therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic
effect. A typical daily dosage might range from about 0.001 mg/kg,
0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 10 mg/kg to up to 100 mg/kg, 1000
mg/kg, 10000 mg/kg or more, depending on the factors mentioned
above. Typically, the clinician will administer the therapeutic
antibody until a dosage is reached that achieves the desired
effect. The progress of this therapy is easily monitored by
conventional assays or as described herein.
[0184] It will be appreciated that administration of therapeutic
entities in accordance with the compositions and methods herein
will be administered with suitable carriers, excipients, and other
agents that are incorporated into formulations to provide improved
transfer, delivery, tolerance, and the like. These formulations
include, for example, powders, pastes, ointments, jellies, waxes,
oils, lipids, lipid (cationic or anionic) containing vesicles (such
as Lipofectin.TM.), DNA conjugates, anhydrous absorption pastes,
oil-in-water and water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of various molecular weights), semi-solid
gels, and semi-solid mixtures containing carbowax. Any of the
foregoing mixtures may be appropriate in treatments and therapies
in accordance with the present invention, provided that the active
ingredient in the formulation is not inactivated by the formulation
and the formulation is physiologically compatible and tolerable
with the route of administration. See also Baldrick P.
"Pharmaceutical excipient development: the need for preclinical
guidance." Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.
"Lyophilization and development of solid protein pharmaceuticals."
Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N "Lipids,
lipophilic drugs, and oral drug delivery-some emerging concepts." J
Pharm Sci 89(8):967-78 (2000), Powell et al. "Compendium of
excipients for parenteral formulations" PDA J Pharm Sci Technol.
52:238-311 (1998) and the citations therein for additional
information related to formulations, excipients and carriers well
known to pharmaceutical chemists.
Design and Generation of Other Therapeutics
[0185] In accordance with the present invention and based on the
activity of the antibodies that are produced and characterized
herein with respect to uPAR, the design of other therapeutic
modalities beyond antibody moieties is facilitated. Such modalities
include, without limitation, advanced antibody therapeutics, such
as bispecific antibodies, immunotoxins, and radiolabeled
therapeutics, single domain antibodies, antibody fragments, such as
a Fab, Fab', F(ab').sub.2, Fv or Dab, generation of peptide
therapeutics, uPAR binding domains in novel scaffolds, gene
therapies, particularly intrabodies, antisense therapeutics, and
small molecules.
[0186] In connection with the generation of advanced antibody
therapeutics, where complement fixation is a desirable attribute,
it may be possible to sidestep the dependence on complement for
cell killing through the use of bispecific antibodies,
immunotoxins, or radiolabels, for example.
[0187] Bispecific antibodies can be generated that comprise (i) two
antibodies one with a specificity to uPAR and another to a second
molecule that are conjugated together, (ii) a single antibody that
has one chain specific to uPAR and a second chain specific to a
second molecule, or (iii) a single chain antibody that has
specificity to uPAR and the other molecule. Such bispecific
antibodies can be generated using techniques that are well known;
for example, in connection with (i) and (ii) see e.g., Fanger et
al. Immunol Methods 4:72-81 (1994) and Wright and Harris, supra.
and in connection with (iii) see e.g., Traunecker et al. Int. J.
Cancer (Suppl.) 7:51-52 (1992). In each case, the second
specificity can be made to the heavy chain activation receptors,
including, without limitation, CD16 or CD64 (see e.g., Deo et al.
Immunol. Today 18:127 (1997)) or CD89 (see e.g., Valerius et al.
Blood 90:4485-4492 (1997)).
[0188] In connection with immunotoxins, antibodies can be modified
to act as immunotoxins utilizing techniques that are well, known in
the art. See e.g.; Vitetta Immunol Today 14:252 (1993). See also
U.S. Pat. No. 5,194,594. In connection with the preparation of
radiolabeled antibodies, such modified antibodies can also be
readily prepared utilizing techniques that are well known in the
art. See e.g., Junghans et al. in Cancer Chemotherapy and
Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott
Raven (1996)). See also U.S. Pat. Nos. 4,681,581, 4,735,210,
5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902.
Combinations
[0189] The anti-tumor treatment defined herein may be applied as a
sole therapy or may involve, in addition to the compounds of the
invention, conventional surgery or radiotherapy or chemotherapy.
Such chemotherapy may include one or more of the following
categories of anti tumor agents: [0190] (i) other
antiproliferative/antineoplastic drugs and combinations thereof, as
used in medical oncology, such as alkylating agents (for example
cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen
mustard, melphalan, chlorambucil, busulphan, temozolamide and
nitrosoureas); antimetabolites (for example gemcitabine and
antifolates such as fluoropyrimidines like 5-fluorouracil and
tegafur, raltitrexed, methotrexate, cytosine arabinoside, and
hydroxyurea); antitumor antibiotics (for example anthracyclines
like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,
idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic
agents (for example vinca alkaloids like vincristine, vinblastine,
vindesine and vinorelbine and taxoids like taxol and taxotere and
polokinase inhibitors); and topoisomerase inhibitors (for example
epipodophyllotoxins like etoposide and teniposide, amsacrine,
topotecan and camptothecin); [0191] (ii) cytostatic agents such as
antioestrogens (for example tamoxifen, fulvestrant, toremifene,
raloxifene, droloxifene and iodoxyfene), antiandrogens (for example
bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH
antagonists or LHRH agonists (for example goserelin, leuprorelin
and buserelin), progestogens (for example megestrol acetate),
aromatase inhibitors (for example as anastrozole, letrozole,
vorazole and exemestane) and inhibitors of 5.alpha.-reductase such
as finasteride; [0192] (iii) anti-invasion agents (for example
c-Src kinase family inhibitors like
4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethox-
y]-5-tetrahydropyran-4-yloxyquinazoline (AZD0530; International
Patent. Application WO 01/94341) and
N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-met-
hylpyrimidin-4-ylamino}thiazole-5-carboxamide (dasatinib,
BMS-354825; J. Med. Chem. 2004, 47, 6658-6661), and
metalloproteinase inhibitors like marimastat, other inhibitors of
urokinase plasminogen activator receptor function or, inhibitors of
cathepsins, inhibitors of serine proteases for example matriptase,
hepsin, urokinase, inhibitors of heparanase); [0193] (iv)
inhibitors of growth factor function: for example such inhibitors
include growth factor antibodies and growth factor receptor
antibodies (for example the anti-erbB2 antibody trastuzumab
[Herceptin.TM. ], the anti-EGFR antibody panitumumab, the anti-erbB
antibody cetuximab [Erbitux, C225] and any growth factor or growth
factor receptor antibodies disclosed by Stern et al. Critical
reviews in oncology/haematology, 2005, Vol. 54, pp 11-29); such
inhibitors also include tyrosine kinase inhibitors, for example
inhibitors of the epidermal growth factor family (for example EGFR
family tyrosine kinase inhibitors such as
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-
-amine (gefitinib, ZD1839),
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine
(erlotinib, OSI-774) and
6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazol-
in-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as
lapatinib, inhibitors of the hepatocyte growth factor family,
inhibitors of the platelet-derived growth factor family such as
imatinib, inhibitors of serine/threonine kinases (for example
Ras/Raf signalling inhibitors such as farnesyl transferase
inhibitors, for example sorafenib (BAY 43-9006)), inhibitors of
cell signalling through MEK and/or AKT kinases, inhibitors of the
hepatocyte growth factor family, c-kit inhibitors, abl kinase
inhibitors, IGF receptor (insulin-like growth factor) kinase
inhibitors; aurora kinase inhibitors (for example AZD1152,
PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459)
and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4
inhibitors; [0194] (v) antiangiogenic agents such as those which
inhibit the effects of vascular endothelial growth factor, [for
example the anti-vascular endothelial cell growth factor antibody
bevacizumab (Avastin.TM.) and VEGF receptor tyrosine kinase
inhibitors such as
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)qu-
inazoline (ZD6474; Example 2 within WO 01/32651),
4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-
quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib
(PTK787; WO 98/35985) and SU11248 (sunitinib; WO 01/60814),
compounds such as those disclosed in International Patent
Applications WO97/22596, WO 97/30035, WO 97/32856 and WO 98/13354
and compounds that work by other mechanisms (for example linomide,
inhibitors of integrin .alpha.v.beta.3 function and angiostatin)];
[0195] (vi) vascular damaging agents such as Combretastatin A4 and
compounds disclosed in International Patent Applications WO
99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO
02/08213; [0196] (vii) antisense therapies, for example those which
are directed to the targets listed above, such as ISIS 2503, an
anti-ras antisense; [0197] (viii) gene therapy approaches,
including for example approaches to replace aberrant genes such as
aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed
enzyme pro-drug therapy) approaches such as those using cytosine
deaminase, thymidine kinase or a bacterial nitroreductase enzyme
and approaches to increase patient tolerance to chemotherapy or
radiotherapy such as multi-drug resistance gene therapy; and [0198]
(ix) immunotherapy approaches, including for example ex-vivo and
in-vivo approaches to increase the immunogenicity of patient tumor
cells, such as transfection with cytokines such as interleukin 2,
interleukin 4 or granulocyte-macrophage colony stimulating factor,
approaches to decrease T-cell anergy, approaches using transfected,
immune cells such as cytokine-transfected dendritic cells,
approaches using cytokine-transfected tumor cell lines and
approaches using anti-idiotypic antibodies.
[0199] In one embodiment the anti-tumor treatment defined herein
may involve, in addition to the compounds of the invention,
treatment with other antiproliferative/antineoplastic drugs and
combinations thereof, as used in medical oncology, such as
alkylating agents (for example cis-platin, oxaliplatin,
carboplatin, cyclophosphamide, nitrogen mustard, melphalan,
chlorambucil, busulphan, temozolamide and nitrosoureas);
antimetabolites (for example gemcitabine and antifolates such as
fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,
methotrexate, cytosine arabinoside, and hydroxyurea); antitumor
antibiotics (for example anthracyclines like adriamycin, bleomycin,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin and mithramycin); antimitotic agents (for example
vinca alkaloids like vincristine, vinblastine, vindesine and
vinorelbine and taxoids like taxol and taxotere and polokinase
inhibitors); and topoisomerase inhibitors (for example
epipodophyllotoxins like etoposide and teniposide, amsacrine,
topotecan and camptothecin).
[0200] In one embodiment the anti-tumor treatment defined herein
may involve, in addition to the compounds of the invention,
treatment with gemcitabine.
[0201] Such conjoint treatment may be achieved by way of the
simultaneous, sequential or separate dosing of the individual
components of the treatment. Such combination products employ the
compounds of this invention, or pharmaceutically acceptable salts
thereof, within the dosage range described hereinbefore and the
other pharmaceutically active agent within its approved dosage
range.
EXAMPLES
[0202] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting upon the teachings
herein.
Example 1
Immunization and Titering
Immunization
[0203] Immunizations were conducted using soluble uPAR (recombinant
human uPAR/His6, Cat. #807-UK-100, R&D Systems, Inc.) and
cell-bound uPAR (B300.19 transfectants expressing human uPAR at the
cell surface), respectively. For the generation of a B300.19
transfectant, human full length uPAR cDNA was inserted into the
pcDNA 3 expression vector. B300.19 cells were transiently
transfected via electroporation. Expression of human uPAR on the
cell surface at a level suitable for immunization was confirmed by
Fluorescence-Activated Cell Sorter (FACS) analysis. Ten .mu.g/mouse
for soluble protein for Campaign 1, and 1.times.10.sup.7
cells/mouse of B300.19 transfected cells for Campaign 2, were used
for immunization in XenoMouse.TM. according to the methods
disclosed in U.S. patent application Ser. No. 08/759,620, filed
Dec. 3, 1996 and International Patent Application Nos. WO 98/24893,
published Jun. 11, 1998 and WO 00/76310, published Dec. 21, 2000,
the disclosures of which are hereby incorporated by reference. The
immunization programs are summarized in Table 2.
Selection of Animals for Harvest by Titer
[0204] Titers of the antibody against human uPAR were tested by
FACS. At the end of the immunization program, fusions were
performed using mouse myeloma cells and lymphocytes isolated from
the spleens and lymph nodes of the immunized mice by means of
electroporation, as described in Example 2.
TABLE-US-00002 TABLE 2 Summary of Immunization Programs No of
Immunization Campaign Group Immunogen Strain mice routes 1 1
Soluble uPAR IgG2 10 Fp, twice/wk, .times. (R&D 807-UK) 4 wks 1
2 Soluble uPAR IgG4 10 Fp, twice/wk, .times. (R&D 807-UK) 4 wks
2 3 Cell-bound IgG2 10 Fp, twice/wk, .times. uPAR (B300.19 4 wks
transfectants) 2 4 Cell-bound IgG4 10 Fp, twice/wk, .times.
uPAR(B300.19) 4 wks transfectants) "Fp" refers to "foot pad"
Example 2
Recovery of Lymphocytes, B-Cell Isolations, Fusions and Generation
of Hybridomas
[0205] Immunized mice were sacrificed by cervical dislocation, and
the draining lymph nodes harvested and pooled from each cohort. The
lymphoid cells were dissociated by grinding in DMEM to release the
cells from the tissues and the cells were suspended in DMEM. The
cells were counted, and 0.9 ml DMEM per 100 million lymphocytes
added to the cell pellet to resuspend the cells gently but
completely. Using 100 .mu.l of CD90+ magnetic beads per 100 million
cells, the cells were labeled by incubating the cells with the
magnetic beads at 4.degree. C. for 15 minutes. The magnetically
labeled cell suspension containing up to 10.sup.8 positive cells
(or up to 2.times.10.sup.9 total cells) was loaded onto a LS+
column and the column washed with DMEM. The total effluent was
collected as the CD90-negative fraction (most of these cells were
expected to be B cells).
[0206] The fusion was performed by mixing washed enriched B cells
from above and nonsecretory myeloma P3X63Ag8.653 cells purchased
from ATCC, cat. #CRL 1580 (Kearney et al, J. Immunol. 123, 1979,
1548-1550) at a ratio of 1:1. The cell mixture was gently pelleted
by centrifugation at 800.times.g. After complete removal of the
supernatant, the cells were treated with 2-4 mL of Pronase solution
(CalBiochem, cat. #53702; 0.5 mg/ml in PBS) for no more than 2
minutes. Then 3-5 ml of FBS was added to stop the enzyme activity
and the suspension was adjusted to ml total volume using electro
cell fusion solution, (ECFS, 0.3M Sucrose, Sigma, Cat#S7903, 0.1 mM
Magnesium Acetate, Sigma, Cat#M2545, 0.1 mM Calcium Acetate, Sigma,
Cat#C4705). The supernatant was removed after centrifugation and
the cells were resuspended in 40 ml ECFS. This wash step was
repeated and the cells again were resuspended in ECFS to a
concentration of 2.times.10.sup.6 cells/ml.
[0207] Electro-cell fusion was performed using a fusion generator
(model ECM2001, Genetronic, Inc., San Diego, Calif.). The fusion
chamber size used was 2.0 ml, using the following instrument
settings:
[0208] Alignment condition: voltage: 50 V, time: 50 sec.
[0209] Membrane breaking at: voltage: 3000 V, time: 30 .mu.sec
[0210] Post-fusion holding time: 3 sec
[0211] After ECF, the cell suspensions were carefully removed from
the fusion chamber under sterile conditions and transferred into a
sterile tube containing the same volume of Hybridoma Culture Medium
(DMEM, JRH Biosciences), 15% FBS (Hyclone), supplemented with
L-glutamine, pen/strep, OPI (oxaloacetate, pyruvate, bovine
insulin) (all from Sigma) and IL-6 (Boehringer Mannheim). The cells
were incubated for 15-30 minutes at 37.degree. C., and then
centrifuged at 400.times.g (1000 rpm) for five minutes. The cells
were gently resuspended in a small volume of Hybridoma Selection
Medium (Hybridoma Culture Medium supplemented with 0.5.times.HA
(Sigma, cat. #A9666)), and the volume adjusted appropriately with
more Hybridoma Selection Medium, based on a final plating of
5.times.10.sup.6 B cells total per 96-well plate and 200 .mu.l per
well. The cells were mixed gently and pipetted into 96-well plates
and allowed to grow. On day 7 or 10, one-half the medium was
removed, and the cells re-fed with Hybridoma Selection Medium.
[0212] Hybridomas were grown as routine in the selective medium.
Exhaustive supernatants collected from the hybridomas that
potentially produce anti-human uPAR antibodies were subjected to
subsequent screening assays.
Example 3
Binding to Cell-Bound hu-uPAR
[0213] Binding of the antibodies to cell-bound uPAR was tested with
the murine pre-B cell line B300.19 (ATCC) transfected to express
human uPAR, and B300.19 parental cells as a control, using
Fluorometric Microvolume Assay Technology (FMAT). 5,000 cells/well
were employed for the assay. Mouse anti-human uPAR mAb (R & E
Systems, MAB807) was used as positive control reagent, whereas, G2
KLH supernatant (1:10) was used as negative control reagent. The
bound antibodies were detected using goat anti-human IgG Cy5
conjugate at 400 ng/ml (goat anti-mouse IgG Cy5 conjugate for mouse
antibody control). The positive lines were confirmed by FACS
analysis by detecting the bound antibodies with both goat
anti-human .kappa.-PE or goat anti-human .gamma.-Cy5 conjugates, at
5 .mu.g/ml and 2.5 .mu.g/ml (both were purchased from Jackson
Immune Research), respectively. For each dye stained, the ratio of
the GeoMean values between positive and negative cells were
tabulated. Ratios above 1.95 were considered as hits. The number of
human .gamma. and .kappa. specific hits from each fusion are listed
in Table 3.
TABLE-US-00003 TABLE 3 Number of Antibodies that bound to
cell-bound hu-uPAR as confirmed by FACS analysis No. specific
.gamma./.kappa. Fusions positive lines 1 168 2 37 3 169 4 69 Total
443
Example 4
Competitive Binding
[0214] The ability of the antibodies to inhibit binding of human
uPA to cell-bound uPAR was tested by FACS analysis in this assay.
Briefly, murine fibroblast L929 cells (ATCC) were transfected to
express human uPAR. Transfectants with 99% viability were incubated
with 1:2 dilution of the supernatants at 4.degree. C. overnight.
After washing, the cells were incubated with 500 ng/ml
FITC-labelled tc-uPA (purchased from Molecular Systems) for an
hour. Bound FITC-uPA was detected using rabbit anti-FITC conjugated
with biotin (2.5 .mu.g/ml, purchased from Molecular Probes). To
amplify the signal, a tertiary detection reagent, streptavidin-Cy5
conjugate (SA-Cy5, 2.5 .mu.g/ml, purchased from Jackson immune
Research), was employed. Monoclonal anti-human uPAR antibody MAB807
(R&D Systems, neutralizing receptor-ligand binding) was used as
positive control reagent.
[0215] A total of 433 supernatants from hybridoma lines (binders)
were tested, and 50% of inhibition was considered as positive. The
positive lines (neutralizers) from each fusion are summarized Table
4. Among the four fusions, there are 102 lines in total that were
able to inhibit binding of uPA to uPAR.
TABLE-US-00004 TABLE 4 Number of Abs that inhibited uPA/uPAR
binding Fusion No. tested (binders) No. neutralizers 1 168 63 2 37
11 3 169 17 4 69 11 Total 443 102
Example 5
Cross-Reactivity to Non-Human Primates
[0216] uPAR derived from cynomolgus monkey was cloned and expressed
on the surface of CHOK1 cells. The binding of the antibodies to
cell-bound cynomolgus uPAR (with parental cells as negative
control) was tested by FACS analysis in this assay. A murine
anti-human uPAR antibody MAB807 (R&D Systems) was employed as a
positive control reagent. Briefly, CHOK1/cyno-uPAR transfectants
with 99% viability were incubated with 1:2 dilution of the
supernatants at 4.degree. C. The bound human antibodies were
detected using both goat anti-human .gamma. chain Cy5 conjugate (5
.mu.g/ml) and anti-human K chain PE conjugate (2.5 .mu.g/ml), both
purchased from Jackson Immunology Research. For each dye stained,
the ratio of the GeoMean values between positive and negative cells
were found. The ratios above 1.95 were considered hits. Only the
lines that demonstrated inhibitory activity to uPA/uPAR binding
(neutralizers) were subjected to this assay. As summarized in Table
5, 90 out of 102 lines that were tested showed binding ability to
cyno-uPAR expressed on the surface of CHOK1 cells. The
cross-reactivity to cynomolgus monkey uPAR was confirmed after 30
candidates were cloned and purified, as shown in Example 12
below.
TABLE-US-00005 TABLE 5 Number of Abs that bound to cells expressing
cynomolgus uPAR No. tested No. of .gamma./.kappa. positives Fusion
(neutralizers) (cyno reactive) 1 63 59 2 11 8 3 17 14 4 11 9
Totals: 102 90
Example 6
Kinetic Assays
High Antigen (HA) Quantitation (ELISA)
[0217] ELISA plates were coated with a greater amount of uPAR in
comparison with the Limited Antigen Quantitation assay described
below (4.2 .mu.g/mL). Sample containing antibody (Ab) was titrated
on the uPAR-coated ELISA plates and was incubated overnight to
allow Ab binding to approach equilibrium. Titration of Ab in sample
covered a dilution range of 1:200 to 1:19, 531. A standard curve of
uPAR-specific antibody of known concentration was used to define
the linear range of the assay. Data within the linear range were
used to derive the relative concentration of uPAR-specific Ab in
each titrated sample. The high uPAR concentration and the overnight
incubation limited the effect of Ab affinity, allowing quantitation
of the relative amount of uPAR-specific Ab present in each
sample.
Limited Antigen (LA) Quantitation (ELISA)
[0218] ELISA plates were coated with a lower amount of uPAR in
comparison with the High Antigen Quantitation assay described below
(320, 160, 80, and 20 ng/mL). Samples containing one concentration
of antibody (Ab) (1:25 dilution) were incubated overnight to allow
Ab binding to approach equilibrium. The low antigen concentration
limited the effect of antibody concentration, allowing ranking of
the antibodies based on their relative affinity.
[0219] 102 lines that had the ability to inhibit uPA/uPAR binding
(neutralizers), were tested by the HA/LA kinetics assay. The
results were analyzed, in combination with the efficacy of uPA/uPAR
binding inhibition and FACS-based binding. Table 6 summarizes the
results for the 30 hybridoma lines that had the desired
neutralizing activity and preferred binding kinetics.
