U.S. patent application number 14/228367 was filed with the patent office on 2014-09-11 for antibodies against tl1a and uses thereof.
This patent application is currently assigned to Teva Pharmaceuticals Australia Pty Ltd. The applicant listed for this patent is Teva Pharmaceuticals Australia Pty Ltd. Invention is credited to Adam Clarke, Anthony Gerard Doyle, Philip Anthony Jennings, George Kopsidas, Matthew Pollard, Lynn Dorothy Poulton, Andrew James Pow, Debra Tamvakis.
Application Number | 20140255302 14/228367 |
Document ID | / |
Family ID | 47994017 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140255302 |
Kind Code |
A1 |
Poulton; Lynn Dorothy ; et
al. |
September 11, 2014 |
ANTIBODIES AGAINST TL1a AND USES THEREOF
Abstract
The disclosure provides TNF-like ligand 1a (TL1a)-binding
proteins comprising an antigen binding domain of an antibody which
binds specifically to TL1a and inhibits interaction of TL1a and
Death Receptor 3 (DR3) and which does not inhibit the interaction
of TL1a and Decoy Receptor 3 (DcR3). The disclosure also provides
uses of the TL1a-binding proteins.
Inventors: |
Poulton; Lynn Dorothy;
(Macquarie Park, AU) ; Clarke; Adam; (Five Dock,
AU) ; Pow; Andrew James; (Pascoe Vale, AU) ;
Tamvakis; Debra; (Camberwell, AU) ; Kopsidas;
George; (Preston, AU) ; Doyle; Anthony Gerard;
(Drummoyne, AU) ; Jennings; Philip Anthony;
(Warrawee, AU) ; Pollard; Matthew; (Dural,
AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Teva Pharmaceuticals Australia Pty Ltd |
Macquarie Park |
|
AU |
|
|
Assignee: |
Teva Pharmaceuticals Australia Pty
Ltd
Macquarie Park
AU
|
Family ID: |
47994017 |
Appl. No.: |
14/228367 |
Filed: |
March 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/AU2012/001161 |
Sep 28, 2012 |
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14228367 |
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61541590 |
Sep 30, 2011 |
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61541590 |
Sep 30, 2011 |
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Current U.S.
Class: |
424/1.49 ;
424/139.1; 424/9.6; 435/252.3; 435/252.31; 435/252.32; 435/252.33;
435/252.34; 435/254.2; 435/254.21; 435/254.23; 435/331; 435/7.21;
435/7.92; 436/501; 530/387.3; 530/387.9; 536/23.53 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 2039/505 20130101; G01N 33/6863 20130101; C07K 2317/76
20130101; A61P 1/04 20180101; C07K 2317/622 20130101; C07K 2317/94
20130101; C07K 2317/55 20130101; A61P 37/06 20180101; A61P 1/00
20180101; A61P 29/00 20180101; A61P 27/02 20180101; C07K 16/2875
20130101; C07K 2317/92 20130101; A61P 19/02 20180101; C07K 2317/21
20130101; C07K 2317/34 20130101; A61P 11/00 20180101; C07K 16/241
20130101; A61P 11/06 20180101; C07K 2317/62 20130101 |
Class at
Publication: |
424/1.49 ;
530/387.9; 530/387.3; 536/23.53; 424/139.1; 435/7.21; 435/7.92;
436/501; 424/9.6; 435/252.3; 435/252.33; 435/252.32; 435/252.31;
435/252.34; 435/254.2; 435/254.23; 435/254.21; 435/331 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/68 20060101 G01N033/68; C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2011 |
AU |
2011904042 |
Claims
1. An isolated or recombinant TNF-like ligand 1a (TL1a)-binding
protein, comprising an antigen binding domain of an antibody that
specifically binds to TL1a and inhibits interaction of TL1a and
Death Receptor 3 (DR3) and does not inhibit the interaction of TL1a
with Decoy Receptor 3 (DcR3).
2. The TL1a-binding protein of claim 1, wherein: (i) the
TL1a-binding protein reduces the level of apoptosis of TF-1 cells
cultured in the presence of human TL1a and cycloheximide with an
effective concentration (EC.sub.50) of from about 1.5 nM to about
10 fM; (ii) the antigen binding domain binds specifically to TL1a
and inhibits the interaction of biotinylated TL1a and a polypeptide
comprising DR3 fused to a Fc region of an antibody ("DR3/Fc") with
an EC.sub.50 of from about 2.5 nM to about 10 fM, or about 1.0 nM
to about 10 fM, or about 0.5 nM to about 10 fM, or about 0.1 nM to
about 10 fM in a competition ELISA; or (iii) the TL1a-binding
protein binds to TL1a on the surface of a cell with an EC.sub.50 of
from about 10 nM to about 10 fM or 5.0 nM to about 10 fM or 1.0 nM
to about 10 fM or 0.5 nM to about 10 fM or 0.1 nM to about 10 fM,
as determined using flow cytometry.
3. The TL1a-binding protein of claim 1, wherein the TL1a-binding
protein binds a mutant form of soluble human TL1a comprising the
amino acid sequence of SEQ ID NO: 202 in which the arginine at
position 32 has been substituted with alanine and/or the arginine
at position 85 has been substituted with alanine at a level that is
at least 75% lower than the level with which the protein binds to
soluble human TL1a comprising the sequence of SEQ ID NO: 202,
wherein the mutant form of TL1a is immobilized on a solid or
semi-solid substrate at a concentration of about 1 .mu.g/mL, and
wherein the TL1a binding protein at a concentration of 10 .mu.g/mL
is then contacted to the immobilized mutant TL1a.
4. The TL1a binding protein of claim 1, wherein the TL1a binding
protein binds at least the amino acid residues arginine at position
32 and arginine at position 85 of a human TL1a comprising the amino
acid sequence of SEQ ID NO: 202.
5. The TL1a-binding protein of claim 1, wherein the antigen binding
domain comprises at least a heavy chain variable region (VH) and a
light chain variable region (VL), wherein the VH and VL form an
Fv.
6. The TL1a-binding protein of claim 1, wherein the antigen binding
domain comprises (a) a heavy chain variable region (VH) comprising:
(i) a FR1 comprising the amino acid sequence of SEQ ID NO: 144;
(ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 43;
(iii) a FR2 comprising the amino acid sequence of SEQ ID NO: 145;
(iv) a CDR2 comprising the amino acid sequence of SEQ ID NO: 142;
(v) a FR3 comprising the amino acid sequence of SEQ ID NO: 146;
(vi) a CDR3 comprising the amino acid sequence of SEQ ID NO: 143;
and (vii) a FR4 comprising the amino acid sequence of SEQ ID NO:
147; and (b) a light chain variable region comprising: (i) a FR1
comprising the amino acid sequence of SEQ ID NO: 148; (ii) a CDR1
comprising the amino acid sequence of SEQ ID NO: 139; (iii) a FR2
comprising the amino acid sequence of SEQ ID NO: 149; (iv) a CDR2
comprising the amino acid sequence of SEQ ID NO: 140; (v) a FR3
comprising the amino acid sequence of SEQ ID NO: 150; (vi) a CDR3
comprising the amino acid sequence of SEQ ID NO: 141; and (vii) a
FR4 comprising the amino acid sequence of SEQ ID NO: 151.
7. The TL1a-binding protein of claim 5, wherein the VH and the VL
are in a single polypeptide chain, and the TL1a-binding protein is:
(i) a single chain Fv fragment (scFv); (ii) a dimeric scFv
(di-scFv); (iii) a scFv or di-scFv linked to a Fc or a heavy chain
constant domain (CH) 2 and/or CH3; or (iv) a scFv or di-scFv linked
to a protein that binds to an immune effector cell; or wherein the
VL and VH are in separate polypeptide chains and the TL1a-binding
protein is: (i) a diabody; (ii) a triabody; (iii) a tetrabody; (iv)
a Fab; (v) a F(ab')2; (vi) a Fv; (vii) a diabody, triabody,
tetrabody, Fab, F(ab')2, or Fv linked to a Fc or a heavy chain
constant domain (CH) 2 and/or CH3; (viii) a diabody, triabody,
tetrabody, Fab, F(ab')2, or Fv linked to a protein that binds to an
immune effector cell; or (ix) an antibody.
8. The TL1a-binding protein of claim 1, wherein the antigen binding
domain is comprised in a chimeric, de-immunized, humanized,
synhumanized, human, or primatized antibody.
9. A nucleic acid encoding the TL1a-binding protein of claim 1.
10. A cell expressing the TL1a-binding protein of claim 1.
11. A composition, comprising the TL1a-binding protein of claim 1
and a suitable carrier.
12. A method, comprising administering the TL1a-binding protein of
claim 1 to a cell, tissue, organ or subject, thereby treating a
TL1a-mediated condition in the cell, tissue, organ or subject.
13. A method for detecting TL1a in a sample, comprising contacting
a sample with the TL1a-binding protein of claim 1, thereby forming
a complex of the TL1a-binding protein and TL1a if Tl1a is in the
sample, and detecting the complex.
14. A method for detecting TL1a in a subject, comprising
administering the TL1a-binding protein of claim 1 conjugated to a
detectable label to the subject, and detecting a complex of the
TL1a-binding protein in the subject.
15. The method of claim 12, wherein the TL1a-mediated condition is
an autoimmune disease.
16. The method of claim 12, wherein the TL1a-mediated condition is
ulcerative colitis, Crohn's disease, irritable bowel syndrome,
rheumatoid arthritis, polyarthritis, multiple sclerosis, uveitis,
asthma or chronic obstructive pulmonary disease.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT/AU2012/001161
filed on Sep. 28, 2012, and claims priority to U.S. Provisional
Application No. 61/541,590 filed on Sep. 30, 2011 and Australian
Patent Application No. 2011904042 filed on Sep. 30, 2011. Each of
these applications is incorporated by reference herein, in its
entirety and for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
electronically as a text file named 512500 Sequence Listing 27 Sep.
2012_ST25.txt, created on Sep. 27, 2012, with a size of 173,000
bytes. The Sequence Listing is incorporated by reference
herein.
FIELD OF THE INVENTION
[0003] The disclosure relates to proteins that bind to TL1a and
uses thereof, e.g., in therapy, prophylaxis, diagnosis or
prognosis.
BACKGROUND OF THE INVENTION
[0004] TNF-like ligand 1a (TL1a, syn. TNF superfamily member 15
(TNFSF15); TL1 and VEGI) is a member of the tumor necrosis factor
superfamily, which is expressed by antigen presenting cells
(including dendritic cells, B cells and macrophages), CD4.sup.+ and
CD8.sup.+ T cells and endothelial cells and can be expressed on the
cell surface or secreted as a soluble cytokine. The receptor for
TL1a, Death Receptor 3 (DR3) is expressed by a variety of cells,
including CD4.sup.+ and CD8.sup.+ T cells, NK cells, NKT cells and
FOXP3.sup.+ regulatory T (Treg) cells.
[0005] TL1a can also bind a decoy receptor (DcR3), which is a
competitive inhibitor of DR3. DcR3 also acts as a decoy receptor
for Fas-ligand (Fas-L) and lymphotoxin-like inducible protein that
competes with glycoprotein D for binding herpesvirus entry mediator
on T-cells (LIGHT). Accordingly, DcR3 is an important regulator of
several signal transduction pathways.
[0006] The TL1a/DR3 signaling pathway has been implicated in
several biological systems, which are associated with human
diseases. For example, TL1a has been shown to play a role in
immunity and in angiogenesis.
[0007] Using mice deficient in TL1a and/or DR3, researchers have
also shown that inhibiting this pathway can provide prophylactic or
therapeutic benefit in several immune-mediated conditions, such as,
experimental autoimmune encephalomyelitis (EAE; a model of multiple
sclerosis), colitis, inflammatory bowel disease, asthma and
arthritis. TL1a has also been shown to promote formation of foam
cells and atherosclerotic plaques.
[0008] It will be apparent to the skilled artisan from the
foregoing that TL1a plays an important role in biological processes
involved in several important human diseases. Accordingly,
compounds that inhibit TL1a activity are desirable, e.g., for their
therapeutic, prophylactic, diagnostic and prognostic uses.
SUMMARY OF THE INVENTION
[0009] The inventors have produced TL1a-binding proteins comprising
antigen binding domains of antibodies which are capable of
specifically binding to TL1a and inhibiting interaction of TL1a and
DR3 (thereby neutralizing TL1a activity(ies)) without inhibiting
interaction of TL1a and DcR3. Without being bound by any theory or
mode of action, the inventors reasoned that such TL1a-binding
proteins may be capable of reducing or preventing signaling of TL1a
through DR3 without significantly disturbing the homeostatic
interaction of DcR3 and TL1a. This preserves the natural
antagonistic effects of DcR3 on TL1a-DR3 interactions, which may be
advantageous because DcR3 also regulates the amount of free Fas-L
and LIGHT available for binding to their receptors (Fas and H-VEM,
respectively). Since Fas-mediated killing plays a role in cancer
surveillance, potential downstream consequences of increasing the
amount of DcR3 to bind to Fas-L could include increased
susceptibility to cancer. Again, without being bound by theory or
mode of action, proteins that specifically inhibit interaction of
TL1a and DR3, but not DcR3, could be advantageous in treating
disease but without compromising safety.
[0010] A subclass of the TL1a-binding proteins identified by the
inventors was also found to inhibit or prevent apoptosis of TF-1
cells induced by human TL1a at low concentrations, i.e., the
antibodies had a low effective concentration or EC.sub.50.
TL1a-binding proteins capable of inhibiting or preventing TL1a
activity (e.g., TL1a-induced apoptosis of TF-1 cells) are sometimes
referred to herein as highly potent TL1a-binding proteins.
[0011] The inventors have also identified a region of TL1a which is
bound by a highly potent TL1a-binding protein which binds
specifically to TL1a and inhibits interaction of TL1a with DR3
without inhibiting the ability of TL1a to interact with DcR3.
[0012] The TL1a-binding proteins identified by the inventors form
the basis for various
therapeutic/prophylactic/diagnostic/prognostic uses. This is
demonstrated by the inventors' use of a TL1a-binding protein of the
disclosure to treat accepted models of colitis, with the protein
showing efficacy at least equal to the current standard of care for
this condition.
[0013] Accordingly, the disclosure provides an isolated or
recombinant TL1a-binding protein comprising an antigen binding
domain of an antibody, wherein the antigen binding domain
specifically binds to TL1a and, wherein the TL1a-binding protein
inhibits interaction of TL1a and DR3 and does not inhibit
interaction of TL1a and DcR3.
[0014] In one example, the TL1a-binding protein does not detectably
reduce interaction of TL1a and DcR3. For example, the effect of the
TL1a-binding protein on interaction of TL1a and DcR3 is assessed
using a competition enzyme linked immunosorbent assay (ELISA). For
example, the TL1a-binding protein is incubated with TL1a (e.g.,
human TL1a) and then contacted with a polypeptide comprising DcR3
(e.g., human DcR3 (hDcR3)) fused to an Fc region of an antibody
("DcR3/Fc") and the level of bound TL1a is detected. In one
example, the level of bound TL1a in the presence or absence of the
protein is not significantly different and/or is insufficiently
different to permit calculation of an EC.sub.50.
[0015] In one example, the level of inhibition of interaction of
TL1a and DcR3 (or DcR3/Fc) in the presence of the TL1a-binding
protein expressed as a percentage of the level of binding in the
absence of the protein is 25% or less, or 22% or less, or 20% or
less, or 18% or less, or 15% or less, or 12% or less, or 10% or
less, or 7% or less, or 5% or less.
[0016] In one example, the ability of a TL1a-binding protein to
inhibit interaction of TL1a and DR3 or DcR3 is assessed by
immobilizing DcR3/Fc or a polypeptide comprising DR3 (e.g., human
DR3 (hDR3)) fused to a Fc region of an antibody (DR3/Fc) on a solid
or semi-solid surface (e.g., an ELISA plate) at a concentration of
about 2 .mu.g/ml. The TL1a-binding protein is then contacted with
biotinylated human TL1a (at a concentration of about 1 .mu.g/ml)
for about 30 minutes then added to the immobilized DcR3/Fc or
DR3/Fc. Following washing, bound TL1a is detected. To determine
percentage binding or inhibition, data are normalized by expression
as a percentage of maximum binding of TL1a to the immobilized
DcR3/Fc or DR3/Fc in the absence of a TL1a-binding protein. By
calculating the level of inhibition at multiple concentrations of
the TL1a-binding protein, an EC.sub.50 can be determined.
[0017] In one example, the TL1a-binding protein inhibits
interaction of TL1a and DR3 (or DR3/Fc) but not TL1a and DcR3 (or
DcR3/Fc).
[0018] For example, the TL1a-binding protein inhibits interaction
of DR3/Fc and TL1a with an EC.sub.50 of from about 20 nM to about
10 fM, or an EC.sub.50 of 20 nM or less, such as, 15 nM or less,
for example, 11 nM or less, for example 5 nM or less. In one
example, the EC.sub.50 is 5 nM or less. For example, the EC.sub.50
is 3 nM or less. For example, the EC.sub.50 is 2.5 nM or less. For
example, the EC.sub.50 is 1 nM or less. For example, the EC.sub.50
is 0.5 nM or less. In one example, the EC.sub.50 is assessed using
a competition enzyme linked immunosorbent assay (ELISA). For
example, various concentrations of the TL1a-binding protein are
incubated with TL1a (e.g., human TL1a) (e.g., about 1 .mu.g/ml of
TL1a) and then contacted with the DR3/Fc (e.g., about 2 .mu.g/ml of
the DR3/Fc) and the level of bound TL1a is detected. The
concentration of protein at which half maximal inhibition of
binding to TL1a is detected is considered the EC.sub.50.
[0019] In one example, the TL1a-binding protein neutralizes TL1a
activity in or on a cell by interfering with TL1a and DR3
interactions.
[0020] In one example, the TL1a-binding protein binds to the
extracellular domain of TL1a, such as the extracellular domain of
human TL1a.
[0021] In one example, the TL1a-binding protein binds to human TL1a
produced by mammalian cells, such as human cells.
[0022] Exemplary TL1a-binding proteins of the disclosure reduce the
level of apoptosis of TF-1 cells cultured in the presence of human
TL1a, such as human TL1a produced by mammalian cells (e.g., human
cells) (e.g., about 100 ng human TL1a per mL of culture) and
cycloheximide. For example, about 7.times.10.sup.4 to
8.times.10.sup.4 TF-1 cells (e.g., 7.5.times.10.sup.4 cells) are
contacted with about 1 .mu.g human TL1a per mL of culture and
cycloheximide. For example, the TL1a-binding protein reduces the
level of apoptosis of the TF-1 cells with an EC.sub.50 (i.e., a
concentration of the TL1a-binding protein that achieves 50% of the
maximum inhibition of TL1a-induced apoptosis of TF-1 cells achieved
by the TL1a-binding protein) of 25 nM or less. In one example, the
EC.sub.50 is 5 nM or less. In one example, the EC.sub.50 is 2 nM or
less. In one example, the EC.sub.50 is 1.5 nM or less or 1.2 nM or
less or 1.1 nM or less. In one example, the EC.sub.50 is 1 nM or
less. In one example, the EC.sub.50 is 0.75 nM or less. In one
example, the EC.sub.50 is 0.3 nM or less. In one example, the
EC.sub.50 is 0.1 nM or less. In one example, the EC.sub.50 is from
about 1.5 nM to about 10 fM, such as from about 1 nM to about 50
fM, for example, from about 1 nM to about 100 fM.
[0023] In one example, the TL1a-binding protein binds to TL1a on
the surface of a cell with an EC.sub.50 (i.e., a concentration of
the TL1a-binding protein that achieves 50% of the maximum binding
to the cell achieved by the TL1a-binding protein) of about 10 nM or
less, e.g., as determined using flow cytometry. In one example, the
flow cytometry is performed with about 2.times.10.sup.5 to
3.times.10.sup.5 cells (e.g., 2.5.times.10.sup.5 cells). In one
example, the EC.sub.50 is 5 nM or less. In one example, the
EC.sub.50 is 2 nM or less. In one example, the EC.sub.50 is from
about 10.0 nM or 5.0 nM or 1.0 nM or 0.5 nM or 0.1 nM to about 10
fM.
[0024] The disclosure also provides an isolated or recombinant
TL1a-binding protein comprising an antigen binding domain of an
antibody, wherein the antigen binding domain binds specifically to
TL1a and inhibits the interaction of biotinylated TL1a and DR3/Fc
with an EC.sub.50 of about 2.5 nM or less, such as 1 nM or less or
from about 2.5 nM, or about 1.0 nM, or about 0.5 nM, or about 0.1
nM to about 10 fM in a competition ELISA,
[0025] wherein the DR3/Fc is immobilized on a solid or semi-solid
substrate (e.g., a solid substrate such as an ELISA plate) at a
concentration of about 2 .mu.g/mL, and wherein the biotinylated
TL1a is contacted for about 30 minutes at a concentration of about
1 .mu.g/mL with the TL1a binding protein at a concentration range
of from about 10 .mu.g/mL to about 0.01 .mu.g/mL and is then
contacted to the immobilized DR3/Fc,
[0026] and wherein the TL1a-binding protein does not detectably
reduce interaction of biotinylated TL1a and DcR3/Fc in a
competition ELISA compared to the level of the binding of
biotinylated TL1a to DcR3/Fc in the absence of the TL1a-binding
protein, wherein the DcR3/Fc is immobilized on a solid or
semi-solid substrate (e.g., a solid substrate such as an ELISA
plate) at a concentration of about 2 .mu.g/mL, wherein the
biotinylated TL1a is contacted for about 30 minutes at a
concentration of about 1 .mu.g/mL with the TL1a-binding protein at
a concentration of from about 10 .mu.g/mL to 0.1 .mu.g/mL and is
then contacted to the immobilized DcR3/Fc.