Example 7
Specificity Study
[0220] The closest homologue to uPAR is human Ly6e (about 36%
similarity). In this study, all antibodies that cross-reacted to
Ly6e were identified and excluded.
[0221] Human Ly6e (Genbank Accession No. BC119709) was cloned and
expressed in HEK293T cells according to standard protocols. Due to
the lack of the appropriate detection antibody, the authenticity of
the expression of Ly6e was confirmed by probing a myc tag fused on
the molecule, as well as demonstrating the right molecular size of
the expressed protein on a Western Blot using the same anti-myc
antibody.
[0222] The reactivity of the antibodies to the HEK293T transfected
to express Ly6e was determined by FACS analysis, using 1:2
hybridoma supernatants, and probed with goat anti-human .gamma.
chain conjugated with Cy5 (2.5 mg/ml, Jackson Immune Research).
Binding of an anti-myc antibody (10 .mu.g/ml, Invitrogen) to the
transfectants served as a positive control, and binding of this
antibody to the (Ly6e negative) parental cells served as the
negative control. The GeoMean ratio to the positive vs negative at
1.95 was used as a threshold. Readings above 1.95 were considered
as binders. As a result, among 102 neutralizers, only lines 4.67
and 4.50 were found to bind to Ly6e-expression cells. The others
had no cross-reactivity to Ly6e. Table 6 summarizes properties of
30 lines selected for cloning.
TABLE-US-00006 TABLE 6 Summary on the Selection of 30 Lines for
Cellular Cloning Cell-based Efficacy Binding Ab Affinity Ranking
uPA/uPAR gCy5 Geo concentration LA at 80 LA at 20 binding Mean Pos
Cyno Cross Line ID Avg (ug/ml) ng/ml ng/ml Inhibition % Cells
Reactivity 1 1.100 65.95 2.15 0.27 78 521 Yes 2 1.113 25.2 1.8 0.53
75 511 Yes 3 1.159 15.9 2.12 0.33 75 491 Yes 4 1.163 33.87 2.64
0.34 73 410 Yes 5 1.2 13.53 1.89 0.56 74 388 Yes 6 1.20 35.22 1.37
0.2 74 467 Yes 7 1.35 8.61 1.1 0.34 75 497 Yes 8 1.4 9.01 0.66 0.16
69 419 Yes 9 1.41 13.1 1.48 0.27 74 422 Yes 10 1.52 3.7 2 0.27 74
516 Yes 11 1.61 62.64 1.99 0.27 69 559 Yes 12 1.76 13.06 1.47 0.4
75 538 Yes 13 1.85 5.74 1.61 0.26 74 494 Yes 14 2.19 6.88 2.65 0.65
85 445 Yes 15 2.6 2.8 3.58 0.63 80 455 Yes 16 3.104 31.79 0.25 0.16
74 302 Yes 17 3.133 113.62 0.32 0.07 72 440 No 18 3.14 33.27 0.51
0.08 74 317 Yes 19 3.140 68.55 1.6 0.4 69 418 Yes 20 3.167 1230.59
0.28 0.09 74 336 No 21 3.176 22.09 0.96 0.22 76 392 Yes 22 3.179
13.95 0.09 0.07 72 371 Yes 23 3.8 19.2 2.57 0.67 70 451 Yes 24 4.12
48.62 0.21 0.06 71 317 Yes 25 4.13 89.59 0.24 0.09 78 282 Yes 28
4.18 2.44 2.06 0.27 75 251 Yes 27 4.19 4.24 1.03 0.16 77 412 Yes 28
4.50 1.08 0.64 0.21 67 402 Yes 29 4.65 0.46 0.13 0.06 80 431 Yes 30
4.68 33.33 0.11 0.07 78 208 Yes
[0223] Through the primary and secondary screening assays, a total
of 30 hybridoma lines were identified based on their ability to
bind to the cell-bound human uPAR, inhibition of uPA/uPAR binding,
and kinetics ranking, as summarized in Table 6. Among them, two
lines did not bind on cyno-uPAR on the cell surface. None of the
antibodies reacted with murine uPAR and none of the selected lines
reacted with Ly6e, the closest homologue of uPAR.
[0224] These 30 lines were cloned and further characterized as
described below.
Example 8
Plasminogen Activation Assay
[0225] Human histocytic lymphoma cell line U937 (ATCC), which is
known to express uPAR, was employed for this assay. Upon binding to
the cell surface uPAR, single chain urokinase type plasminogen
activator (scuPA) can be activated into uPA, and can cleave
plasminogen to plasmin. The latter cleaves D-Val-Leu-Lys
7-Amido-4-methylcoumarin (VLK-AMC, Sigma catalog number V3138) and
releases the fluorescent AMC dye. Hence, the intensity of
Fluorescence Unit in the assay system indicates the quantity of
bound uPA to cell surface uPAR. The neutralizing mAbs against uPAR
were expected to inhibit binding of scuPA, and thus, suppress the
fluorescence.
[0226] U937 cells were maintained and propagated in RPMI. 1640
medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium
bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate,
and 10% FBS at 37.degree. C. with 5% CO.sub.2. To perform the
assay, U937 cells were seeded at the density of 60,000 cells/well
into a V-bottom plate, and were washed with cold reaction buffer
(0.05 M Tris.HCl, 0.1 M NaCl, pH=7.4, with 2% BSA) by spinning
cells down at 1,500 rpm for 3 min. After resuspension, the cells
were treated with mAbs at indicated final concentrations for one
hour on ice. The treated cell suspensions were spiked with scuPA at
a final concentration of 3 nM. The cell suspensions were incubated
on ice for one hour prior to being washed with cold reaction
buffer. 50 .mu.l of 1 .mu.M Glu-plasminogen and 400 .mu.M VLK-AMC
substrate were added to the cell suspensions whose volumes were
adjusted to 200 .mu.l. The mixtures were transferred to a FMAT
plate for fluorescence determination with a Tecan reader.
[0227] 30 mAbs were initially tested in the plasminogen activation
assay. 15 candidates with higher plasminogen activation inhibitory
activity than others were then selected for further testing. The
dose-response relationship curves for the 15 selected mAbs were
generated by testing their inhibitory activities at various
concentrations, as indicated in FIG. 1. Tests were run in
triplicate for each mAb concentration. A quantitative index, IC50,
for each mAb is included in Table 11.
[0228] It was noticed that there was an obvious gap between the
curves for antibodies 4.19.1 and 1.41.1. The curves on the left of
the gap (including 8 mAbs, namely, 4.18.1, 4.50.2, 4.13.2, 4.12.2,
2.6.1, 1.99.1, 2.19.2, and 4.19.1) had higher efficacy and potency.
They inhibited plasminogen activation at greater than 90% at
concentrations as low as 1 .mu.g/ml. Therefore, these 8 mAbs were
selected for further characterization.
Example 9
Adhesion Assay
[0229] A 96-well microtiter plate was coated with vitronectin (5
.mu.g/ml) in sodium phosphate (pH 9) at 4.degree. C. overnight. The
plate was then blocked with 3% BSA in room temperature for at least
an hour. U937 cells were acid-stripped using 2.5 ml of 50 mM
glycine-HCl and 100 mM NaCl (pH 3.0) on ice for 3 min. The acidic
cell suspension was then neutralized by 0.5 ml of 500 mM HEPES and
100 mM NaCl (pH 7.0).
[0230] The cells were then washed twice with pre-warmed serum-free
medium, and twice again with HBBS containing 1% BSA and free of
Mn.sup.2+, before they were finally resuspended in HBBS at 180,000
cells/100 .mu.L. The acid-stripped cells were then incubated with a
pre-chosen mAb at 10 .mu.g/ml for 1 hour at 4.degree. C. The cells
were subsequently spiked with 25 nM scuPA.
[0231] 50 .mu.l of cells and their pre-chosen mAb were added into
each of the vitronectin-coated wells in the microtiter plate. The
cells were allowed to adhere to the plate at 37.degree. C. for 40
min. Unbound cells were washed off using warm HBSS. The bound cells
were frozen by placing the plate in -80.degree. C. for 1 hour. The
cells were than thawed at room temperature. 100 ml of the CyQuant
dye/lysis buffer were added to each well, and the fluorescence was
read at 485 nm excitation and 530 nm emission.
[0232] The 15 selected mAbs from Example 8 were subjected to the
adhesion assay at 2 .mu.g/ml initially. When 8 mAbs that inhibited
adhesion were found, the dose-response relationship curves for them
were obtained in triplicate, as illustrated in FIG. 2. The
remaining 7 mAbs did not show an obvious inhibitory effect under
the defined experimental conditions.
[0233] Neither human isotype IgGs nor goat IgG control inhibited
adhesion of U937 to vitronectin. Similar to the goat anti-uPAR,
which is a known neutralizer (black thick dot line, FIG. 2), these
inhibitory 8 mAbs inhibited cell adhesion to vitronectin in a
dose-dependent manner. The left-most group of the curves in the
graph is composed of mAb 4.18.1, 2.6.1, 2.19.2, and 4.50.2. The
maximum mAb concentration tested in the assay was 2 .mu.g/ml. At
this concentration, at least mAb 2.19.2 and 2.6.1 reach 90% of
inhibition, and mAb 4.18.1 approaches 90% inhibition of
uPAR-mediated cell adhesion to vitronectin. This experiment was
repeated three times with similar results.
[0234] The correlation of the anti-adhesion activity to other
features of the antibodies is summarized in Table 11.
Example 10
Invasion Assay
[0235] Matrigel.TM. (BD Biosciences Cat#356237 Lot #007024--7.7
mg/ml) was defrosted on ice in a 4.degree. C. refrigerator
overnight. 80 .mu.l of the gel was added to each well in a BD
24-well Transwell Plate with pore size of 8 .mu.m, and incubated at
37.degree. C. for 90 min prior to cell attachment. HT-1080 cells
were re-suspended using cell-dissociation solution (Sigma) at
1.times.10.sup.5 cell/ml in DMEM (0.2% FCS) media. 100 .mu.l
(1.times.10.sup.4 cells) of the cell suspension was aliquoted to
sterile eppendorf tubes and pre-incubated with the mAbs at final
concentrations of 0.1, 1.0, and 10 .mu.g/ml at 37.degree. C. for 1
hour with occasional mixing. Untreated control samples were also
pre-incubated at 37.degree. C. for 1 hour with occasional
mixing.
[0236] 750 .mu.l of DMEM (10% FCS) media supplemented with the mAbs
at 0.1, 1.0, and 10 .mu.g/ml was added to the appropriate wells of
lower transwell chamber. 750 .mu.l of DMEM (10% FCS) media was
added to lower chamber of control samples. The upper chambers
containing solidified Matrigel.TM. were inserted into lower
transwell chambers, and 100 .mu.l (1.times.10.sup.4 cells) of
pre-incubated cell suspension was aliquoted to the upper chamber of
appropriate wells, and the chambers were incubated at 37.degree. C.
for 3 days. The transwells were fixed by immersing in -20.degree.
C. methanol for 20 minutes at room temperature. The transwells were
washed twice in PBS before being stained with Hoechst 33342 (Sigma
catalog number B2261) (10 .mu.M). After staining, the wells were
washed twice with PBS. Using a 20.times. objective, optical images
of cells on top of Matrigel.TM. and invaded cells at sequential 20
.mu.m sections through the Matrigel.TM. were obtained.
Image-ProPlus (Media Cybernetics, Bethesda, Md.) image analysis
software package was employed to calculate cell number at each
section. For each sample, data were presented as the percentage
number of cells present in all sections that had invaded over a
specified distance (60-200 .mu.m).
[0237] The 15 mAbs from Example 8, together with isotype controls
(PK16.3.1 for IgG2, 108.2.1 for IgG4), were tested in the assay at
the mAb concentration of 0.1, 1.0, and 10 .mu.g/ml. The number of
cells invading beyond 60 .mu.m in distance was normalized to the
"fold change over IgG2 and IgG4 isotype control." The three panels
in FIG. 3 represent the data collected with mAb concentration at
0.1, 1.0, and 10 .mu.g/ml, respectively, from top to bottom. Most
of the mAbs tested were found to inhibit cell invasion. Antibodies
2.19.1, 2.6.1 and 4.18.1 had the best efficacy and potency. At a
concentration of 10 .mu.g/ml, mAb 2.19.1 completely inhibited
invasion beyond a 60 .mu.m distance, and both mAbs 2.6.1 and 4.18.1
achieved 90% inhibition. Subsequently, analysis of 2.6.1, 2.19.2
and 4:18.1 was carried out over the concentration range 0.1-100 ng
per ml and invasion was determined by the number of cells invading
through Matrigel.TM. at a depth of 80 .mu.M. As demonstrated in
FIG. 5, mAbs 2.6.1, 2.19.2 and 4.18.1 at a concentration of 100
ng/ml were found to significantly (>70% inhibition) inhibit cell
migration to 80 .mu.m.
Example 11
Determination of Affinity Using Low and High Resolution
Biacore.TM.
Low Resolution Biacore.TM.
[0238] For the low resolution Biacore.TM. tests, a selection of 31
hybridoma cell line products or hybridoma supernatants were tested
for affinity determination. A high-density goat anti-human antibody
surface over a CM5 Biacore.TM. chip was prepared using routine
amine coupling. All mAbs were diluted to approximately 6 .mu.g/mL
in HBS-P running buffer (10 mM HEPES [pH 7.4], 150 nM NaCl, 0.005%
surfactant P20) containing 100 .mu.g/ml BSA. Each mAb was captured
on a separate flow cell using a 30-second contact time at 10
.mu.L/min followed by a 5-minute wash at a 100 .mu.L/min flow rate
for stabilization of the mAb baseline. Next, hu-uPAR (R&D
Systems, Lot ALH01402A) was injected at 312 nM (23.degree. C.) over
all surfaces for 90 sec., followed by a 5-minute dissociation (100
.mu.L/min flow rate). Four-mAbs were re-analyzed with a 4-minute
hu-uPAR injection (312 nM, 50 .mu.L/min flow rate). All antigen
samples were prepared in the running buffer. The surfaces were
regenerated after every capture/injection cycle with one 15-second
pulse of 146 mM phosphoric acid (pH 1.5).
[0239] The baseline drift of a buffer injection prior to each
antigen injection was subtracted for each cycle.
[0240] Data were fit globally to a 1:1 interaction model using
CLAMP (David G. Myszka and Thomas Morton (1998) "CLAMP.COPYRGT.: a
biosensor kinetic data analysis program," TIBS 23, 149-150) to
determine the binding kinetics. A mass transport coefficient was
used in fitting the data. The kinetic analysis results are listed
in Table 7a. The mAb supernatants used in the analysis are also
indicated. MAbs are ranked from high to low affinity.
TABLE-US-00007 TABLE 7a Affinity Determination Results for the
Hybridoma Lines Derived from Low Resolution Biacore .TM. Amt.
Captured Sample (RU) k.sub.a (M.sup.-1s.sup.-1) k.sub.d (s.sup.-1)
K.sub.D (nM) 2.19 299 1.4 .times. 10.sup.5 4.3 .times. 10.sup.-5
0.31 2.6 290 8.1 .times. 10.sup.4 4.6 .times. 10.sup.-5 0.57 3.29
249 2.7 .times. 10.sup.4 3.1 .times. 10.sup.-5 1.1 4.18 384 1.1
.times. 10.sup.5 1.6 .times. 10.sup.-4 1.5 4.50 298 1.2 .times.
10.sup.5 2.6 .times. 10.sup.-4 2.2 1.100 566 7.5 .times. 10.sup.4
1.7 .times. 10.sup.-4 2.3 4.65 291 1.1 .times. 10.sup.5 2.5 .times.
10.sup.-4 2.3 1.61 459 9.4 .times. 10.sup.4 2.5 .times. 10.sup.-4
2.7 4.19 227 1.2 .times. 10.sup.5 3.2 .times. 10.sup.-4 2.7 3.140
443 7.0 .times. 10.sup.4 2.1 .times. 10.sup.-4 3.0 3.8 427 2.2
.times. 10.sup.4 6.8 .times. 10.sup.-5 3.1 3.104 228 8.7 .times.
10.sup.4 3.0 .times. 10.sup.-4 3.4 1.99 509 1.2 .times. 10.sup.5
4.2 .times. 10.sup.-4 3.5 3.14 274 9.8 .times. 10.sup.4 3.4 .times.
10.sup.-4 3.5 1.35 546 3.8 .times. 10.sup.4 1.5 .times. 10.sup.-4
3.9 1.41 718 3.3 .times. 10.sup.4 1.3 .times. 10.sup.-4 3.9
3.176(supe) 239 5.3 .times. 10.sup.4 2.3 .times. 10.sup.-4 4.3
3.117 (supe) 459 9.8 .times. 10.sup.4 4.3 .times. 10.sup.-4 4.4
1.113 694 2.8 .times. 10.sup.4 1.3 .times. 10.sup.-4 4.6 1.85 434
3.0 .times. 10.sup.4 1.4 .times. 10.sup.-4 4.7 4.68 209 5.3 .times.
10.sup.4 2.9 .times. 10.sup.-4 5.5 1.20 224 7.7 .times. 10.sup.4
4.5 .times. 10.sup.-4 5.8 1.163 662 7.4 .times. 10.sup.3 4.6
.times. 10.sup.-5 6.2 1.2 (supe) 464 1.7 .times. 10.sup.4 1.3
.times. 10.sup.-4 7.6 1.4 321 1.1 .times. 10.sup.5 9.6 .times.
10.sup.-4 8.7 1.159 975 1.6 .times. 10.sup.4 1.4 .times. 10.sup.-4
8.7 4.13 161 9.5 .times. 10.sup.4 1.2 .times. 10.sup.-3 12.6 4.12
154 6.3 .times. 10.sup.4 1.3 .times. 10.sup.-3 20.6 3.133 324 9.5
.times. 10.sup.4 3.6 .times. 10.sup.-3 37.9 3.179 308 5.4 .times.
10.sup.4 2.2 .times. 10.sup.-3 40.7 3.167 414 6.6 .times. 10.sup.4
3.9 .times. 10.sup.-3 59.1
[0241] Most Abs fit a 1:1 model very well. The off-rate data for
mAbs 3.133 and 3.167 showed some complexity, but the kinetic
parameters are acceptable estimates. The longer 4-minute injection
time in the analysis of mAb 1.163 still did not produce significant
curvature in the on-rate data because the association rate was slow
(on the order of 10.sup.3 M.sup.-1s.sup.-1).
High Resolution Biacore.TM.
[0242] Considering that most mAbs fit a 1:1 model very well, the
affinity ranking derived from the line product low resolution
Biacore.TM. was believed to be reliable. The Kd determination using
high resolution Biacore.TM. with three (3) cloned and purified mAbs
was conducted in separate experiments.
[0243] Each of three purified mAbs (2.19.2, 2.6.1 and 4.18.1) were
amine coupled on a different flow cell surface of a CM5 Biacore.TM.
chip and tested for their binding affinity to human uPAR. All mAbs
were diluted into 10 mM sodium acetate, pH 4.0 for immobilization.
The running buffer and sample preparation buffer for all
experiments were degassed HBS-P (10 mM HEPES [pH 7.4], 150 nM NaCl,
0.005% surfactant P20) containing 100 .mu.g/mL BSA. All experiments
were run at 23.degree. C. with a flow rate of 100 .mu.L/min.
Serially diluted (2-fold) uPAR samples were randomly injected in
triplicate for 90 seconds with several buffer injections
interspersed for double referencing. A Biacore.TM. 2000 biosensor
instrument was used for all high resolution experiments. Data were
fit globally to a 1:1 interaction model with a term for mass
transport included. The resulting K.sub.D values are summarized in
Table 7b.
TABLE-US-00008 TABLE 7b Affinity Determination Results for Cloned
and Purified mAbs Derived from High Resolution Biacore .TM. mAb
K.sub.D (pM) 2.19.2 175 2.6.1 249 4.18.1 446
[0244] The Kd values for antibodies 2.19.2, 2.6.1, and 4.18.1 were
175 pM, 249 pM, and 446 pM, respectively. The Kd ranking of each
antibody with high resolution Biacore.TM. was consistent with that
obtained via low resolution Biacore.TM..
Example 12
Cross-Reactivity to Mouse and Monkey uPAR
[0245] For determination of the mouse cross-reactivity, the mAb's
binding to human uPAR expressed on the surface of U937 cells was
detected in a competition assay using excess soluble mouse uPAR. In
the assay, excess amounts of the recombinant mouse uPAR were
preincubated with the mAbs before the mAbs were exposed to the
cells expressing uPAR. If a mAb cross-reacted with the mouse uPAR,
the preincubation would mask the mAb, thus, it would not bind to
human uPAR.
[0246] A mouse uPAR/human IgG Fc chimera (uPARHuIgG) (R&D
Systems, catalog number 531-PA-100) was used for the mouse uPAR. 50
.mu.l of each mAb at 1 .mu.g/mL was mixed with equal volume of the
mouse uPARHuIgG 40 .mu.g/mL. The final concentrations of the mAbs
and mouse uPAR were 500 ng/mL and 20 .mu.g/mL, respectively
(Approx. M, W, of Mo-uPARHuIg is 66-90 Kd). The mixture was in FACS
buffer (PBS supplemented with 1% BSA and 0.05% NaN.sub.3), and was
incubated for 30 min. Subsequently, 20,000 U397 cells were added
into the mixture for an additional 30 minute incubation on ice. At
the end of incubation, unbound mAbs were intensively eliminated by
washing five times using FACS buffer. The bound mAbs were detected
with Cy5 (Cy5-bis-OSU,
N,N'-biscarboxypentyl-5,5'-disulfonatoindodicarbocyanine, Amersham
Life Science, catalog No. PA15000)-labeled goat anti-Hu IgG Fc (5.0
.mu.g/mL) and 7AAD (5.0 .mu.g/mL) using a FACSCalibur flow
cytometer (BD Biosciences, San Jose, Calif.).
[0247] The results showed that the excess amounts of soluble
recombinant mouse uPAR did not block the binding of any mAb to
cell-bound human uPAR (data not shown), indicating that none of the
mAbs cross-reacted to the mouse uPAR. To make the mouse
cross-reactivity results conclusive, direct binding of the mAbs to
cell-bound mouse uPAR was measured using FACS analysis; The mouse
cell line derived from the connective tissue, NCTC L929, was
employed for FACS analysis. The harvested cells were resuspended at
50,000 cells/well using FACS buffer into a V-bottom plate which was
put on ice. The uPAR mAbs at the final concentration of 6.25
.mu.g/ml were added into the wells, and incubated for 1 hour on
ice. After washing, Cy5-labeled goat anti-human IgG Fc at 5
.mu.g/ml and 7AAD at 5 .mu.g/ml was added. The mixtures were
incubated for 15 min on ice, washed and resuspended in cold FACS
buffer for FACS analysis.
[0248] Table 8 summarizes the FACS results. The rat anti-uPAR,
which cross-reacts with mouse antigen, generated a great shift with
GeoMean of 248.07. In contrast, the GeoMean values for the mouse
anti-uPAR mAb (lacks reactivity to mouse antigen), isotype controls
from the rat, mouse, and human, together with the secondary
antibodies for detection, ranged between 3.14 and 13.07. All the
fully human uPAR mAbs showed a GeoMean between 9 and 12, comparable
to the background seen in the above negative controls. It was
therefore concluded that none of the mAbs bound to the cell surface
mouse uPAR.
TABLE-US-00009 TABLE 8 Summary of the FACS Analysis Results. Sample
X Geo X Geo Sample ID Mean Controls mean 1 1.100.1 9.89 rat
anti-uPAR 248.07 2 1.99.1 11.65 Mo anti-uPAR 3.14 4 4.19.1 9.59 Hu
IgG2 8.88 isotype 5 4.50.2 8.92 Hu IgG4 8.5 isotype 6 4.12.2 8.97
Hu 2' 7.63 7 4.18.1 8.18 rat isotype 12.31 12 2.19.2 9.55 rat 2'
13.07 13 2.6.1 12.95 Mo isotype 3.31 14 1.61.1 9.7 Mo 2' 3.22 15
4.13.2 11.24 17 1.4.2 12.38 24 1.113.2 10.19 26 1.41.1 9.59 29
1.2.3.3 9.89 30 3.176.2.2 8.36
[0249] Prior to the cloning, two out of the 30 selected hybridoma
lines, (3.1.33 and 3.167) were shown to lack cross-reactivity to
the monkey uPAR. After cloning, the cross-reactivity to the monkey
uPAR expressed recombinantly on the surface of 293T cells was
confirmed using FACS analysis. The FACS analysis results are
summarized in FIG. 4.
[0250] In comparison to the negative control antibodies, all the
anti-uPAR mAbs bound to the cell-bound monkey uPAR, although the
GeoMean values varied widely. Weak binders included mAbs 1.159.1,
1.163.1, 3.14.1 and mAb 1.100.1. Mabs 2.19.2, 2.6.1, and 4.18.1
were found to cross-react to the monkey antigen very strongly (FIG.
4).
Example 13
Cynomolgus Affinity
[0251] Kinetic measurements of several of the antibodies were
evaluated using the KinExA.RTM. method. This method involves
solution-based determination of formal affinity measurements at
equilibrium. Recombinant human uPAR (amino acids 1-303) purchased
from R&D systems was coupled to NHS-Sepharose beads. The
remaining active groups on the beads were blocked with 10 mg/ml BSA
in 1 M Tris and stored in the blocking solution as recommended by
the manufacturer. KinExA experiments were performed using an
automated flow immunoassay system, KinExA 3000, in which beads
coupled with human uPAR served as the solid phase.
[0252] The nucleotide sequence shown as SEQ ID NO: 81 encodes the
extracellular domain (ECD) of the cynomolgus protein with an
in-frame His tag at the C-terminus (SEQ ID NO: 82). The cynomolgus
uPAR was produced by transient transfection of an expression vector
containing this nucleotide sequence in HEK293 cells using the
Freestyle expression system (Invitrogen) of protein production as
per manufacturer's recommendations. The conditioned media from this
transient transfection was used for the affinity determination as
described above.
[0253] Briefly, a constant amount of 2.6.1 or 2.19.2 anti-uPAR
antibody (5 nM or 500 pM) was incubated with titrating
concentrations of conditioned media containing recombinant
cynomolgus uPAR ECD starting at approximately 625 nM.
Antigen/antibody complexes were incubated at room temperature for
48 hrs to 168 hrs to allow equilibrium to be reached. The mixture
was drawn through the human uPAR-coupled beads to accumulate
unbound antibody. The captured antibody was detected using
solutions containing a Cy5-conjugated goat anti-human IgG heavy and
light chain specific secondary antibody in sample buffer. The bound
signals were converted into relative values as a proportion of
control in the absence of hIL-13. Three replicates of each sample
were measured for all equilibrium experiments. The results are
summarized in Table 9 below. The equilibrium dissociation constant
(K.sub.D) was obtained from nonlinear regression analysis of the
data using a one-site homogeneous binding model with an unknown
ligand concentration contained within the KinExA software. The
software calculates the K.sub.D and determines the 95% confidence
interval by fitting the data points to a theoretical K.sub.D curve.
The 95% confidence interval is given as K.sub.D low and K.sub.D
high.
TABLE-US-00010 TABLE 9 Summary of KinExA Results for Cynomolgus
Binding Activity Antibody K.sub.D K.sub.D low K.sub.D high 2.6.1
1.09 nM 478 pM 2.46 nM 2.19.2 346 pM 234 pM 508 pM
Example 14
Internalization Assay
[0254] Eight of the mAbs from Example 8 were tested for the ability
to induce uPAR internalization using U937 cells. A cell suspension
containing 100,000 cells was pipetted into each well of a V-bottom
plates in duplicate. Two V-bottom plates were prepared, one to be
kept at 37.degree. C. during internalization with antibody complex,
and the other to be incubated at 4.degree. C. Each of the eight
uPAR mAbs was incubated with U937 cells at 5 .mu.g/ml on ice for 15
min. Incubation on ice was necessary to prevent internalization of
the primary mAbs. The cells were then washed with cold FACS buffer.
Cells were subsequently incubated on ice in 100 .mu.l of 5 .mu.g/ml
alexa647-labelled goat anti-humanF(ab) (Invitrogen catalogue number
A21249) in cold FACS buffer for 15 min. This allowed the goat FAB
bind to the bound uPAR mAbs without permitting any internalization.
After a 15 minute incubation on ice, one plate was kept on ice,
while the other plate was incubated at 37.degree. C. for 1
hour.
[0255] Following the 1 hr incubation with the mAb-FAB complex, the
cells were washed twice with cold 5% FACS buffer, and 200 .mu.l of
Tris(2-carboxyethyl)phosphine HCl (TCEP) was added to each well as
a stripping agent. The plates were both incubated with TCEP on ice
for 1 hour, followed by washing and resuspending with FACS buffer.
4 .mu.l of 7-Amino-Actinomycin D (7AAD) was added to each well,
followed by 10 min incubation on ice before FACS analysis was
conducted.
[0256] The percentage of internalization was calculated using the
following equation:
[(37 strip-4 strip)/(4 no strip-4 strip)].times.100
[0257] where `37 strip` is the geo mean at 37.degree. C. with
TCEP;
[0258] `4 strip` is the geo mean on ice with TCEP;
[0259] `4 no strip` is the geo mean on ice without TCEP (see Table
10).
TABLE-US-00011 TABLE 10 Summary of the Internalization Assay geo
mean geo mean geo mean cold + cold - 37.degree. C. + % Antibody
TCEP TCEP TCEP Internalized 2.19.2 2 42.67 3.54 3.7 4.50.2 2.01
42.12 3.2 2.9 4.12.2 2 33.35 3.02 3.2 1.99.1 1.95 42.49 2.69 1.8
2.6.1 2.07 44.34 3.02 2.2 4.13.2 1.95 37.42 2.95 2.8 4.19.1 2.34
45.95 5.31 6.8 4.18.1 2.05 41.2 3.25 3.0
[0260] As summarized in Table 10, very little, if any
internalization was detected after 1 hour incubation at 37.degree.
C. The experiment was repeated twice with similar results.
[0261] Table 11 summarizes the results of the in vitro
characterization for the 15 candidates from Example 8.
TABLE-US-00012 TABLE 11 Summary of the in vitro Characterization
Assay Results for Fifteen uPAR mAbs. Human Plasmin- Inva- uPAR/
ogen sion Ab to uPA activa- Low High inhibi- cell binding tion
Resolu- Resolu- tion Anti- bound inhibi- inhibi- Cyno Murine tion
tion Vitro- (Po- body Ag (line tion tion X- X- Affin- Affin- nectin
tency (Clone material, (%, line (IC50, reac- reac- ity (KD, ity
(Kd, adhe- Rank- no.) FACS) material) ug/ml) tivity tivity nM) nM)
sion ing) 2.19.2 445 85 (1) 0.053(1) Y N 0.31(1) 0.175(1) +(1) Y(1)
2.6.1 455 80 (2) 0.073(4) Y N 0.57(2) 0.249(2) +(4) Y(2) 4.18.1 251
75 (7) 0.067(3) Y N 1.5(3) 0.446(3) +(3) Y(3) 1.99.1 514 78 (3)
0.066(2) Y N 3.5 ND - Y 4.13.2 28.2 78 (4) 0.077(5) Y N 12.6 ND + Y
4.50.2 402 67 0.104 Y N 2.2 ND +(2) Y 4.12.2 317 71 0.067 Y N 20.6
ND - Y 4.19.1 412 77 0.056 Y N 2.7 ND + Y 1.41.1 422 74 0.181 Y N
3.9 ND + Y 3.8.3 451 70 0.328 Y N 3.1 ND - Y 1.4.2 419 69 0.075 Y N
8.7 ND - Y 1.61.1 559 69 0.119 Y N 2.7 ND - Y 1.100.1 521 78 0.061
Y N 2.3 ND - Y 3.176.2 392 76 0.270 Y N 4.3 ND - Y 1.113.2 511 75
0.775 Y N 4.6 ND - Y *The numbers in the parentheses indicate
ranking order for each category.
Example 15
Structural Analysis of uPAR Antibodies
[0262] The variable heavy chains and the variable light chains of
the antibodies were sequenced to determine their DNA sequences. The
complete sequence information for the anti-uPAR antibodies is
provided in the sequence listing with nucleotide and amino acid
sequences for each gamma and kappa chain combination. The variable
heavy sequences were analyzed to determine the VH family, the
D-region sequence and the J-region sequence. The sequences were
then translated to determine the primary amino acid sequence and
compared to the germline VH, D and J-region sequences to assess
somatic hypermutations.
[0263] Table 18 is a table comparing the antibody heavy chain
regions to their cognate germ line heavy chain region. Table 19 is
a table comparing the antibody kappa light chain regions to their
cognate germ line light chain region.
[0264] The variable (V) regions of immunoglobulin chains are
encoded by multiple germ line DNA segments, which are joined into
functional variable regions (V.sub.HDJ.sub.H or V.sub.KJ.sub.K)
during B-cell ontogeny. The molecular and genetic diversity of the
antibody response to uPAR was studied in detail. These assays
revealed several points specific to anti-uPAR antibodies.
[0265] It should be appreciated that amino acid sequences among the
sister clones collected from each hybridoma are identical. As an
example, the heavy chain and light chain sequences for mAb 2.19.3
would be identical to the sequences for mAbs 2.19 and 2.19.1.
[0266] According the sequencing data, the primary structure of the
heavy chains of 2.6.1 and 4.18.1 are similar, but not identical.
2.19.2 is structurally different from the other two.
[0267] It should also be appreciated that where a particular
antibody differs from its respective germline sequence at the amino
acid level, the antibody sequence can be mutated back to the
germline sequence. Such corrective mutations can occur at one, two,
three or more positions, or a combination of any of the mutated
positions, using standard molecular biological techniques. By way
of non-limiting example, Table 19 shows that the light chain
sequence of mAb 4.18.1 (SEQ ID NO.: 52) differs from the
corresponding germline sequence (SEQ ID NO.:66) through a Ser to
Phe mutation (mutation 1) in the FR1 region, a Val to Met mutation
(mutation 2) in the FR1 region, a Lys to Thr mutation (mutation 3)
in the FR2 region, a Ile to Phe mutation (mutation 4) in the FR3
region and a Ser to Ile mutation (mutation 5) in the FR3 region.
Thus, the amino acid or nucleotide sequence encoding the light
chain of mAb 4.18.1 can be modified to change mutation 1 to yield
the germline sequence at the site of mutation 1. Further, the amino
acid or nucleotide sequence encoding the light chain of mAb 4.18.1
can be modified to change mutation 2 or mutation 3 to yield the
germline sequence at the site of mutation 2 or mutation 3. Still
further, the amino acid or nucleotide sequence encoding the light
chain of mAb 4.18.1 can be modified to change both mutation I and
mutation 2, or any other combination of two or more mutations to
yield the germline sequence at those particular sites. Tables 12-17
below illustrate the position of such variations from the germline
for mAb 2.19.2, 2.6.1 and 4.18.1. Each row represents a unique
combination of germline and non-germline residues at the position
indicated by bold type.
TABLE-US-00013 TABLE 12 Exemplary Mutations of mAb 2.19.2 Light
Chain (SEQ ID NO: 32) to Germline at the Indicated Residue Number.
12 32 36 38 100 101 103 A S K Y DELETED T P A S K Y DELETED T W A S
K Y DELETED F P A S K Y DELETED F W A S K Y I T P A S K Y I T W A S
K Y I F P A S K Y I F W A S K S DELETED T P A S K S DELETED T W A S
K S DELETED F P A S K S DELETED F W A S K S I T P A S K S I T W A S
K S I F P A S K S I F W A S R Y DELETED T P A S R Y DELETED T W A S
R Y DELETED F P A S R Y DELETED F W A S R Y I T P A S R Y I T W A S
R Y I F P A S R Y I F W A S R S DELETED T P A S R S DELETED T W A S
R S DELETED F P A S R S DELETED F W A S R S I T P A S R S I T W A S
R S I F P A S R S I F W A G K Y DELETED T P A G K Y DELETED T W A G
K Y DELETED F P A G K Y DELETED F W A G K Y I T P A G K Y I T W A G
K Y I F P A G K Y I F W A G K S DELETED T P A G K S DELETED T W A G
K S DELETED F P A G K S DELETED F W A G K S I T P A G K S I T W A G
K S I F P A G K S I F W A G R Y DELETED T P A G R Y DELETED T W A G
R Y DELETED F P A G R Y DELETED F W A G R Y I T P A G R Y I T W A G
R Y I F P A G R Y I F W A G R S DELETED T P A G R S DELETED T W A G
R S DELETED F P A G R S DELETED F W A G R S I T P A G R S I T W A G
R S I F P A G R S I F W S S K Y DELETED T P S S K Y DELETED T W S S
K Y DELETED F P S S K Y DELETED F W S S K Y I T P S S K Y I T W S S
K Y I F P S S K Y I F W S S K S DELETED T P S S K S DELETED T W S S
K S DELETED F P S S K S DELETED F W S S K S I T P S S K S I T W S S
K S I F P S S K S I F W S S R Y DELETED T P S S R Y DELETED T W S S
R Y DELETED F P S S R Y DELETED F W S S R Y I T P S S R Y I T W S S
R Y I F P S S R Y I F W S S R S DELETED T P S S R S DELETED T W S S
R S DELETED F P S S R S DELETED F W S S R S I T P S S R S I T W S S
R S I F P S S R S I F W S G K Y DELETED T P S G K Y DELETED T W S G
K Y DELETED F P S G K Y DELETED F W S G K Y I T P S G K Y I T W S G
K Y I F P S G K Y I F W S G K S DELETED T P S G K S DELETED T W S G
K S DELETED F P S G K S DELETED F W S G K S I T P S G K S I T W S G
K S I F P S G K S I F W S G R Y DELETED T P S G R Y DELETED T W S G
R Y DELETED F P S G R Y DELETED F W S G R Y I T P S G R Y I T W S G
R Y I F P S G R Y I F W S G R S DELETED T P S G R S DELETED T W S G
R S DELETED F P S G R S DELETED F W S G R S I T P S G R S I T W S G
R S I F P S G R S I F W
TABLE-US-00014 TABLE 13 Exemplary Mutations of mAb 2.19.2 Heavy
Chain (SEQ ID NO: 30) to Germline at the Indicated Residue Number.
99-100 DQ DELETED
TABLE-US-00015 TABLE 14 Exemplary Mutations of mAb 2.6.1 Light
Chain (SEQ ID NO: 28) to Germline at the Indicated Residue Number.
95-96 103 PL K PL R DELETED K DELETED R
TABLE-US-00016 TABLE 15 Exemplary Mutations of mAb 2.6.1 Heavy
Chain (SEQ ID NO: 26) to Germline at the Indicated Residue Number.