[0027] The disclosure also provides an isolated or recombinant
TL1a-binding protein comprising an antigen binding domain of an
antibody, wherein the antigen binding domain binds specifically to
TL1a and inhibits the interaction of biotinylated TL1a and DR3/Fc
with an EC.sub.50 of about 2.5 nM or less, such as 1 nM or less or
from about 2.5 nM, or about 1.0 nM, or about 0.5 nM, or about 0.1
nM to about 10 fM in a competition ELISA,
[0028] wherein the DR3/Fc is immobilized on a solid or semi-solid
substrate (e.g., a solid substrate such as an ELISA plate) at a
concentration of about 2 .mu.g/mL, and wherein the biotinylated
TL1a is contacted for about 30 minutes at a concentration of about
1 .mu.g/mL with the TL1-a binding protein at a concentration range
of from about 10 .mu.g/mL to about 0.01 .mu.g/mL and is then
contacted to the immobilized DR3/Fc,
[0029] and wherein the TL1a-binding protein does not detectably
reduce interaction of biotinylated TL1a and DcR3/Fc in a
competition ELISA compared to the level of the binding of
biotinylated TL1a to DcR3/Fc in the absence of the TL1a-binding
protein, and wherein the DcR3/Fc is immobilized on a solid or
semi-solid substrate (e.g., a solid substrate such as an ELISA
plate) at a concentration of about 2 .mu.g/mL, wherein the
biotinylated TL1a is contacted for about 30 minutes at a
concentration of about 1 .mu.g/mL with the TL1a-binding protein at
a concentration of from about 10 .mu.g/mL to 0.1 .mu.g/mL and is
then contacted to the immobilized DcR3/Fc.
[0030] and wherein the TL1a-binding protein reduces the level of
apoptosis of TF-1 cells cultured in the presence of human TL1a
produced by human cells and cycloheximide with an EC.sub.50 (i.e.,
a concentration of the TL1a-binding protein that achieves 50% of
the maximum inhibition of TL1a-induced apoptosis of TF-1 cells
achieved by the TL1a-binding protein) of 25 nM or less, or from
about 1.5 nM or 1.0 nM or 0.5 nM or 0.1 nM or 0.05 nM to about 10
fM
[0031] wherein about 7.5.times.10.sup.4 TF-1 cells are contacted
with about 100 ng human TL1a per mL of culture and about 10
.mu.g/ml cycloheximide with the TL1a-binding protein at a
concentration of about 5 .mu.g/mL or less for about 4 to 5
hours.
[0032] In one example, the TL1a is biotinylated at one site, i.e.,
the biotin is linked to only one amino acid in TL1a.
[0033] In one example, the TL1a-binding protein does not detectably
reduce interaction of the biotinylated TL1a and DcR3/Fc in the
competition ELISA compared to the level of the binding of
biotinylated TL1a to DcR3/Fc in the absence of the TL1a-binding
protein, wherein the biotinylated TL1a is contacted for about 30
minutes at a concentration of about 1 .mu.g/mL with the
TL1a-binding protein at a concentration of about 100 .mu.g/mL and
is then contacted to the immobilized DcR3/Fc.
[0034] In one example, the TL1a-binding protein does not detectably
reduce interaction of the biotinylated TL1a and DcR3/Fc in the
competition ELISA compared to the level of the binding of
biotinylated TL1a to DcR3/Fc in the absence of the TL1a-binding
protein, wherein the biotinylated TL1a is contacted for about 30
minutes at a concentration of about 1 .mu.g/mL with the
TL1a-binding protein at a concentration of about 10 .mu.g/mL and is
then contacted to the immobilized DcR3/Fc.
[0035] In one example, the TL1a-binding protein reduces the level
of apoptosis of the TF-1 cells with an EC.sub.50 of 22 nM or less.
In one example, the EC.sub.50 is 10 nM or less. In one example, the
EC.sub.50 is 5 nM or less. In one example, the EC.sub.50 is 2 nM or
less. In one example, the EC.sub.50 is 1.5 nM or less or 1.2 nM or
less or 1.1 nM or less. In one example, the EC.sub.50 is 1 nM or
less. In one example, the EC.sub.50 is 0.75 nM or less. In one
example, the EC.sub.50 is 0.3 nM or less. In one example, the
EC.sub.50 is 0.1 nM or less.
[0036] The disclosure additionally, or alternatively, provides an
isolated or recombinant TL1a-binding protein comprising an antigen
binding domain of an antibody, wherein the antigen binding domain
specifically binds to TL1a and, wherein the TL1a-binding protein
inhibits interaction of TL1a and DR3 and does not inhibit
interaction of TL1a and DcR3, and wherein the TL1a-binding protein
binds a mutant form of soluble human TL1a comprising a sequence set
forth in SEQ ID NO: 202 in which the arginine at position 32 has
been substituted with alanine and/or the arginine at position 85
has been substituted with alanine at a level that is at least 75%
lower than the level with which the TL1a-binding protein binds to
soluble human TL1a comprising a sequence set forth in SEQ ID NO:
202.
[0037] In one example, the mutant form of soluble human TL1a is
immobilized on a solid or semi-solid substrate (e.g., a solid
substrate such as an ELISA plate) at a concentration of about 1
.mu.g/mL, and wherein the TL1a binding protein at a concentration
range of from about 10 .mu.g/mL to about 0.01 .mu.g/ml is then
contacted to the immobilized mutant TL1a.
[0038] In one example, the TL1a-binding protein binds a mutant form
of soluble human TL1a comprising a sequence set forth in SEQ ID NO:
202 in which the arginine at position 32 has been substituted with
alanine and/or the arginine at position 85 has been substituted
with alanine at a level that is no greater than 25% of the level
with which the protein binds to soluble human TL1a comprising a
sequence set forth in SEQ ID NO: 202. For example, the level of
binding of the TL1a-binding protein to the mutant form of soluble
human TL1a is no greater than 25% of the level with which the
protein binds to soluble human TL1a, when the TL1a-binding protein
is tested at a concentration of 10 .mu.g/mL.
[0039] The disclosure additionally, or alternatively, provides an
isolated or recombinant TL1a-binding protein comprising an antigen
binding domain of an antibody, wherein the antigen binding domain
specifically binds to TL1a and, wherein the TL1a-binding protein
inhibits interaction of TL1a and DR3 and does not inhibit
interaction of TL1a and DcR3, and wherein the TL1a-binding protein
binds a mutant form of soluble human TL1a comprising a sequence set
forth in SEQ ID NO: 202 in which the arginine at position 32 has
been substituted with alanine and/or the arginine at position 85
has been substituted with alanine at a level that is at least 75%
lower than the level with which the protein binds to soluble human
TL1a comprising a sequence set forth in SEQ ID NO: 202,
[0040] wherein the mutant form of TL1a is immobilized on a solid or
semi-solid substrate at a concentration of about 1 .mu.g/mL, and
wherein the TL1a binding protein at a concentration of 10 .mu.g/mL
is then contacted to the immobilized mutant TL1a.
[0041] In one example, the mutant form of soluble human TL1a
comprises a sequence set forth in SEQ ID NO: 202 in which the
arginine at position 32 has been substituted with alanine.
[0042] In one example, the mutant form of soluble human TL1a
comprises a sequence set forth in SEQ ID NO: 202 in which the
arginine at position 85 has been substituted with alanine.
[0043] In one example, the mutant form of soluble human TL1a
comprises a sequence set forth in SEQ ID NO: 202 in which the
arginine at position 32 has been substituted with alanine and in
which the arginine at position 85 has been substituted with
alanine.
[0044] In one example, the level of binding of the TL1a-binding
protein to the mutant form of soluble human TL1a is at least 80% or
85% or 90% or 95% lower than the level with which the protein binds
to soluble human TL1a comprising a sequence set forth in SEQ ID NO:
202.
[0045] In one example, the TL1a-binding protein does not detectably
bind to the mutant form of soluble human TL1a.
[0046] In one example, the binding of the TL1a-binding protein to
soluble human TL1a or a mutant form thereof is assessed using
Surface Plasmon Resonance. For example, the soluble human TL1a or a
mutant form thereof is immobilized (e.g., at a concentration of
about 1 .mu.g/mL) and the TL1a-binding protein (e.g., at a
concentration of about 500 ng/mL) contacted to the immobilized
soluble human TL1a or a mutant form thereof and binding detected by
Surface Plasmon Resonance. By comparing the level of binding to the
soluble human TL1a or a mutant form thereof a comparison can be
made to determine a TL1a-binding protein that binds at a level that
is at least 75% lower than the level with which the protein binds
to soluble human TL1a.
[0047] In one example, the binding of the TL1a-binding protein to
soluble human TL1a or a mutant form thereof is assessed using
ELISA. For example, the soluble human TL1a or a mutant form thereof
is immobilized (e.g., at a concentration of about 1 .mu.g/mL) and
the TL1a-binding protein (e.g., at a concentration of about 10
.mu.g/mL) contacted to the immobilized soluble human TL1a or a
mutant form thereof and binding detected by ELISA (e.g., using
standard methods in the art).
[0048] In one example, the TL1a-binding protein binds to an epitope
within TL1a comprising residues corresponding to arginine at
position 32 of SEQ ID NO: 202 and the arginine at position 85 of
SEQ ID NO: 202. In one example, the epitope is a conformational
epitope.
[0049] In one example, the TL1a-binding protein binds at least at
amino acid residues arginine at position 32 and arginine at
position 85 of a human TL1a which comprises an amino acid sequence
as set forth in SEQ ID NO:202.
[0050] Exemplary TL1a-binding proteins having the binding
characteristics set forth in the foregoing paragraphs will be
apparent to the skilled artisan from the description herein and
include those comprising the following pairs of V.sub.H and
V.sub.L:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 94 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 95; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 137 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 138; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 162 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; or (iv)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 173 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 174.
[0051] V.sub.H and V.sub.L falling within the foregoing sequences
will be apparent to the skilled person from the description herein
and are to be taken to apply mutatis mutandis to the present
example of the disclosure.
[0052] In one example, a TL1a-binding protein of the disclosure
inhibits interaction of TL1a from human, cynomolgus monkey or
rhesus monkey and DR3. Such TL1a-binding proteins are useful for
characterization in animal models of human disease.
[0053] In one example, a TL1a-binding protein of the disclosure
does not detectably inhibit interaction of TL1a from mouse, pig,
rabbit or guinea pig and DR3, e.g., the TL1a-binding protein does
not detectably inhibit the level of apoptosis of TF-1 cells
cultured in the presence of the relevant TL1a, e.g. as determined
using an assay described herein.
[0054] In one example, a TL1a-binding protein of the disclosure
detectably inhibits interaction of TL1a from rat and DR3. For
example, the TL1a-binding protein detectably inhibits the level of
apoptosis of TF-1 cells cultured in the presence of the relevant
TL1a, e.g. as determined using an assay described herein.
[0055] In one example, a TL1a-binding protein of the disclosure
detectably binds to an isoform of TL1a consisting of amino acids
72-251 of SEQ ID NO: 123 and/or to an isoform of TL1a consisting of
amino acids 84-251 of SEQ ID NO: 123.
[0056] For example, binding is assessed by an ELISA in which the
isoform of TL1a is immobilized at a concentration of 1 .mu.g/ml and
the TL1a-binding protein is contacted to the isoform and the level
of binding assessed.
[0057] The disclosure additionally or alternatively provides an
isolated or recombinant TL1a-binding protein comprising an antigen
binding domain of an antibody comprising any one or more of the
following:
(i) aV.sub.H comprising a sequence set forth in SEQ ID NO: 94 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 95; (ii)
aV.sub.H comprising a sequence set forth in SEQ ID NO: 137 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 138; (iii)
aV.sub.H comprising a sequence set forth in SEQ ID NO: 162 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; or (iv)
aV.sub.H comprising a sequence set forth in SEQ ID NO: 173 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 174.
[0058] The disclosure additionally or alternatively provides an
isolated or recombinant TL1a-binding protein comprising an antigen
binding domain of an antibody comprising any one or more of the
following:
(i) aV.sub.H comprising a sequence set forth in SEQ ID NO: 2 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 6; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 10 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 14; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 18 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 22; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 50 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 54; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xviii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 163; (xx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 166; (xxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 167; (xxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xxxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165;
(xxxviii) a V.sub.H comprising a sequence set forth in SEQ ID NO:
42 and a V.sub.L comprising a sequence set forth in SEQ ID NO: 166;
(xxxix) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 167;
(xl) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xli)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xlii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xliii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 171; (xliv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; (xlv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (xlvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (xlvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (xlviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (xlix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (l) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (li) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (lii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 182 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 195; (liii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (liv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 184 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 197; (lv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (lvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; or
(lvii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 187
and a V.sub.L comprising a sequence set forth in SEQ ID NO:
200.
[0059] In a particular example, there is provided an isolated or
recombinant TL1a-binding protein comprising an antigen binding
domain of an antibody comprising a V.sub.H and a V.sub.L, wherein
the V.sub.H and V.sub.L respectively comprise sequences selected
from the group consisting of
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ii) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 106 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 107 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (iv) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 222 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (v) a V.sub.H
comprising a sequence set forth in SEQ ID NO: 176 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 189; (vi) a V.sub.H
encoded by a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 223 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions and a V.sub.L encoded by a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 228 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions; (vii) a V.sub.H comprising
a sequence set forth in SEQ ID NO: 177 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 190; (viii) a V.sub.H encoded by a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 224 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions and
a V.sub.L encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 229 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions; (ix) a V.sub.H comprising a sequence set
forth in SEQ ID NO: 178 and a V.sub.L comprising a sequence set
forth in SEQ ID NO: 191; (x) a V.sub.H encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 224 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions and a V.sub.L
encoded by a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 230 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions; (xi) a V.sub.H comprising a sequence set forth in SEQ
ID NO: 179 and a V.sub.L comprising a sequence set forth in SEQ ID
NO: 192; (xii) a V.sub.H encoded by a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 224 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions and a V.sub.L encoded by a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 231 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions;
(xiii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 180
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 193;
(xiv) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
224 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (xvi) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 225 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 230 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (xviii)
a V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 226 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 230 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (xx) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 226 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; (xxii) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 227 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xxiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 187 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 200; and
(xxiv) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
227 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 233 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions.
[0060] The disclosure additionally or alternatively provides an
isolated or recombinant TL1a-binding protein comprising an antigen
binding domain of an antibody comprising a V.sub.H comprising a
sequence set forth in SEQ ID NO: 42 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 46, wherein the V.sub.H and/or
V.sub.L comprise one or more of the following substitutions or
groups of substitutions:
(i) the V.sub.H comprises an alanine at position 16 of SEQ ID NO:
42; (ii) the V.sub.H comprises an alanine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a (iii) the V.sub.H comprises a
serine at position 100 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (iv) the V.sub.H
comprises a histidine at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (v)
the V.sub.H comprises a leucine at position 100 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (vi) the V.sub.H comprises an aspartic acid at position 100 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (vii) the V.sub.H comprises a tyrosine at
position 100 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (viii) the V.sub.H comprises a
proline at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (ix) the V.sub.H
comprises a glutamine at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (x)
the V.sub.H comprises a lysine at position 100 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xi) the V.sub.H comprises an alanine at position 101 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xii) the V.sub.H comprises a serine at position 101 of SEQ
ID NO: 42 and the V.sub.L comprises a threonine at position 76 of
SEQ ID NO: 46; (xiii) the V.sub.H comprises a histidine at position
101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xiv) the V.sub.H comprises a leucine
at position 101 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xv) the V.sub.H
comprises an aspartic acid at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xvi) the V.sub.H comprises a tyrosine at position 101 of SEQ ID
NO: 42 and the V.sub.L comprises a (xvii) the V.sub.H comprises a
glutamine at position 101 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xviii) the
V.sub.H comprises a lysine at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xix) the V.sub.H comprises an alanine at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xx) the V.sub.H comprises a serine at position 102 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxi) the V.sub.H comprises a histidine at
position 102 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxii) the V.sub.H comprises a
leucine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxiii) the V.sub.H
comprises a tyrosine at position 102 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxiv) the V.sub.H comprises a proline at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxv) the V.sub.H comprises a glutamine at position 102
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xxvi) the V.sub.H comprises a lysine at
position 102 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxvii) the V.sub.H comprises an
alanine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxviii) the V.sub.H
comprises a serine at position 103 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxix) the
V.sub.H comprises a histidine at position 103 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxx) the V.sub.H comprises a leucine at position 103 of SEQ ID NO:
42 and the V.sub.L comprises a (xxxi) the V.sub.H comprises an
aspartic acid at position 103 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxxii) the
V.sub.H comprises a tyrosine at position 103 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxiii) the V.sub.H comprises a proline at position 103 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxiv) the V.sub.H comprises a glutamine at position
103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xxxv) the V.sub.H comprises a lysine
at position 103 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xxxvi) the V.sub.H
comprises a serine at position 104 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxxvii) the
V.sub.H comprises a histidine at position 104 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxviii) the V.sub.H comprises a leucine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxix) the V.sub.H comprises an aspartic acid at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xl) the V.sub.H comprises a
tyrosine at position 104 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xli) the V.sub.H
comprises a proline at position 104 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlii) the V.sub.H comprises a glutamine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xliii) the V.sub.H comprises a lysine at position 104
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xliv) the V.sub.H comprises an alanine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a (xlv) the
V.sub.H comprises a histidine at position 105 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlvi) the V.sub.H comprises a leucine at position 105 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xlvii) the V.sub.H comprises an aspartic acid at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlviii) the V.sub.H comprises a
tyrosine at position 105 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xlix) the V.sub.H
comprises a proline at position 105 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (l)
the V.sub.H comprises a glutamine at position 105 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (li) the V.sub.H comprises a lysine at position 105 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (lii) the V.sub.H comprises an alanine at position 107
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (liii) the V.sub.H comprises a serine at
position 107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (liv) the V.sub.H comprises a
histidine at position 107 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (lv) the
V.sub.H comprises a leucine at position 107 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lvi) the V.sub.H comprises an aspartic acid at position 107 of SEQ
ID NO: 42 and the V.sub.L comprises a threonine at position 76 of
SEQ ID NO: 46; (lvii) the V.sub.H comprises a tyrosine at position
107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lviii) the V.sub.H comprises a
proline at position 107 of SEQ ID NO: 42 and the V.sub.L comprises
a (lix) the V.sub.H comprises a glutamine at position 107 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (lx) the V.sub.H comprises a lysine at position 107 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lxi) the V.sub.H comprises a threonine at
position 41 of SEQ ID NO: 42 and the V.sub.L comprises an alanine
at position 23 of SEQ ID NO: 46; (lxii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
an aspartic acid at position 28 of SEQ ID NO: 46; (lxiii) the
V.sub.H comprises a threonine at position 41 of SEQ ID NO: 42 and
the V.sub.L comprises a tyrosine at position 33 of SEQ ID NO: 46;
(lxiv) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an aspartic acid at position 34 of
SEQ ID NO: 46; (lxv) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises an asparagine at
position 53 of SEQ ID NO: 46; (lxvi) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a serine at position 54 of SEQ ID NO: 46; (lxvii) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises an alanine at position 82 of SEQ ID NO: 46;
(lxviii) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises a serine at position 95 of SEQ ID
NO: 46; (lxix) the V.sub.H comprises a threonine at position 41 of
SEQ ID NO: 42 and the V.sub.L comprises a serine at position 96 of
SEQ ID NO: 46; (lxx) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lxxi) the V.sub.H comprises a serine
at position 47 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23 of SEQ ID NO: 46; (lxxii) the V.sub.H
comprises a proline at position 41, an alanine at position 72, an
aspartic acid at position 73, an arginine at position 74 and a
threonine at position 76 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxxiii) the V.sub.H comprises a proline at position 41, a leucine
at position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a serine at position 24 and
a threonine at position 76 each relative to SEQ ID NO: 46; (lxxiv)
the V.sub.H comprises a proline at position 41, a leucine at
position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 23,
a serine at position 24 and a threonine at position 76 each
relative to SEQ ID NO: 46; (lxxv) the V.sub.H comprises a proline
at position 41, a leucine at position 51 and a glutamic acid at
position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23 and a threonine at position 76
each relative to SEQ ID NO: 46; (lxxvi) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (lxxvii) the
V.sub.H comprises a proline at position 41, a leucine at position
51, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24 and a threonine
at position 76 each relative to SEQ ID NO: 46; (lxxviii) the
V.sub.H comprises a proline at position 41, a leucine at position
51, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (lxxix) the V.sub.H
comprises a proline at position 41, an alanine at position 72, an
aspartic acid at position 73, an arginine at position 74 and a
threonine at position 76 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 23, a serine at position
24 and a threonine at position 76 each relative to SEQ ID NO: 46;
(lxxx) the V.sub.H comprises a proline at position 41, an alanine
at position 72, an aspartic acid at position 73, an arginine at
position 74 and a threonine at position 76 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glutamic
acid at position 51 each relative to SEQ ID NO: 46; (lxxxi) the
V.sub.H comprises a proline at position 41, a leucine at position
51, an alanine at position 72, an aspartic acid at position 73, an
arginine at position 74, a threonine at position 76, a glutamic
acid at position 102 and an alanine at position 105 each relative
to SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
23, a serine at position 24, a threonine at position 76 and a
glutamic acid at position 51 each relative to SEQ ID NO: 46; and
(lxxxii) the V.sub.H comprises a proline at position 41, a leucine
at position 51, an alanine at position 72, an aspartic acid at
position 73, an arginine at position 74, a threonine at position
76, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24, a threonine at
position 76 and a glycine at position 51 each relative to SEQ ID
NO: 46.
[0061] In one example, the TL1a-binding protein comprises an
antigen binding domain of an antibody comprising any one or more of
the following:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; and (iii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46.
[0062] In one particular example, there is provided an isolated or
recombinant TL1a-binding protein comprising an antigen binding
domain of an antibody comprising a V.sub.H and a V.sub.L, wherein
the V.sub.H and V.sub.L respectively comprise sequences selected
from the group consisting of
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; and
(xii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 187
and a V.sub.L comprising a sequence set forth in SEQ ID NO:
200.
[0063] In one example, a TL1a-binding protein of the disclosure
comprises an antigen binding domain comprising a CDR3 of a variable
region of an antibody recited above. For example, the CDR3 is
defined according to the Kabat numbering system and comprises a
sequence set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 25,
29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93
or a sequence labeled as "CDR3" and shown in bold text in FIGS. 1A
to 1H or in FIG. 9B or 9C or a sequence comprising amino acids 99
to 108 of any one of SEQ ID NOs: 175 to 187 or amino acids 91 to
100 of any one of SEQ ID NOs: 188 to 200 or 234.
[0064] For example, the CDR3 is defined according to the Kabat
numbering system and comprises a sequence set forth in SEQ ID NO:
45 or 49 or a sequence comprising amino acids 99 to 108 of any one
of SEQ ID NOs: 175 to 181, 183 or 185 to 187 or amino acids 91 to
100 of any one of SEQ ID NOs: 188 to 194, 196 or 198 to 200.
[0065] In another example, the CDR3 (e.g., a HCDR3) is defined
according to the enhanced Chothia numbering system and comprises a
sequence labeled as "CDR3" and shown in underlined text in any one
of FIG. 1A, 1C, 1D or 1E or 9B.
[0066] In one example, the CDR3 comprises a sequence
EVPX.sub.1TAX.sub.2FEY (SEQ ID NO: 143), wherein X.sub.1 is
aspartic acid or glutamic acid and X.sub.2 is serine or
alanine.
[0067] In one example, the CDR3 comprises a sequence
EX.sub.1PX.sub.2X.sub.3AX.sub.4FX.sub.5Y (SEQ ID NO: 235),
wherein:
X.sub.1 is an amino acid selected from the group consisting of
valine, alanine, serine, histidine, aspartic acid, leucine,
tyrosine, proline, glutamine or lysine; X.sub.2 is an amino acid
selected from the group consisting of alanine, serine, histidine,
lysine, glutamic acid or aspartic acid; X.sub.3 is an amino acid
selected from the group consisting of alanine, serine, aspartic
acid, tyrosine or threonine; X.sub.4 is an amino acid selected from
the group consisting of serine, alanine, histidine, leucine,
aspartic acid or tyrosine; and X.sub.5 is an amino acid selected
from the group consisting of alanine, serine, histidine, leucine,
aspartic acid, proline, glutamine, glutamic acid or lysine.
[0068] In one example, the CDR3 (e.g., a LCDR3) comprises a
sequence set forth in SEQ ID NO: 141 (or sequence labeled as CDR3
in bold text of the sequence labeled "Consensus" in FIG. 1F or
9C).
[0069] For example, the antigen binding domain comprises three CDRs
of a variable region of the antibody.
[0070] In some examples of the disclosure, the antigen binding
domain is an antibody variable region comprising three CDRs of a
variable region comprising an amino acid sequence set forth in any
one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46,
50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 95, 137, 138, 152
to 200 or 234.
[0071] In some examples, the antigen binding domain is an antibody
variable region comprising three CDRs of a variable region
comprising:
(a) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
comprising one or more of the following substitutions or groups of
substitutions: (i) an alanine at position 16 of SEQ ID NO: 42; (ii)
an alanine at position 100 of SEQ ID NO: 42; (iii) a serine at
position 100 of SEQ ID NO: 42; (iv) a histidine at position 100 of
SEQ ID NO: 42; (v) a leucine at position 100 of SEQ ID NO: 42; (vi)
an aspartic acid at position 100 of SEQ ID NO: 42; (vii) a tyrosine
at position 100 of SEQ ID NO: 42; (viii) a proline at position 100
of SEQ ID NO: 42; (ix) a glutamine at position 100 of SEQ ID NO:
42; (x) a lysine at position 100 of SEQ ID NO: 42; (xi) an alanine
at position 101 of SEQ ID NO: 42; (xii) a serine at position 101 of
SEQ ID NO: 42; (xiii) a histidine at position 101 of SEQ ID NO: 42;
(xiv) a leucine at position 101 of SEQ ID NO: 42; (xv) an aspartic
acid at position 101 of SEQ ID NO: 42; (xvi) a tyrosine at position
101 of SEQ ID NO: 42; (xvii) a glutamine at position 101 of SEQ ID
NO: 42; (xviii) a lysine at position 101 of SEQ ID NO: 42; (xix) an
alanine at position 102 of SEQ ID NO: 42; (xx) a serine at position
102 of SEQ ID NO: 42; (xxi) a histidine at position 102 of SEQ ID
NO: 42; (xxii) a leucine at position 102 of SEQ ID NO: 42; (xxiii)
a tyrosine at position 102 of SEQ ID NO: 42; (xxiv) a proline at
position 102 of SEQ ID NO: 42; (xxv) a glutamine at position 102 of
SEQ ID NO: 42; (xxvi) a lysine at position 102 of SEQ ID NO: 42;
(xxvii) an alanine at position 103 of SEQ ID NO: 42; (xxviii) a
serine at position 103 of SEQ ID NO: 42; (xxix) a histidine at
position 103 of SEQ ID NO: 42; (xxx) a leucine at position 103 of
SEQ ID NO: 42; (xxxi) an aspartic acid at position 103 of SEQ ID
NO: 42; (xxxii) a tyrosine at position 103 of SEQ ID NO: 42;
(xxxiii) a proline at position 103 of SEQ ID NO: 42; (xxxiv) a
glutamine at position 103 of SEQ ID NO: 42; (xxxv) a lysine at
position 103 of SEQ ID NO: 42; (xxxvi) a serine at position 104 of
SEQ ID NO: 42; (xxxvii) a histidine at position 104 of SEQ ID NO:
42; (xxxviii) a leucine at position 104 of SEQ ID NO: 42; (xxxix)
an aspartic acid at position 104 of SEQ ID NO: 42; (xl) a tyrosine
at position 104 of SEQ ID NO: 42; (xli) a proline at position 104
of SEQ ID NO: 42; (xlii) a glutamine at position 104 of SEQ ID NO:
42; (xliii) a lysine at position 104 of SEQ ID NO: 42; (xliv) an
alanine at position 105 of SEQ ID NO: 42; (xlv) a histidine at
position 105 of SEQ ID NO: 42; (xlvi) a leucine at position 105 of
SEQ ID NO: 42; (xlvii) an aspartic acid at position 105 of SEQ ID
NO: 42; (xlviii) a tyrosine at position 105 of SEQ ID NO: 42;
(xlix) a proline at position 105 of SEQ ID NO: 42; (l) a glutamine
at position 105 of SEQ ID NO: 42; (li) a lysine at position 105 of
SEQ ID NO: 42; (lii) an alanine at position 107 of SEQ ID NO: 42;
(liii) a serine at position 107 of SEQ ID NO: 42; (liv) a histidine
at position 107 of SEQ ID NO: 42; (lv) a leucine at position 107 of
SEQ ID NO: 42; (lvi) an aspartic acid at position 107 of SEQ ID NO:
42; (lvii) a tyrosine at position 107 of SEQ ID NO: 42; (lviii) a
proline at position 107 of SEQ ID NO: 42; (lix) a glutamine at
position 107 of SEQ ID NO: 42; (lx) a lysine at position 107 of SEQ
ID NO: 42; (lxi) a threonine at position 41 of SEQ ID NO: 42;
(lxii) a serine at position 47 of SEQ ID NO: 42; (lxiii) a proline
at position 41, an alanine at position 72, an aspartic acid at
position 73 and an arginine at position 74 and a threonine at
position 76 each relative to SEQ ID NO: 42; (lxiv) a proline at
position 41, a leucine at position 51 and a glutamic acid at
position 102 each relative to SEQ ID NO: 42; (lxv) a proline at
position 41, a leucine at position 51, a glutamic acid at position
102 and an alanine at position 105 each relative to SEQ ID NO: 42;
(lxvi) a proline at position 41, a leucine at position 51, an
alanine at position 72, an aspartic acid at position 73, an
arginine at position 74, a threonine at position 76, a glutamic
acid at position 102 and an alanine at position 105; or (b) a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46 and
comprising one or more of the following substitutions or groups of
substitutions: (i) a threonine at position 76 of SEQ ID NO: 46;
(ii) a threonine at position 23 of SEQ ID NO: 46; (iii) an
asparagine at position 28 of SEQ ID NO: 46; (iv) a tyrosine at
position 33 of SEQ ID NO: 46; (v) an aspartic acid at position 34
of SEQ ID NO: 46; (vi) an asparagine at position 53 of SEQ ID NO:
46; (vii) a serine at position 54 of SEQ ID NO: 46; (viii) an
alanine at position 82 of SEQ ID NO: 46; (ix) a serine at position
95 of SEQ ID NO: 46; (x) a serine at position 96 of SEQ ID NO: 46;
(xi) a threonine at position 23, a serine at position 24 and a
threonine at position 76 each relative to SEQ ID NO: 46; (xii) a
threonine at position 23 and a threonine at position 76 each
relative to SEQ ID NO: 46; (xiii) a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glutamic
acid at position 51 each relative to SEQ ID NO: 46; or (xiv) a
threonine at position 23, a serine at position 24, a threonine at
position 76 and a glycine at position 51 each relative to SEQ ID
NO: 46.
[0072] For example, the antigen binding domain is a V.sub.H
comprising three CDRs of an amino acid sequence set forth in any
one of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 70, 74, 78,
86, 90, 94, 137, 152, 154 to 162, 173, 175 to 187 or 234.
[0073] In one example, the CDRs are defined according to the Kabat
numbering system.
[0074] For example, the TL1a-binding protein comprises a V.sub.H
including CDRs as follows:
(i) a CDR1 comprising a sequence set forth in SEQ ID NO: 3, a CDR2
comprising a sequence set forth in SEQ ID NO: 4 (wherein any one or
more of the five C-terminal amino acids of the CDR2 amino acid
sequence are substituted with any other naturally-occurring amino
acid) and a CDR3 comprising a sequence set forth in SEQ ID NO: 5
(or sequences labeled as CDRs 1, 2 and 3 in bold text of antibody
C336 in FIG. 1A); (ii) a CDR1 comprising a sequence set forth in
SEQ ID NO: 11, a CDR2 comprising a sequence set forth in SEQ ID NO:
12 (wherein any one or more of the five C-terminal amino acids of
the CDR2 amino acid sequence are substituted with any other
naturally-occurring amino acid) and a CDR3 comprising a sequence
set forth in SEQ ID NO: 13 (or sequences labeled as CDRs 1, 2 and 3
in bold text of antibody C334 in FIG. 1A); (iii) a CDR1 comprising
a sequence set forth in SEQ ID NO: 19, a CDR2 comprising a sequence
set forth in SEQ ID NO: 20 (wherein any one or more of the five
C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and a
CDR3 comprising a sequence set forth in SEQ ID NO: 21 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C333 in FIG.
1A); (iv) a CDR1 comprising a sequence set forth in SEQ ID NO: 27,
a CDR2 comprising a sequence set forth in SEQ ID NO: 28 (wherein
any one or more of the five C-terminal amino acids of the CDR2
amino acid sequence are substituted with any other
naturally-occurring amino acid) and a CDR3 comprising a sequence
set forth in SEQ ID NO: 29 (or sequences labeled as CDRs 1, 2 and 3
in bold text of antibody C323 in FIG. 1A); (v) a CDR1 comprising a
sequence set forth in SEQ ID NO: 35, a CDR2 comprising a sequence
set forth in SEQ ID NO: 36 (wherein any one or more of the five
C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and a
CDR3 comprising a sequence set forth in SEQ ID NO: 37 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C321 in FIG.
1A); (vi) a CDR1 comprising a sequence set forth in SEQ ID NO: 43,
a CDR2 comprising a sequence set forth in SEQ ID NO: 44 (wherein
any one or more of the five C-terminal amino acids of the CDR2
amino acid sequence are substituted with any other
naturally-occurring amino acid) and a CDR3 comprising a sequence
set forth in SEQ ID NO: 45 (or sequences labeled as CDRs 1, 2 and 3
in bold text of antibody C320 in FIG. 1A); (vii) a CDR1 comprising
a sequence set forth in SEQ ID NO: 51, a CDR2 comprising a sequence
set forth in SEQ ID NO: 52 (wherein any one or more of the five
C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and a
CDR3 comprising a sequence set forth in SEQ ID NO: 53 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C319 in FIG.
1A); (viii) a CDR1 comprising a sequence set forth in SEQ ID NO:
67, a CDR2 comprising a sequence set forth in SEQ ID NO: 68
(wherein any one or more of the five C-terminal amino acids of the
CDR2 amino acid sequence are substituted with any other
naturally-occurring amino acid) and a CDR3 comprising a sequence
set forth in SEQ ID NO: 69 (or sequences labeled as CDRs 1, 2 and 3
in bold text of antibody C320-90 in FIG. 1C); (ix) a CDR1
comprising a sequence set forth in SEQ ID NO: 71, a CDR2 comprising
a sequence set forth in SEQ ID NO: 72 (wherein any one or more of
the five C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and a
CDR3 comprising a sequence set forth in SEQ ID NO: 73 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C320-103 in
FIG. 1C); (x) a CDR1 comprising a sequence set forth in SEQ ID NO:
75, a CDR2 comprising a sequence set forth in SEQ ID NO: 76
(wherein any one or more of the five C-terminal amino acids of the
CDR2 amino acid sequence are substituted with any other
naturally-occurring amino acid) and a CDR3 comprising a sequence
set forth in SEQ ID NO: 77 (or sequences labeled as CDRs 1, 2 and 3
in bold text of antibody C320-114 in FIG. 1C); (xi) a CDR1
comprising a sequence set forth in SEQ ID NO: 79, a CDR2 comprising
a sequence set forth in SEQ ID NO: 80 (wherein any one or more of
the five C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and a
CDR3 comprising a sequence set forth in SEQ ID NO: 81 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C320-115 in
FIG. 1C); (xii) a CDR1 comprising a sequence set forth in SEQ ID
NO: 87, a CDR2 comprising a sequence set forth in SEQ ID NO: 88
(wherein any one or more of the five C-terminal amino acids of the
CDR2 amino acid sequence are substituted with any other
naturally-occurring amino acid) and a CDR3 comprising a sequence
set forth in SEQ ID NO: 89 (or sequences labeled as CDRs 1, 2 and 3
in bold text of antibody C320-129 in FIG. 1C); (xiii) a CDR1
comprising a sequence set forth in SEQ ID NO: 91, a CDR2 comprising
a sequence set forth in SEQ ID NO: 92 (wherein any one or more of
the five C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and a
CDR3 comprising a sequence set forth in SEQ ID NO: 93 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C320-130 in
FIG. 1C); (xiv) a CDR1 comprising a sequence set forth in amino
acids 31 to 35 of any one of SEQ ID NOs: 175 to 187, a CDR2
comprising amino acids 50 to 66 of any one of SEQ ID NOs: 175 to
187 (wherein any one or more of the five C-terminal amino acids of
the CDR2 amino acid sequence are substituted with any other
naturally-occurring amino acid) and amino acids 99 to 108 of any
one of SEQ ID NOs: 175 to 187; and (xv) a CDR1 comprising a
sequence set forth in amino acids 26 to 35 of any one of SEQ ID
NOs: 175 to 187, a CDR2 comprising amino acids 50 to 66 of any one
of SEQ ID NOs: 175 to 187 (wherein any one or more of the five
C-terminal amino acids of the CDR2 amino acid sequence are
substituted with any other naturally-occurring amino acid) and
amino acids 99 to 108 of any one of SEQ ID NOs: 175 to 187.
[0075] For example, the TL1a-binding protein comprises a V.sub.H
including CDRs as follows:
(i) a CDR1 comprising a sequence set forth in SEQ ID NO: 43, a CDR2
comprising a sequence set forth in SEQ ID NO: 44 (wherein any one
or more of the five C-terminal amino acids of the CDR2 amino acid
sequence are substituted with any other naturally-occurring amino
acid) and a CDR3 comprising a sequence set forth in SEQ ID NO: 45
(or sequences labeled as CDRs 1, 2 and 3 in bold text of antibody
C320 in FIG. 1A); (ii) a CDR1 comprising a sequence set forth in
amino acids 31 to 35 of any one of SEQ ID NOs: 175 to 181, 183 or
185 to 187, a CDR2 comprising amino acids 50 to 66 of any one of
SEQ ID NOs: 175 to 181, 183 or 185 to 187 (wherein any one or more
of the five C-terminal amino acids of the CDR2 amino acid sequence
are substituted with any other naturally-occurring amino acid) and
amino acids 99 to 108 of any one of SEQ ID NOs: 175 to 181, 183 or
185 to 187; or (iii) a CDR1 comprising a sequence set forth in
amino acids 26 to 35 of any one of SEQ ID NOs: 175 to 181, 183 or
185 to 187, a CDR2 comprising amino acids 50 to 66 of any one of
SEQ ID NOs: 175 to 181, 183 or 185 to 187 (wherein any one or more
of the five C-terminal amino acids of the CDR2 amino acid sequence
are substituted with any other naturally-occurring amino acid) and
amino acids 99 to 108 of any one of SEQ ID NOs: 175 to 181, 183 or
185 to 187
[0076] In one example, the TL1a-binding protein comprises a V.sub.H
including CDRs as follows:
(i) a CDR1 comprising a sequence set forth in SEQ ID NO: 43 (or
sequence labeled as CDR 1 in bold text of the sequence labeled
"Consensus" in FIG. 1E); (ii) a CDR2 comprising a sequence
WX.sub.1NPNSGNTGYAQKFQG (SEQ ID NO: 142), wherein X.sub.1 is
methionine or leucine (or sequence labeled as CDR 2 in bold text of
the sequence labeled "Consensus" in FIG. 1E or FIG. 9B); and (iii)
a CDR3 comprising a sequence EVPX.sub.1TAX.sub.2FEY (SEQ ID NO:
143), wherein X.sub.1 is aspartic acid or glutamic acid and X.sub.2
is serine or alanine. (or sequence labeled as CDR3 in bold text of
the sequence labeled "Consensus" in FIG. 1E or FIG. 9B) or
comprising a sequence EX.sub.1PX.sub.2X.sub.3AX.sub.4FX.sub.5Y (SEQ
ID NO: 235), wherein: X.sub.1 is an amino acid selected from the
group consisting of valine, alanine, serine, histidine, aspartic
acid, leucine, tyrosine, proline, glutamine or lysine; X.sub.2 is
an amino acid selected from the group consisting of alanine,
serine, histidine, lysine, glutamic acid or aspartic acid; X.sub.3
is an amino acid selected from the group consisting of alanine,
serine, aspartic acid, tyrosine or threonine; X.sub.4 is an amino
acid selected from the group consisting of serine, alanine,
histidine, leucine, aspartic acid or tyrosine; and X.sub.5 is an
amino acid selected from the group consisting of alanine, serine,
histidine, leucine, aspartic acid, proline, glutamine, glutamic
acid or lysine.
[0077] Additional residues suitable for inclusion in CDR3 are
described herein and are to be taken to apply mutatis mutandis to
the present example of the disclosure.
[0078] In one example, the CDRs are defined according to the
enhanced Chothia numbering system. For example, the TL1a-binding
protein comprises a V.sub.H including CDRs labeled as CDRs 1, 2 and
3 in underlined text of antibody 336, 334, 333, 323, 321, 320 or
319 in FIG. 1A or of antibody C320-90, C320-103, C320-114,
C320-115, C320-129 or C320-130 in FIG. 1C or of the sequence
labeled "Consensus" in FIG. 1C or FIG. 1E or FIG. 9B.
[0079] In one example, the TL1a-binding protein additionally
comprises the following:
(i) a heavy chain FR1 comprising an amino acid sequence set forth
in SEQ ID NO: 144; (ii) a heavy chain FR2 comprising an amino acid
sequence set forth in SEQ ID NO: 145; (iii) a heavy chain FR3
comprising an amino acid sequence set forth in SEQ ID NO: 146; and
(iv) a heavy chain FR4 comprising an amino acid sequence set forth
in SEQ ID NO: 147.