30 31 35 50 53 58 69 81 87 98 S S S V S Y I Q A DELETED S S S V S Y
I Q A E S S S V S Y I Q V DELETED S S S V S Y I Q V E S S S V S Y I
H A DELETED S S S V S Y I H A E S S S V S Y I H V DELETED S S S V S
Y I H V E S S S V S Y V Q A DELETED S S S V S Y V Q A E S S S V S Y
V Q V DELETED S S S V S Y V Q V E S S S V S Y V H A DELETED S S S V
S Y V H A E S S S V S Y V H V DELETED S S S V S Y V H V E S S S V S
S I Q A DELETED S S S V S S I Q A E S S S V S S I Q V DELETED S S S
V S S I Q V E S S S V S S I H A DELETED S S S V S S I H A E S S S V
S S I H V DELETED S S S V S S I H V E S S S V S S V Q A DELETED S S
S V S S V Q A E S S S V S S V Q V DELETED S S S V S S V Q V E S S S
V S S V H A DELETED S S S V S S V H A E S S S V S S V H V DELETED S
S S V S S V H V E S S S V T Y I Q A DELETED S S S V T Y I Q A E S S
S V T Y I Q V DELETED S S S V T Y I Q V E S S S V T Y I H A DELETED
S S S V T Y I H A E S S S V T Y I H V DELETED S S S V T Y I H V E S
S S V T Y V Q A DELETED S S S V T Y V Q A E S S S V T Y V Q V
DELETED S S S V T Y V Q V E S S S V T Y V H A DELETED S S S V T Y V
H A E S S S V T Y V H V DELETED S S S V T Y V H V E S S S V T S I Q
A DELETED S S S V T S I Q A E S S S V T S I Q V DELETED S S S V T S
I Q V E S S S V T S I H A DELETED S S S V T S I H A E S S S V T S I
H V DELETED S S S V T S I H V E S S S V T S V Q A DELETED S S S V T
S V Q A E S S S V T S V Q V DELETED S S S V T S V Q V E S S S V T S
V H A DELETED S S S V T S V H A E S S S V T S V H V DELETED S S S V
T S V H V E S S S I S Y I Q A DELETED S S S I S Y I Q A E S S S I S
Y I Q V DELETED S S S I S Y I Q V E S S S I S Y I H A DELETED S S S
I S Y I H A E S S S I S Y I H V DELETED S S S I S Y I H V E S S S I
S Y V Q A DELETED S S S I S Y V Q A E S S S I S Y V Q V DELETED S S
S I S Y V Q V E S S S I S Y V H A DELETED S S S I S Y V H A E S S S
I S Y V H V DELETED S S S I S Y V H V E S S S I S S I Q A DELETED S
S S I S S I Q A E S S S I S S I Q V DELETED S S S I S S I Q V E S S
S I S S I H A DELETED S S S I S S I H A E S S S I S S I H V DELETED
S S S I S S I H V E S S S I S S V Q A DELETED S S S I S S V Q A E S
S S I S S V Q V DELETED S S S I S S V Q V E S S S I S S V H A
DELETED S S S I S S V H A E S S S I S S V H V DELETED S S S I S S V
H V E S S S I T Y I Q A DELETED S S S I T Y I Q A E S S S I T Y I Q
V DELETED S S S I T Y I Q V E S S S I T Y I H A DELETED S S S I T Y
I H A E S S S I T Y I H V DELETED S S S I T Y I H V E S S S I T Y V
Q A DELETED S S S I T Y V Q A E S S S I T Y V Q V DELETED S S S I T
Y V Q V E S S S I T Y V H A DELETED S S S I T Y V H A E S S S I T Y
V H V DELETED S S S I T Y V H V E S S S I T S I Q A DELETED S S S I
T S I Q A E S S S I T S I Q V DELETED S S S I T S I Q V E S S S I T
S I H A DELETED S S S I T S I H A E S S S I T S I H V DELETED S S S
I T S I H V E S S S I T S V Q A DELETED S S S I T S V Q A E S S S I
T S V Q V DELETED S S S I T S V Q V E S S S I T S V H A DELETED S S
S I T S V H A E S S S I T S V H V DELETED S S S I T S V H V E S S N
V S Y I Q A DELETED S S N V S Y I Q A E S S N V S Y I Q V DELETED S
S N V S Y I Q V E S S N V S Y I H A DELETED S S N V S Y I H A E S S
N V S Y I H V DELETED S S N V S Y I H V E S S N V S Y V Q A DELETED
S S N V S Y V Q A E S S N V S Y V Q V DELETED S S N V S Y V Q V E S
S N V S Y V H A DELETED S S N V S Y V H A E S S N V S Y V H V
DELETED S S N V S Y V H V E S S N V S S I Q A DELETED S S N V S S I
Q A E S S N V S S I Q V DELETED S S N V S S I Q V E S S N V S S I H
A DELETED S S N V S S I H A E S S N V S S I H V DELETED S S N V S S
I H V E S S N V S S V Q A DELETED S S N V S S V Q A E S S N V S S V
Q V DELETED S S N V S S V Q V E S S N V S S V H A DELETED S S N V S
S V H A E S S N V S S V H V DELETED S S N V S S V H V E S S N V T Y
I Q A DELETED S S N V T Y I Q A E S S N V T Y I Q V DELETED S S N V
T Y I Q V E S S N V T Y I H A DELETED S S N V T Y I H A E S S N V T
Y I H V DELETED S S N V T Y I H V E S S N V T Y V Q A DELETED S S N
V T Y V Q A E S S N V T Y V Q V DELETED S S N V T Y V Q V E S S N V
T Y V H A DELETED S S N V T Y V H A E S S N V T Y V H V DELETED S S
N V T Y V H V E S S N V T S I Q A DELETED S S N V T S I Q A E S S N
V T S I Q V DELETED S S N V T S I Q V E S S N V T S I H A DELETED S
S N V T S I H A E S S N V T S I H V DELETED S S N V T S I H V E S S
N V T S V Q A DELETED S S N V T S V Q A E S S N V T S V Q V DELETED
S S N V T S V Q V E S S N V T S V H A DELETED S S N V T S V H A E S
S N V T S V H V DELETED S S N V T S V H V E S S N I S Y I Q A
DELETED S S N I S Y I Q A E S S N I S Y I Q V DELETED S S N I S Y I
Q V E S S N I S Y I H A DELETED S S N I S Y I H A E S S N I S Y I H
V DELETED S S N I S Y I H V E S S N I S Y V Q A DELETED S S N I S Y
V Q A E S S N I S Y V Q V DELETED S S N I S Y V Q V E S S N I S Y V
H A DELETED S S N I S Y V H A E S S N I S Y V H V DELETED S S N I S
Y V H V E S S N I S S I Q A DELETED S S N I S S I Q A E S S N I S S
I Q V DELETED S S N I S S I Q V E S S N I S S I H A DELETED S S N I
S S I H A E S S N I S S I H V DELETED S S N I S S I H V E S S N I S
S V Q A DELETED S S N I S S V Q A E S S N I S S V Q V DELETED S S N
I S S V Q V E S S N I S S V H A DELETED S S N I S S V H A E S S N I
S S V H V DELETED S S N I S S V H V E S S N I T Y I Q A DELETED S S
N I T Y I Q A E S S N I T Y I Q V DELETED S S N I T Y I Q V E S S N
I T Y I H A DELETED S S N I T Y I H A E S S N I T Y I H V DELETED S
S N I T Y I H V E S S N I T Y V Q A DELETED S S N I T Y V Q A E S S
N I T Y V Q V DELETED S S N I T Y V Q V E S S N I T Y V H A DELETED
S S N I T Y V H A E S S N I T Y V H V DELETED S S N I T Y V H V E S
S N I T S I Q A DELETED S S N I T S I Q A E S S N I T S I Q V
DELETED S S N I T S I Q V E
S S N I T S I H A DELETED S S N I T S I H A E S S N I T S I H V
DELETED S S N I T S I H V E S S N I T S V Q A DELETED S S N I T S V
Q A E S S N I T S V Q V DELETED S S N I T S V Q V E S S N I T S V H
A DELETED S S N I T S V H A E S S N I T S V H V DELETED S S N I T S
V H V E S T S V S Y I Q A DELETED S T S V S Y I Q A E S T S V S Y I
Q V DELETED S T S V S Y I Q V E S T S V S Y I H A DELETED S T S V S
Y I H A E S T S V S Y I H V DELETED S T S V S Y I H V E S T S V S Y
V Q A DELETED S T S V S Y V Q A E S T S V S Y V Q V DELETED S T S V
S Y V Q V E S T S V S Y V H A DELETED S T S V S Y V H A E S T S V S
Y V H V DELETED S T S V S Y V H V E S T S V S S I Q A DELETED S T S
V S S I Q A E S T S V S S I Q V DELETED S T S V S S I Q V E S T S V
S S I H A DELETED S T S V S S I H A E S T S V S S I H V DELETED S T
S V S S I H V E S T S V S S V Q A DELETED S T S V S S V Q A E S T S
V S S V Q V DELETED S T S V S S V Q V E S T S V S S V H A DELETED S
T S V S S V H A E S T S V S S V H V DELETED S T S V S S V H V E S T
S V T Y I Q A DELETED S T S V T Y I Q A E S T S V T Y I Q V DELETED
S T S V T Y I Q V E S T S V T Y I H A DELETED S T S V T Y I H A E S
T S V T Y I H V DELETED S T S V T Y I H V E S T S V T Y V Q A
DELETED S T S V T Y V Q A E S T S V T Y V Q V DELETED S T S V T Y V
Q V E S T S V T Y V H A DELETED S T S V T Y V H A E S T S V T Y V H
V DELETED S T S V T Y V H V E S T S V T S I Q A DELETED S T S V T S
I Q A E S T S V T S I Q V DELETED S T S V T S I Q V E S T S V T S I
H A DELETED S T S V T S I H A E S T S V T S I H V DELETED S T S V T
S I H V E S T S V T S V Q A DELETED S T S V T S V Q A E S T S V T S
V Q V DELETED S T S V T S V Q V E S T S V T S V H A DELETED S T S V
T S V H A E S T S V T S V H V DELETED S T S V T S V H V E S T S I S
Y I Q A DELETED S T S I S Y I Q A E S T S I S Y I Q V DELETED S T S
I S Y I Q V E S T S I S Y I H A DELETED S T S I S Y I H A E S T S I
S Y I H V DELETED S T S I S Y I H V E S T S I S Y V Q A DELETED S T
S I S Y V Q A E S T S I S Y V Q V DELETED S T S I S Y V Q V E S T S
I S Y V H A DELETED S T S I S Y V H A E S T S I S Y V H V DELETED S
T S I S Y V H V E S T S I S S I Q A DELETED S T S I S S I Q A E S T
S I S S I Q V DELETED S T S I S S I Q V E S T S I S S I H A DELETED
S T S I S S I H A E S T S I S S I H V DELETED S T S I S S I H V E S
T S I S S V Q A DELETED S T S I S S V Q A E S T S I S S V Q V
DELETED S T S I S S V Q V E S T S I S S V H A DELETED S T S I S S V
H A E S T S I S S V H V DELETED S T S I S S V H V E S T S I T Y I Q
A DELETED S T S I T Y I Q A E S T S I T Y I Q V DELETED S T S I T Y
I Q V E S T S I T Y I H A DELETED S T S I T Y I H A E S T S I T Y I
H V DELETED S T S I T Y I H V E S T S I T Y V Q A DELETED S T S I T
Y V Q A E S T S I T Y V Q V DELETED S T S I T Y V Q V E S T S I T Y
V H A DELETED S T S I T Y V H A E S T S I T Y V H V DELETED S T S I
T Y V H V E S T S I T S I Q A DELETED S T S I T S I Q A E S T S I T
S I Q V DELETED S T S I T S I Q V E S T S I T S I H A DELETED S T S
I T S I H A E S T S I T S I H V DELETED S T S I T S I H V E S T S I
T S V Q A DELETED S T S I T S V Q A E S T S I T S V Q V DELETED S T
S I T S V Q V E S T S I T S V H A DELETED S T S I T S V H A E S T S
I T S V H V DELETED S T S I T S V H V E S T N V S Y I Q A DELETED S
T N V S Y I Q A E S T N V S Y I Q V DELETED S T N V S Y I Q V E S T
N V S Y I H A DELETED S T N V S Y I H A E S T N V S Y I H V DELETED
S T N V S Y I H V E S T N V S Y V Q A DELETED S T N V S Y V Q A E S
T N V S Y V Q V DELETED S T N V S Y V Q V E S T N V S Y V H A
DELETED S T N V S Y V H A E S T N V S Y V H V DELETED S T N V S Y V
H V E S T N V S S I Q A DELETED S T N V S S I Q A E S T N V S S I Q
V DELETED S T N V S S I Q V E S T N V S S I H A DELETED S T N V S S
I H A E S T N V S S I H V DELETED S T N V S S I H V E S T N V S S V
Q A DELETED S T N V S S V Q A E S T N V S S V Q V DELETED S T N V S
S V Q V E S T N V S S V H A DELETED S T N V S S V H A E S T N V S S
V H V DELETED S T N V S S V H V E S T N V T Y I Q A DELETED S T N V
T Y I Q A E S T N V T Y I Q V DELETED S T N V T Y I Q V E S T N V T
Y I H A DELETED S T N V T Y I H A E S T N V T Y I H V DELETED S T N
V T Y I H V E S T N V T Y V Q A DELETED S T N V T Y V Q A E S T N V
T Y V Q V DELETED S T N V T Y V Q V E S T N V T Y V H A DELETED S T
N V T Y V H A E S T N V T Y V H V DELETED S T N V T Y V H V E S T N
V T S I Q A DELETED S T N V T S I Q A E S T N V T S I Q V DELETED S
T N V T S I Q V E S T N V T S I H A DELETED S T N V T S I H A E S T
N V T S I H V DELETED S T N V T S I H V E S T N V T S V Q A DELETED
S T N V T S V Q A E S T N V T S V Q V DELETED S T N V T S V Q V E S
T N V T S V H A DELETED S T N V T S V H A E S T N V T S V H V
DELETED S T N V T S V H V E S T N I S Y I Q A DELETED S T N I S Y I
Q A E S T N I S Y I Q V DELETED S T N I S Y I Q V E S T N I S Y I H
A DELETED S T N I S Y I H A E S T N I S Y I H V DELETED S T N I S Y
I H V E S T N I S Y V Q A DELETED S T N I S Y V Q A E S T N I S Y V
Q V DELETED S T N I S Y V Q V E S T N I S Y V H A DELETED S T N I S
Y V H A E S T N I S Y V H V DELETED S T N I S Y V H V E S T N I S S
I Q A DELETED S T N I S S I Q A E S T N I S S I Q V DELETED S T N I
S S I Q V E S T N I S S I H A DELETED S T N I S S I H A E S T N I S
S I H V DELETED S T N I S S I H V E S T N I S S V Q A DELETED S T N
I S S V Q A E S T N I S S V Q V DELETED S T N I S S V Q V E S T N I
S S V H A DELETED S T N I S S V H A E S T N I S S V H V DELETED S T
N I S S V H V E S T N I T Y I Q A DELETED S T N I T Y I Q A E S T N
I T Y I Q V DELETED S T N I T Y I Q V E S T N I T Y I H A DELETED S
T N I T Y I H A E S T N I T Y I H V DELETED S T N I T Y I H V E S T
N I T Y V Q A DELETED S T N I T Y V Q A E S T N I T Y V Q V DELETED
S T N I T Y V Q V E S T N I T Y V H A DELETED S T N I T Y V H A E S
T N I T Y V H V DELETED
S T N I T Y V H V E S T N I T S I Q A DELETED S T N I T S I Q A E S
T N I T S I Q V DELETED S T N I T S I Q V E S T N I T S I H A
DELETED S T N I T S I H A E S T N I T S I H V DELETED S T N I T S I
H V E S T N I T S V Q A DELETED S T N I T S V Q A E S T N I T S V Q
V DELETED S T N I T S V Q V E S T N I T S V H A DELETED S T N I T S
V H A E S T N I T S V H V DELETED S T N I T S V H V E I S S V S Y I
Q A DELETED I S S V S Y I Q A E I S S V S Y I Q V DELETED I S S V S
Y I Q V E I S S V S Y I H A DELETED I S S V S Y I H A E I S S V S Y
I H V DELETED I S S V S Y I H V E I S S V S Y V Q A DELETED I S S V
S Y V Q A E I S S V S Y V Q V DELETED I S S V S Y V Q V E I S S V S
Y V H A DELETED I S S V S Y V H A E I S S V S Y V H V DELETED I S S
V S Y V H V E I S S V S S I Q A DELETED I S S V S S I Q A E I S S V
S S I Q V DELETED I S S V S S I Q V E I S S V S S I H A DELETED I S
S V S S I H A E I S S V S S I H V DELETED I S S V S S I H V E I S S
V S S V Q A DELETED I S S V S S V Q A E I S S V S S V Q V DELETED I
S S V S S V Q V E I S S V S S V H A DELETED I S S V S S V H A E I S
S V S S V H V DELETED I S S V S S V H V E I S S V T Y I Q A DELETED
I S S V T Y I Q A E I S S V T Y I Q V DELETED I S S V T Y I Q V E I
S S V T Y I H A DELETED I S S V T Y I H A E I S S V T Y I H V
DELETED I S S V T Y I H V E I S S V T Y V Q A DELETED I S S V T Y V
Q A E I S S V T Y V Q V DELETED I S S V T Y V Q V E I S S V T Y V H
A DELETED I S S V T Y V H A E I S S V T Y V H V DELETED I S S V T Y
V H V E I S S V T S I Q A DELETED I S S V T S I Q A E I S S V T S I
Q V DELETED I S S V T S I Q V E I S S V T S I H A DELETED I S S V T
S I H A E I S S V T S I H V DELETED I S S V T S I H V E I S S V T S
V Q A DELETED I S S V T S V Q A E I S S V T S V Q V DELETED I S S V
T S V Q V E I S S V T S V H A DELETED I S S V T S V H A E I S S V T
S V H V DELETED I S S V T S V H V E I S S I S Y I Q A DELETED I S S
I S Y I Q A E I S S I S Y I Q V DELETED I S S I S Y I Q V E I S S I
S Y I H A DELETED I S S I S Y I H A E I S S I S Y I H V DELETED I S
S I S Y I H V E I S S I S Y V Q A DELETED I S S I S Y V Q A E I S S
I S Y V Q V DELETED I S S I S Y V Q V E I S S I S Y V H A DELETED I
S S I S Y V H A E I S S I S Y V H V DELETED I S S I S Y V H V E I S
S I S S I Q A DELETED I S S I S S I Q A E I S S I S S I Q V DELETED
I S S I S S I Q V E I S S I S S I H A DELETED I S S I S S I H A E I
S S I S S I H V DELETED I S S I S S I H V E I S S I S S V Q A
DELETED I S S I S S V Q A E I S S I S S V Q V DELETED I S S I S S V
Q V E I S S I S S V H A DELETED I S S I S S V H A E I S S I S S V H
V DELETED I S S I S S V H V E I S S I T Y I Q A DELETED I S S I T Y
I Q A E I S S I T Y I Q V DELETED I S S I T Y I Q V E I S S I T Y I
H A DELETED I S S I T Y I H A E I S S I T Y I H V DELETED I S S I T
Y I H V E I S S I T Y V Q A DELETED I S S I T Y V Q A E I S S I T Y
V Q V DELETED I S S I T Y V Q V E I S S I T Y V H A DELETED I S S I
T Y V H A E I S S I T Y V H V DELETED I S S I T Y V H V E I S S I T
S I Q A DELETED I S S I T S I Q A E I S S I T S I Q V DELETED I S S
I T S I Q V E I S S I T S I H A DELETED I S S I T S I H A E I S S I
T S I H V DELETED I S S I T S I H V E I S S I T S V Q A DELETED I S
S I T S V Q A E I S S I T S V Q V DELETED I S S I T S V Q V E I S S
I T S V H A DELETED I S S I T S V H A E I S S I T S V H V DELETED I
S S I T S V H V E I S N V S Y I Q A DELETED I S N V S Y I Q A E I S
N V S Y I Q V DELETED I S N V S Y I Q V E I S N V S Y I H A DELETED
I S N V S Y I H A E I S N V S Y I H V DELETED I S N V S Y I H V E I
S N V S Y V Q A DELETED I S N V S Y V Q A E I S N V S Y V Q V
DELETED I S N V S Y V Q V E I S N V S Y V H A DELETED I S N V S Y V
H A E I S N V S Y V H V DELETED I S N V S Y V H V E I S N V S S I Q
A DELETED I S N V S S I Q A E I S N V S S I Q V DELETED I S N V S S
I Q V E I S N V S S I H A DELETED I S N V S S I H A E I S N V S S I
H V DELETED I S N V S S I H V E I S N V S S V Q A DELETED I S N V S
S V Q A E I S N V S S V Q V DELETED I S N V S S V Q V E I S N V S S
V H A DELETED I S N V S S V H A E I S N V S S V H V DELETED I S N V
S S V H V E I S N V T Y I Q A DELETED I S N V T Y I Q A E I S N V T
Y I Q V DELETED I S N V T Y I Q V E I S N V T Y I H A DELETED I S N
V T Y I H A E I S N V T Y I H V DELETED I S N V T Y I H V E I S N V
T Y V Q A DELETED I S N V T Y V Q A E I S N V T Y V Q V DELETED I S
N V T Y V Q V E I S N V T Y V H A DELETED I S N V T Y V H A E I S N
V T Y V H V DELETED I S N V T Y V H V E I S N V T S I Q A DELETED I
S N V T S I Q A E I S N V T S I Q V DELETED I S N V T S I Q V E I S
N V T S I H A DELETED I S N V T S I H A E I S N V T S I H V DELETED
I S N V T S I H V E I S N V T S V Q A DELETED I S N V T S V Q A E I
S N V T S V Q V DELETED I S N V T S V Q V E I S N V T S V H A
DELETED I S N V T S V H A E I S N V T S V H V DELETED I S N V T S V
H V E I S N I S Y I Q A DELETED I S N I S Y I Q A E I S N I S Y I Q
V DELETED I S N I S Y I Q V E I S N I S Y I H A DELETED I S N I S Y
I H A E I S N I S Y I H V DELETED I S N I S Y I H V E I S N I S Y V
Q A DELETED I S N I S Y V Q A E I S N I S Y V Q V DELETED I S N I S
Y V Q V E I S N I S Y V H A DELETED I S N I S Y V H A E I S N I S Y
V H V DELETED I S N I S Y V H V E I S N I S S I Q A DELETED I S N I
S S I Q A E I S N I S S I Q V DELETED I S N I S S I Q V E I S N I S
S I H A DELETED I S N I S S I H A E I S N I S S I H V DELETED I S N
I S S I H V E I S N I S S V Q A DELETED I S N I S S V Q A E I S N I
S S V Q V DELETED I S N I S S V Q V E I S N I S S V H A DELETED I S
N I S S V H A E I S N I S S V H V DELETED I S N I S S V H V E I S N
I T Y I Q A DELETED I S N I T Y I Q A E I S N I T Y I Q V DELETED I
S N I T Y I Q V E I S N I T Y I H A DELETED I S N I T Y I H A E I S
N I T Y I H V DELETED I S N I T Y I H V E I S N I T Y V Q A DELETED
I S N I T Y V Q A E
I S N I T Y V Q V DELETED I S N I T Y V Q V E I S N I T Y V H A
DELETED I S N I T Y V H A E I S N I T Y V H V DELETED I S N I T Y V
H V E I S N I T S I Q A DELETED I S N I T S I Q A E I S N I T S I Q
V DELETED I S N I T S I Q V E I S N I T S I H A DELETED I S N I T S
I H A E I S N I T S I H V DELETED I S N I T S I H V E I S N I T S V
Q A DELETED I S N I T S V Q A E I S N I T S V Q V DELETED I S N I T
S V Q V E I S N I T S V H A DELETED I S N I T S V H A E I S N I T S
V H V DELETED I S N I T S V H V E I T S V S Y I Q A DELETED I T S V
S Y I Q A E I T S V S Y I Q V DELETED I T S V S Y I Q V E I T S V S
Y I H A DELETED I T S V S Y I H A E I T S V S Y I H V DELETED I T S
V S Y I H V E I T S V S Y V Q A DELETED I T S V S Y V Q A E I T S V
S Y V Q V DELETED I T S V S Y V Q V E I T S V S Y V H A DELETED I T
S V S Y V H A E I T S V S Y V H V DELETED I T S V S Y V H V E I T S
V S S I Q A DELETED I T S V S S I Q A E I T S V S S I Q V DELETED I
T S V S S I Q V E I T S V S S I H A DELETED I T S V S S I H A E I T
S V S S I H V DELETED I T S V S S I H V E I T S V S S V Q A DELETED
I T S V S S V Q A E I T S V S S V Q V DELETED I T S V S S V Q V E I
T S V S S V H A DELETED I T S V S S V H A E I T S V S S V H V
DELETED I T S V S S V H V E I T S V T Y I Q A DELETED I T S V T Y I
Q A E I T S V T Y I Q V DELETED I T S V T Y I Q V E I T S V T Y I H
A DELETED I T S V T Y I H A E I T S V T Y I H V DELETED I T S V T Y
I H V E I T S V T Y V Q A DELETED I T S V T Y V Q A E I T S V T Y V
Q V DELETED I T S V T Y V Q V E I T S V T Y V H A DELETED I T S V T
Y V H A E I T S V T Y V H V DELETED I T S V T Y V H V E I T S V T S
I Q A DELETED I T S V T S I Q A E I T S V T S I Q V DELETED I T S V
T S I Q V E I T S V T S I H A DELETED I T S V T S I H A E I T S V T
S I H V DELETED I T S V T S I H V E I T S V T S V Q A DELETED I T S
V T S V Q A E I T S V T S V Q V DELETED I T S V T S V Q V E I T S V
T S V H A DELETED I T S V T S V H A E I T S V T S V H V DELETED I T
S V T S V H V E I T S I S Y I Q A DELETED I T S I S Y I Q A E I T S
I S Y I Q V DELETED I T S I S Y I Q V E I T S I S Y I H A DELETED I
T S I S Y I H A E I T S I S Y I H V DELETED I T S I S Y I H V E I T
S I S Y V Q A DELETED I T S I S Y V Q A E I T S I S Y V Q V DELETED
I T S I S Y V Q V E I T S I S Y V H A DELETED I T S I S Y V H A E I
T S I S Y V H V DELETED I T S I S Y V H V E I T S I S S I Q A
DELETED I T S I S S I Q A E I T S I S S I Q V DELETED I T S I S S I
Q V E I T S I S S I H A DELETED I T S I S S I H A E I T S I S S I H
V DELETED I T S I S S I H V E I T S I S S V Q A DELETED I T S I S S
V Q A E I T S I S S V Q V DELETED I T S I S S V Q V E I T S I S S V
H A DELETED I T S I S S V H A E I T S I S S V H V DELETED I T S I S
S V H V E I T S I T Y I Q A DELETED I T S I T Y I Q A E I T S I T Y
I Q V DELETED I T S I T Y I Q V E I T S I T Y I H A DELETED I T S I
T Y I H A E I T S I T Y I H V DELETED I T S I T Y I H V E I T S I T
Y V Q A DELETED I T S I T Y V Q A E I T S I T Y V Q V DELETED I T S
I T Y V Q V E I T S I T Y V H A DELETED I T S I T Y V H A E I T S I
T Y V H V DELETED I T S I T Y V H V E I T S I T S I Q A DELETED I T
S I T S I Q A E I T S I T S I Q V DELETED I T S I T S I Q V E I T S
I T S I H A DELETED I T S I T S I H A E I T S I T S I H V DELETED I
T S I T S I H V E I T S I T S V Q A DELETED I T S I T S V Q A E I T
S I T S V Q V DELETED I T S I T S V Q V E I T S I T S V H A DELETED
I T S I T S V H A E I T S I T S V H V DELETED I T S I T S V H V E I
T N V S Y I Q A DELETED I T N V S Y I Q A E I T N V S Y I Q V
DELETED I T N V S Y I Q V E I T N V S Y I H A DELETED I T N V S Y I
H A E I T N V S Y I H V DELETED I T N V S Y I H V E I T N V S Y V Q
A DELETED I T N V S Y V Q A E I T N V S Y V Q V DELETED I T N V S Y
V Q V E I T N V S Y V H A DELETED I T N V S Y V H A E I T N V S Y V
H V DELETED I T N V S Y V H V E I T N V S S I Q A DELETED I T N V S
S I Q A E I T N V S S I Q V DELETED I T N V S S I Q V E I T N V S S
I H A DELETED I T N V S S I H A E I T N V S S I H V DELETED I T N V
S S I H V E I T N V S S V Q A DELETED I T N V S S V Q A E I T N V S
S V Q V DELETED I T N V S S V Q V E I T N V S S V H A DELETED I T N
V S S V H A E I T N V S S V H V DELETED I T N V S S V H V E I T N V
T Y I Q A DELETED I T N V T Y I Q A E I T N V T Y I Q V DELETED I T
N V T Y I Q V E I T N V T Y I H A DELETED I T N V T Y I H A E I T N
V T Y I H V DELETED I T N V T Y I H V E I T N V T Y V Q A DELETED I
T N V T Y V Q A E I T N V T Y V Q V DELETED I T N V T Y V Q V E I T
N V T Y V H A DELETED I T N V T Y V H A E I T N V T Y V H V DELETED
I T N V T Y V H V E I T N V T S I Q A DELETED I T N V T S I Q A E I
T N V T S I Q V DELETED I T N V T S I Q V E I T N V T S I H A
DELETED I T N V T S I H A E I T N V T S I H V DELETED I T N V T S I
H V E I T N V T S V Q A DELETED I T N V T S V Q A E I T N V T S V Q
V DELETED I T N V T S V Q V E I T N V T S V H A DELETED I T N V T S
V H A E I T N V T S V H V DELETED I T N V T S V H V E I T N I S Y I
Q A DELETED I T N I S Y I Q A E I T N I S Y I Q V DELETED I T N I S
Y I Q V E I T N I S Y I H A DELETED I T N I S Y I H A E I T N I S Y
I H V DELETED I T N I S Y I H V E I T N I S Y V Q A DELETED I T N I
S Y V Q A E I T N I S Y V Q V DELETED I T N I S Y V Q V E I T N I S
Y V H A DELETED I T N I S Y V H A E I T N I S Y V H V DELETED I T N
I S Y V H V E I T N I S S I Q A DELETED I T N I S S I Q A E I T N I
S S I Q V DELETED I T N I S S I Q V E I T N I S S I H A DELETED I T
N I S S I H A E I T N I S S I H V DELETED I T N I S S I H V E I T N
I S S V Q A DELETED I T N I S S V Q A E I T N I S S V Q V DELETED I
T N I S S V Q V E I T N I S S V H A DELETED I T N I S S V H A E I T
N I S S V H V DELETED I T N I S S V H V E I T N I T Y I Q A DELETED
I T N I T Y I Q A E I T N I T Y I Q V DELETED I T N I T Y I Q V E I
T N I T Y I H A DELETED
I T N I T Y I H A E I T N I T Y I H V DELETED I T N I T Y I H V E I
T N I T Y V Q A DELETED I T N I T Y V Q A E I T N I T Y V Q V
DELETED I T N I T Y V Q V E I T N I T Y V H A DELETED I T N I T Y V
H A E I T N I T Y V H V DELETED I T N I T Y V H V E I T N I T S I Q
A DELETED I T N I T S I Q A E I T N I T S I Q V DELETED I T N I T S
I Q V E I T N I T S I H A DELETED I T N I T S I H A E I T N I T S I
H V DELETED I T N I T S I H V E I T N I T S V Q A DELETED I T N I T
S V Q A E I T N I T S V Q V DELETED I T N I T S V Q V E I T N I T S
V H A DELETED I T N I T S V H A E I T N I T S V H V DELETED I T N I
T S V H V E
TABLE-US-00017 TABLE 16 Exemplary Mutations of mAb 4.18.1 Light
Chain (SEQ ID NO: 52) to Germline at the Indicated Residue Number.