[0080] In one example, the TL1a-binding protein comprises the
following:
(i) a heavy chain FR1 comprising an amino acid sequence set forth
in SEQ ID NO: 144; (ii) a heavy chain CDR1 comprising a sequence
set forth in SEQ ID NO: 43; (iii) a heavy chain FR2 comprising an
amino acid sequence set forth in SEQ ID NO: 145; (iv) a heavy chain
CDR2 comprising a sequence set forth in SEQ ID NO: 142; (v) a heavy
chain FR3 comprising an amino acid sequence set forth in SEQ ID NO:
146; (vi) a heavy chain CDR3 comprising a sequence set forth in SEQ
ID NO: 143 or 235; and (vii) a heavy chain FR4 comprising an amino
acid sequence set forth in SEQ ID NO: 147.
[0081] For example, the antigen binding domain is a V.sub.L
comprising three CDRs of an amino acid sequence set forth in any
one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 82, 95, 153, 163
to 172, 174, or 188 to 200. In one example, the CDRs are defined
according to the Kabat numbering system. For example, the
TL1a-binding protein comprises a V.sub.L including CDRs as
follows:
(i) a CDR1 comprising a sequence set forth in SEQ ID NO: 7, a CDR2
comprising a sequence set forth in SEQ ID NO: 8 and a CDR3
comprising a sequence set forth in SEQ ID NO: 9 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C336 in FIG.
1B); (ii) a CDR1 comprising a sequence set forth in SEQ ID NO: 15,
a CDR2 comprising a sequence set forth in SEQ ID NO: 16 and a CDR3
comprising a sequence set forth in SEQ ID NO: 17 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C334 in FIG.
1B); (iii) a CDR1 comprising a sequence set forth in SEQ ID NO: 23,
a CDR2 comprising a sequence set forth in SEQ ID NO: 24 and a CDR3
comprising a sequence set forth in SEQ ID NO: 25 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C333 in FIG.
1B); (iv) a CDR1 comprising a sequence set forth in SEQ ID NO: 31,
a CDR2 comprising a sequence set forth in SEQ ID NO: 32 and a CDR3
comprising a sequence set forth in SEQ ID NO: 33 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C323 in FIG.
1B); (v) a CDR1 comprising a sequence set forth in SEQ ID NO: 39, a
CDR2 comprising a sequence set forth in SEQ ID NO: 40 and a CDR3
comprising a sequence set forth in SEQ ID NO: 41 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C321 in FIG.
1B); (vi) a CDR1 comprising a sequence set forth in SEQ ID NO: 47,
a CDR2 comprising a sequence set forth in SEQ ID NO: 48 and a CDR3
comprising a sequence set forth in SEQ ID NO: 49 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C320 in FIG.
1B); (vii) a CDR1 comprising a sequence set forth in SEQ ID NO: 55,
a CDR2 comprising a sequence set forth in SEQ ID NO: 56 and a CDR3
comprising a sequence set forth in SEQ ID NO: 57 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C319 in FIG.
1B); (viii) a CDR1 comprising a sequence set forth in SEQ ID NO:
83, a CDR2 comprising a sequence set forth in SEQ ID NO: 84 and a
CDR3 comprising a sequence set forth in SEQ ID NO: 85 (or sequences
labeled as CDRs 1, 2 and 3 in bold text of antibody C320-120 in
FIG. 1F); or (ix) a CDR1 comprising amino acids 23 to 36 of any one
of SEQ ID NOs: 188 to 200, a CDR2 comprising amino acids 52 to 58
of any one of SEQ ID NOs: 188 to 200 and a CDR3 comprising amino
acids 91 to 100 of any one of SEQ ID NOs: 188 to 200.
[0082] In one example, the TL1a-binding protein comprises a V.sub.L
including CDRs as follows:
(i) a CDR1 comprising a sequence X.sub.1X.sub.2SSSDIGAGLGVH (SEQ ID
NO: 139), wherein X.sub.1 is alanine or threonine; X.sub.2 is
glycine or serine (or sequence labeled as CDR 1 in bold text of the
sequence labeled "Consensus" in FIG. 9C); (ii) a CDR2 comprising a
sequence set forth in SEQ ID NO: 140; and (iii) a CDR3 comprising a
sequence set forth in SEQ ID NO: 141.
[0083] In one example, the TL1a-binding protein additionally
comprises the following:
(i) a light chain FR1 comprising an amino acid sequence set forth
in SEQ ID NO: 148; (ii) a light chain FR2 comprising an amino acid
sequence set forth in SEQ ID NO: 149; (iii) a light chain FR3
comprising an amino acid sequence set forth in SEQ ID NO: 150; and
(iv) a light chain FR4 comprising an amino acid sequence set forth
in SEQ ID NO: 151.
[0084] In one example, the TL1a-binding protein comprises the
following:
(i) a light chain FR1 comprising an amino acid sequence set forth
in SEQ ID NO: 148; (ii) a light chain CDR1 comprising a sequence
set forth in SEQ ID NO: 139; (iii) a light chain FR2 comprising an
amino acid sequence set forth in SEQ ID NO: 149; (iv) a light chain
CDR2 comprising a sequence set forth in SEQ ID NO: 140; (v) a light
chain FR3 comprising an amino acid sequence set forth in SEQ ID NO:
150; (vi) a light chain CDR3 comprising a sequence set forth in SEQ
ID NO: 141; and (vii) a light chain FR4 comprising an amino acid
sequence set forth in SEQ ID NO: 151.
[0085] In one example, the CDRs are defined according to the
enhanced Chothia numbering system. For example, the TL1a-binding
protein comprises a V.sub.L including CDRs labeled as CDRs 1, 2 and
3 in underlined text of antibody 336, 334, 333, 323, 321, 320 or
319 in FIG. 1B or of antibody C320-120 in FIG. 1F or of the
sequence labeled "Consensus" in FIG. 1H and FIG. 9.
[0086] In one example, the antigen binding domain comprises six
CDRs of one of the following pairs of variable regions:
(i) aV.sub.H comprising a sequence set forth in SEQ ID NO: 2 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 6; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 10 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 14; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 18 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 22; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 50 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 54; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xviii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 163; (xx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 166; (xxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 167; (xxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xxxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165;
(xxxviii) a V.sub.H comprising a sequence set forth in SEQ ID NO:
42 and a V.sub.L comprising a sequence set forth in SEQ ID NO: 166;
(xxxix) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 167;
(xl) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xli)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xlii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xliii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 171; (xliv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; (xlv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (xlvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (xlvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (xlviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (xlix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (l) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (li) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (lii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 182 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 195; (liii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (liv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 184 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 197; (lv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (lvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; or
(lvii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 187
and a V.sub.L comprising a sequence set forth in SEQ ID NO:
200.
[0087] In one example, the antigen binding domain comprises six
CDRs of an antibody comprising a V.sub.H comprising a sequence set
forth in SEQ ID NO: 42 and a V.sub.L comprising a sequence set
forth in SEQ ID NO: 46, wherein the V.sub.H and/or V.sub.L comprise
one or more of the following substitutions or groups of
substitutions:
(i) the V.sub.H comprises an alanine at position 16 of SEQ ID NO:
42; (ii) the V.sub.H comprises an alanine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (iii) the V.sub.H comprises a serine at position 100 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (iv) the V.sub.H comprises a histidine at
position 100 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (v) the V.sub.H comprises a
leucine at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (vi) the V.sub.H
comprises an aspartic acid at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(vii) the V.sub.H comprises a tyrosine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (viii) the V.sub.H comprises a proline at position 100
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (ix) the V.sub.H comprises a glutamine at
position 100 of SEQ ID NO: 42 and the V.sub.1 comprises a threonine
at position 76 of SEQ ID NO: 46; (x) the V.sub.H comprises a lysine
at position 100 of SEQ ID NO: 42 and the V.sub.1 comprises a
threonine at position 76 of SEQ ID NO: 46; (xi) the V.sub.H
comprises an alanine at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xii) the V.sub.H comprises a serine at position 101 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xiii) the V.sub.H comprises a histidine at position 101 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xiv) the V.sub.H comprises a leucine at position
101 of SEQ ID NO: 42 and the V.sub.1 comprises a (xv) the V.sub.H
comprises an aspartic acid at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xvi) the V.sub.H comprises a tyrosine at position 101 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xvii) the V.sub.H comprises a glutamine at position 101
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xviii) the V.sub.H comprises a lysine at
position 101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xix) the V.sub.H comprises an
alanine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xx) the V.sub.H
comprises a serine at position 102 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxi) the
V.sub.H comprises a histidine at position 102 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxii) the V.sub.H comprises a leucine at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxiii) the V.sub.H comprises a tyrosine at position 102
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xxiv) the V.sub.H comprises a proline at
position 102 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxv) the V.sub.H comprises a
glutamine at position 102 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxvi) the
V.sub.H comprises a lysine at position 102 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxvii) the V.sub.H comprises an alanine at position 103 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxviii) the V.sub.H comprises a serine at position 103
of SEQ ID NO: 42 and the V.sub.L comprises a (xxix) the V.sub.H
comprises a histidine at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxx) the V.sub.H comprises a leucine at position 103 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xxxi) the V.sub.H comprises an aspartic acid at position
103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xxxii) the V.sub.H comprises a
tyrosine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxxiii) the V.sub.H
comprises a proline at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxiv) the V.sub.H comprises a glutamine at position 103 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxv) the V.sub.H comprises a lysine at position 103 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxxvi) the V.sub.H comprises a serine at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxxvii) the V.sub.H comprises a
histidine at position 104 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxxviii)
the V.sub.H comprises a leucine at position 104 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (xxxix) the V.sub.H comprises an aspartic acid at position 104
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xl) the V.sub.H comprises a tyrosine at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xli) the V.sub.H comprises a
proline at position 104 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xlii) the V.sub.H
comprises a glutamine at position 104 of SEQ ID NO: 42 and the
V.sub.L comprises a (xliii) the V.sub.H comprises a lysine at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xliv) the V.sub.H comprises an
alanine at position 105 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xlv) the V.sub.H
comprises a histidine at position 105 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlvi) the V.sub.H comprises a leucine at position 105 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xlvii) the V.sub.H comprises an aspartic acid at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlviii) the V.sub.H comprises a
tyrosine at position 105 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xlix) the V.sub.H
comprises a proline at position 105 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (l)
the V.sub.H comprises a glutamine at position 105 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (li) the V.sub.H comprises a lysine at position 105 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (lii) the V.sub.H comprises an alanine at position 107
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (liii) the V.sub.H comprises a serine at
position 107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (liv) the V.sub.H comprises a
histidine at position 107 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (lv) the
V.sub.H comprises a leucine at position 107 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lvi) the V.sub.H comprises an aspartic acid at position 107 of SEQ
ID NO: 42 and the V.sub.L comprises a (lvii) the V.sub.H comprises
a tyrosine at position 107 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (lviii) the
V.sub.H comprises a proline at position 107 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lix) the V.sub.H comprises a glutamine at position 107 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (lx) the V.sub.H comprises a lysine at position 107 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lxi) the V.sub.H comprises a threonine at
position 41 of SEQ ID NO: 42 and the V.sub.L comprises an alanine
at position 23 of SEQ ID NO: 46; (lxii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
an aspartic acid at position 28 of SEQ ID NO: 46; (lxiii) the
V.sub.H comprises a threonine at position 41 of SEQ ID NO: 42 and
the V.sub.L comprises a tyrosine at position 33 of SEQ ID NO: 46;
(lxiv) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an aspartic acid at position 34 of
SEQ ID NO: 46; (lxv) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises an asparagine at
position 53 of SEQ ID NO: 46; (lxvi) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a serine at position 54 of SEQ ID NO: 46; (lxvii) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises an alanine at position 82 of SEQ ID NO: 46;
(lxviii) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises a serine at position 95 of SEQ ID
NO: 46; (lxix) the V.sub.H comprises a threonine at position 41 of
SEQ ID NO: 42 and the V.sub.L comprises a serine at position 96 of
SEQ ID NO: 46; (lxx) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises a (lxxi) the V.sub.H
comprises a serine at position 47 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23 of SEQ ID NO: 46; (lxxii) the
V.sub.H comprises a proline at position 41, an alanine at position
72, an aspartic acid at position 73 and an arginine at position 74
and a threonine at position 76 each relative to SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxxiii) the V.sub.H comprises a proline at position 41, a leucine
at position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a serine at position 24 and
a threonine at position 76 each relative to SEQ ID NO: 46; (lxxiv)
the V.sub.H comprises a proline at position 41, a leucine at
position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 23,
a serine at position 24 and a threonine at position 76 each
relative to SEQ ID NO: 46; (lxxv) the V.sub.H comprises a proline
at position 41, a leucine at position 51 and a glutamic acid at
position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23 and a threonine at position 76
each relative to SEQ ID NO: 46; (lxxvi) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (lxxvii) the
V.sub.H comprises a proline at position 41, a leucine at position
51, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24 and a threonine
at position 76 each relative to SEQ ID NO: 46; (lxxviii) the
V.sub.H comprises a proline at position 41, a leucine at position
51, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (lxxix) the V.sub.H
comprises a proline at position 41, an alanine at position 72, an
aspartic acid at position 73 an arginine at position 74 and a
threonine at position 76 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 23, a serine at position
24 and a threonine at position 76 each relative to SEQ ID NO: 46;
(lxxx) the V.sub.H comprises a proline at position 41, an alanine
at position 72, an aspartic acid at position 73 an arginine at
position 74 and a threonine at position 76 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glutamic
acid at position 51 each relative to SEQ ID NO: 46; (lxxxi) the
V.sub.H comprises a proline at position 41, a leucine at position
51, an alanine at position 72, an aspartic acid at position 73, an
arginine at position 74, a threonine at position 76, a glutamic
acid at position 102 and an alanine at position 105 each relative
to SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
23, a serine at position 24, a threonine at position 76 and a
glutamic acid at position 51 each relative to SEQ ID NO: 46; or
(lxxxii) the V.sub.H comprises a proline at position 41, a leucine
at position 51, an alanine at position 72, an aspartic acid at
position 73, an arginine at position 74, a threonine at position
76, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24, a threonine at
position 76 and a glycine at position 51 each relative to SEQ ID
NO: 46.
[0088] In one example, the antigen binding domain comprises six
CDRs of one of the following pairs of variable regions:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ii) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 106 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 107 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (iv) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 222 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (v) a V.sub.H
comprising a sequence set forth in SEQ ID NO: 176 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 189; (vi) a V.sub.H
encoded by a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 223 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions and a V.sub.L encoded by a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 228 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions; (vii) a V.sub.H comprising
a sequence set forth in SEQ ID NO: 177 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 190; (viii) a V.sub.H encoded by a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 224 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions and
a V.sub.L encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 229 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions; (ix) a V.sub.H comprising a sequence set
forth in SEQ ID NO: 178 and a V.sub.L comprising a sequence set
forth in SEQ ID NO: 191; (x) a V.sub.H encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 224 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions and a V.sub.L
encoded by a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 230 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions; (xi) a V.sub.H comprising a sequence set forth in SEQ
ID NO: 179 and a V.sub.L comprising a sequence set forth in SEQ ID
NO: 192; (xii) a V.sub.H encoded by a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 224 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions and a V.sub.L encoded by a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 231 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions;
(xiii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 180
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 193;
(xiv) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
224 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (xvi) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 225 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 230 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (xviii)
a V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 226 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 230 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (xx) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 226 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; and
(xxii) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
227 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xxiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 187 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 200; or
(xxiv) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
227 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 233 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions.
[0089] In one example, the TL1a-binding protein comprises the
following six CDRs:
(i) a heavy chain CDR1 comprising a sequence set forth in SEQ ID
NO: 43 (or sequence labeled as CDR 1 in bold text of the sequence
labeled "Consensus" in FIG. 1E); (ii) a heavy chain CDR2 comprising
a sequence WX.sub.1NPNSGNTGYAQKFQG (SEQ ID NO: 142), wherein
X.sub.1 is methionine or leucine (or sequence labeled as CDR 2 in
bold text of the sequence labeled "Consensus" in FIG. 1E); (iii) a
heavy chain CDR3 comprising a sequence EVPX.sub.1TAX.sub.2FEY (SEQ
ID NO: 143), wherein X.sub.1 is aspartic acid or glutamic acid and
X.sub.2 is serine or alanine. (or the sequence labeled as CDR 3 in
bold text of the sequence labeled "Consensus" in FIG. 9B) or a
sequence EX.sub.1PX.sub.2X.sub.3AX.sub.4FX.sub.5Y (SEQ ID NO: 235),
wherein: X.sub.1 is an amino acid selected from the group
consisting of valine, alanine, serine, histidine, aspartic acid,
leucine, tyrosine, proline, glutamine or lysine; X.sub.2 is an
amino acid selected from the group consisting of alanine, serine,
histidine, lysine, glutamic acid or aspartic acid; X.sub.3 is an
amino acid selected from the group consisting of alanine, serine,
aspartic acid, tyrosine or threonine; X.sub.4 is an amino acid
selected from the group consisting of serine, alanine, histidine,
leucine, aspartic acid or tyrosine; and X.sub.5 is an amino acid
selected from the group consisting of alanine, serine, histidine,
leucine, aspartic acid, proline, glutamine, glutamic acid or
lysine; (iv) a CDR1 comprising a sequence
X.sub.1X.sub.2SSSDIGAGLGVH (SEQ ID NO: 139), wherein X.sub.1 is
alanine or threonine; X.sub.2 is glycine or serine (or sequence
labeled as CDR 1 in bold text of the sequence labeled "Consensus"
in FIG. 9C); (v) a CDR2 comprising a sequence set forth in SEQ ID
NO: 48; and (vi) a CDR3 comprising a sequence set forth in SEQ ID
NO: 49
[0090] In one example, the TL1a-binding protein comprises the
following:
(a) a V.sub.H comprising: (i) a heavy chain FR1 comprising an amino
acid sequence set forth in SEQ ID NO: 144; (ii) a heavy chain CDR1
comprising a sequence set forth in SEQ ID NO: 43; (iii) a heavy
chain FR2 comprising an amino acid sequence set forth in SEQ ID NO:
145; (iv) a heavy chain CDR2 comprising a sequence set forth in SEQ
ID NO: 142; (v) a heavy chain FR3 comprising an amino acid sequence
set forth in SEQ ID NO: 146; (vi) a heavy chain CDR3 comprising a
sequence set forth in SEQ ID NO: 143 or 235; and (vii) a heavy
chain FR4 comprising an amino acid sequence set forth in SEQ ID NO:
147; and (b) a V.sub.L comprising: (i) a light chain FR1 comprising
an amino acid sequence set forth in SEQ ID NO: 148; (ii) a light
chain CDR1 comprising a sequence set forth in SEQ ID NO: 139; (iii)
a light chain FR2 comprising an amino acid sequence set forth in
SEQ ID NO: 149; (iv) a light chain CDR2 comprising a sequence set
forth in SEQ ID NO: 48; (v) a light chain FR3 comprising an amino
acid sequence set forth in SEQ ID NO: 150; (vi) a light chain CDR3
comprising a sequence set forth in SEQ ID NO: 49; and (vii) a light
chain FR4 comprising an amino acid sequence set forth in SEQ ID NO:
151.
[0091] Additional residues suitable for inclusion in heavy chain
CDR3 are described herein and are to be taken to apply mutatis
mutandis to the present example of the disclosure.
[0092] In one example, the CDRs are defined according to the Kabat
numbering system. Exemplary CDRs defined according to the Kabat
numbering system are described above and/or in FIGS. 1A to 1H, 9B
or 9C labeled as CDRs 1 to 3 in bold text and are taken to apply
mutatis mutandis to the present example of the disclosure.
[0093] In one example, the CDRs are defined according to the
enhanced Chothia numbering system. Exemplary CDRs defined according
to the enhanced Chothia numbering system are described above and/or
in FIGS. 1A to 1H labeled as CDRs 1 to 3 in underlined text and are
taken to apply mutatis mutandis to the present example of the
disclosure.
[0094] In one example, the TL1a-binding protein comprises a
variable region of the antibody.
[0095] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in any one of SEQ ID NOs: 2, 10,
18, 26, 34, 42, 50, 58, 66, 70, 74, 78, 86, 90, 94, 137, 152, 154
to 162, 173, 175 to 187 or 234 or a sequence having at least about
80% identity to any one of the foregoing. In one example, the
V.sub.H comprises a sequence set forth in any one of SEQ ID NOs:
26, 34, 42 or 94 or a sequence having at least about 80% identity
to any one of the foregoing. In one example, the V.sub.H comprises
a sequence set forth in any one of SEQ ID NOs: 42, 175 to 181, 183
or 185 to 187 or a sequence having at least about 80% identity to
any one of the foregoing.
[0096] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 94, 137, 152, 162 or
173. In one example, the V.sub.H comprises a sequence set forth in
any one of SEQ ID NOs: 42, 58, 66, 70, 74, 78, 86 or 90. In one
example, the V.sub.H comprises a sequence set forth in SEQ ID NO:
42.