7 13 32 39 55 58 65 92 S V N K A I S N S V N K A I S G S V N K A I
I N S V N K A I I G S V N K A F S N S V N K A F S G S V N K A F I N
S V N K A F I G S V N K S I S N S V N K S I S G S V N K S I I N S V
N K S I I G S V N K S F S N S V N K S F S G S V N K S F I N S V N K
S F I G S V N T A I S N S V N T A I S G S V N T A I I N S V N T A I
I G S V N T A F S N S V N T A F S G S V N T A F I N S V N T A F I G
S V N T S I S N S V N T S I S G S V N T S I I N S V N T S I I G S V
N T S F S N S V N T S F S G S V N T S F I N S V N T S F I G S V S K
A I S N S V S K A I S G S V S K A I I N S V S K A I I G S V S K A F
S N S V S K A F S G S V S K A F I N S V S K A F I G S V S K S I S N
S V S K S I S G S V S K S I I N S V S K S I I G S V S K S F S N S V
S K S F S G S V S K S F I N S V S K S F I G S V S T A I S N S V S T
A I S G S V S T A I I N S V S T A I I G S V S T A F S N S V S T A F
S G S V S T A F I N S V S T A F I G S V S T S I S N S V S T S I S G
S V S T S I I N S V S T S I I G S V S T S F S N S V S T S F S G S V
S T S F I N S V S T S F I G S M N K A I S N S M N K A I S G S M N K
A I I N S M N K A I I G S M N K A F S N S M N K A F S G S M N K A F
I N S M N K A F I G S M N K S I S N S M N K S I S G S M N K S I I N
S M N K S I I G S M N K S F S N S M N K S F S G S M N K S F I N S M
N K S F I G S M N T A I S N S M N T A I S G S M N T A I I N S M N T
A I I G S M N T A F S N S M N T A F S G S M N T A F I N S M N T A F
I G S M N T S I S N S M N T S I S G S M N T S I I N S M N T S I I G
S M N T S F S N S M N T S F S G S M N T S F I N S M N T S F I G S M
S K A I S N S M S K A I S G S M S K A I I N S M S K A I I G S M S K
A F S N S M S K A F S G S M S K A F I N S M S K A F I G S M S K S I
S N S M S K S I S G S M S K S I I N S M S K S I I G S M S K S F S N
S M S K S F S G S M S K S F I N S M S K S F I G S M S T A I S N S M
S T A I S G S M S T A I I N S M S T A I I G S M S T A F S N S M S T
A F S G S M S T A F I N S M S T A F I G S M S T S I S N S M S T S I
S G S M S T S I I N S M S T S I I G S M S T S F S N S M S T S F S G
S M S T S F I N S M S T S F I G F V N K A I S N F V N K A I S G F V
N K A I I N F V N K A I I G F V N K A F S N F V N K A F S G F V N K
A F I N F V N K A F I G F V N K S I S N F V N K S I S G F V N K S I
I N F V N K S I I G F V N K S F S N F V N K S F S G F V N K S F I N
F V N K S F I G F V N T A I S N F V N T A I S G F V N T A I I N F V
N T A I I G F V N T A F S N F V N T A F S G F V N T A F I N F V N T
A F I G F V N T S I S N F V N T S I S G F V N T S I I N F V N T S I
I G F V N T S F S N F V N T S F S G F V N T S F I N F V N T S F I G
F V S K A I S N F V S K A I S G F V S K A I I N F V S K A I I G F V
S K A F S N F V S K A F S G F V S K A F I N F V S K A F I G F V S K
S I S N F V S K S I S G F V S K S I I N F V S K S I I G F V S K S F
S N F V S K S F S G F V S K S F I N F V S K S F I G F V S T A I S N
F V S T A I S G F V S T A I I N F V S T A I I G F V S T A F S N F V
S T A F S G F V S T A F I N F V S T A F I G F V S T S I S N F V S T
S I S G F V S T S I I N F V S T S I I G F V S T S F S N F V S T S F
S G F V S T S F I N F V S T S F I G F M N K A I S N F M N K A I S G
F M N K A I I N F M N K A I I G F M N K A F S N F M N K A F S G F M
N K A F I N F M N K A F I G F M N K S I S N F M N K S I S G F M N K
S I I N F M N K S I I G F M N K S F S N F M N K S F S G F M N K S F
I N F M N K S F I G F M N T A I S N F M N T A I S G F M N T A I I N
F M N T A I I G F M N T A F S N F M N T A F S G F M N T A F I N F M
N T A F I G F M N T S I S N F M N T S I S G F M N T S I I N F M N T
S I I G F M N T S F S N F M N T S F S G F M N T S F I N F M N T S F
I G F M S K A I S N F M S K A I S G F M S K A I I N F M S K A I I G
F M S K A F S N F M S K A F S G F M S K A F I N F M S K A F I G F M
S K S I S N F M S K S I S G F M S K S I I N F M S K S I I G F M S K
S F S N F M S K S F S G F M S K S F I N F M S K S F I G F M S T A I
S N F M S T A I S G F M S T A I I N F M S T A I I G
F M S T A F S N F M S T A F S G F M S T A F I N F M S T A F I G F M
S T S I S N F M S T S I S G F M S T S I I N F M S T S I I G F M S T
S F S N F M S T S F S G F M S T S F I N F M S T S F I G
TABLE-US-00018 TABLE 17 Exemplary Mutations of mAb 4.18.1 Heavy
Chain (SEQ ID NO: 50) to Germline at the Indicated Residue Number.
30 31 35 53 75 98-99 103 117 S S S S K EG G S S S S S K EG G L S S
S S K EG DELETED S S S S S K EG DELETED L S S S S K DELETED G S S S
S S K DELETED G L S S S S K DELETED DELETED S S S S S K DELETED
DELETED L S S S S R EG G S S S S S R EG G L S S S S R EG DELETED S
S S S S R EG DELETED L S S S S R DELETED G S S S S S R DELETED G L
S S S S R DELETED DELETED S S S S S R DELETED DELETED L S S S T K
EG G S S S S T K EG G L S S S T K EG DELETED S S S S T K EG DELETED
L S S S T K DELETED G S S S S T K DELETED G L S S S T K DELETED
DELETED S S S S T K DELETED DELETED L S S S T R EG G S S S S T R EG
G L S S S T R EG DELETED S S S S T R EG DELETED L S S S T R DELETED
G S S S S T R DELETED G L S S S T R DELETED DELETED S S S S T R
DELETED DELETED L S S N S K EG G S S S N S K EG G L S S N S K EG
DELETED S S S N S K EG DELETED L S S N S K DELETED G S S S N S K
DELETED G L S S N S K DELETED DELETED S S S N S K DELETED DELETED L
S S N S R EG G S S S N S R EG G L S S N S R EG DELETED S S S N S R
EG DELETED L S S N S R DELETED G S S S N S R DELETED G L S S N S R
DELETED DELETED S S S N S R DELETED DELETED L S S N T K EG G S S S
N T K EG G L S S N T K EG DELETED S S S N T K EG DELETED L S S N T
K DELETED G S S S N T K DELETED G L S S N T K DELETED DELETED S S S
N T K DELETED DELETED L S S N T R EG G S S S N T R EG G L S S N T R
EG DELETED S S S N T R EG DELETED L S S N T R DELETED G S S S N T R
DELETED G L S S N T R DELETED DELETED S S S N T R DELETED DELETED L
S N S S K EG G S S N S S K EG G L S N S S K EG DELETED S S N S S K
EG DELETED L S N S S K DELETED G S S N S S K DELETED G L S N S S K
DELETED DELETED S S N S S K DELETED DELETED L S N S S R EG G S S N
S S R EG G L S N S S R EG DELETED S S N S S R EG DELETED L S N S S
R DELETED G S S N S S R DELETED G L S N S S R DELETED DELETED S S N
S S R DELETED DELETED L S N S T K EG G S S N S T K EG G L S N S T K
EG DELETED S S N S T K EG DELETED L S N S T K DELETED G S S N S T K
DELETED G L S N S T K DELETED DELETED S S N S T K DELETED DELETED L
S N S T R EG G S S N S T R EG G L S N S T R EG DELETED S S N S T R
EG DELETED L S N S T R DELETED G S S N S T R DELETED G L S N S T R
DELETED DELETED S S N S T R DELETED DELETED L S N N S K EG G S S N
N S K EG G L S N N S K EG DELETED S S N N S K EG DELETED L S N N S
K DELETED G S S N N S K DELETED G L S N N S K DELETED DELETED S S N
N S K DELETED DELETED L S N N S R EG G S S N N S R EG G L S N N S R
EG DELETED S S N N S R EG DELETED L S N N S R DELETED G S S N N S R
DELETED G L S N N S R DELETED DELETED S S N N S R DELETED DELETED L
S N N T K EG G S S N N T K EG G L S N N T K EG DELETED S S N N T K
EG DELETED L S N N T K DELETED G S S N N T K DELETED G L S N N T K
DELETED DELETED S S N N T K DELETED DELETED L S N N T R EG G S S N
N T R EG G L S N N T R EG DELETED S S N N T R EG DELETED L S N N T
R DELETED G S S N N T R DELETED G L S N N T R DELETED DELETED S S N
N T R DELETED DELETED L R S S S K EG G S R S S S K EG G L R S S S K
EG DELETED S R S S S K EG DELETED L R S S S K DELETED G S R S S S K
DELETED G L R S S S K DELETED DELETED S R S S S K DELETED DELETED L
R S S S R EG G S R S S S R EG G L R S S S R EG DELETED S R S S S R
EG DELETED L R S S S R DELETED G S R S S S R DELETED G L R S S S R
DELETED DELETED S R S S S R DELETED DELETED L R S S T K EG G S R S
S T K EG G L R S S T K EG DELETED S R S S T K EG DELETED L R S S T
K DELETED G S R S S T K DELETED G L R S S T K DELETED DELETED S R S
S T K DELETED DELETED L R S S T R EG G S R S S T R EG G L R S S T R
EG DELETED S R S S T R EG DELETED L R S S T R DELETED G S R S S T R
DELETED G L R S S T R DELETED DELETED S R S S T R DELETED DELETED L
R S N S K EG G S R S N S K EG G L R S N S K EG DELETED S R S N S K
EG DELETED L R S N S K DELETED G S R S N S K DELETED G L R S N S K
DELETED DELETED S R S N S K DELETED DELETED L R S N S R EG G S R S
N S R EG G L R S N S R EG DELETED S R S N S R EG DELETED L R S N S
R DELETED G S R S N S R DELETED G L R S N S R DELETED DELETED S R S
N S R DELETED DELETED L R S N T K EG G S R S N T K EG G L R S N T K
EG DELETED S R S N T K EG DELETED L R S N T K DELETED G S R S N T K
DELETED G L R S N T K DELETED DELETED S R S N T K DELETED DELETED L
R S N T R EG G S R S N T R EG G L R S N T R EG DELETED S R S N T R
EG DELETED L R S N T R DELETED G S R S N T R DELETED G L R S N T R
DELETED DELETED S R S N T R DELETED DELETED L R N S S K EG G S R N
S S K EG G L R N S S K EG DELETED S R N S S K EG DELETED L R N S S
K DELETED G S R N S S K DELETED G L R N S S K DELETED DELETED S R N
S S K DELETED DELETED L R N S S R EG G S R N S S R EG G L R N S S R
EG DELETED S R N S S R EG DELETED L R N S S R DELETED G S R N S S R
DELETED G L R N S S R DELETED DELETED S R N S S R DELETED DELETED L
R N S T K EG G S R N S T K EG G L R N S T K EG DELETED S R N S T K
EG DELETED L R N S T K DELETED G S R N S T K DELETED G L R N S T K
DELETED DELETED S R N S T K DELETED DELETED L R N S T R EG G S R N
S T R EG G L R N S T R EG DELETED S R N S T R EG DELETED L R N S T
R DELETED G S R N S T R DELETED G L R N S T R DELETED DELETED S R N
S T R DELETED DELETED L R N N S K EG G S R N N S K EG G L R N N S K
EG DELETED S R N N S K EG DELETED L R N N S K DELETED G S R N N S K
DELETED G L R N N S K DELETED DELETED S R N N S K DELETED DELETED L
R N N S R EG G S R N N S R EG G L R N N S R EG DELETED S R N N S R
EG DELETED L R N N S R DELETED G S R N N S R DELETED G L R N N S R
DELETED DELETED S R N N S R DELETED DELETED L R N N T K EG G S R N
N T K EG G L R N N T K EG DELETED S R N N T K EG DELETED L
R N N T K DELETED G S R N N T K DELETED G L R N N T K DELETED
DELETED S R N N T K DELETED DELETED L R N N T R EG G S R N N T R EG
G L R N N T R EG DELETED S R N N T R EG DELETED L R N N T R DELETED
G S R N N T R DELETED G L R N N T R DELETED DELETED S R N N T R
DELETED DELETED L
TABLE-US-00019 TABLE 18 Heavy chain analysis SEQ Chain ID Name NO:
V D J FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 70 Germline EVQLVESGGGL SYDMH
WVRQATG AIGTAGDT RFTISRENAKNSLYLQ DYSNYTM WGQGTT VQPGGSLRLSC
KGLEWVS YYPGSVKG MNSLRAGDTAVYYYAR VRGVYGM VTVSS AASGFTFS DV 1.4.2g2
2 VH3-13 D3-10 JH6B EVQLVESGGGL SYDMH WVRQATG AIGTAGDT
RFTISRENAKNSLYLQ EGDYSNY WGQGTT VQPGGSLRLSC KGLEWVS YYPGSVKG
MNSLRAGDTAVYYCAR FTMVRGV VTVSS AASGFTFS TYGMDV 1.61.1g2 10 VH3-13
D3-10 JH6B EVQLVESGGGL SYDMH WVRQATG AIGTAGDT RFTISRENAKNSLYLQ
EGDYSNY WGQGTT VQPGGSLRLSC KGLEWVS YYPGSVKG MNSLRAGDTAVYYCAR
FTRVRGV VTVSS AASGFTFS TYGMDV 71 Germline EVQLLESGGGL SYAMS WVRQAPG
AISGSGGS RFTISRDNSKNTLYLQ YGDYWYF WGRGTL VQPGGSLRLSC KGLEWVS
TYYADSVK MNSLRAEDTAVYYCAK DL VTVSS AASGFTFS G 4.13.1g4 46 VH3-23
D4-17 JH2 EVQLLESGGGL SYAMS WVRQAPG GISGSGGS RFTISRDNSKNTLYLQ
DGYGDYV WGRGTL VQPGGSLRLSC KGLEWVL TYYADSVK MNSLRAEDTAVYYCAK GWYFDL
VTVSS AASGFTFS G 72 Germline QVQLVESGGGV SYGMH WVRQAPG VIWYDGSN
RFTISRDNSKNTLYLQ DMIVVVI WGQGTM VQPGRSLRLSC KGLEWVA KYYADSVK
MNSLRAEDTAVYYCAR TAFDI VTVSS AASGFTFS G 4.50.1g4 58 VH3-33 D3-22
JH3B QVQLVESGGGV SYGMH WVRQAPG VIWYDGSN RFTISRDNSKNTLYLQ DMIVVVI
WGQGTM VQPGRSLRLSC KGLEWVA KDYADSVK MNSLRAEDTAVYYCAR TYAFDI VTVSS
AASGFTFS G 73 Germline QVQLVESGGGV SYGMH WVRQAPG VIWYDGSN
RFTISRDNSKNTLYLQ YYYYYGM WGQGTT VQPGRSLRLSC KGLEWVA KYYADSVK
MNSLRAEDTAVYYCAR DV VTVSS AASGFTFS G 1.41.1g2 6 VH3-33 D6-6 JH6B
QVQLVESGGGV SYGMH WVRQAPG VIWYDGSQ RFTISRDNSKNTLYLQ GMTYYYY WGQGTT
VQPGRSLRLSC KGLEWVA KYYADSVK MNSLRAEDTAVYYCAR YGMDV VTVSS AAPGFTFS
G 1.113. 22 VH3-33 D6-6 JH6B QVQLVESGGGV SYGMH WVRQAPG VIWYDGSN
RFTISRDNSKNTLYLQ DLAALYY WGQGTT 2g2 VQPGRSLRLSC KGLEWVA KYYADSVK
MNSLRAEDTAVYYCAR YYGMHV VTVSS AASGFTFS G 74 Germline EVQLVESGGGL
SNYMS WVRQAPG VIYSGGST RFTISRDNSKNTLYLQ AVAFDY WGQGTL IQPGGSLRLSC
KGLEWVS YYADSVKG MNSLRAEDTAVYYCAR VTVSS AASGFTVS 1.99.1g2 14 VH3-53
D6-19 JH4B EVQLVESGGGL SNYMS WVRQAPG VIYTGGST RFSISRDNSKNTLYLQ
EGAVAGF WGQGTL IQPGGSLRLSC KGLEWIS YYADSVKG MNSLRAEDTAVYYCAR DY
VTVSS AASGFTVR 4.18.1g4 50 VH3-53 D6-19 JH4B EVQLVESGGGL NNYMN
WVRQAPG VIYTGGST RFTISRDNSRNTLYLQ EGAVAGF WGQGTL IQPGGSLRLSC
KGLEWVS YYADSVKG MNSLRAEDTAVYYCAR DY VTVSL AASGFTVR 75 Germline
EVQLVESGGGL SNYMS WVRQAPG VIYSGGST RFTISRDNSKNTLYLQ YYYYYGM WGQGTT
IQPGGSINLSC KGLEWVS YYADSVKG MNSLRAEDTAVYYCAR DV VTVSS AASGFTVS
1.100. 18 VH3-53 JH6B EVQLVESGGGL NNYMN WVRQAPG VIYTGGST
RFTISRDNSKNTLYLQ EGKLAGM WGQGTT 1g2 LQPGGSLRLSC KGLEWVS YYADSVKG
MNSLRAEDTAVYYCAR DV VTVSS AASGFTVR 4.19.1g4 54 VH3-53 JH6B
EVQLVESGGGL SNYMN WVRQAPG VIYTGGST RFTISRDNSKNTLYLQ DTYLYGM WGQGTT
IQPGGSLRLSC KGLEWVS YYADSVKG MNSLRAEDTAVYYCAR DV VTVSS AASGFSVS 76
Germline EVQLVESGGGL SYWMS WVRQAPG NIKQDGSE RFTISRDNAKNSLYLQ
GWYWYFD WGRGTL VQPGGSLRLSC KGLEWVA KYYVDSVK MNSLRAEDTAVYYCAR L
VTVSS AASGFTFS G 4.12.1g4 42 VH3-7 D6-19 JH2 EVQLVESGGGL SYWMS
WVRQAPG NINGDGSE RFTISRDNAKNSPHLQ EGSGGRY WGRGTL VQPGGSLRLSC
TGLDWVA RYYVDSVK MNSLRAEDTAVYYCAR GGWYFDL VTVSS AASGFTFS G 77
Germline QVQLVESGGGV SYGMH WVRQAPG VIWYDGSN RFTISRDNSKNTLYLQ
HIVVVTA WGQGTL VQPGRSLRLSC KGLEWVA KYYADSVK MNSLRAEDTAVYYCAR ILDY
VTVSS AASGFTFS G 2.19.2g2 30 VH3-33 D2-21 JH4B QVQLVESGGGV SYGMH
WVRQAPG VIWYDGSN RFTISRDNSKNTLYLQ DQHIVVV WGQGTL VQPGRSLRLSC
KGLEWVA KYYADSVK MNSLRAEDTAVYYCAR TAILDY VTVSS AASGFTFS G 78
Germline EVQLVESGGGL SNYMS WVRQAPG VIYSGGST RFTISRDNSKNTLYLQ
IAVAGYY WGQGTT IQPGGSLRLSC KGLEWVS YYADSVKG MNSLRAEDTAVYYCAR GMDV
VTVS AASGFTVS 2.6.1g2 26 VH3-53 D6-19 JH6B EVQLVESGGGL TNYMN
WVRQAPG IIYTGGST RFTVSRDNSKNTLYLH EIAVAGY WGQGTT IQPGGSLRLSC
KGLEWVS SYADSVKG MNSLRVEDTAVYYCAR YGMDV VTVS AASGFTVI 79 Germline
EVQLVESGGGL SYWMS WVRQAPG NIKQDGSE RFTISRDNAKNSLYLQ YYYDSSG WGRGTL
VQPGGSLRLSC KGLEWVA KYYVDSVK MNSLRAEDTAVYYCAR YYWFDL VTVSS AASGFTFS
G 3.8.3g2 34 VH3-7 D3-22 JH2 EVQLVESGGGL SYWMS WVRQAPG NIKQHGSB
RFTISRDNAXWSLYLQ EEAYYYD WARGTL VQPGGSLRLSC KGLEWVA KYYVDSVK
MNSLRAEDTAVYYCAR NSGYYWY VTVSS AASGFTFS G FDL 80 Germline
QVQLQESGPGL SGGYY WIRGHPG YIYYSGST RVTISVDTSXNQFSLK YYYYYGM WGQGTT
VKPSQTLSLTC WS KGLEWIG YYNPSLKS LSSVTAADTAVYYCAR DV VTVSS TVSGGSIS
3.176. 38 VH4-31 D2-21 JH6B QVQLQESGPGL SGGHY WIRQHPG YIYYSGRT
RVTISVDTSKNQFSLK DGTGDYY WGQGTT 2g2 VKPSQTLSLTC WS KGLEWIG YYNPSLKS
LSSVTAADTAVYYCAR YYGMDV VTVSS TVSGGSIS
TABLE-US-00020 TABLE 19 Light chain analysis SEQ Chain ID Name NO:
V J FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 61 Germline DIVMTQSPDSLA
KSSQSVLYS WYQQKPGQ WAST GVPDRFSGSGSGTDFT QQYYS FGQGTK VSLGERATINC
SNNKNYLA PPKLLIY RES LTISSLQAEDVAVYYC TPPT VEIK 4.13.1k 48 B3 JK1
DIVMTQSPDSLA KSSQSVLYS WYQQKPGQ WAST GVPDRFSGSGSGTDFT QQFYS FGQGTK
VSLGERATINC SNNKNFLA PPKLLIY RES LTISNLQAEDVAVYYC TPPT VEIR 4.50.1k
60 B3 JK1 DIVMTQSPDSLA KSSQSVLYS WYQQKPGQ WAST GVPDRFSGSGSGTDFT
QQYYR FGQGTR VSLGERATINC SNNKNYLA PPKLLIY RES LTISSLQAEDVAVYYC TPPT
VEIK 2.19.2k 32 B3 JK1 DIVMTQSPDSLS KSSQSVLYG WYQQKPGQ WAST
GVPDRFSGSGSGTDFT QQYYS FGQGTK VSLGERATINC SNNRNSLA PPKLLIY RES
LTISSLQAEDVAVYYC IFPWT VEIK 62 Germline DIVMTQSPDSLA KSSQSVLYS
WYQQKPGQ WAST GVPDRFSGSGSGTDFT QQYYS FGQGTR VSLGERATINC SNNKNYLA
PPKLLIY RES LTISSLQAEDVAVYYC TPPIT LEIK 4.12.1k 44 B3 JK5
DIVMTQSPDSLA KSSQSVLYS WYQQKPGQ WAST GVPDRFSGSGSGTDFT QQYYS FGQGTR
VSLGERATINC SNNKNFLA PPKLLIY RES LTISSLQAEDVAVYYC TPPIT LEIK 63
Germline DIQMTQSPSSLS RASQGISNY WFQQKPGK AASS GVPSRFSGSGSGTDFT
QQYNS FGGGTK ASVGDRVTITC LA APKSLIY LQS LTISSLQPEDFATYYC YPLT VEIK
1.4.2k 4 L1 JK4 DIQLTQSPSSLC RASQGISKY WFQQKPGK GASS
GVPAKFSGSGSGTDFT QQYNS FGGGTK ASVGDRITITC LA APKSLIY LQS
LTISSLQPEDFATYYC YPLT VEIK 1.61.1k 12 L1 JK4 DIQMTQSPSSLS RASQGISKY
WFQQKPGK GASS GVPSKFSGSGSGTDFT QQYNS FGGGTK ASVGDRVTITC LA APKSLIY
LQS LTISSLQPEDFATYYC YPLT VEIK 64 Germline EIVMTQSPATLS RASQSVSSN
WYQQKPGQ GAST GIPARFSGSGSGTEFT QQYNN FGQGTK VSPGERATLSC LA APRLLIY
RAT LTISSLQSEDFAVYYC WPRT VEIK 1.113.2k 24 L2 JK1 EIVMTQSPATLS
RASQSFSSN WYQQKPGQ GAST GIPARFSGSGSGTEFT QQNNN FGQGTK VSPGERATLSC
LA APRLLIY RAT LTISSLQSEDFAVYYC WPRT VEIK 1.41.1k 8 L2 JK1
EIVMTQSPAILS RASQSVSSK WYQQKPGQ GAST GIPARFSGSGSGTEFT QQYNN FGQGTK
VSPGERATLSC LD APRLLIY RAT LTISSLQSEDFAVYYC WPRT VEIK 65 Germline
EIVMTQSPATLS RASQSVSSN WYQQKPGQ GAST GIPARFSGSGSGTEFT QQYNN FGGGTK
VSPGERATLSC LA APRLLIY RAT LTISSLQSEDFAVYYC WPLT VEIK 1.99.1k 16 L2
JK4 EIVMTQSPATLS RASQSISSN WYQQKPGQ GAST GIPARFSGSGSGTEFT QQYNN
FGGGTK VSPGERATLSC LA APRLLIY RAT LTISSLQSEDFAVYYC WPLT VEIK
4.19.1k 56 L2 JK4 EIVMTQSPATLS RTSQSVSSN WYQQKPGQ GASA
GVPARFSGSGSGTEFT QQYYN FGGGTK VSPGESATLSC LA TPRLLIY RAT
LTISSLQSEDFAVYYC WPLT VEIK 1.100.1k 20 L2 JK4 ETVMTQSPATLS
RASQSVSSN WYQQKPGQ GAST GIPARFSGSGSGTEFT QQYNN FGGGTK VSPGERATLSC
LA APRLLIY RAT LTISSLQSEEFAVYYC WPLT VEIK 66 Germline EIVMTQSPATLS
RASQSVSSN WYQQKPGQ GAST GIPARFSGSGSGTEFT QQYNN FGQGTR VSPGERATLSC
LA APRLLIY RAT LTISSLQSEDFAVYYC WPLT LEIK 4.18.1k 52 L2 JK5
EIVMTQFPATLS RASQSVSSS WYQQTPGQ GAST GFPARFSGIGSGTEFT QQYGN FGQGTR
MSPGERATLSC LA APRLLIY RST LTISSLQSEDFAVYYC WPLT LEIK 67 Germline
DIQMTQSPSSLS RASQGIRND WYQQKPGK AASS GVPSRFSGSGSGTEFT LQHNS FGGGTK
ASVGDRVTITC LG APKRLIY LQS LTISSLQPEDFATYYC YT VEIK 2.6.1k 28 A30
JK4 DIQMTQSPSSLS RASQGIRND WYQQKPGK AASS GVPSRFSGSGSGTEFT LQHNS
FGGGTR ASVGDRVTITC LG APKRLIY LQS LTISSLQPEDFATYYC YPLT VEIK 68
Germline DIVMTQSPDSLA KSSQSVLYS WYQQKPGQ WAST GVPDRFSGSGSGTDFT
QQYYS FGGGTK VSLGERATINC SNNKNYLA PPKLLIY RES LTISSLQAEDVAVYYC TT
VEIK 3.176.2k 40 B3 JK4 DIVMTQSPTSLT KSSQSVLYS WYQQKPRQ WAST
GVPDRFSGSGSGTDFT QQYYT FGGGTK VSLGBRATINC SNNRNFLA PPKLLIY RES
LTISSLQAEDVAVYYC TPLT VEIK 69 Germline DIQMTQSPSSVS RASQGISSW
WYQQKPGK AASS GVPSRFSGSGSGTDFT QQANS FGQGTK ASVGDRVTITC LA APKLLIY
LQS LTISSLQPEDFATYYC FPWT VEIK 3.8.3k 36 L5 JK1 DIQMTQSPSSVS
RASQGISSW WYQQKPQK AASS GVPSRFSGSGSGTDFT QQADS FGQGTK ASIGDRVTITC
LA TPKLLIY LQS LTISSLQPEDFATYYC FPWT VEIK
Example 16
Identification of uPAR Binding Epitopes
[0268] As the anti-uPAR antibodies failed to cross-react with mouse
uPAR, we generated a number of mouse uPAR-human uPAR chimeric
proteins to identify the domains involved in antibody binding.
Chimeric human-murine uPAR comprising murine D1/humanD2D3; murine
D1/human D2/MuD3; and human D1D2/murineD3uPAR each with a
C-terminal His tag were expressed as secreted proteins in HEK293T
cells. Binding sites for 2.6.1, 2.19.2, and 4.18.1 were determined
by Western blotting of culture supernatants; expression of the
appropriate recombinant proteins was demonstrated by detection
using anti-His mAb. The binding epitope for 2.19.2 lies in Domain
I, and 2.6.1 interacts primarily with an epitope within Domain
II/III and 4.18.1 with an epitope in Domain II (see FIG. 6).
[0269] The nucleotide sequences encoding the proteins used for the
epitope binning and the translated amino acid sequence are set
forth in Table 20 below.
TABLE-US-00021 TABLE 20 Nucleotide and Amino Acid Sequences Used
for Epitope Binning uPAR ECD Chimera Sequence SEQ ID NO: Murine
D1/Human D2D3 Nucleotide Sequence SEQ ID NO: 83 Amino Acid Sequence
SEQ ID NO: 84 Human D1D2/Murine D3 Nucleotide Sequence SEQ ID NO:
85 Amino Acid Sequence SEQ ID NO: 86 Murine D1/Human Nucleotide
Sequence SEQ ID NO: 87 D2/Murine D3 Amino Acid Sequence SEQ ID NO:
88 Murine D1D2/Human D3 Nucleotide Sequence SEQ ID NO: 89 Amino
Acid Sequence SEQ ID NO: 90
Example 17
Inhibition of Tumor Growth and Metastasis In Vivo
[0270] MDA MB231 cells stably expressing GFP (MDA MB231-GFP cells)
were grown in culture and harvested at 85% confluence. A total of
5.times.10.sup.5 tumor cells were inoculated in saline/20%
Matrigel.TM. into the mammary fat pad of female. Balb/c nu/nu mice.
Three days post transplantation mice received the following;
control non-specific isotype matched IgG4; doxorubicin Rx, 5 mg per
kg i.p. once per week for two weeks; control or anti-human uPAR
mAbs 0.5 mg per mouse i.p. once per week for the duration of the
experiment. Tumor growth was measured once per week using calipers
and tumor volumes calculated according to formula
(a.times.b.sup.2/2), where a--is length of the long diameter,
b--width of the perpendicular small diameter (FIG. 7). Untreated
control tumor growth (not shown in FIG. 7) was not significantly
different compared to IgG4 isotype controls.
[0271] At the end of the study, primary tumors were fixed, and
lung, liver and spleen from control and experimental animals were
removed for further analysis. Tissue sections were stained for the
following proteins using standard immunohistochemical techniques:
Ki67; CD31; MAPK; phospho-MAPK; FAK; phospho-FAK. Staining
intensity was measured using BioQuant image analysis software and
data was recorded from 6 animals per treatment group (FIG. 8).
[0272] Lung, liver and spleen tissues were similarly treated and
tumor cells were identified by fluorescence microscopy and the
numbers of metastases were recorded (FIG. 9).
[0273] As demonstrated by FIGS. 7-9, the anti-uPAR antibodies
inhibited primary tumor growth and significantly inhibited the
incidence of metastases to distant organs. Immunohistochemical
examination of the primary tumors showed no effect against the
proliferation marker Ki67 but there was a consistent inhibition in
the levels of pMAPK and pFAK compared to controls. Unexpectedly,
CD31 staining also showed some reduction in staining intensity
although in the absence of antibody cross reactivity with murine
uPAR the mechanism must be assumed to be indirect as a result of
local tumor associated uPA activity.
Example 18
Immunohistochemistry
[0274] The binding profile of 2.6.1, 2.19.2 and 4.18.1 were
evaluated together with an IgG4 isotype control antibody on frozen
cell pellets and normal human tissue microarrays. (TriStar
Technology Group). Mouse anti uPAR from American Diagnostica Inc.,
(Cat #: 3936, Lot #: 041223) was a positive control Ab in the
study. Staining intensity was estimated by eye using a 0-3+ scale
(.+-. equivocal staining; 1+ weak staining; 2+ moderate staining;
3+ strong staining). Frozen cell pellets from PC3 human prostate
cancer and U937 human histocytic lymphoma U937 cell pellets used as
positive controls and both membrane and cytoplasmic staining was
observed (See Table 21). Positive staining was reported only when
staining was greater than and/or distinguishable from the
background if the background staining was observed with the
negative control antibody, or staining was in cells within the test
tissue that did not stain with the negative control antibody (see
Table 23 below).
TABLE-US-00022 TABLE 21 Staining Intenstity in Cell Pellets Abs
IgG4 control 2.6.1 2.19.2 4.18.1 PC-3 -- 2+ .+-. 3+ U937 -- 2+ 2+
1+
[0275] For staining of human tissue microarrays, 2.6.1, 2.19.2 and
4.18.1 were conjugated with Biotin, reassessed for binding to U937
cells (see Table 22 below) and then used to stain tissue
microarrays.
TABLE-US-00023 TABLE 22 Staining Intenstity in Cell Pellets Using
Biotin-Conjugated mAbs Abs IgG4 control 2.6.1 2.19.2 4.18.1 U937 --
2+ 2+ 2+
[0276] Tissue microarrays were stained at a concentration of 20
.mu.g per ml biotin conjugated antibody. The results are shown in
Table 23. Where indicated c=cytoplasmic and m=membrane staining
pattern.
TABLE-US-00024 TABLE 23 Tissue Microarray Staining Pattern Tissue
Type 2.6.1 20 ug 2.19.2 20 ug 4.18.1 20 ug liver inflammation
cells3+ (c) inflammation cells 3+ (c) inflammation cells 2+ (m, c)
liver inflammation cells3+ (c) inflammation cells 3+ (c)
inflammation cells 2+ (m, c) liver inflammation cells3+ (c)
inflammation cells 2+ (c) inflammation cells 1+ (c) liver
inflammation cells2+ (c), inflammation cells 1+ (c), inflammation
cells 1+ (c) stroma1+ stroma1+ liver inflammation cells 3+ (c)
inflammation cells 2+ (c) inflammation cells 1+ (c) liver
inflammation cells 3+ (c) inflammation cells 2+ (c) inflammation
cells 1+ (c) pancreas acinar cells 2+ (c) acinar cells 2+ (c)
acinar cells 1+ (c) pancreas acinar cells 3+ (c) acinar cells 2+
(c) acinar cells 1+ (c) pancreas acinar cells 2+ (c) acinar cells
2+ (c) acinar cells 1+ (c) pancreas acinar cells 1+ (c) acinar
cells 2+ (c) acinar cells 1+ (c) pancreas acinar cells 1+ (c)
fibroblast 1+ (c) inflammation cells 2+ (c) pancreas acinar cells
1+ (c) acinar cells 1+ (c) inflammation cells 1+ (c) colon mucosa
epithelium1+ negative inflammation cells 2+ (c) colon mucosa
epithelium1+ epithelium1+ (c) inflammation cells 2+ (m, c) colon
mucosa negative negative negative colon mucosa epithelium1+
inflammation cells 1+ (c) inflammation cells 2+ (m, c) colon mucosa
epithelium1+ epithelium 1+ (c) epithelium1+ (c), inflammation cells
1+ (c) colon mucosa epithelium1+ epithelium 1+ (c) epithelium1+
(c), inflammation cells 1+ (c) colon muscularis negative negative
inflammation cells 1+ (c) colon muscularis lymphocytes 1+ (c)
negative inflammation cells 1+ (c) colon muscularis negative
inflammation cells 1+ (c) negative colon muscularis negative nerve
1+ negative colon muscularis negative inflammation cells 1+ (c)
inflammation cells 1+ (c) colon muscularis nerve 1+ nerve 1+
negative kidney cortex negative negative negative kidney cortex
negative negative negative kidney cortex glomerulus 1+ (c)
glomerulus 1+ (c) glomerulus 1+ (c) kidney cortex glomerulus 1+ (c)
glomerulus 1+ (c) glomerulus 1+ (c) kidney cortex glomerulus 1+ (c)
negative inflammation cells 1+ (c) kidney cortex glomerulus 1+ (c)
inflammation cells 1+ (c) inflammation cells 1+ (c) kidney medulla
negative negative negative kidney medulla negative inflammation
cells 1+ (c) inflammation cells 1+ (c) kidney medulla negative
negative negative kidney medulla negative negative negative kidney
medulla negative negative negative kidney medulla negative negative
inflammation cells 1+ (c) lung alveoli epithelium 2+ (m) epithelium
2+ (m, c) epithelium 1+ (m, c), inflammation cells 1+ (c) lung
alveoli epithelium 3+ (m, c) epithelium 3+ (m, c) epithelium 2+ (m,
c), inflammation cells 2+ (c) lung alveoli epithelium 3+ (m, c)
epithelium 3+ (m, c) epithelium 2+ (m, c), inflammation cells 2+
(c) lung bronchus epithelium 1+ (c) lung bronchus negative negative
lung bronchus epithelium 1+ (c) epithelium 1+ (c) inflammation
cells 1+ (c) lung bronchus non-specific staining inflammation cells
2+ (c) inflammation cells 1+ (c) lung bronchus epithelium 1+ (c)
inflammation cells 2+ (c) inflammation cells 1+ (c) lymph node
lymphocytes 3+ (m.c) lymphocytes 2+ (m.c) lymphocytes 1+ (m.c)
lymph node lymphocytes 3+ (m.c) lymphocytes 3+ (m.c) lymphocytes 1+
(m.c) lymph node lymphocytes 2+ (m.c) lymphocytes 2+ (m.c)
lymphocytes 1+ (m.c) lymph node lymphocytes 2+ (m.c) lymphocytes 2+
(m.c) lymphocytes 1+ (m.c) lymph node lymphocytes 2+ (m.c)
lymphocytes 3+ (m.c) lymphocytes 1+ (m.c) heart negative negative
negative heart negative negative inflammation cells 1+ (c) heart
negative negative inflammation cells 1+ (c) heart negative negative
inflammation cells 1+ (c) heart negative negative negative
Example 19
Inhibition of Tumor Cell Growth in Human Patients
[0277] A group of human cancer patients diagnosed with pancreatic
cancer is randomized into treatment groups. Each patient group is
treated with weekly intravenous injections of mAb 2.6.1, 2.19.2 or
4.18.1 described herein. Each patient is dosed with an effective
amount of the antibody ranging from 5 mg/kg/week to 15 mg/kg/week
for 4-8 months. A control group is given only the standard
chemotherapeutic regimen.
[0278] At periodic times during and after the treatment regimen,
tumor-burden is assessed by magnetic resonance imaging (MRI). It is
found that the patients who have received weekly antibody treatment
with mAb 2.6.1, 2.19.2 or 4.18.1 show significant reductions in
tumor size, time delay to progression or prolonged survival
compared to patients that do not receive antibody treatment. In
some treated patients, the tumors are no longer detectable. In
contrast, tumor size increases or remains substantially the same in
the control group.
Example 20
Inhibition of Tumor Cell Growth in a Human Patient
[0279] A group of human cancer patients diagnosed with thyroid
cancer is randomized into treatment groups. Each patient group is
treated 3-weekly with intravenous injections of mAb 2.6.1, 2.19.2
or 4.18.1 described herein and gemcitabine. Each patient is dosed
with an effective amount of the antibody ranging from 5 mg/kg/week
to 15 mg/kg/week for 4-8 months and an effective therapeutic dose
of gemcitabine. A control group is given only the standard
chemotherapeutic regimen. At periodic times during and after the
treatment regimen, tumor burden is assessed by magnetic resonance
imaging (MRI). It is found that the patients who have received
3-weekly antibody treatment with mAb 2.6.1, 2.19.2 or 4.18.1 and
gemcitabine show significant reductions in tumor size, time delay
to progression or prolonged survival compared to patients that do
not receive antibody and gemcitabine treatment. In some treated
patients, the tumors are no longer detectable. In contrast, tumor
size increases or remains substantially the same in the control
group.
Example 21
Inhibition of Tumor Metastasis in Human Patients
[0280] A group of human cancer patients diagnosed with non-small
cell lung cancer is randomized into treatment groups. In addition
to a standard chemotherapeutic regimen, each patient group is
treated with weekly intravenous injections of mAb 2.6.1, 2.19.2 or
4.18.1 described herein. Each patient is dosed with an effective
amount of the antibody ranging from 5 mg/kg/week to 15 mg/kg/week
for 2-8 months. A control group is given only the standard
chemotherapeutic regimen.
[0281] At periodic times during and after the treatment regimen,
metastatic lesions are assessed by positron emission tomography
(PET). It is found that the patients who have received weekly
antibody treatment with mAb 2.6.1, 2.19.2 or 4.18.1 show
significant reductions in the number and size of metastatic
lesions, compared to patients that do not receive antibody
treatment.