[0097] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 42 and comprising one
or more of the following substitutions or groups of
substitutions:
(i) an alanine at position 16 of SEQ ID NO: 42; (ii) an alanine at
position 100 of SEQ ID NO: 42; (iii) a serine at position 100 of
SEQ ID NO: 42; (iv) a histidine at position 100 of SEQ ID NO: 42;
(v) a leucine at position 100 of SEQ ID NO: 42; (vi) an aspartic
acid at position 100 of SEQ ID NO: 42; (vii) a tyrosine at position
100 of SEQ ID NO: 42; (viii) a proline at position 100 of SEQ ID
NO: 42; (ix) a glutamine at position 100 of SEQ ID NO: 42; (x) a
lysine at position 100 of SEQ ID NO: 42; (xi) an alanine at
position 101 of SEQ ID NO: 42; (xii) a serine at position 101 of
SEQ ID NO: 42; (xiii) a histidine at position 101 of SEQ ID NO: 42;
(xiv) a leucine at position 101 of SEQ ID NO: 42; (xv) an aspartic
acid at position 101 of SEQ ID NO: 42; (xvi) a tyrosine at position
101 of SEQ ID NO: 42; (xvii) a glutamine at position 101 of SEQ ID
NO: 42; (xviii) a lysine at position 101 of SEQ ID NO: 42; (xix) an
alanine at position 102 of SEQ ID NO: 42; (xx) a serine at position
102 of SEQ ID NO: 42; (xxi) a histidine at position 102 of SEQ ID
NO: 42; (xxii) a leucine at position 102 of SEQ ID NO: 42; (xxiii)
a tyrosine at position 102 of SEQ ID NO: 42; (xxiv) a proline at
position 102 of SEQ ID NO: 42; (xxv) a glutamine at position 102 of
SEQ ID NO: 42; (xxvi) a lysine at position 102 of SEQ ID NO: 42;
(xxvii) an alanine at position 103 of SEQ ID NO: 42; (xxviii) a
serine at position 103 of SEQ ID NO: 42; (xxix) a histidine at
position 103 of SEQ ID NO: 42; (xxx) a leucine at position 103 of
SEQ ID NO: 42; (xxxi) an aspartic acid at position 103 of SEQ ID
NO: 42; (xxxii) a tyrosine at position 103 of SEQ ID NO: 42;
(xxxiii) a proline at position 103 of SEQ ID NO: 42; (xxxiv) a
glutamine at position 103 of SEQ ID NO: 42; (xxxv) a lysine at
position 103 of SEQ ID NO: 42; (xxxvi) a serine at position 104 of
SEQ ID NO: 42; (xxxvii) a histidine at position 104 of SEQ ID NO:
42; (xxxviii) a leucine at position 104 of SEQ ID NO: 42; (xxxix)
an aspartic acid at position 104 of SEQ ID NO: 42; (xl) a tyrosine
at position 104 of SEQ ID NO: 42; (xli) a proline at position 104
of SEQ ID NO: 42; (xlii) a glutamine at position 104 of SEQ ID NO:
42; (xliii) a lysine at position 104 of SEQ ID NO: 42; (xliv) an
alanine at position 105 of SEQ ID NO: 42; (xlv) a histidine at
position 105 of SEQ ID NO: 42; (xlvi) a leucine at position 105 of
SEQ ID NO: 42; (xlvii) an aspartic acid at position 105 of SEQ ID
NO: 42; (xlviii) a tyrosine at position 105 of SEQ ID NO: 42;
(xlix) a proline at position 105 of SEQ ID NO: 42; (l) a glutamine
at position 105 of SEQ ID NO: 42; (li) a lysine at position 105 of
SEQ ID NO: 42; (lii) an alanine at position 107 of SEQ ID NO: 42;
(liii) a serine at position 107 of SEQ ID NO: 42; (liv) a histidine
at position 107 of SEQ ID NO: 42; (lv) a leucine at position 107 of
SEQ ID NO: 42; (lvi) an aspartic acid at position 107 of SEQ ID NO:
42; (lvii) a tyrosine at position 107 of SEQ ID NO: 42; (lviii) a
proline at position 107 of SEQ ID NO: 42; (lix) a glutamine at
position 107 of SEQ ID NO: 42; (lx) a lysine at position 107 of SEQ
ID NO: 42; (lxi) a threonine at position 41 of SEQ ID NO: 42;
(lxii) a serine at position 47 of SEQ ID NO: 42; (lxiii) a proline
at position 41, an alanine at position 72, a aspartic acid at
position 73 an arginine at position 74 and a threonine at position
76 each relative to SEQ ID NO: 42; (lxiv) a proline at position 41,
a leucine at position 51 and a glutamic acid at position 102 each
relative to SEQ ID NO: 42; (lxv) a proline at position 41, a
leucine at position 51, a glutamic acid at position 102 and an
alanine at position 105 each relative to SEQ ID NO: 42; (lxvi) a
proline at position 41, a leucine at position 51, an alanine at
position 72, a aspartic acid at position 73, an arginine at
position 74, a threonine at position 76 a glutamic acid at position
102 and an alanine at position 105; or (lxvii) a sequence having at
least about 80% identity to any one of the foregoing.
[0098] In one example, the TL1a-binding protein comprises a V.sub.H
encoded by a nucleic acid comprising a sequence set forth in any
one of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, 113,
114, 115, 117, 118 or 222 to 227 or a sequence at least about 80%
identical thereto or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions.
[0099] In one example, the TL1a-binding protein comprises a V.sub.H
encoded by a nucleic acid comprising a sequence set forth in SEQ ID
NO: 106 or 222 to 227 or a sequence at least about 80% identical
thereto or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions.
[0100] In one example, the TL1a-binding protein comprises a V.sub.L
comprising a sequence set forth in any one of SEQ ID NOs: 6, 14,
22, 30, 38, 46, 54, 62, 82, 95, 138, 153, 163 or 174, or a sequence
having at least about 80% identity to any one of the foregoing. In
one example, the V.sub.L comprises a sequence set forth in any one
of SEQ ID NOs: 30, 38, 46, 188 to 194, 196 or 198 to 200 or a
sequence having at least about 80% identity to any one of the
foregoing.
[0101] In one example, the TL1a-binding protein comprises a V.sub.L
comprising a sequence set forth in SEQ ID NO: 95, 138, 153, 163 or
174. In one example, the TL1a-binding protein comprises a V.sub.L
comprising a sequence set forth in any one of SEQ ID NOs: 46, 62 or
82. In one example, the TL1a-binding protein comprises a V.sub.L
comprising a sequence set forth in SEQ ID NO: 46.
[0102] In one example, the TL1a-binding protein comprises a V.sub.L
comprising a sequence set forth in SEQ ID NO: 46 and comprising one
or more of the following substitutions or groups of
substitutions:
(i) a threonine at position 76 of SEQ ID NO: 46; (ii) an threonine
at position 23 of SEQ ID NO: 46; (iii) an asparagine at position 28
of SEQ ID NO: 46; (iv) a tyrosine at position 33 of SEQ ID NO: 46;
(v) an aspartic acid at position 34 of SEQ ID NO: 46; (vi) an
asparagine at position 53 of SEQ ID NO: 46; (vii) a serine at
position 54 of SEQ ID NO: 46; (viii) an alanine at position 82 of
SEQ ID NO: 46; (ix) a serine at position 95 of SEQ ID NO: 46; (x) a
serine at position 96 of SEQ ID NO: 46; (xi) a threonine at
position 23, a serine at position 24 and a threonine at position 76
each relative to SEQ ID NO: 46; (xii) a threonine at position 23
and a threonine at position 76 each relative to SEQ ID NO: 46;
(xiii) a threonine at position 23, a serine at position 24, a
threonine at position 76 and a glutamic acid at position 51 each
relative to SEQ ID NO: 46; (xiv) a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glycine at
position 51 each relative to SEQ ID NO: 46; or (xv) a sequence
having at least about 80% identity to any one of the foregoing.
[0103] In one example, the TL1a-binding protein comprises a V.sub.L
encoded by a nucleic acid comprising a sequence set forth in any
one of SEQ ID NOs: 97, 99, 101, 103, 105, 107, 109, 111, 116 or 228
to 233 or a sequence at least about 80% identical thereto or a
nucleic acid that hybridizes thereto under moderate to high
stringency conditions.
[0104] In one example, the protein comprises a V.sub.L encoded by a
nucleic acid comprising a sequence set forth in SEQ ID NO: 107 or
228 to 233 or a sequence at least about 80% identical thereto or a
nucleic acid that hybridizes thereto under moderate to high
stringency conditions.
[0105] In one example, the TL1a-binding protein is a domain
antibody, optionally linked to a heavy chain constant region or a
Fc or a heavy chain constant domain (C.sub.H) 2 and/or C.sub.H3 or
a protein that binds to an immune effector cell.
[0106] In one example, a TL1a-binding protein of the disclosure
comprises at least a V.sub.H and a V.sub.L, wherein the V.sub.H and
V.sub.L bind to form a Fv comprising the antigen binding domain.
For example, the TL1a-binding protein comprises any one of the
following pairs of V.sub.H and V.sub.L:
(i) aV.sub.H comprising a sequence set forth in SEQ ID NO: 2 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 6; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 10 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 14; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 18 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 22; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 50 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 54; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xviii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 163; (xx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 166; (xxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 167; (xxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xxxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165;
(xxxviii) a V.sub.H comprising a sequence set forth in SEQ ID NO:
42 and a V.sub.L comprising a sequence set forth in SEQ ID NO: 166;
(xxxix) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 167;
(xl) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xli)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xlii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xliii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 171; (xliv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; (xlv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (xlvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (xlvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (xlviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (xlix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (l) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (li) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (lii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 182 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 195; (liii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (liv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 184 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 197; (lv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (lvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; or
(lvii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 187
and a V.sub.L comprising a sequence set forth in SEQ ID NO:
200.
[0107] In one example, the TL1a-binding protein comprises any one
of the following pairs of V.sub.H and V.sub.L:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (xii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (xiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; or (xiv)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 187 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 200;
[0108] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 94 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 95.
[0109] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 137 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 138.
[0110] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 152 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 153.
[0111] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 173 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 174.
[0112] In one example, the TL1a-binding protein comprises any one
of the following pairs of V.sub.H and V.sub.L:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; or (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62.
[0113] For example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 42 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 46, wherein the
V.sub.H and/or V.sub.L comprise one or more of the following
substitutions or groups of substitutions:
(i) the V.sub.H comprises an alanine at position 16 of SEQ ID NO:
42; (ii) the V.sub.H comprises an alanine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (iii) the V.sub.H comprises a serine at position 100 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (iv) the V.sub.H comprises a histidine at
position 100 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (v) the V.sub.H comprises a
leucine at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (vi) the V.sub.H
comprises an aspartic acid at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(vii) the V.sub.H comprises a tyrosine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a (viii) the V.sub.H comprises a
proline at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (ix) the V.sub.H
comprises a glutamine at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (x)
the V.sub.H comprises a lysine at position 100 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xi) the V.sub.H comprises an alanine at position 101 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xii) the V.sub.H comprises a serine at position 101 of SEQ
ID NO: 42 and the V.sub.L comprises a threonine at position 76 of
SEQ ID NO: 46; (xiii) the V.sub.H comprises a histidine at position
101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xiv) the V.sub.H comprises a leucine
at position 101 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xv) the V.sub.H
comprises an aspartic acid at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xvi) the V.sub.H comprises a tyrosine at position 101 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xvii) the V.sub.H comprises a glutamine at position 101
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xviii) the V.sub.H comprises a lysine at
position 101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xix) the V.sub.H comprises an
alanine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xx) the V.sub.H
comprises a serine at position 102 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxi) the
V.sub.H comprises a histidine at position 102 of SEQ ID NO: 42 and
the V.sub.L comprises a (xxii) the V.sub.H comprises a leucine at
position 102 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxiii) the V.sub.H comprises a
tyrosine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxiv) the V.sub.H
comprises a proline at position 102 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxv) the V.sub.H comprises a glutamine at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxvi) the V.sub.H comprises a lysine at position 102 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxvii) the V.sub.H comprises an alanine at
position 103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxviii) the V.sub.H comprises a
serine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xxix) the V.sub.H
comprises a histidine at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxx) the V.sub.H comprises a leucine at position 103 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xxxi) the V.sub.H comprises an aspartic acid at position
103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xxxii) the V.sub.H comprises a
tyrosine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxxiii) the V.sub.H
comprises a proline at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxiv) the V.sub.H comprises a glutamine at position 103 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxv) the V.sub.H comprises a lysine at position 103 of
SEQ ID NO: 42 and the V.sub.L comprises a (xxxvi) the V.sub.H
comprises a serine at position 104 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxxvii) the
V.sub.H comprises a histidine at position 104 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxviii) the V.sub.H comprises a leucine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxix) the V.sub.H comprises an aspartic acid at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xl) the V.sub.H comprises a
tyrosine at position 104 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xli) the V.sub.H
comprises a proline at position 104 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlii) the V.sub.H comprises a glutamine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xliii) the V.sub.H comprises a lysine at position 104
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xliv) the V.sub.H comprises an alanine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlv) the V.sub.H comprises a
histidine at position 105 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xlvi) the
V.sub.H comprises a leucine at position 105 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlvii) the V.sub.H comprises an aspartic acid at position 105 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xlviii) the V.sub.H comprises a tyrosine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlix) the V.sub.H comprises a
proline at position 105 of SEQ ID NO: 42 and the V.sub.L comprises
a (l) the V.sub.H comprises a glutamine at position 105 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (li) the V.sub.H comprises a lysine at position 105 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lii) the V.sub.H comprises an alanine at
position 107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (liii) the V.sub.H comprises a
serine at position 107 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (liv) the V.sub.H
comprises a histidine at position 107 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (lv)
the V.sub.H comprises a leucine at position 107 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (lvi) the V.sub.H comprises an aspartic acid at position 107 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lvii) the V.sub.H comprises a tyrosine at
position 107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (lviii) the V.sub.H comprises a
proline at position 107 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (lix) the V.sub.H
comprises a glutamine at position 107 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (lx)
the V.sub.H comprises a lysine at position 107 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxi) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an alanine at position 23 of SEQ
ID NO: 46; (lxii) the V.sub.H comprises a threonine at position 41
of SEQ ID NO: 42 and the V.sub.L comprises an aspartic acid at
position 28 of SEQ ID NO: 46; (lxiii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a tyrosine at position 33 of SEQ ID NO: 46; (lxiv) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises an aspartic acid at position 34 of SEQ ID NO: 46;
(lxv) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an asparagine at position 53 of
SEQ ID NO: 46; (lxvi) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises a serine at position
54 of SEQ ID NO: 46; (lxvii) the V.sub.H comprises a threonine at
position 41 of SEQ ID NO: 42 and the V.sub.L comprises a alanine at
position 82 of SEQ ID NO: 46; (lxviii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a serine at position 95 of SEQ ID NO: 46; (lxix) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises a serine at position 96 of SEQ ID NO: 46; (lxx)
the V.sub.H comprises a threonine at position 41 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (lxxi) the V.sub.H comprises a serine at position 47 of SEQ ID
NO: 42 and the V.sub.L comprises an threonine at position 23 of SEQ
ID NO: 46; (lxxii) the V.sub.H comprises a proline at position 41,
an alanine at position 72, an aspartic acid at position 73 an
arginine at position 74 and a threonine at position 76 each
relative to SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lxxiii) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a serine at position 24 and a threonine at position 76
each relative to SEQ ID NO: 46; (lxxiv) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23, a serine at position 24 and a
threonine at position 76 each relative to SEQ ID NO: 46; (lxxv) the
V.sub.H comprises a proline at position 41, a leucine at position
51 and a glutamic acid at position 102 each relative to SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 23 and a
threonine at position 76 each relative to SEQ ID NO: 46; (lxxvi)
the V.sub.H comprises a proline at position 41, a leucine at
position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lxxvii) the V.sub.H comprises a proline at
position 41, a leucine at position 51, a glutamic acid at position
102 and an alanine at position 105 each relative to SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 23, a serine at
position 24 and a threonine at position 76 each relative to SEQ ID
NO: 46; (lxxviii) the V.sub.H comprises a proline at position 41, a
leucine at position 51, a glutamic acid at position 102 and an
alanine at position 105 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxxix) the V.sub.H comprises a proline at position 41, an alanine
at position 72, an aspartic acid at position 73 an arginine at
position 74 and a threonine at position 76 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24 and a threonine at position 76 each relative
to SEQ ID NO: 46; (lxxx) the V.sub.H comprises a proline at
position 41, an alanine at position 72, an aspartic acid at
position 73 an arginine at position 74 and a threonine at position
76 each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24, a threonine at
position 76 and a glutamic acid at position 51 each relative to SEQ
ID NO: 46; (lxxxi) the V.sub.H comprises a proline at position 41,
a leucine at position 51, an alanine at position 72, an aspartic
acid at position 73, an arginine at position 74, a threonine at
position 76, a glutamic acid at position 102 and an alanine at
position 105 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23, a serine at position 24, a
threonine at position 76 and a glutamic acid at position 51 each
relative to SEQ ID NO: 46; or (lxxxii) the V.sub.H comprises a
proline at position 41, a leucine at position 51, an alanine at
position 72, an aspartic acid at position 73, an arginine at
position 74, a threonine at position 76, a glutamic acid at
position 102 and an alanine at position 105 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glycine at
position 51 each relative to SEQ ID NO: 46.
[0114] In one example, the V.sub.H and the V.sub.L are in a single
polypeptide chain. For example, the TL1a-binding protein is:
(i) a single chain Fv fragment (scFv); (ii) a dimeric scFv
(di-scFv); (iii) one of (i) or (ii) linked to a heavy chain
constant region or a Fc or a heavy chain constant domain (C.sub.H)
2 and/or C.sub.H3; or (iv) one of (i) or (ii) linked to a protein
that binds to an immune effector cell.
[0115] In another example, the V.sub.L and V.sub.H are in separate
polypeptide chains. For example, the TL1a-binding protein is:
(i) a diabody; (ii) a triabody; (iii) a tetrabody;
(iv) a Fab;
(v) a F(ab').sub.2;
(vi) a Fv;
[0116] (vii) one of (i) to (vi) linked to a heavy chain constant
region or a Fc or a heavy chain constant domain (C.sub.H) 2 and/or
C.sub.H3; or (viii) one of (i) to (vi) linked to a protein that
binds to an immune effector cell.
[0117] In an exemplary form of the disclosure, the TL1a-binding
protein is an antibody.
[0118] Exemplary TL1a-binding proteins of the disclosure are
chimeric, de-immunized, humanized, human, synhumanized or
primatized.
[0119] In one example, the disclosure provides an antibody
comprising an antigen binding domain, wherein the antigen binding
domain specifically binds to TL1a and, wherein the antibody
inhibits interaction of TL1a and DR3 and does not inhibit
interaction of TL1a and DcR3, the antigen binding domain comprising
any one of:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 2 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 6; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 10 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 14; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 18 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 22; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 50 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 54; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xviii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 163; (xx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 166; (xxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 167; (xxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xxxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165;
(xxxviii) a V.sub.H comprising a sequence set forth in SEQ ID NO:
42 and a V.sub.L comprising a sequence set forth in SEQ ID NO: 166;
(xxxix) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 167;
(xl) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xli)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xlii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xliii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 171; (xliv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; (xlv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (xlvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (xlvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (xlviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (xlix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (l) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (li) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (lii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 182 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 195; (liii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (liv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 184 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 197; (lv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (lvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; or
(lvii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 187
and a V.sub.L comprising a sequence set forth in SEQ ID NO:
200.
[0120] In one example, the antibody comprises any one of the
following pairs of V.sub.H and V.sub.L:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; and (iii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46.
[0121] In one example, the antibody comprises any one of the
following pairs of V.sub.H and V.sub.L:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ii) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 106 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 107 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (iv) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 222 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (v) a V.sub.H
comprising a sequence set forth in SEQ ID NO: 176 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 189; (vi) a V.sub.H
encoded by a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 223 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions and a V.sub.L encoded by a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 228 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions; (vii) a V.sub.H comprising
a sequence set forth in SEQ ID NO: 177 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 190; (viii) a V.sub.H encoded by a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 224 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions and
a V.sub.L encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 229 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions; (ix) a V.sub.H comprising a sequence set
forth in SEQ ID NO: 178 and a V.sub.L comprising a sequence set
forth in SEQ ID NO: 191; (x) a V.sub.H encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 224 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions and a V.sub.L
encoded by a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 230 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions; (xi) a V.sub.H comprising a sequence set forth in SEQ
ID NO: 179 and a V.sub.L comprising a sequence set forth in SEQ ID
NO: 192; (xii) a V.sub.H encoded by a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 224 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions and a V.sub.L encoded by a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 231 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions;
(xiii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 180
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 193;
(xiv) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
224 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (xvi) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 225 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 230 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (xviii)
a V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 226 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 230 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (xx) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 226 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; (xxii) a
V.sub.H encoded by a nucleic acid comprising a sequence at least
about 95% identical to the sequence set forth in SEQ ID NO: 227 or
a nucleic acid that hybridizes thereto under moderate to high
stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (xxiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 187 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 200; or
(xxiv) a V.sub.H encoded by a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
227 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a V.sub.L encoded by a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 233 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions.
[0122] In one example, the antibody comprises a V.sub.H comprising
a sequence set forth in SEQ ID NO: 94 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 95.
[0123] In one example, the antibody comprises any one of the
following pairs of V.sub.H and V.sub.L:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; or (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62.
[0124] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO:42 and a V.sub.L of SEQ ID
NO:46.
[0125] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 175 and a V.sub.L of SEQ ID NO:
188.
[0126] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 176 and a V.sub.L of SEQ ID NO:
189.
[0127] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 177 and a V.sub.L of SEQ ID NO:
190.
[0128] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 178 and a V.sub.L of SEQ ID NO:
191.
[0129] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 179 and a V.sub.L of SEQ ID NO:
192.
[0130] The disclosure also provides a A TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 180 and a V.sub.L of SEQ ID NO:
193.
[0131] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 181 and a V.sub.L of SEQ ID NO:
194.
[0132] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 183 and a V.sub.L of SEQ ID NO:
196.