Example 22
Treatment of Estrogen Receptor Positive Breast Cancer in a Human
Patient
[0282] An adult female is diagnosed with estrogen receptor positive
breast cancer. The patient is treated every 2 weeks with
intravenous injections of mAb 2.19.2 and an anti-hormonal agent
over a period of 2-8 months. As a result, the patient experiences
prolonged progression free survival.
Example 23
Treatment of HER2 Positive Breast Cancer in a Human Patient
[0283] An adult female is diagnosed with HER2 positive breast
cancer. The patient is treated every week with intravenous
injections of mAb 2.19.2 and an anti-her2 agent over a period of
2-8 months. As a result, the patient experiences a significant
reduction in tumor size.
Example 24
Treatment of EGFR-Expressing Lung Cancer in a Human Patient
[0284] An adult male is diagnosed with EGFR-expressing lung cancer.
The patient is treated weekly with intravenous injections of mAb
2.19.2 and a receptor tyrosine kinase inhibitor over a period of
2-8 months. As a result, the patient experiences a significant
delay in time to progression.
Example 25
Treatment of Arthritis in a Human Patient
[0285] An adult female is diagnosed with severe arthritis. The
patient is treated with intravenous injections every 2 weeks of mAb
2.19.2 over a period of 3 weeks. As a result, the patient
experiences a significant reduction in the symptoms of
arthritis.
Example 27
Treatment of Atherosclerosis in a Human Patient
[0286] An adult male is diagnosed with atherosclerosis. The patient
is treated with intravenous injections every other week of mAb
4.18.1 over a period of a year. As a result, the patient
experiences a reduction in the symptoms of atherosclerosis, such as
angina pectoris.
INCORPORATION BY REFERENCE
[0287] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated herein by reference in their entirety.
EQUIVALENTS
[0288] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The foregoing description and Examples detail certain
preferred embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
1081384DNAHuman 1gaggtgcagc tggtggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctacgaca
tgcactgggt ccgccaagct 120acaggaaaag gtctggagtg ggtctcagct
attggtactg ctggtgacac atactatcca 180ggctccgtga agggccgatt
caccatctcc agagaaaatg ccaagaactc cttgtatctt 240caaatgaaca
gcctgagagc cggggacacg gctgtgtatt actgtgcaag agagggggac
300tacagtaact actttactat ggttcgggga gttacctacg gtatggacgt
ctggggccaa 360gggaccacgg tcaccgtctc ctca 3842128PRTHuman 2Glu Val
Gln Leu Val 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 Ser Tyr 20 25
30Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Arg Glu Gly Asp Tyr Ser Asn Tyr Phe Thr Met
Val Arg Gly Val Thr 100 105 110Tyr Gly Met Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 1253321DNAHuman 3gacatccagt
tgacccagtc tccatcctca ctgtgtgcat ctgttggaga cagaatcacc 60atcacttgtc
gggcgagtca gggcattagc aaatatttag cctggtttca gcagaaacca
120gggaaagccc ctaagtccct gatctatggt gcatccagtt tgcaaagtgg
ggtcccagca 180aagttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt accctctcac tttcggcgga 300gggaccaagg tggagatcaa a
3214107PRTHuman 4Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Cys
Ala Ser Val Gly1 5 10 15Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Ser Lys Tyr 20 25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys
Ala Pro Lys Ser Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly
Val Pro Ala Lys 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 Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 1055363DNAHuman 5caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgcctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaagtca gaaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagggatg 300acttactact actactacgg tatggacgtc
tggggccaag ggaccacggt caccgtctcc 360tca 3636121PRTHuman 6Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Pro Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Gln Lys 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 Arg Gly Met Thr Tyr Tyr Tyr Tyr Tyr Gly
Met Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser
115 1207321DNAHuman 7gaaatagtga tgacgcagtc tccagccatc ctgtctgtgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaagttag
actggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt
gcatccacca gggccactgg tatcccagcc 180aggttcagcg gcagtgggtc
cgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg
cagtttatta ctgtcagcag tataataact ggccgcggac gttcggccaa
300gggaccaagg tggaaatcaa a 3218107PRTHuman 8Glu Ile Val Met Thr Gln
Ser Pro Ala Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Lys 20 25 30Leu Asp Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala
Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1059384DNAHuman 9gaggtgcagc tggtggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctacgaca
tgcactgggt ccgccaagct 120acaggaaaag gtctggagtg ggtctcagct
attggtactg ctggtgacac atactatcca 180ggctccgtga agggccgatt
caccatctcc agagaaaatg ccaagaactc cttgtatctt 240caaatgaaca
gcctgagagc cggggacacg gctgtgtatt actgtgcaag agagggggac
300tacagcaact attttactag ggttcgggga gttacctacg gtatggacgt
ctggggccaa 360gggaccacgg tcaccgtctc ctca 38410128PRTHuman 10Glu Val
Gln Leu Val 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 Ser Tyr 20 25
30Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Arg Glu Gly Asp Tyr Ser Asn Tyr Phe Thr Arg
Val Arg Gly Val Thr 100 105 110Tyr Gly Met Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 12511321DNAHuman 11gacatccaga
tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggcattagc aaatatttgg cctggtttca gcagaaacca
120gggaaagccc ctaagtccct gatctatggt gcatccagtt tgcaaagtgg
ggtcccatca 180aagttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt accctctcac tttcggcgga 300gggaccaagg tggagatcaa a
32112107PRTHuman 12Asp 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
Gly Ile Ser Lys Tyr 20 25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys
Ala Pro Lys Ser Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Lys 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 Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 10513351DNAHuman 13gaggtgcagc
tggtggagtc tggaggaggc ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag
cctcagggtt caccgtcaga agcaactaca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg gatctcagtt atttataccg gtggtagcac
atactacgca 180gactccgtga agggccgatt ctccatctcc agagacaatt
ccaagaacac gctgtatctt 240caaatgaaca gcctgagagc cgaggacacg
gccgtgtatt actgtgcgag agagggagca 300gtggctggat ttgactactg
gggccaggga accctggtca ccgtctcctc a 35114117PRTHuman 14Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Arg Ser Asn 20 25 30Tyr
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Ser Val Ile Tyr Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Glu Gly Ala Val Ala Gly Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser 11515321DNAHuman
15gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtattagc agcaacttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg
tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag
tatactaact ggcctctcac tttcggcgga 300gggaccaagg tggagatcaa a
32116107PRTHuman 16Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser
Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Ile Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Thr Asn Trp Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 10517351DNAHuman 17gaggtgcagc
tggtggagtc tggaggaggc ttgctccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctgggtt caccgtcaga aacaattaca tgaactgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagtt atttataccg gtggtagcac
atactacgca 180gactccgtga agggccgctt caccatctcc agagacaatt
ccaagaacac gctgtatctt 240caaatgaaca gcctgagagc cgaggacacg
gccgtgtatt actgtgcgag agaggggaag 300cttgccggta tggacgtctg
gggccaaggg accacggtca ccgtctcctc a 35118117PRTHuman 18Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Leu Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Arg Asn Asn 20 25 30Tyr
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Tyr Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Glu Gly Lys Leu Ala Gly Met Asp Val Trp Gly
Gln Gly Thr Thr 100 105 110Val Thr Val Ser Ser 11519321DNAHuman
19gaaacagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg
tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 240gaagaatttg cagtttatta ctgtcagcag
tataataact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa a
32120107PRTHuman 20Glu Thr Val Met Thr Gln Ser Pro Ala Thr Leu Ser
Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Glu Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 10521366DNAHuman 21caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa
taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagatcta 300gcagctctct actactacta
cggtatgcac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
36622122PRTHuman 22Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys 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 Arg Asp Leu Ala
Ala Leu Tyr Tyr Tyr Tyr Gly Met His Val Trp 100 105 110Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 12023321DNAHuman 23gaaatagtga
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagttttagc agcaacttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg
tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag
aataataact ggcctcggac gttcggccaa 300gggaccaagg tggaaatcaa a
32124107PRTHuman 24Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser
Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Phe Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Asn Asn Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 10525359DNAHuman 25gaggtgcagc
tggtggagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctgggtt caccgtcatt accaactaca tgaactgggt ccgccaggct
120ccaggaaagg ggctggagtg ggtctcaatt atttataccg gtggtagcac
atcctacgca 180gactccgtga agggccgatt caccgtctcc agagacaatt
ccaagaacac gctgtatctt 240cacatgaaca gcctgagagt cgaggacacg
gccgtgtatt actgtgcgag agagatagca 300gtggctggct actacggtat
ggacgtctgg ggccaaggga ccacggtcac cgtctcctc 35926119PRTHuman 26Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ile Thr Asn
20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ile Ile Tyr Thr Gly Gly Ser Thr Ser Tyr Ala Asp Ser
Val Lys 50 55 60Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu65 70 75 80His Met Asn Ser Leu Arg Val Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Arg Glu Ile Ala Val Ala Gly Tyr Tyr Gly
Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser
11527321DNAHuman 27gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga aatgatttag
gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg
caacttatta ctgtctacag cataatagtt acccgctcac tttcggcgga
300gggaccaggg tggagatcaa a 32128107PRTHuman 28Asp 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 Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg 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 Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro
Leu 85 90 95Thr Phe Gly Gly Gly Thr Arg Val Glu Ile Lys 100
10529365DNAHuman 29caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt
atatggtatg atggaagtaa taaatactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agccgaggac acggctgtgt attactgtgc gagagatcag
300catattgtgg tggtgactgc tattcttgac tactggggcc agggaaccct
ggtcaccgtc 360tcctc 36530122PRTHuman 30Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys 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 Arg Asp Gln His Ile Val Val Val Thr Ala Ile Leu Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12031341DNAHuman 31gacatcgtga tgacccagtc tccagactcc ctgtctgtgt
ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtgtttta tatggctcca
acaataggaa ctccttggct 120tggtaccagc agaaaccagg acagcctccc
aagctgctca tttactgggc atctacccgg 180gaatccgggg tccctgaccg
attcagtggc agcgggtctg ggacagattt cactctcacc 240atcagcagcc
tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtatt
300tttccgtgga cgttcggcca agggaccaag gtggaaatca a 34132114PRTHuman
32Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly1
5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
Gly 20 25 30Ser Asn Asn Arg Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Ile Phe Pro Trp Thr Phe
Gly Gln Gly Thr Lys Val Glu 100 105 110Ile Lys33377DNAHuman
33gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacttttagt agctattgga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtggccaac ataaagcaac atggaagtga
gaaatactat 180gtggactctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagaggag 300gcttattatt atgataatag
tggttactac tggtacttcg atctctgggc tcgtggcacc 360ctggtcactg tctcctc
37734126PRTHuman 34Glu Val Gln Leu Val 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 Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln His Gly Ser Glu
Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ala
Tyr Tyr Tyr Asp Asn Ser Gly Tyr Tyr Trp Tyr 100 105 110Phe Asp Leu
Trp Ala Arg Gly Thr Leu Val Thr Val Ser Ser 115 120
12535322DNAHuman 35gacatccaga tgacccagtc tccatcttcc gtgtctgcat
ctataggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc agctggttag
cctggtatca gcagaaacca 120gggaaaaccc ctaagctcct gatctatgct
gcatccagtt tacaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttacta ttgtcaacag gctgacagtt tcccgtggac gttcggccaa
300gggaccaagg tggaaatcaa ac 32236107PRTHuman 36Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Thr 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 Ala Asp Ser Phe Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10537372DNAHuman 37caggtgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtggtggtc
actactggag ctggatccgc 120cagcacccag ggaagggcct ggagtggatt
gggtacatct attacagtgg gagaacctac 180tacaacccgt ccctcaagag
tcgagttacc atatcagtag acacgtctaa gaaccagttc 240tccctgaagc
tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat
300ggggggactg gggattacta ctactacggt atggacgtct ggggccaagg
gaccacggtc 360accgtctcct ca 37238123PRTHuman 38Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30Gly His Tyr
Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile
Gly Tyr Ile Tyr Tyr Ser Gly Arg Thr Tyr Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75
80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95Cys Ala Arg Asp Gly Thr Gly Asp Tyr Tyr Tyr Tyr Gly Met Asp
Val 100 105 110Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
12039338DNAHuman 39gacatcgtga tgacccagtc tccaacctcc ctgactgtgt
ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtgtttta tacagctcca
acaataggaa cttcttagct 120tggtaccagc agaaaccaag acagcctcct
aagctgctca tttactgggc atctacccgg 180gaatccgggg tccctgaccg
attcagtggc agcgggtctg ggacagattt cactctcacc 240atcagcagcc
tgcaggctga agatgtggca gtttattact gtcagcaata ttatactact
300ccgctcactt tcggcggagg gaccaaggtg gagatcaa 33840113PRTHuman 40Asp
Ile Val Met Thr Gln Ser Pro Thr Ser Leu Thr Val Ser Leu Gly1 5 10
15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30Ser Asn Asn Arg Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Arg
Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Thr Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile 100 105 110Lys41369DNAHuman 41gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacctttagt agctattgga tgagctgggt ccgccaggct
120ccagggacgg ggctggattg ggtggccaac ataaaccaag atggaagtga
gagatactat 180gtggactctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcaccgcat 240ctgcaaatga acagcctgag agccgaggac
acggctgttt attactgtgc gagagaggga 300agcggtggca ggtacggagg
ctggtacttc gatctctggg gccgtggcac cctggtcact 360gtctcctca
36942123PRTHuman 42Glu Val Gln Leu Val 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 Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Thr Gly Leu Asp Trp Val 35 40 45Ala Asn Ile Asn Gln Asp Gly Ser Glu
Arg Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Pro His65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Ser
Gly Gly Arg Tyr Gly Gly Trp Tyr Phe Asp Leu 100 105 110Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser 115 12043342DNAHuman 43gacatcgtga
tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca
agtccagcca gagtgtttta tacagttcca acaataagaa cttcttagct
120tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc
atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcagcagtc tgcaggctga agatgtggca
gtttattact gtcagcaata ttatagtact 300cctccgatca ccttcggcca
agggacacga ctggagatta aa 34244114PRTHuman 44Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile
Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys
Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95Tyr Tyr Ser Thr Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu
Glu 100 105 110Ile Lys45366DNAHuman 45gaggtgcagt tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtcttaggt attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaagatggc 300tacggtgact acgtaggctg gtatttcgat
ctctggggcc gtggcaccct ggtcactgtc 360tcctca 36646122PRTHuman 46Glu
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 Ser Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Leu Gly Ile Ser Gly Ser Gly Gly 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 Asp Gly Tyr Gly Asp Tyr Val Gly
Trp Tyr Phe Asp Leu Trp 100 105 110Gly Arg Gly Thr Leu Val Thr Val
Ser Ser 115 12047339DNAHuman 47gacatcgtga tgacccagtc tccagactcc
ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtgtttta
tacagctcca acaataagaa cttcttagct 120tggtaccagc agaaaccagg
acagcctcct aagctactca tttactgggc atctacccgg 180gaatccgggg
tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
240atcagcaacc tgcaggctga agatgtggca gtttattact gtcaacaatt
ttatagtact 300cctccgacgt tcggccaagg gaccaaggtg gagatcaga
33948113PRTHuman 48Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn Phe Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Asn Leu Gln Ala
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Phe Tyr Ser Thr Pro
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Arg49351DNAHuman 49gaggtgcagc tggtggagtc tggaggaggc ttgatccagc
ctggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccgtcagg aataactaca
tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagtt
atttataccg gtggtagcac atactacgca 180gactccgtga agggccgatt
caccatctcc agagacaatt ccaggaacac gctgtatctt 240caaatgaaca
gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agaaggggca
300gtggctggtt ttgactactg gggccaggga accctggtca ccgtctcttt a
35150117PRTHuman 50Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Val Arg Asn Asn 20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Thr Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Arg Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Glu Gly Ala Val
Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Leu 11551321DNAHuman 51gaaatagtga tgacgcagtt tccagccacc
ctgtctatgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agcagcttag cctggtacca gcagacacct 120ggccaggctc ccaggctcct
catctatggt gcatccacca ggtccactgg tttcccagcc 180aggttcagtg
gcattgggtc tgggacagag ttcactctca ccatcagcag ccttcagtct
240gaagattttg cagtttatta ctgtcagcag tatggtaact ggcctctcac
cttcggccaa 300gggacacgac tggagattaa a 32152107PRTHuman 52Glu Ile
Val Met Thr Gln Phe Pro Ala Thr Leu Ser Met Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Leu Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Gly Ala Ser Thr Arg Ser Thr Gly Phe Pro Ala Arg Phe Ser
Gly 50 55 60Ile Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Asn Trp Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 10553351DNAHuman 53gaggtgcagc tggtggagtc tggaggaggc ttgatccagc
ctggggggtc cctgagactc 60tcctgtgcag cctctgggtt ctccgtcagt agcaactaca
tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagtt
atttataccg gtggtagcac atactacgca 180gactccgtga agggccgatt
caccatctcc agagacaatt ccaagaacac cctgtatctt 240caaatgaaca
gcctgagagc cgaggacacg gccgtatatt actgtgcgag agatacttac
300ctctacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc a
35154117PRTHuman 54Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Val Ser Ser Asn 20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Thr Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Asp Thr Tyr Leu
Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110Val Thr Val
Ser Ser 11555321DNAHuman 55gaaatagtga tgacgcagtc tccagccacc
ctgtctgtgt ctccagggga aagcgccacc 60ctctcctgca ggaccagtca gagtgttagc
agcaacttag cctggtacca gcagaaacct 120ggccagactc ccaggctcct
catctatggt gcatccgcca gggccactgg tgtcccagcc 180aggttcagtg
gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct
240gaagattttg cagtttatta
ctgtcagcag tattataact ggcctctcac tttcggcgga 300gggaccaagg
tggagatcaa a 32156107PRTHuman 56Glu Ile Val Met Thr Gln Ser Pro Ala
Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Ser Ala Thr Leu Ser Cys Arg
Thr Ser Gln Ser Val Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Thr Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ala Arg