[0133] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 185 and a V.sub.L of SEQ ID NO:
198.
[0134] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 186 and a V.sub.L of SEQ ID NO:
199.
[0135] The disclosure also provides a TL1a-binding antibody
comprising a V.sub.H of SEQ ID NO: 187 and a V.sub.L of SEQ ID NO:
200.
[0136] The antibodies set out in the foregoing list provide one or
more of the following advantages:
(i) inhibit TL1a interaction with DR3 and do not inhibit
interaction of TL1a and DcR3 as determined by the methods described
herein; (ii) reduce the level of apoptosis of the TF-1 cells (e.g.,
about 7.times.10.sup.4 cells-8.times.10.sup.4 cells (e.g.,
7.5.times.10.sup.4 cells)) with an EC.sub.50 of less than about 2
nM (such as less than about 1.5 nM or 1.2 nM or 1.1 nM; or 1 nM or
less) compared to the level of apoptosis in the absence of the
TL1a-binding protein; or (iii) bind to TL1a on the surface of a
cell with an EC.sub.50 of less than about 10 nM, such as less than
about 5 nM or 3 nM or 2 nM, e.g., as assessed using flow cytometry
performed with about 2.times.10.sup.5 to 3.times.10.sup.5 cells
(e.g., 2.5.times.10.sup.5 cells).
[0137] In one example, the antibody comprises a V.sub.H comprising
a sequence set forth in SEQ ID NO: 137 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 138.
[0138] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 152 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 153.
[0139] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 162 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 172.
[0140] In one example, the TL1a-binding protein comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 163 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 174.
[0141] For example, the antibody comprises a V.sub.H comprising a
sequence set forth in SEQ ID NO: 42 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 46, wherein the V.sub.H and/or
V.sub.L comprise one or more of the following substitutions or
groups of substitutions:
(i) the V.sub.H comprises an alanine at position 16 of SEQ ID NO:
42; (ii) the V.sub.H comprises an alanine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (iii) the V.sub.H comprises a serine at position 100 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (iv) the V.sub.H comprises a histidine at
position 100 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (v) the V.sub.H comprises a
leucine at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (vi) the V.sub.H
comprises an aspartic acid at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a (vii) the V.sub.H comprises a tyrosine at
position 100 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (viii) the V.sub.H comprises a
proline at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (ix) the V.sub.H
comprises a glutamine at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (x)
the V.sub.H comprises a lysine at position 100 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xi) the V.sub.H comprises an alanine at position 101 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xii) the V.sub.H comprises a serine at position 101 of SEQ
ID NO: 42 and the V.sub.L comprises a threonine at position 76 of
SEQ ID NO: 46; (xiii) the V.sub.H comprises a histidine at position
101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xiv) the V.sub.H comprises a leucine
at position 101 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xv) the V.sub.H
comprises an aspartic acid at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xvi) the V.sub.H comprises a tyrosine at position 101 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xvii) the V.sub.H comprises a glutamine at position 101
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xviii) the V.sub.H comprises a lysine at
position 101 of SEQ ID NO: 42 and the V.sub.1 comprises a threonine
at position 76 of SEQ ID NO: 46; (xix) the V.sub.H comprises an
alanine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xx) the V.sub.H
comprises a serine at position 102 of SEQ ID NO: 42 and the V.sub.L
comprises a (xxi) the V.sub.H comprises a histidine at position 102
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xxii) the V.sub.H comprises a leucine at
position 102 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxiii) the V.sub.H comprises a
tyrosine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxiv) the V.sub.H
comprises a proline at position 102 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxv) the V.sub.H comprises a glutamine at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxvi) the V.sub.H comprises a lysine at position 102 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxvii) the V.sub.H comprises an alanine at
position 103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxviii) the V.sub.H comprises a
serine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xxix) the V.sub.H
comprises a histidine at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxx) the V.sub.H comprises a leucine at position 103 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xxxi) the V.sub.H comprises an aspartic acid at position
103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xxxii) the V.sub.H comprises a
tyrosine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxxiii) the V.sub.H
comprises a proline at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxiv) the V.sub.H comprises a glutamine at position 103 of SEQ ID
NO: 42 and the V.sub.L comprises a (xxxv) the V.sub.H comprises a
lysine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xxxvi) the V.sub.H
comprises a serine at position 104 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxxvii) the
V.sub.H comprises a histidine at position 104 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxviii) the V.sub.H comprises a leucine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxix) the V.sub.H comprises an aspartic acid at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xl) the V.sub.H comprises a
tyrosine at position 104 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xli) the V.sub.H
comprises a proline at position 104 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlii) the V.sub.H comprises a glutamine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xliii) the V.sub.H comprises a lysine at position 104
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xliv) the V.sub.H comprises an alanine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlv) the V.sub.H comprises a
histidine at position 105 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xlvi) the
V.sub.H comprises a leucine at position 105 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlvii) the V.sub.H comprises an aspartic acid at position 105 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xlviii) the V.sub.H comprises a tyrosine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a (xlix)
the V.sub.H comprises a proline at position 105 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (l) the V.sub.H comprises a glutamine at position 105 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (li) the V.sub.H comprises a lysine at position 105 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lii) the V.sub.H comprises an alanine at
position 107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (liii) the V.sub.H comprises a
serine at position 107 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (liv) the V.sub.H
comprises a histidine at position 107 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (lv)
the V.sub.H comprises a leucine at position 107 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (lvi) the V.sub.H comprises an aspartic acid at position 107 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lvii) the V.sub.H comprises a tyrosine at
position 107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (lviii) the V.sub.H comprises a
proline at position 107 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (lix) the V.sub.H
comprises a glutamine at position 107 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (lx)
the V.sub.H comprises a lysine at position 107 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxi) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an alanine at position 23 of SEQ
ID NO: 46; (lxii) the V.sub.H comprises a threonine at position 41
of SEQ ID NO: 42 and the V.sub.L comprises an aspartic acid at
position 28 of SEQ ID NO: 46; (lxiii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a tyrosine at position 33 of SEQ ID NO: 46; (lxiv) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises an aspartic acid at position 34 of SEQ ID NO: 46;
(lxv) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an asparagine at position 53 of
SEQ ID NO: 46; (lxvi) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises a serine at position
54 of SEQ ID NO: 46; (lxvii) the V.sub.H comprises a threonine at
position 41 of SEQ ID NO: 42 and the V.sub.L comprises a alanine at
position 82 of SEQ ID NO: 46; (lxviii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a serine at position 95 of SEQ ID NO: 46; (lxix) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises a serine at position 96 of SEQ ID NO: 46; (lxx)
the V.sub.H comprises a threonine at position 41 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (lxxi) the V.sub.H comprises a serine at position 47 of SEQ ID
NO: 42 and the V.sub.L comprises an threonine at position 23 of SEQ
ID NO: 46; (lxxii) the V.sub.H comprises a proline at position 41,
an alanine at position 72, an aspartic acid at position 73 an
arginine at position 74 and a threonine at position 76 each
relative to SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lxxiii) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a serine at position 24 and a threonine at position 76
each relative to SEQ ID NO: 46; (lxxiv) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23, a serine at position 24 and a
threonine at position 76 each relative to SEQ ID NO: 46; (lxxv) the
V.sub.H comprises a proline at position 41, a leucine at position
51 and a glutamic acid at position 102 each relative to SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 23 and a
threonine at position 76 each relative to SEQ ID NO: 46; (lxxvi)
the V.sub.H comprises a proline at position 41, a leucine at
position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lxxvii) the V.sub.H comprises a proline at
position 41, a leucine at position 51, a glutamic acid at position
102 and an alanine at position 105 each relative to SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 23, a serine at
position 24 and a threonine at position 76 each relative to SEQ ID
NO: 46; (lxxviii) the V.sub.H comprises a proline at position 41, a
leucine at position 51, a glutamic acid at position 102 and an
alanine at position 105 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxxix) the V.sub.H comprises a proline at position 41, an alanine
at position 72, an aspartic acid at position 73 an arginine at
position 74 and a threonine at position 76 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24 and a threonine at position 76 each relative
to SEQ ID NO: 46; (lxxx) the V.sub.H comprises a proline at
position 41, an alanine at position 72, an aspartic acid at
position 73 an arginine at position 74 and a threonine at position
76 each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24, a threonine at
position 76 and a glutamic acid at position 51 each relative to SEQ
ID NO: 46; (lxxxi) the V.sub.H comprises a proline at position 41,
a leucine at position 51, an alanine at position 72, an aspartic
acid at position 73, an arginine at position 74, a threonine at
position 76, a glutamic acid at position 102 and an alanine at
position 105 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23, a serine at position 24, a
threonine at position 76 and a glutamic acid at position 51 each
relative to SEQ ID NO: 46; or (lxxxii) the V.sub.H comprises a
proline at position 41, a leucine at position 51, an alanine at
position 72, an aspartic acid at position 73, an arginine at
position 74, a threonine at position 76, a glutamic acid at
position 102 and an alanine at position 105 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glycine at
position 51 each relative to SEQ ID NO: 46.
[0142] In one example, a TL1a-binding protein of the disclosure
comprises a human or non-human primate heavy chain immunoglobulin
constant region selected from a group consisting of IgG1, IgG2,
IgG3, IgG4, IgD, IgM, IgE and IgA. An exemplary heavy chain
immunoglobulin constant region is an IgG constant region, e.g., an
IgG1 constant region, such as a human IgG1 constant region. For
example, a human IgG1 constant region comprises a Fc region
comprising a sequence set forth in SEQ ID NO: 134. In one example,
the human or non-human primate heavy chain immunoglobulin constant
region lacks a C-terminal lysine residue.
[0143] In another example, a TL1a-binding protein of the disclosure
comprises a human or non-human primate light chain immunoglobulin
constant region selected from a group consisting of kappa or
lambda. In one example, the TL1a-binding protein comprises a human
light chain constant region comprising a sequence set forth in SEQ
ID NO: 135 (kappa) or SEQ ID NO: 136 (lambda).
[0144] The disclosure also provides an isolated or recombinant
nucleic acid encoding the TL1a-binding protein of the disclosure or
a sequence having at least about 80% identity thereto or a sequence
that hybridizes thereto under moderate or high stringency
conditions. In this regard, the disclosure is not limited to the
specific exemplified nucleic acids described herein, but also
encompasses any nucleic acid that encodes a TL1a-binding protein of
the disclosure as a result of degeneracy of the genetic code. For
example, the nucleic acid may be codon optimized for expression in
a particular cell type.
[0145] In one example, the nucleic acid comprises a sequence set
forth in any one of SEQ ID NOs: 96 to 118 or a sequence having at
least about 80% identity thereto or a sequence that hybridizes
thereto under moderate or high stringency conditions.
[0146] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 102 or a sequence having at least about 80%
identity thereto or a sequence that hybridizes thereto under
moderate or high stringency conditions.
[0147] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 103 or a sequence having at least about 80%
identity thereto or a sequence that hybridizes thereto under
moderate or high stringency conditions.
[0148] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 102 and a sequence set forth in SEQ ID NO: 103
or a sequence having at least about 80% identity thereto or a
sequence that hybridizes thereto under moderate or high stringency
conditions.
[0149] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 104 or a sequence having at least about 80%
identity thereto or a sequence that hybridizes thereto under
moderate or high stringency conditions.
[0150] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 105 or a sequence having at least about 80%
identity thereto or a sequence that hybridizes thereto under
moderate or high stringency conditions.
[0151] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 104 and a sequence set forth in SEQ ID NO: 105
or a sequence having at least about 80% identity thereto or a
sequence that hybridizes thereto under moderate or high stringency
conditions.
[0152] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 106 or a sequence having at least about 80%
identity thereto or a sequence that hybridizes thereto under
moderate or high stringency conditions.
[0153] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 107 or a sequence having at least about 80%
identity thereto or a sequence that hybridizes thereto under
moderate or high stringency conditions.
[0154] In one example, the nucleic acid comprises a sequence set
forth in SEQ ID NO: 106 and a sequence set forth in SEQ ID NO: 107
or a sequence having at least about 80% identity thereto or a
sequence that hybridizes thereto under moderate or high stringency
conditions.
[0155] In one example, the nucleic acid comprises a sequence at
least 95% identical to a sequence set forth in any one of SEQ ID
NOs: 106, 107 or 222 to 233 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions.
[0156] In one example, the nucleic acid comprises one or more of
the following:
(i) a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 106 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions and a nucleic acid comprising a sequence at least about
95% identical to the sequence set forth in SEQ ID NO: 107 or a
nucleic acid that hybridizes thereto under moderate to high
stringency conditions; (ii) a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
222 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a nucleic acid comprising a sequence
at least about 95% identical to the sequence set forth in SEQ ID
NO: 228 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions; (iii) a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 223 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions and a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (iv) a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 224 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions and
a nucleic acid comprising a sequence at least about 95% identical
to the sequence set forth in SEQ ID NO: 229 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions;
(v) a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 224 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions and a nucleic acid comprising a sequence at least about
95% identical to the sequence set forth in SEQ ID NO: 230 or a
nucleic acid that hybridizes thereto under moderate to high
stringency conditions; (vi) a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
224 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a nucleic acid comprising a sequence
at least about 95% identical to the sequence set forth in SEQ ID
NO: 231 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions; (vii) a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 224 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions and a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 228 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; (viii) a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 225 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions and
a nucleic acid comprising a sequence at least about 95% identical
to the sequence set forth in SEQ ID NO: 230 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions;
(ix) a nucleic acid comprising a sequence at least about 95%
identical to the sequence set forth in SEQ ID NO: 226 or a nucleic
acid that hybridizes thereto under moderate to high stringency
conditions and a nucleic acid comprising a sequence at least about
95% identical to the sequence set forth in SEQ ID NO: 230 or a
nucleic acid that hybridizes thereto under moderate to high
stringency conditions; (x) a nucleic acid comprising a sequence at
least about 95% identical to the sequence set forth in SEQ ID NO:
226 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions and a nucleic acid comprising a sequence
at least about 95% identical to the sequence set forth in SEQ ID
NO: 232 or a nucleic acid that hybridizes thereto under moderate to
high stringency conditions; (xi) a nucleic acid comprising a
sequence at least about 95% identical to the sequence set forth in
SEQ ID NO: 227 or a nucleic acid that hybridizes thereto under
moderate to high stringency conditions and a nucleic acid
comprising a sequence at least about 95% identical to the sequence
set forth in SEQ ID NO: 232 or a nucleic acid that hybridizes
thereto under moderate to high stringency conditions; or (xii) a
nucleic acid comprising a sequence at least about 95% identical to
the sequence set forth in SEQ ID NO: 227 or a nucleic acid that
hybridizes thereto under moderate to high stringency conditions and
a nucleic acid comprising a sequence at least about 95% identical
to the sequence set forth in SEQ ID NO: 233 or a nucleic acid that
hybridizes thereto under moderate to high stringency
conditions.
[0157] Sequences of exemplary nucleic acids and combinations
thereof are set out in Table 1 and are to be taken to provide
literal support for each individual sequence and combination
thereof.
[0158] In one example, such a nucleic acid is included in an
expression construct in which the nucleic acid is operably linked
to a promoter. Such an expression construct can be in a vector,
e.g., a plasmid.
[0159] In examples of the disclosure directed to single polypeptide
TL1a-binding proteins, the expression construct may comprise a
promoter linked to a nucleic acid encoding that polypeptide
chain.
[0160] In examples directed to multiple polypeptides that form a
TL1a-binding protein, an expression construct of the disclosure
comprises a nucleic acid encoding one of the polypeptides (e.g.,
comprising a V.sub.H) operably linked to a promoter and a nucleic
acid encoding another of the polypeptides (e.g., comprising a
V.sub.L) operably linked to a promoter.
[0161] In another example, the expression construct is a
bicistronic expression construct, e.g., comprising the following
operably linked components in 5' to 3' order:
(i) a promoter (ii) a nucleic acid encoding a first polypeptide;
(iii) an internal ribosome entry site; and (iv) a nucleic acid
encoding a second polypeptide.
[0162] For example, the first polypeptide comprises a V.sub.H and
the second polypeptide comprises a V.sub.L, or the first
polypeptide comprises a V.sub.L and the second polypeptide
comprises a V.sub.H.
[0163] The disclosure also contemplates separate expression
constructs one of which encodes a first polypeptide (e.g.,
comprising a V.sub.H) and another of which encodes a second
polypeptide (e.g., comprising a V.sub.L). For example, the
disclosure also provides a composition comprising:
(i) a first expression construct comprising a nucleic acid encoding
a polypeptide (e.g., comprising a V.sub.H operably linked to a
promoter); and (ii) a second expression construct comprising a
nucleic acid encoding a polypeptide (e.g., comprising a V.sub.L
operably linked to a promoter), wherein the first and second
polypeptides associate to form a TL1a-binding protein of the
disclosure.
[0164] The disclosure also provides an isolated cell expressing a
TL1a-binding protein of the disclosure or a recombinant cell
genetically-modified to express a TL1a-binding protein of the
disclosure.
[0165] In one example, the cell comprises the expression construct
of the disclosure or:
(i) a first expression construct comprising a nucleic acid encoding
a polypeptide (e.g., comprising a V.sub.H) operably linked to a
promoter; and (ii) a second expression construct comprising a
nucleic acid encoding a polypeptide (e.g., comprising a V.sub.L)
operably linked to a promoter, wherein the first and second
polypeptides associate to form a TL1a-binding protein of the
disclosure.
[0166] Examples of cells of the disclosure include bacterial cells,
yeast cells, insect cells or mammalian cells. Exemplary cells are
mammalian.
[0167] The disclosure additionally provides methods for producing a
TL1a-binding protein of the disclosure. For example, such a method
involves maintaining the expression construct(s) of the disclosure
under conditions sufficient for the TL1a-binding protein to be
produced.
[0168] In one example, a method for producing a TL1a-binding
protein of the disclosure comprises culturing the cell of the
disclosure under conditions sufficient for the protein to be
produced and, optionally, secreted.
[0169] In one example, the method for producing a TL1a-binding
protein of the disclosure additionally comprises isolating the
protein.
[0170] The disclosure also provides a composition comprising the
TL1a-binding protein, nucleic acid, expression construct or cell of
the disclosure and a suitable carrier. In one example, the
composition comprises the TL1a-binding protein of the disclosure
and a suitable carrier. In one example, the carrier is
pharmaceutically acceptable, e.g., the composition is a
pharmaceutical composition.
[0171] The disclosure also provides a method for treating or
preventing symptoms of a condition (e.g., a TL1a-mediated
condition) in a cell, tissue, organ or subject, the method
comprising administering the TL1a-binding protein, nucleic acid,
expression construct, cell or composition of the disclosure to the
cell, tissue, organ or subject. In one example, the disclosure
provides a method for treating or preventing a condition (e.g., a
TL1a-mediated condition) in a subject, the method comprising
administering the TL1a-binding protein, nucleic acid, expression
construct, cell or composition of the disclosure to the subject. In
this regard, a method of preventing a condition can prevent a
relapse of a condition having a relapsing-remitting form, such as
multiple sclerosis, e.g., the protein is administered during
remission to thereby prevent a relapse.
[0172] The disclosure also provides a method for inducing or
enhancing angiogenesis in a subject, the method comprising
administering the TL1a-binding protein, nucleic acid, expression
construct, cell or composition of the disclosure to the
subject.
[0173] The disclosure also provides for use of the TL1a-binding
protein, nucleic acid, expression construct, cell or composition of
the disclosure in medicine.
[0174] The disclosure also provides for use of the TL1a-binding
protein, nucleic acid, expression construct, or cell in the
manufacture of a medicament or prevention for treating or
preventing symptoms of a condition (e.g., a TL1a-mediated
condition) or for treating or preventing a condition (e.g., a
TL1a-mediated condition) or for inducing or enhancing angiogenesis
in a subject.
[0175] The disclosure also provides the TL1a-binding protein,
nucleic acid, expression construct, or cell for use in treating or
preventing symptoms of a condition (e.g., a TL1a-mediated
condition) or for treating or preventing a condition (e.g., a
TL1a-mediated condition) or for inducing or enhancing angiogenesis
in a subject.
[0176] In one example, a method of treatment or prophylaxis of the
disclosure additionally comprises diagnosing the condition, e.g.,
by performing a method described herein.
[0177] In one example, a method of treatment or prophylaxis of the
disclosure additionally comprises detecting the level of TL1a in a
subject and administering a further dose of the TL1a-binding
protein, nucleic acid, expression construct, cell or composition of
the disclosure if the level of TL1a is not significantly reduced or
is not reduced to a level not associated with a condition.
[0178] The disclosure also provides a method for inhibiting
interaction of TL1a and DR3 (and in one example, not inhibiting
interaction of TL1a and DcR3) in a cell, tissue, organ or subject,
the method comprising administering the TL1a-binding protein,
nucleic acid, expression construct, cell or composition of the
disclosure to the cell, tissue, organ or subject. In one example,
the subject suffers from a condition (e.g., a TL1a-mediated
condition).
[0179] The disclosure also provides a method for detecting TL1a in
a sample, the method comprising contacting a sample with the
TL1a-binding protein of the disclosure such that an antigen-protein
complex forms and detecting the complex, wherein detecting the
complex is indicative of TL1a in the sample.
[0180] The disclosure also provides a method for detecting TL1a in
a subject, the method comprising detecting the TL1a-binding protein
of the disclosure in the subject, wherein the protein is conjugated
to a detectable label. In one example, the method comprises
administering the protein to the subject.
[0181] The disclosure also provides a method for diagnosing a
TL1a-mediated condition in a subject, the method comprising
performing the method of the disclosure to detect TL1a in a sample
from the subject, wherein detection of TL1a in the sample is
indicative of the TL1a-mediated condition.
[0182] In one example, the method comprises determining the level
of TL1a in the sample, wherein an increased or decreased level of
TL1a in the sample compared to a control sample is indicative of
the TL1a-mediated condition.
[0183] In one example, the results of a method to detect TL1a or
diagnose/prognose a condition are provided, e.g., in paper or
machine-readable form.
[0184] The disclosure also provides a method comprising obtaining
the results of a method to detect TL1a or diagnose/prognose a
condition of the disclosure and administering a therapeutic or
prophylactic composition or recommending such administration. In
one example, the composition is a composition of the
disclosure.
[0185] Exemplary conditions to be treated, prevented, diagnosed or
prognosed are T.sub.H17-mediated conditions, inflammatory
conditions, autoimmune conditions or conditions associated with or
caused by insufficient angiogenesis. Suitable conditions are
described herein.
[0186] In one example, a condition to be treated, prevented,
diagnosed or prognosed is an autoimmune disease. For example, the
condition is uveitis, ulcerative colitis, Crohn's disease,
irritable bowel syndrome, rheumatoid arthritis, polyarthritis,
multiple sclerosis, asthma or chronic obstructive pulmonary
disease.
[0187] In one example, the condition is an inflammatory bowel
condition, such as, colitis, e.g., ulcerative colitis or Crohn's
disease.
[0188] The disclosure also provides a method of selecting a
TL1a-binding protein which binds specifically to TL1a and inhibits
interaction of TL1a and DR3 and which does not inhibit interaction
of TL1a and DcR3 from a plurality of TL1a-binding proteins, the
method comprising:
[0189] contacting the plurality of TL1a-binding proteins to a TL1a
mutein in which the arginine at amino acid position 32 of SEQ ID
NO:202 has been substituted with an alanine and/or the arginine at
amino acid position 85 has been substituted with alanine under
conditions sufficient to allow binding of TL1a-binding proteins to
the mutein to form a TL1a-binding protein-TL1a mutein complex and a
depleted plurality of TL1a-binding proteins which do not bind the
TL1a mutein,
[0190] and collecting TL1a-binding proteins which do not bind to
the TL1a mutein from the depleted plurality of TL1a-binding
proteins, wherein the collected TL1a-binding proteins bind
specifically to TL1a and inhibit interaction of TL1a and DR3 and do
not inhibit interaction of TL1a and DcR3.
[0191] The disclosure also provides a method of isolating a
TL1a-binding protein which binds specifically to TL1a and inhibits
interaction of TL1a and DR3 and which does not inhibit interaction
of TL1a and DcR3 from a plurality of TL1a-binding proteins, the
method comprising isolating from the plurality of TL1a-binding
proteins one or more TL1a-binding proteins that do not bind to a
TL1a mutein in which the arginine at amino acid position 32 of SEQ
ID NO:202 has been substituted with an alanine and/or the arginine
at amino acid position 85 has been substituted with alanine.
[0192] The plurality of TL1a-binding proteins may be present in a
library of antibodies or antigen binding domains, such as, a phage
display, ribosoime display or yeast display library. The plurality
of TL1a-binding proteins may be present in an antiserum. The
plurality of TL1a-binding proteins may be present in hybridoma
culture supernatants.
[0193] The disclosure also provides an isolated polypeptide
comprising a sequence set forth in SEQ ID NO: 202 in which the
arginine at amino acid position 32 as been substituted with an
alanine and/or the arginine at amino acid position 85 has been
substituted with alanine.
BRIEF DESCRIPTION OF THE FIGURES
[0194] FIGS. 1A to 1H are diagrammatic representations showing
sequences of variable regions of antibodies. In alignments shown in
FIGS. 1D to 1H any identical amino acids are indicated by a period,
i.e., ".". FIG. 1A shows sequences of V.sub.H regions of human
anti-TL1a antibodies. FIG. 1B shows sequences of V.sub.L regions of
human anti-TL1a antibodies. FIG. 1C shows sequences of V.sub.H
regions of anti-TL1a antibody C320 and some derivatives thereof.
FIG. 1D shows alignment of V.sub.H sequences of selected human
antibodies into which the V.sub.H CDRs 1, 2 and 3 of C320 were
grafted. FIG. 1E shows a consensus sequence of V.sub.H regions of
derivatives of C320. FIG. 1F shows sequences of V.sub.L regions of
anti-TL1a antibody C320 and derivatives thereof. FIG. 1G shows an
alignment of human V.sub.L sequences of antibodies into which the
V.sub.L CDRs 1, 2 and 3 of C320 were grafted. FIG. 1H shows a
consensus sequence of V.sub.L regions of derivatives of C320. Boxed
regions contain CDRs (as indicated) as defined by the Kabat
numbering system and the enhanced Chothia numbering system. CDRs
defined by the Kabat numbering system are shown in bold. CDRs
defined by the enhanced Chothia numbering system are
underlined.
[0195] FIG. 2 is a graphical representation showing results of a
potency assay demonstrating the ability of a range of anti-TL1a
antibodies to inhibit TL1a-induced apoptosis of TF-1 cells. Five
.mu.g/ml of anti-TL1a antibodies were screened for their ability to
inhibit TL1a-induced apoptosis in TF-1 cells.
[0196] FIG. 3 is a graphical representation of results of an assay
to identify highly potent anti-TL1a antibodies. Results depicted
show the level of inhibition of TL1a-induced apoptosis of TF-1
cells achieved at various concentrations of test antibodies
(maximum concentration tested 5 .mu.g/mL). These levels permit
determination of the EC.sub.50 of anti-TL1a antibodies for the
inhibition of TL1a-induced apoptosis of TF-1 cells, which allows
for functional comparisons based on relative potency.
[0197] FIG. 4A is a graphical representation showing the level of
inhibition of interaction of TL1a with DR3 achieved at various
concentrations of test antibodies (maximum concentration tested 25
.mu.g/mL). Antibodies C320, C321 and C323 inhibited the interaction
between TL1a and DR3 at numerous concentrations.
[0198] FIG. 4B is a graphical representation showing the level of
inhibition of interaction of TL1a with DcR3 achieved at various
concentrations of test antibodies (maximum concentration tested 25
.mu.g/mL). Antibodies C320, C321 and C323 did not detectably
inhibit the interaction between TL1a and DcR3 at antibody
concentrations of 10 .mu.g/mL or less.
[0199] FIG. 4C is a graphical representation showing the level of
inhibition of interaction of TL1a with DR3 achieved at various
concentrations of test antibodies (maximum concentration tested 10
.mu.g/mL). Antibodies C320-0, C320-168 and C320-179 inhibited the
interaction between TL1a and DR3 at numerous concentrations.
Antibodies 1B4 and C300-25 are included for the purposes of
comparison
[0200] FIG. 4D is a graphical representation showing the level of
inhibition of interaction of TL1a and DcR3 achieved at various
concentrations of test antibodies (maximum concentration tested 100
.mu.g/mL). Antibodies C320-0, C320-168 and C320-179 did not
detectably inhibit the interaction between TL1a and DcR3.
Antibodies 1B4 and C300-25 inhibited the interaction between TL1a
and DcR3 at various concentrations and are included for the
purposes of comparison
[0201] FIG. 5A includes two a graphical representations showing the
binding of various concentrations of test antibodies to soluble
human TL1a (SEQ ID NO: 202; top panel) and soluble human TL1a in
which arginine at residue 32 has been substituted with alanine
(R32A mutein, bottom panel). Antibodies C320-0, C320-168 and
C320-179 all bound TL1a at various concentrations but not R32A
mutein TL1a. Anti-TL1a antibodies 1B4 and 16H2 (as described in
US20090280116) bound both TL1a and R32A mutein TL1a at various
concentrations. The isotype control antibody did not bind any form
of TL1a
[0202] FIG. 5B is a graphical representation showing the binding of
various concentrations of test antibodies to soluble human TL1a in
which arginine at residue 85 has been substituted with alanine
(R85A mutein, top panel) and soluble human TL1a in which arginines
at residue 32 and residue 85 have been substituted with alanine
(R32A+R85A mutein, bottom panel). Antibodies C320-0, C320-168 and
C320-179 did not bind either R85A or R32A+R85A mutein TL1a.
Anti-TL1a antibodies 1B4 and 16H2 (as described in US20090280116)
bound both R85A or R32A+R85A mutein TL1a at various concentrations.
The isotype control antibody did not bind any form of TL1a
[0203] FIG. 5C is a graphical representation showing the binding of
soluble human TL1a, R32A mutein TL1a and R85A mutein TL1a at a
concentration of 1 .mu.g/ml to receptors DR3 and DcR3. TL1a bound
both receptors equally well. R32A and R85A mutein TL1a bound DcR3
to a similar extent as did TL1a but bound DR3 at a level
approximately 50% or more lower than TL1a binding to DcR3
[0204] FIG. 5D is a diagrammatic representation depicting the X-ray
crystal structure of trimeric human TL1a (PDB: 3K51) (gray) with
the residues R32 and R85 highlighted in black on each monomer.
[0205] FIG. 6 is a graphical representation showing the ability of
antibodies C320, C321, C323 and 1B4 to bind to cell surface TL1a.
Binding was assessed using flow cytometry, in which results are
presented as mean fluorescence intensity (MFI). All antibodies,
except the isotype control, bound TL1a
[0206] FIG. 7A is a graphical representation showing production of
cell surface TL1a by mitogen (Concanavalin A)-stimulated peripheral
blood mononuclear cells (PBMCs). The shaded graph represents an
isotype control antibody and the line graph represents cell surface
TL1a as detected by a chimeric rat/human anti-human TL1a.
[0207] FIG. 7B is a graphical representation showing increased
production of secreted TL1a in PBMCs as mitogen (Concanavalin
A)-stimulation is increased.
[0208] FIG. 8A is a graphical representation showing the ability of
anti-TL1a antibodies (maximum concentration tested 50 .mu.g/mL) to
inhibit interferon gamma (IFN-.gamma.) production induced by
endogenous human TL1a. Endogenous TL1a enhanced cytokine production
by stimulated PBMCs. Antibodies C320 and C323 inhibited the
production of IFN-.gamma.. Antibody 1B4 is included for the
purposes of comparison
[0209] FIG. 8B is a graphical representation showing the ability of
anti-TL1a antibodies (maximum concentration tested 50 .mu.g/mL) to
inhibit IL-13 production induced by endogenous human TL1a.
Endogenous TL1a enhanced cytokine production by stimulated PBMCs.
Antibodies C320 and C323 inhibited the production of IL-13 Antibody
1B4 is included for the purposes of comparison.
[0210] FIG. 9A is a diagrammatic representation showing an
alignment if the light chain sequence of C320 against the germline
sequence of highest homology, IGLV1-40*01. Any identical amino
acids are indicated by a period, i.e., ".". Differences in amino
acid sequences in the CDR regions are identified.
[0211] FIG. 9B is a diagrammatic representation showing an
alignment of V.sub.H regions of antibodies identified herein.
[0212] FIG. 9C is a diagrammatic representation showing an
alignment of V.sub.L regions of antibodies identified herein.
[0213] FIG. 9D is a copy of a photographic representation showing
results of isoelectric focusing gel comparing C320-168, C320-163
and C320-170. C320-168 has 5-6 distinct charged isoforms compared
to 1-2 isoforms visualized for C320-163 and C320-170.
[0214] FIG. 9E is a graphical representation showing the ability of
antibodies C320-168, C320-179 and C320-183 to neutralize
TL1a-induced apoptosis of TF-1 cells at various concentrations
(maximum concentration tested 10 .mu.g/mL). Antibodies C320-0 and
1B4 are included for comparison. All antibodies inhibit
TL1a-induced apoptosis of TF-1 cells at multiple
concentrations.
[0215] FIG. 10A is a graphical representation showing the total
area of colon ulcerated (cm.sup.2) in rats in the days following
DNBS-induced colitis. Rats were treated with antibody C320-168 (10
mg/kg), vehicle (negative control) days 0 and 4 or sulfasalazine
(standard of care compound) daily from day 0 (with results for each
treatment group indicated). C320-168 reduced average ulcer area
compared to vehicle treated animals to a comparable extent as
sulfasalazine.
[0216] FIG. 10B is a graphical representation showing the weight
change (g) in rats in the days following oxazalone-induced colitis.
Rats were treated with antibody C320-168 (10 mg/kg) or isotype
control (10 mg/kg) on days 0 and 4 or sulfasalazine (SoC (5-ASA);
standard of care compound) daily from day 0 (with results for each
treatment group indicated.) C320-168 ameliorated weight loss
relative to the isotype control antibody to a comparable extent as
sulfasalazine.
[0217] FIG. 10C is a graphical representation showing stool
consistency (DAI--Disease Activity Index) in rats in the days
following oxazalone-induced colitis. Rats were treated with
antibody C320-168 (10 mg/kg) or isotype control (10 mg/kg) on days
0 and 4 or sulfasalazine (SoC (5-ASA); standard of care compound)
daily from day 0 (with results for each treatment group indicated).
C320-168 improved the clinical signs of disease (stool consistency)
relative to the isotype control antibody.
[0218] FIG. 11A is a graphical representation showing stool
consistency (DAI--Disease Activity Index) in rats in the days
following dextran sulphate sodium (DSS)-induced colitis. Rats were
treated with antibody C320-168 (10 mg/kg), or isotype control (10
mg/kg) twice weekly from day 4 after disease induction or
sulfasalazine (SoC (5-ASA); standard of care compound) daily from
day 4 after disease induction (with results for each treatment
group indicated). C320-168 improved the clinical signs of disease
(stool consistency) relative to the isotype control antibody to a
similar extent as sulfasalazine.
[0219] FIG. 11B is a graphical representation showing the weight
change (%) in rats in the days following DSS-induced colitis. Rats
were treated with antibody C320-168 (10 mg/kg or isotype control
(10 mg/kg) twice weekly from day 4 after disease induction or
sulfasalazine (SoC (5-ASA); standard of care compound) daily from
day 4 after disease induction (with results for each treatment
group indicated). C320-168 ameliorated weight loss relative to the
isotype control antibody to a similar extent as sulfasalazine
[0220] FIG. 12 is a graphical representation showing the results of
an ELISA assay to identify the binding of antibodies to different
TL1a isoforms. Both C320-168 and C320-179 bound the longer (72-251)
and shorter (84-251) isoforms of soluble, cleaved TL1a.
DETAILED DESCRIPTION OF THE INVENTION
[0221] Throughout this specification, unless specifically stated
otherwise or the context requires otherwise, reference to a single
step, composition of matter, group of steps or group of
compositions of matter shall be taken to encompass one and a
plurality (i.e. one or more) of those steps, compositions of
matter, groups of steps or groups of compositions of matter. Thus,
as used herein, the singular forms "a", "an" and "the" include
plural aspects unless the context clearly dictates otherwise. For
example, reference to "a" includes a single as well as two or more;
reference to "an" includes a single as well as two or more;
reference to "the" includes a single as well as two or more and so
forth.
[0222] Each example of the disclosure described herein is to be
applied mutatis mutandis to each and every other example unless
specifically stated otherwise.
[0223] Those skilled in the art will appreciate that the disclosure
herein is susceptible to variations and modifications other than
those specifically described. It is to be understood that the
disclosure includes all such variations and modifications. The
disclosure also includes all of the steps, features, compositions
and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations or any
two or more of said steps or features.
[0224] The disclosure is not to be limited in scope by the specific
examples described herein, which are intended for the purpose of
exemplification only. Functionally-equivalent products,
compositions and methods are clearly within the scope of the
disclosure, as described herein.
[0225] The disclosure is performed without undue experimentation
using, unless otherwise indicated, conventional techniques of
molecular biology, microbiology, virology, recombinant DNA
technology, peptide synthesis in solution, solid phase peptide
synthesis, and immunology. Such procedures are described, for
example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratories, New York,
Second Edition (1989), whole of Vols I, II, and III; Benny K. C.
Lo, Antibody Engineering: Methods and Protocols, (2004) Humana
Press, Vol. 248; DNA Cloning: A Practical Approach, Vols. I and II
(D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;
Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed,
1984) IRL Press, Oxford, whole of text, and particularly the papers
therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al.,
pp 83-115; and Wu et al., pp 135-151; 4. Nucleic Acid
Hybridization: A Practical Approach (B. D. Hames & S. J.
Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized
Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford,
whole of text; Perbal, B., A Practical Guide to Molecular Cloning
(1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,
Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, "The
Chemistry of Peptide Synthesis" In: Knowledge database of Access to
Virtual Laboratory website (Interactiva, Germany); Sakakibara
Biochem. Biophys. Res. Commun. 73: 336-342, 1976; Merrifield J. Am.
Chem. Soc. 85: 2149-2154, 1963; Barany and Merrifield (1979) in The
Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284,
Academic Press, New York. 12. Wunsch, E., ed. (1974) Synthese von
Peptiden in Houben-Weyls Metoden der Organischen Chemieler, E.,
ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart;
Bodanszky, M. (1984) Principles of Peptide Synthesis,
Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A.
(1984) The Practice of Peptide Synthesis, Springer-Verlag,
Heidelberg; Bodanszky Int. J. Peptide Protein Res. 25: 449-474,
1985; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir
and C. C. Blackwell, eds., 1986, Blackwell Scientific
Publications); and Animal Cell Culture: Practical Approach, Third
Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of
text.
[0226] The term "and/or", e.g., "X and/or Y" shall be understood to
mean either "X and Y" or "X or Y" and shall be taken to provide
explicit support for both meanings or for either meaning.