Ala Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Trp Pro Leu 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10557366DNAHuman
57caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa
taaagactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagatatg 300atagtagtgg ttattactta
tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360tcttca
36658122PRTHuman 58Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Asp 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 Arg Asp Met Ile
Val Val Val Ile Thr Tyr Ala Phe Asp Ile Trp 100 105 110Gly Gln Gly
Thr Met Val Thr Val Ser Ser 115 12059339DNAHuman 59gacatcgtga
tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca
agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct
120tggtaccagc agaaatcagg acagcctcct aagctgctca tttactgggc
atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtggca
gtttactact gtcagcaata ttataggact 300cctccgacgt tcggccaagg
gaccagggtg gaaatcaaa 33960113PRTHuman 60Asp Ile Val Met Thr Gln Ser
Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn
Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn
Tyr Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln 35 40 45Pro Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile
Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90
95Tyr Tyr Arg Thr Pro Pro Thr Phe Gly Gln Gly Thr Arg Val Glu Ile
100 105 110Lys61113PRTHuman 61Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr
Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys62114PRTHuman 62Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Thr
Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu 100 105 110Ile
Lys63107PRTHuman 63Asp 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
Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys
Ala Pro Lys Ser 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 Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 10564107PRTHuman 64Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10565107PRTHuman 65Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser
Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 10566107PRTHuman 66Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
10567105PRTHuman 67Asp 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
Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Arg 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 Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln His Asn Ser Tyr Thr Phe 85 90 95Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 10568111PRTHuman 68Asp Ile Val Met Thr Gln Ser
Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn
Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile
Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90
95Tyr Tyr Ser Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105 11069107PRTHuman 69Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala 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 Ala Asn Ser Phe Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 10570124PRTHuman 70Glu Val Gln Leu
Val 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 Ser Tyr 20 25 30Asp Met
His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ala Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Arg Asp Tyr Ser Asn Tyr Thr Met Val Arg Gly Val Tyr Gly
Met Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 12071118PRTHuman 71Glu 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 Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
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 Tyr Gly
Asp Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110Leu Val
Thr Val Ser Ser 11572121PRTHuman 72Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr
Asp Gly Ser Asn Lys 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
Arg Asp Met Ile Val Val Val Ile Thr Ala Phe Asp Ile Trp Gly 100 105
110Gln Gly Thr Met Val Thr Val Ser Ser 115 12073118PRTHuman 73Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys 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 Arg Tyr Tyr Tyr Tyr Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11574114PRTHuman 74Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Val Ser Ser Asn 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Ser Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Val Ala Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110Ser
Ser75112PRTHuman 75Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Val Ser Ser Asn 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Ser Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105
11076117PRTHuman 76Glu Val Gln Leu Val 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 Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu
Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Trp Tyr
Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu 100 105 110Val Thr Val
Ser Ser 11577120PRTHuman 77Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly
Ser Asn Lys 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 Arg His
Ile Val Val Val Thr Ala Ile Leu Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 12078118PRTHuman 78Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25
30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Arg Ile Ala Val Ala Gly Tyr Tyr Gly Met Asp
Val Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser
11579123PRTHuman 79Glu Val Gln Leu Val 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 Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu
Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Tyr Tyr
Asp Ser Ser Gly Tyr Tyr Trp Tyr Phe Asp Leu 100 105 110Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser 115 12080119PRTHuman 80Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30Gly
Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40
45Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr 85 90 95Cys Ala Arg Thr Tyr Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11581990DNACynomolgus 81atgggtcacc cgctgctgct gccactgctg ctgctgctcc
acacctgcgt cccagcctct 60tggggcctgc ggtgcatgca gtgtaagagc aacggggatt
gccgggtgga agagtgcgcc 120ctaggacagg acctctgcag gaccacgatc
gtgcgcatgt gggaagaagg ggaagagctg 180gagctggtgg agaaaagctg
tacccactca gagaagacca acaggaccat gagctatcgg 240actggcttga
agatcaccag ccttaccgag gttgtgtgtg ggttagactt gtgcaaccag
300ggcaactctg gccgggctgt caccttttcc cgaagccgtt accttgaatg
catttcctgt 360ggctcatcag acatgagctg tgagaggggc cggcaccaga
gcctgcaatg ccgcagccct 420gaagaacagt gcctggacgt ggtgacccac
tggatccagg agggtgaaga agggcgtcca 480aaggatgacc gccacctccg
aggctgtggc taccttccca gctgcccagg ctccagtggt 540ttccacaaca
acgacacctt ccacttcctg aagtgttgca acaccaccaa atgcaacgag
600ggcccaatcc tggagcttga aaatctgcca cagaatggcc accagtgtta
cagctgcaag 660gggaacagca cccatggatg ctcctctgaa gagactttcc
tcattgactg ccgaggcccc 720atgaatcaat gtctggtagc caccggcact
tacgaaccga aaaaccaaag ctatatggta 780agaggctgtg taactgcctc
aatgtgccaa cgtgcccacc tgggtgacgc cttcagcatg 840caccacatca
atgtctcctg ctgtactgaa agtggctgta accacccaga cctggatatc
900cagtaccgcg ctcgaggagg gcccgaacaa aaactcatct cagaagagga
tctgaatagc 960gccgtcgacc atcatcatca tcatcattga
99082329PRTCynomolgus 82Met Gly His Pro Leu Leu Leu Pro Leu Leu Leu
Leu Leu His Thr Cys1 5 10 15Val Pro Ala Ser Trp Gly Leu Arg Cys Met
Gln Cys Lys Ser Asn Gly 20 25 30Asp Cys Arg Val Glu Glu Cys Ala Leu
Gly Gln Asp Leu Cys Arg Thr 35 40 45Thr Ile Val Arg Met Trp Glu Glu
Gly Glu Glu Leu Glu Leu Val Glu 50 55 60Lys Ser Cys Thr His Ser Glu
Lys Thr Asn Arg Thr Met Ser Tyr Arg65 70 75 80Thr Gly Leu Lys Ile
Thr Ser Leu Thr Glu Val Val Cys Gly Leu Asp 85 90 95Leu Cys Asn Gln
Gly Asn Ser Gly Arg Ala Val Thr Phe Ser Arg Ser 100 105 110Arg Tyr
Leu Glu Cys Ile Ser Cys Gly Ser Ser Asp Met Ser Cys Glu 115 120
125Arg Gly Arg His Gln Ser Leu Gln Cys Arg Ser Pro Glu Glu Gln Cys
130 135 140Leu Asp Val Val Thr His Trp Ile Gln Glu Gly Glu Glu Gly
Arg Pro145 150 155 160Lys Asp Asp Arg His Leu Arg Gly Cys Gly Tyr
Leu Pro Ser Cys Pro 165 170 175Gly Ser Ser Gly Phe His Asn Asn Asp
Thr Phe His Phe Leu Lys Cys 180 185 190Cys Asn Thr Thr Lys Cys Asn
Glu Gly Pro Ile Leu Glu Leu Glu Asn 195 200 205Leu Pro Gln Asn Gly
His Gln Cys Tyr Ser Cys Lys Gly Asn Ser Thr 210 215 220His Gly Cys
Ser Ser Glu Glu Thr Phe Leu Ile Asp Cys Arg Gly Pro225 230 235
240Met Asn Gln Cys Leu Val Ala Thr Gly Thr Tyr Glu Pro Lys Asn Gln
245 250 255Ser Tyr Met Val Arg Gly Cys Val Thr Ala Ser Met Cys Gln
Arg Ala 260 265 270His Leu Gly Asp Ala Phe Ser Met His His Ile Asn
Val Ser Cys Cys 275 280 285Thr Glu Ser Gly Cys Asn His Pro Asp Leu
Asp Ile Gln Tyr Arg Ala 290 295 300Arg Gly Gly Pro Glu Gln Lys Leu
Ile Ser Glu Glu Asp Leu Asn Ser305 310 315 320Ala Val Asp His His
His His His His 32583996DNAChimera 83atgggactcc caaggcggct
gctgctgctg ctgttgctgg cgactacctg tgtcccagcc 60tcccagggcc tgcagtgcat
gcagtgtgag agtaaccaga gctgcctggt agaggagtgt 120gctctgggcc
aggacctctg caggactacc gtgcttcggg aatggcaaga tgatagagag
180ctggaggtgg tgacaagagg ctgtgcccac agcgaaaaga ccaacaggac
catgagttac 240cgcatgggct ccatgatcat cagcctgaca gagaccgtgt
gcgccacaaa cctctgcaac 300aggcccagac ccggagcccg aggccgtgct
ttccctcagg gccgttacct cgagtgtatt 360tcctgtggct catcagacat
gagctgtgag aggggccggc accagagcct gcagtgccgc 420agccctgaag
aacagtgcct ggatgtggtg acccactgga tccaggaagg tgaagaaggg
480cgtccaaagg atgaccgcca cctccgtggc tgtggctacc ttcccggctg
cccgggctcc 540aatggtttcc acaacaacga caccttccac ttcctgaaat
gctgcaacac caccaaatgc 600aacgagggcc caatcctgga gcttgaaaat
ctgccgcaga atggccgcca gtgttacagc 660tgcaagggga acagcaccca
tggatgctcc tctgaagaga ctttcctcat tgactgccga 720ggccccatga
atcaatgtct ggtagccacc ggcactcacg aaccgaaaaa ccaaagctat
780atggtaagag gctgtgcaac cgcctcaatg tgccaacatg cccacctggg
tgacgccttc 840agcatgaacc acattgatgt ctcctgctgt actaaaagtg
gctgtaacca cccagacctg 900gatatccagt accgcgctca aggagggccc
gaacaaaaac tcatctcaga agaggatctg 960aatagcgccg tcgaccatca
tcatcatcat cattga 99684331PRTChimera 84Met Gly Leu Pro Arg Arg Leu
Leu Leu Leu Leu Leu Leu Ala Thr Thr1 5 10 15Cys Val Pro Ala Ser Gln
Gly Leu Gln Cys Met Gln Cys Glu Ser Asn 20 25 30Gln Ser Cys Leu Val
Glu Glu Cys Ala Leu Gly Gln Asp Leu Cys Arg 35 40 45Thr Thr Val Leu
Arg Glu Trp Gln Asp Asp Arg Glu Leu Glu Val Val 50 55 60Thr Arg Gly
Cys Ala His Ser Glu Lys Thr Asn Arg Thr Met Ser Tyr65 70 75 80Arg
Met Gly Ser Met Ile Ile Ser Leu Thr Glu Thr Val Cys Ala Thr 85 90
95Asn Leu Cys Asn Arg Pro Arg Pro Gly Ala Arg Gly Arg Ala Phe Pro
100 105 110Gln Gly Arg Tyr Leu Glu Cys Ile Ser Cys Gly Ser Ser Asp
Met Ser 115 120 125Cys Glu Arg Gly Arg His Gln Ser Leu Gln Cys Arg
Ser Pro Glu Glu 130 135 140Gln Cys Leu Asp Val Val Thr His Trp Ile
Gln Glu Gly Glu Glu Gly145 150 155 160Arg Pro Lys Asp Asp Arg His
Leu Arg Gly Cys Gly Tyr Leu Pro Gly 165 170 175Cys Pro Gly Ser Asn
Gly Phe His Asn Asn Asp Thr Phe His Phe Leu 180 185 190Lys Cys Cys
Asn Thr Thr Lys Cys Asn Glu Gly Pro Ile Leu Glu Leu 195 200 205Glu
Asn Leu Pro Gln Asn Gly Arg Gln Cys Tyr Ser Cys Lys Gly Asn 210 215
220Ser Thr His Gly Cys Ser Ser Glu Glu Thr Phe Leu Ile Asp Cys
Arg225 230 235 240Gly Pro Met Asn Gln Cys Leu Val Ala Thr Gly Thr
His Glu Pro Lys 245 250 255Asn Gln Ser Tyr Met Val Arg Gly Cys Ala
Thr Ala Ser Met Cys Gln 260 265 270His Ala His Leu Gly Asp Ala Phe
Ser Met Asn His Ile Asp Val Ser 275 280 285Cys Cys Thr Lys Ser Gly
Cys Asn His Pro Asp Leu Asp Ile Gln Tyr 290 295 300Arg Ala Gln Gly
Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu305 310 315 320Asn
Ser Ala Val Asp His His His His His His 325 33085972DNAChimera
85atgggtcacc cgctgctgct gccgctgctg ctgctgctcc acacctgcgt cccagcctct
60tggggcctgc ggtgcatgca gtgtaagacc aacggggatt gccgtgtgga agagtgcgcc
120ctgggacagg acctctgcag gaccacgatc gtgcgcttgt gggaagaagg
agaagagctg 180gagctggtgg agaaaagctg tacccactca gagaagacca
acaggaccct gagctatcgg 240actggcttga agatcaccag ccttaccgag
gttgtgtgtg ggttagactt gtgcaaccag 300ggcaactctg gccgggctgt
cacctattcc cgaagccgtt acctcgaatg catttcctgt 360ggctcatcag
acatgagctg tgagaggggc cggcaccaga gcctgcagtg ccgcagccct
420gaagaacagt gcctggatgt ggtgacccac tggatccagg aaggtgaaga
agggcgtcca 480aaggatgacc gccacctccg tggctgtggc taccttcccg
gctgcccggg ctccaatggt 540ttccacaaca acgacacctt ccacttcctg
aaatgctgca acaccaccaa atgcaacgag 600ggcccaatcc tggagcttga
aaatctgccg cagaatggcc gccagtgtta cagctgtgag 660gggaacaata
cccttgggtg ttcctccgaa gaggcgtctc tcatcaactg ccggggacca
720atgaatcagt gcctggtggc tacaggctta gatgtgctgg gaaaccggag
ttataccgta 780agaggctgcg ccacggcttc ctggtgccaa ggctcccacg
tggcagactc cttcccgacc 840cacctcaacg tctctgtctc ctgctgccac
ggcagcggct gtaacagccc cacacgagga 900gggcccgaac aaaaactcat
ctcagaagag gatctgaata gcgccgtcga ccatcatcat 960catcatcatt ga
97286323PRTChimera 86Met Gly His Pro Leu Leu Leu Pro Leu Leu Leu
Leu Leu His Thr Cys1 5 10 15Val Pro Ala Ser Trp Gly Leu Arg Cys Met
Gln Cys Lys Thr Asn Gly 20 25 30Asp Cys Arg Val Glu Glu Cys Ala Leu
Gly Gln Asp Leu Cys Arg Thr 35 40 45Thr Ile Val Arg Leu Trp Glu Glu
Gly Glu Glu Leu Glu Leu Val Glu 50 55 60Lys Ser Cys Thr His Ser Glu
Lys Thr Asn Arg Thr Leu Ser Tyr Arg65 70 75 80Thr Gly Leu Lys Ile
Thr Ser Leu Thr Glu Val Val Cys Gly Leu Asp 85 90 95Leu Cys Asn Gln
Gly Asn Ser Gly Arg Ala Val Thr Tyr Ser Arg Ser 100 105 110Arg Tyr
Leu Glu Cys Ile Ser Cys Gly Ser Ser Asp Met Ser Cys Glu 115 120
125Arg Gly Arg His Gln Ser Leu Gln Cys Arg Ser Pro Glu Glu Gln Cys
130 135 140Leu Asp Val Val Thr His Trp Ile Gln Glu Gly Glu Glu Gly
Arg Pro145 150 155 160Lys Asp Asp Arg His Leu Arg Gly Cys Gly Tyr
Leu Pro Gly Cys Pro 165 170 175Gly Ser Asn Gly Phe His Asn Asn Asp
Thr Phe His Phe Leu Lys Cys 180 185 190Cys Asn Thr Thr Lys Cys Asn
Glu Gly Pro Ile Leu Glu Leu Glu Asn 195 200 205Leu Pro Gln Asn Gly
Arg Gln Cys Tyr Ser Cys Glu Gly Asn Asn Thr 210 215 220Leu Gly Cys
Ser Ser Glu Glu Ala Ser Leu Ile Asn Cys Arg Gly Pro225 230 235
240Met Asn Gln Cys Leu Val Ala Thr Gly Leu Asp Val Leu Gly Asn Arg
245 250 255Ser Tyr Thr Val Arg Gly Cys Ala Thr Ala Ser Trp Cys Gln
Gly Ser 260 265 270His Val Ala Asp Ser Phe Pro Thr His Leu Asn Val
Ser Val Ser Cys 275 280 285Cys His Gly Ser Gly Cys Asn Ser Pro Thr
Arg Gly Gly Pro Glu Gln 290 295 300Lys Leu Ile Ser Glu Glu Asp Leu
Asn Ser Ala Val Asp His His His305 310 315 320His His
His87978DNAChimera 87atgggactcc caaggcggct gctgctgctg ctgttgctgg
cgactacctg tgtcccagcc 60tcccagggcc tgcagtgcat gcagtgtgag agtaaccaga
gctgcctggt agaggagtgt 120gctctgggcc aggacctctg caggactacc
gtgcttcggg aatggcaaga tgatagagag 180ctggaggtgg tgacaagagg
ctgtgcccac agcgaaaaga ccaacaggac catgagttac 240cgcatgggct
ccatgatcat cagcctgaca gagaccgtgt gcgccacaaa cctctgcaac
300aggcccagac ccggagcccg aggccgtgct ttccctcagg gccgttacct
cgagtgtatt 360tcctgtggct catcagacat gagctgtgag aggggccggc
accagagcct gcagtgccgc 420agccctgaag aacagtgcct ggatgtggtg
acccactgga tccaggaagg tgaagaaggg 480cgtccaaagg atgaccgcca
cctccgtggc tgtggctacc ttcccggctg cccgggctcc 540aatggtttcc
acaacaacga caccttccac ttcctgaaat gctgcaacac caccaaatgc
600aacgagggcc caatcctgga gcttgaaaat ctgccgcaga atggccgcca
gtgttacagc 660tgtgagggga acaataccct tgggtgttcc tccgaagagg
cgtctctcat caactgccgg 720ggaccaatga atcagtgcct ggtggctaca
ggcttagatg tgctgggaaa ccggagttat 780accgtaagag gctgcgccac
ggcttcctgg tgccaaggct cccacgtggc agactccttc 840ccgacccacc
tcaacgtctc tgtctcctgc tgccacggca gcggctgtaa cagccccaca
900cgaggagggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc
cgtcgaccat 960catcatcatc atcattga 97888325PRTChimera 88Met Gly Leu
Pro Arg Arg Leu Leu Leu Leu Leu Leu Leu Ala Thr Thr1 5 10 15Cys Val
Pro Ala Ser Gln Gly Leu Gln Cys Met Gln Cys Glu Ser Asn 20 25 30Gln
Ser Cys Leu Val Glu Glu Cys Ala Leu Gly Gln Asp Leu Cys Arg 35 40
45Thr Thr Val Leu Arg Glu Trp Gln Asp Asp Arg Glu Leu Glu Val Val
50 55 60Thr Arg Gly Cys Ala His Ser Glu Lys Thr Asn Arg Thr Met Ser
Tyr65 70 75 80Arg Met Gly Ser Met Ile Ile Ser Leu Thr Glu Thr Val
Cys Ala Thr 85 90 95Asn Leu Cys Asn Arg Pro Arg Pro Gly Ala Arg Gly
Arg Ala Phe Pro 100 105 110Gln Gly Arg Tyr Leu Glu Cys Ile Ser Cys
Gly Ser Ser Asp Met Ser 115 120 125Cys Glu Arg Gly Arg His Gln Ser
Leu Gln Cys Arg Ser Pro Glu Glu 130 135 140Gln Cys Leu Asp Val Val
Thr His Trp Ile Gln Glu Gly Glu Glu Gly145 150 155 160Arg Pro Lys
Asp Asp Arg His Leu Arg Gly Cys Gly Tyr Leu Pro Gly 165 170 175Cys
Pro Gly Ser Asn Gly Phe His Asn Asn Asp Thr Phe His Phe Leu 180 185
190Lys Cys Cys Asn Thr Thr Lys Cys Asn Glu Gly Pro Ile Leu Glu Leu
195 200 205Glu Asn Leu Pro Gln Asn Gly Arg Gln Cys Tyr Ser Cys Glu
Gly Asn 210 215 220Asn Thr Leu Gly Cys Ser Ser Glu Glu Ala Ser Leu
Ile Asn Cys Arg225 230 235 240Gly Pro Met Asn Gln Cys Leu Val Ala
Thr Gly Leu Asp Val Leu Gly 245 250 255Asn Arg Ser Tyr Thr Val Arg
Gly Cys Ala Thr Ala Ser Trp Cys Gln 260 265 270Gly Ser His Val Ala
Asp Ser Phe Pro Thr His Leu Asn Val Ser Val 275 280 285Ser Cys Cys
His Gly Ser Gly Cys Asn Ser Pro Thr Arg Gly Gly Pro 290 295 300Glu
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His305 310
315 320His His His His His 32589987DNAChimera 89atgggactcc
caaggcggct gctgctgctg ctgttgctgg cgactacctg tgtcccagcc 60tcccagggcc
tgcagtgcat gcagtgtgag agtaaccaga gctgcctggt agaggagtgt
120gctctgggcc aggacctctg caggactacc gtgcttcggg aatggcaaga
tgatagagag 180ctggaggtgg tgacaagagg ctgtgcccac agcgaaaaga
ccaacaggac catgagttac 240cgcatgggct ccatgatcat cagcctgaca
gagaccgtgt gcgccacaaa cctctgcaac 300aggcccagac ccggagcccg
aggccgtgct ttccctcagg gccgttacct cgagtgtgcg 360tcctgcacct
ctttggacca gagctgtgag aggggccggg agcaaagcct gcaatgccgc
420tatcctacag agcactgtat tgaagtggtg accctccaga gcacagaaag
gagcttgaag 480gatgaggact acacccgagg ctgtggcagt cttcccggat
gcccaggcac agcaggtttc 540catagcaacc agacctttca cttcctgaag
tgttgcaact acacccactg caatggtggc 600ccagttctgg atcttcagag
ctttccaccg aatggcttcc agtgttacag ctgcaagggg 660aacagcaccc
atggatgctc ctctgaagag actttcctca ttgactgccg aggccccatg
720aatcaatgtc tggtagccac cggcactcac gaaccgaaaa accaaagcta
tatggtaaga 780ggctgtgcaa ccgcctcaat gtgccaacat gcccacctgg
gtgacgcctt cagcatgaac 840cacattgatg tctcctgctg tactaaaagt
ggctgtaacc acccagacct ggatatccag
900taccgcgctc taggagggcc cgaacaaaaa ctcatctcag aagaggatct
gaatagcgcc 960gtcgaccatc atcatcatca tcattga 98790328PRTChimera
90Met Gly Leu Pro Arg Arg Leu Leu Leu Leu Leu Leu Leu Ala Thr Thr1
5 10 15Cys Val Pro Ala Ser Gln Gly Leu Gln Cys Met Gln Cys Glu Ser
Asn 20 25 30Gln Ser Cys Leu Val Glu Glu Cys Ala Leu Gly Gln Asp Leu
Cys Arg 35 40 45Thr Thr Val Leu Arg Glu Trp Gln Asp Asp Arg Glu Leu
Glu Val Val 50 55 60Thr Arg Gly Cys Ala His Ser Glu Lys Thr Asn Arg
Thr Met Ser Tyr65 70 75 80Arg Met Gly Ser Met Ile Ile Ser Leu Thr
Glu Thr Val Cys Ala Thr 85 90 95Asn Leu Cys Asn Arg Pro Arg Pro Gly
Ala Arg Gly Arg Ala Phe Pro 100 105 110Gln Gly Arg Tyr Leu Glu Cys
Ala Ser Cys Thr Ser Leu Asp Gln Ser 115 120 125Cys Glu Arg Gly Arg
Glu Gln Ser Leu Gln Cys Arg Tyr Pro Thr Glu 130 135 140His Cys Ile
Glu Val Val Thr Leu Gln Ser Thr Glu Arg Ser Leu Lys145 150 155
160Asp Glu Asp Tyr Thr Arg Gly Cys Gly Ser Leu Pro Gly Cys Pro Gly
165 170 175Thr Ala Gly Phe His Ser Asn Gln Thr Phe His Phe Leu Lys
Cys Cys 180 185 190Asn Tyr Thr His Cys Asn Gly Gly Pro Val Leu Asp
Leu Gln Ser Phe 195 200 205Pro Pro Asn Gly Phe Gln Cys Tyr Ser Cys
Lys Gly Asn Ser Thr His 210 215 220Gly Cys Ser Ser Glu Glu Thr Phe
Leu Ile Asp Cys Arg Gly Pro Met225 230 235 240Asn Gln Cys Leu Val
Ala Thr Gly Thr His Glu Pro Lys Asn Gln Ser 245 250 255Tyr Met Val
Arg Gly Cys Ala Thr Ala Ser Met Cys Gln His Ala His 260 265 270Leu
Gly Asp Ala Phe Ser Met Asn His Ile Asp Val Ser Cys Cys Thr 275 280
285Lys Ser Gly Cys Asn His Pro Asp Leu Asp Ile Gln Tyr Arg Ala Leu
290 295 300Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
Ser Ala305 310 315 320Val Asp His His His His His His 325915PRTHomo
sapiens 91Ser Tyr Gly Met His1 59217PRTHomo sapiens 92Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly9313PRTHomo sapiens 93Asp Gln His Ile Val Val Val Thr Ala Ile
Leu Asp Tyr1 5 10945PRTHomo sapiens 94Thr Asn Tyr Met Asn1
59516PRTHomo sapiens 95Ile Ile Tyr Thr Gly Gly Ser Thr Ser Tyr Ala
Asp Ser Val Lys Gly1 5 10 159612PRTHomo sapiens 96Glu Ile Ala Val
Ala Gly Tyr Tyr Gly Met Asp Val1 5 10975PRTHomo sapiens 97Asn Asn
Tyr Met Asn1 59816PRTHomo sapiens 98Val Ile Tyr Thr Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val Lys Gly1 5 10 15999PRTHomo sapiens 99Glu
Gly Ala Val Ala Gly Phe Asp Tyr1 510017PRTHomo sapiens 100Lys Ser
Ser Gln Ser Val Leu Tyr Gly Ser Asn Asn Arg Asn Ser Leu1 5 10
15Ala1017PRTHomo sapiens 101Trp Ala Ser Thr Arg Glu Ser1
510210PRTHomo sapiens 102Gln Gln Tyr Tyr Ser Ile Phe Pro Trp Thr1 5
1010311PRTHomo sapiens 103Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu
Gly1 5 101047PRTHomo sapiens 104Ala Ala Ser Ser Leu Gln Ser1
51059PRTHomo sapiens 105Leu Gln His Asn Ser Tyr Pro Leu Thr1
510611PRTHomo sapiens 106Arg Ala Ser Gln Ser Val Ser Ser Ser Leu
Ala1 5 101077PRTHomo sapiens 107Gly Ala Ser Thr Arg Ser Thr1
51089PRTHomo sapiens 108Gln Gln Tyr Gly Asn Trp Pro Leu Thr1 5
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