[0227] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
Key to Sequence Listing
[0228] SEQ ID NO: 1: amino acid sequence of human TL1a
extracellular domain with N-terminal HIS and FLAG tags SEQ ID NO 2:
amino acid sequence of C336 V.sub.H SEQ ID NO: 3: amino acid
sequence of C336 HCDR1 SEQ ID NO: 4: amino acid sequence of C336
HCDR2 SEQ ID NO: 5: amino acid sequence of C336 HCDR3 SEQ ID NO: 6:
amino acid sequence of C336 V.sub.L SEQ ID NO: 7: amino acid
sequence of C336 LCDR1 SEQ ID NO: 8: amino acid sequence of C336
LCDR2 SEQ ID NO: 9: amino acid sequence of C336 LCDR3 SEQ ID NO:
10: amino acid sequence of C334 V.sub.H SEQ ID NO: 11: amino acid
sequence of C334 HCDR1 SEQ ID NO: 12: amino acid sequence of C334
HCDR2 SEQ ID NO: 13: amino acid sequence of C334 HCDR3 SEQ ID NO:
14: amino acid sequence of C334 V.sub.L SEQ ID NO: 15: amino acid
sequence of C334 LCDR1 SEQ ID NO: 16: amino acid sequence of C334
LCDR2 SEQ ID NO: 17: amino acid sequence of C334 LCDR3 SEQ ID NO:
18: amino acid sequence of C333 V.sub.H SEQ ID NO: 19: amino acid
sequence of C333 HCDR1 SEQ ID NO: 20: amino acid sequence of C333
HCDR2 SEQ ID NO: 21: amino acid sequence of C333 HCDR3 SEQ ID NO:
22: amino acid sequence of C333 V.sub.L SEQ ID NO: 23: amino acid
sequence of C333 LCDR1 SEQ ID NO: 24: amino acid sequence of C333
LCDR2 SEQ ID NO: 25: amino acid sequence of C333 LCDR3 SEQ ID NO:
26: amino acid sequence of C323 V.sub.H SEQ ID NO: 27: amino acid
sequence of C323 HCDR1 SEQ ID NO: 28: amino acid sequence of C323
HCDR2 SEQ ID NO: 29: amino acid sequence of C323 HCDR3 SEQ ID NO:
30: amino acid sequence of C323 V.sub.L SEQ ID NO: 31: amino acid
sequence of C323 LCDR1 SEQ ID NO: 32: amino acid sequence of C323
LCDR2 SEQ ID NO: 33: amino acid sequence of C323 LCDR3 SEQ ID NO:
34: amino acid sequence of C321 V.sub.H SEQ ID NO: 35: amino acid
sequence of C321 HCDR1 SEQ ID NO: 36: amino acid sequence of C321
HCDR2 SEQ ID NO: 37: amino acid sequence of C321 HCDR3 SEQ ID NO:
38: amino acid sequence of C321 V.sub.L SEQ ID NO: 39: amino acid
sequence of C321 LCDR1 SEQ ID NO: 40: amino acid sequence of C321
LCDR2 SEQ ID NO: 41: amino acid sequence of C321 LCDR3 SEQ ID NO:
42: amino acid sequence of C320 V.sub.H SEQ ID NO: 43: amino acid
sequence of C320 HCDR1 SEQ ID NO: 44: amino acid sequence of C320
HCDR2 SEQ ID NO: 45: amino acid sequence of C320 HCDR3 SEQ ID NO:
46: amino acid sequence of C320 V.sub.L SEQ ID NO: 47: amino acid
sequence of C320 LCDR1 SEQ ID NO: 48: amino acid sequence of C320
LCDR2 SEQ ID NO: 49: amino acid sequence of C320 LCDR3 SEQ ID NO:
50: amino acid sequence of C319 V.sub.H SEQ ID NO: 51: amino acid
sequence of C319 HCDR1 SEQ ID NO: 52: amino acid sequence of C319
HCDR2 SEQ ID NO: 53: amino acid sequence of C319 HCDR3 SEQ ID NO:
54: amino acid sequence of C319 V.sub.L SEQ ID NO: 55: amino acid
sequence of C319 LCDR1 SEQ ID NO: 56: amino acid sequence of C319
LCDR2 SEQ ID NO: 57: amino acid sequence of C319 LCDR3 SEQ ID NO:
58: amino acid sequence of C320-3 V.sub.H SEQ ID NO: 59: amino acid
sequence of C320-3 HCDR1 SEQ ID NO: 60: amino acid sequence of
C320-3 HCDR2 SEQ ID NO: 61: amino acid sequence of C320-3 HCDR3 SEQ
ID NO: 62: amino acid sequence of C320-5 V.sub.L SEQ ID NO: 63:
amino acid sequence of C320-5 LCDR1 SEQ ID NO: 64: amino acid
sequence of C320-5 LCDR2 SEQ ID NO: 65: amino acid sequence of
C320-5 LCDR3 SEQ ID NO: 66: amino acid sequence of C320-90 V.sub.H
SEQ ID NO: 67: amino acid sequence of C320-90 HCDR1 SEQ ID NO: 68:
amino acid sequence of C320-90 HCDR2 SEQ ID NO: 69: amino acid
sequence of C320-90 HCDR3 SEQ ID NO: 70: amino acid sequence of
C320-103 V.sub.H SEQ ID NO: 71: amino acid sequence of C320-103
HCDR1 SEQ ID NO: 72: amino acid sequence of C320-103 HCDR2 SEQ ID
NO: 73: amino acid sequence of C320-103 HCDR3 SEQ ID NO: 74: amino
acid sequence of C320-114 V.sub.H SEQ ID NO: 75: amino acid
sequence of C320-114 HCDR1 SEQ ID NO: 76: amino acid sequence of
C320-114 HCDR2 SEQ ID NO: 77: amino acid sequence of C320-114 HCDR3
SEQ ID NO: 78: amino acid sequence of C320-115 V.sub.H SEQ ID NO:
79: amino acid sequence of C320-115 HCDR1 SEQ ID NO: 80: amino acid
sequence of C320-115 HCDR2 SEQ ID NO: 81: amino acid sequence of
C320-115 HCDR3 SEQ ID NO: 82: amino acid sequence of C320-120
V.sub.L SEQ ID NO: 83: amino acid sequence of C320-120 LCDR1 SEQ ID
NO: 84: amino acid sequence of C320-120 LCDR2 SEQ ID NO: 85: amino
acid sequence of C320-120 LCDR3 SEQ ID NO: 86: amino acid sequence
of C320-129 V.sub.H SEQ ID NO: 87: amino acid sequence of C320-129
HCDR1 SEQ ID NO: 88: amino acid sequence of C320-129 HCDR2 SEQ ID
NO: 89: amino acid sequence of C320-129 HCDR3 SEQ ID NO: 90: amino
acid sequence of C320-130 V.sub.H SEQ ID NO: 91: amino acid
sequence of C320-130 HCDR1 SEQ ID NO: 92: amino acid sequence of
C320-130 HCDR2 SEQ ID NO: 93: amino acid sequence of C320-130 HCDR3
SEQ ID NO: 94: amino acid sequence of V.sub.H consensus sequence of
C320 and derivatives SEQ ID NO: 95: amino acid sequence of V.sub.L
consensus sequence of C320 and derivatives SEQ ID NO: 96:
nucleotide sequence encoding V.sub.H of C336 SEQ ID NO: 97:
nucleotide sequence encoding V.sub.L of C336 SEQ ID NO: 98:
nucleotide sequence encoding V.sub.H of C334 SEQ ID NO: 99:
nucleotide sequence encoding V.sub.L of C334 SEQ ID NO: 100:
nucleotide sequence encoding V.sub.H of C333 SEQ ID NO: 101:
nucleotide sequence encoding V.sub.L of C333 SEQ ID NO: 102:
nucleotide sequence encoding V.sub.H of C323 SEQ ID NO: 103:
nucleotide sequence encoding V.sub.L of C323 SEQ ID NO: 104:
nucleotide sequence encoding V.sub.H of C321 SEQ ID NO: 105:
nucleotide sequence encoding V.sub.L of C321 SEQ ID NO: 106:
nucleotide sequence encoding V.sub.H of C320 SEQ ID NO: 107:
nucleotide sequence encoding V.sub.L of C320 SEQ ID NO: 108:
nucleotide sequence encoding V.sub.H of C319 SEQ ID NO: 109:
nucleotide sequence encoding V.sub.L of C319 SEQ ID NO: 110:
nucleotide sequence encoding V.sub.H of C320-3 SEQ ID NO: 111:
nucleotide sequence encoding V.sub.L of C320-5 SEQ ID NO: 112:
nucleotide sequence encoding V.sub.H of C320-90 SEQ ID NO: 113:
nucleotide sequence encoding V.sub.H of C320-103 SEQ ID NO: 114:
nucleotide sequence encoding V.sub.H of C320-114 SEQ ID NO: 115:
nucleotide sequence encoding V.sub.H of C320-115 SEQ ID NO: 116:
nucleotide sequence encoding V.sub.L of C320-120 SEQ ID NO: 117:
nucleotide sequence encoding V.sub.H of C320-129 SEQ ID NO: 118:
nucleotide sequence encoding V.sub.H of C320-130 SEQ ID NO: 119:
amino acid sequence of V.sub.H of humanized antibody 1B4 SEQ ID NO:
120: amino acid sequence of V.sub.L of humanized antibody 1B4 SEQ
ID NO: 121: nucleotide sequence encoding V.sub.H of humanized
antibody 1B4 SEQ ID NO: 122: nucleotide sequence encoding V.sub.L
of humanized antibody 1B4 SEQ ID NO: 123: amino acid sequence of
human TL1a (derived from GenBank Gene accession no 9966 as of 8 May
2011) SEQ ID NO: 124: amino acid sequence of mouse TL1a
Extracellular Domain SEQ ID NO: 125: amino acid sequence of
cynomolgus/Rhesus TL1a Extracellular Domain SEQ ID NO: 126: amino
acid sequence of rat TL1a Extracellular Domain SEQ ID NO: 127:
amino add sequence of rabbit TL1a Extracellular Domain SEQ ID NO:
128: amino acid sequence of guinea Pig TL1a Extracellular Domain
SEQ ID NO: 129: amino acid sequence of human Death Receptor 3
(derived from Genbank Gene Accession No. 8718 as of 8 May 2011) SEQ
ID NO: 130: amino acid sequence of human Decoy Receptor 3 (derived
from Genbank Gene Accession No. 8771 as of 8 May 2011) SEQ ID NO:
131: sequence of region of TL1a SEQ ID NO: 132: sequence of region
of TL1a SEQ ID NO: 133: sequence of region of TL1a SEQ ID NO: 134:
amino acid sequence of human IgG1 Fc region SEQ ID NO: 135: amino
acid sequence of human kappa constant region SEQ ID NO: 136: amino
acid sequence of human lambda constant region. SEQ ID NO: 137:
amino acid sequence of V.sub.H consensus sequence of C320 and
derivatives SEQ ID NO: 138: amino acid sequence of V.sub.L
consensus sequence of C320 and derivatives SEQ ID NO 139: amino
acid sequence of LCDR1 consensus sequence of C320 and derivatives
SEQ ID NO 140: amino acid sequence of LCDR2 consensus sequence of
C320 and derivatives SEQ ID NO 141: amino acid sequence of LCDR3
consensus sequence of C320 and derivatives SEQ ID NO 142: amino
acid sequence of HCDR2 consensus sequence of C320 and derivatives
SEQ ID NO 143: amino acid sequence of HCDR3 consensus sequence of
C320 and derivatives SEQ ID NO: 144: amino acid sequence of HFR1
consensus sequence of C320 and derivatives SEQ ID NO: 145: amino
acid sequence of HFR2 consensus sequence of C320 and derivatives
SEQ ID NO: 146: amino acid sequence of HFR3 consensus sequence of
C320 and derivatives SEQ ID NO: 147: amino acid sequence of HFR4
consensus sequence of C320 and derivatives SEQ ID NO: 148: amino
acid sequence of LFR1 consensus sequence of C320 and derivatives
SEQ ID NO: 149: amino acid sequence of LFR2 consensus sequence of
C320 and derivatives SEQ ID NO: 150: amino acid sequence of LFR3
consensus sequence of C320 and derivatives SEQ ID NO: 151: amino
acid sequence of LFR4 consensus sequence of C320 and derivatives
SEQ ID NO: 152: amino acid sequence of V.sub.H consensus sequence
of C320 and derivatives SEQ ID NO: 153: amino acid sequence of
V.sub.L consensus sequence of C320 and derivatives SEQ ID NO: 154:
amino acid sequence of V.sub.H comprising CDRs from C320 grafted
onto FRs of antibody 1TZG. SEQ ID NO: 155: amino acid sequence of
V.sub.H comprising CDRs from C320 grafted onto FRs of antibody
1RHH. SEQ ID NO: 156: amino acid sequence of V.sub.H comprising
CDRs from C320 grafted onto FRs of antibody 2DD8. SEQ ID NO: 157:
amino acid sequence of V.sub.H comprising CDRs from C320 grafted
onto FRs of antibody 2JB5. SEQ ID NO: 158: amino acid sequence of
V.sub.H comprising CDRs from C320 grafted onto FRs of antibody
3FKU. SEQ ID NO: 159: amino acid sequence of V.sub.H comprising
CDRs from C320 grafted onto FRs of antibody 3GBM. SEQ ID NO: 160:
amino acid sequence of V.sub.H comprising CDRs from C320 grafted
onto FRs of antibody 3LMJ. SEQ ID NO: 161: amino acid sequence of
V.sub.H comprising CDRs from C320 grafted onto FRs of antibody
3P30. SEQ ID NO: 162: amino acid sequence of V.sub.H consensus
sequence of C320 and derivatives SEQ ID NO: 163: amino acid
sequence of V.sub.L comprising CDRs from C320 grafted onto FRs of
antibody 1RHH. SEQ ID NO: 164: amino acid sequence of V.sub.L
comprising CDRs from C320 grafted onto FRs of antibody 1TZGL. SEQ
ID NO: 165: amino acid sequence of V.sub.L comprising CDRs from
C320 grafted onto FRs of antibody 2DD8. SEQ ID NO: 166: amino acid
sequence of V.sub.L comprising CDRs from C320 grafted onto FRs of
antibody 2JB5. SEQ ID NO: 167: amino acid sequence of V.sub.L
comprising CDRs from C320 grafted onto FRs of antibody 3FKU. SEQ ID
NO: 168: amino acid sequence of V.sub.L comprising CDRs from C320
grafted onto FRs of antibody 3GBM. SEQ ID NO: 169: amino acid
sequence of V.sub.L comprising CDRs from C320 grafted onto FRs of
antibody 3LMJ. SEQ ID NO: 170: amino acid sequence of V.sub.L
comprising CDRs from C320 grafted onto FRs of antibody 3P30. SEQ ID
NO: 171: amino acid sequence of V.sub.L comprising CDRs from C320
grafted onto FRs of antibody 3IYW. SEQ ID NO: 172: amino acid
sequence of V.sub.L consensus sequence of C320 and derivatives SEQ
ID NO: 173: amino acid sequence of V.sub.H consensus sequence of
C320 and derivatives SEQ ID NO: 174: amino acid sequence of V.sub.L
consensus sequence of C320 and derivatives SEQ ID NO: 175: amino
acid sequence of V.sub.H of antibody C320-162 SEQ ID NO: 176: amino
acid sequence of V.sub.H of antibody C320-163 SEQ ID NO: 177: amino
acid sequence of V.sub.H of antibody C320-164 SEQ ID NO: 178: amino
acid sequence of V.sub.H of antibody C320-165 SEQ ID NO: 179: amino
acid sequence of V.sub.H of antibody C320-166 SEQ ID NO: 180: amino
acid sequence of V.sub.H of antibody C320-167 SEQ ID NO: 181: amino
acid sequence of V.sub.H of antibody C320-168 SEQ ID NO: 182: amino
acid sequence of V.sub.H of antibody C320-169 SEQ ID NO: 183: amino
acid sequence of V.sub.H of antibody C320-170 SEQ ID NO: 184: amino
acid sequence of V.sub.H of antibody C320-171 SEQ ID NO: 185: amino
acid sequence of V.sub.H of antibody C320-172 SEQ ID NO: 186: amino
acid sequence of V.sub.H of antibody C320-179 SEQ ID NO: 187: amino
acid sequence of V.sub.H of antibody C320-183 SEQ ID NO: 188: amino
acid sequence of V.sub.L of antibody C320-162 SEQ ID NO: 189: amino
acid sequence of V.sub.L of antibody C320-163 SEQ ID NO: 190: amino
acid sequence of V.sub.L of antibody C320-164 SEQ ID NO: 191: amino
acid sequence of V.sub.L of antibody C320-165 SEQ ID NO: 192: amino
acid sequence of V.sub.L of antibody C320-166 SEQ ID NO: 193: amino
acid sequence of V.sub.L of antibody C320-167 SEQ ID NO: 194: amino
acid sequence of V.sub.L of antibody C320-168 SEQ ID NO: 195: amino
acid sequence of V.sub.L of antibody C320-169 SEQ ID NO: 196: amino
acid sequence of V.sub.L of antibody C320-170 SEQ ID NO: 197: amino
acid sequence of V.sub.L of antibody C320-171 SEQ ID NO: 198: amino
acid sequence of V.sub.L of antibody C320-172 SEQ ID NO: 199: amino
acid sequence of V.sub.L of antibody C320-179 SEQ ID NO: 200: amino
acid sequence of V.sub.L of antibody C320-183 SEQ ID NO: 201: amino
acid sequence of V.sub.L of germline sequence IGLV1-40*1 SEQ ID NO:
202: amino acid sequence of soluble human TL1a SEQ ID NO: 203:
amino acid sequence of N-linked glycosylation site in V.sub.H of
C320 SEQ ID NO: 204: amino add sequence from V.sub.H of 1TZG
corresponding to N-linked glycosylation site in V.sub.H of C320 SEQ
ID NO: 205: amino acid sequence of peptide from V.sub.H of C320-168
SEQ ID NO: 206: amino acid sequence of peptide from V.sub.L of
C320-168 SEQ ID NO: 207: amino acid sequence of influenza peptide
SEQ ID NO: 208: amino acid sequence of mutant peptide from V.sub.L
of C320-168 SEQ ID NO: 209: amino acid sequence of mutant peptide
from V.sub.L of C320-168 SEQ ID NO: 210: amino acid sequence of
mutant peptide from V.sub.L of C320-168 SEQ ID NO: 211: amino acid
sequence of mutant peptide from V.sub.L of C320-168 SEQ ID NO: 212:
amino acid sequence of mutant peptide from V.sub.L of C320-168 SEQ
ID NO: 213: amino acid sequence of mutant peptide from V.sub.L of
C320-168 SEQ ID NO: 214: amino acid sequence of mutant peptide from
V.sub.L of C320-168 SEQ ID NO: 215: amino acid sequence of mutant
peptide from V.sub.L of C320-168 SEQ ID NO: 216: amino acid
sequence of mutant peptide from V.sub.L of C320-168 SEQ ID NO: 217:
amino acid sequence of mutant peptide from V.sub.L of C320-168 SEQ
ID NO: 218: amino acid sequence of mutant peptide from V.sub.L of
C320-168 SEQ ID NO: 219: amino acid sequence of mutant peptide from
V.sub.L of C320-168 SEQ ID NO: 220: amino acid sequence of mutant
peptide from V.sub.L of C320-168 SEQ ID NO: 221: amino acid
sequence of mutant peptide from V.sub.L of C320-168 SEQ ID NO: 222:
nucleotide sequence encoding V.sub.H of antibody C320-162 SEQ ID
NO: 223: nucleotide sequence encoding V
.sub.H of antibody C320-163 SEQ ID NO: 224: nucleotide sequence
encoding V.sub.H of antibodies C320-164, C320-165, C320-166 and
C320-167 SEQ ID NO: 225: nucleotide sequence encoding V.sub.H of
antibodies C320-168 and C320-169 SEQ ID NO: 226: nucleotide
sequence encoding V.sub.H of antibodies C320-170 and C320-172 SEQ
ID NO: 227: nucleotide sequence encoding V.sub.H of antibodies
C320-179 and C320-183 SEQ ID NO: 228: nucleotide sequence encoding
V.sub.L of antibodies C320-162, C320-163, C320-167 and C320-169 SEQ
ID NO: 229: nucleotide sequence encoding V.sub.L of antibody
C320-164 SEQ ID NO: 230: nucleotide sequence encoding V.sub.L of
antibodies C320-165, C320-168 and C320-170 SEQ ID NO: 231:
nucleotide sequence encoding V.sub.L of antibody C320-166 SEQ ID
NO: 232: nucleotide sequence encoding V.sub.L of antibodies
C320-172 and C320-179 SEQ ID NO: 233: nucleotide sequence encoding
V.sub.L of antibody C320-183 SEQ ID NO: 234: amino acid sequence of
C320-13 and C320-22 SEQ ID NO: 235: amino acid sequence of
consensus of HCDR3 of C320 and derivatives
[0229] For the purposes of nomenclature and not limitation the
amino acid sequence of human TL1a is set forth in SEQ ID NO: 123.
Additional sequences of human TL1a are set out in Genbank Gene
Accession No. 9966. Accordingly, in one example, the amino acid
sequence of human TL1a comprises a sequence set forth in SEQ ID NO:
123. Other isoforms of TL1a have been described: 72-251 (Position
72 to 251 of SEQ ID No: 123), 84-251 (Position 84 to 251 of SEQ ID
No: 123); 101-251 or VEGI-174 (Position 101 to 251 of SEQ ID No:
123) and 86-251 or VEGI-192 (Position 86 to 251 in SEQ ID No: 123).
The sequences of the extracellular domain of TL1a from various
species are set forth in SEQ ID NO: 1 (amino acids 16 to 184;
human), SEQ ID NO: 124 (mouse), SEQ ID NO: 125 (cynomolgus/rhesus
monkey), SEQ ID NO: 126 (rat), SEQ ID NO: 127 (rabbit) and SEQ ID
NO: 128 (guinea pig). TL1a generally forms a homotrimer in a
subject and signals through DR3. Exemplary TL1a-binding proteins of
the disclosure bind to or bind specifically to human TL1a
(abbreviated herein as human TL1a), including recombinant forms
thereof.
[0230] For the purposes of nomenclature and not limitation, a
sequence of a human DR3 is set forth in SEQ ID NO: 129. Additional
sequences of human DR3 are set out in Genbank Gene Accession No.
8718. In one example, DR3 encompassed by the disclosure is human
DR3 comprising a sequence set forth in SEQ ID NO: 129.
[0231] For the purposes of nomenclature and not limitation, a
sequence of a human DcR3 is set forth in SEQ ID NO: 130. Additional
sequences of human DcR3 are set out in Genbank Gene Accession No.
8771. In one example, DcR3 encompassed by the disclosure is human
DcR3 comprising a sequence set forth in SEQ ID NO: 130.
[0232] The term "isolated protein" or "isolated polypeptide" is
intended to mean a protein or polypeptide that by virtue of its
origin or source of derivation is not associated with
naturally-associated components that accompany it in its native
state; is substantially free of other proteins from the same
source. A protein may be rendered substantially free of naturally
associated components or substantially purified by isolation, using
protein purification techniques known in the art. By "substantially
purified" is meant the protein is substantially free of
contaminating agents, e.g., at least about 70% or 75% or 80% or 85%
or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating
agents.
[0233] The term "recombinant" shall be understood to mean the
product of artificial genetic recombination. Accordingly, in the
context of a recombinant protein comprising an antigen binding
domain, this term does not encompass an antibody
naturally-occurring within a subject's body that is the product of
natural recombination that occurs during B cell maturation.
However, if such an antibody is isolated, it is to be considered an
isolated protein comprising an antigen binding domain. Similarly,
if nucleic acid encoding the protein is isolated and expressed
using recombinant means, the resulting protein is a recombinant
protein comprising an antibody antigen binding domain. A
recombinant protein also encompasses a protein expressed by
artificial recombinant means when it is within a cell, tissue or
subject, e.g., in which it is expressed.
[0234] The term "TL1a-binding protein" shall be taken to include a
single polypeptide chain (i.e., a series of contiguous amino acids
linked by peptide bonds), or a series of polypeptide chains
covalently or non-covalently linked to one another (i.e., a
polypeptide complex) capable of binding to TL1a in the manner
described and/or claimed herein. For example, the series of
polypeptide chains can be covalently linked using a suitable
chemical or a disulphide bond. Examples of non-covalent bonds
include hydrogen bonds, ionic bonds, Van der Waals forces, and
hydrophobic interactions.
[0235] The term "polypeptide" or "polypeptide chain" will be
understood from the foregoing paragraph to mean a series of
contiguous amino acids linked by peptide bonds.
[0236] As used herein, the term "antigen binding domain" shall be
taken to mean a region of an antibody that is capable of
specifically binding to an antigen, i.e., a V.sub.H or a V.sub.L or
an Fv comprising both a V.sub.H and a V.sub.L. The antigen binding
domain need not be in the context of an entire antibody, e.g., it
can be in isolation (e.g., a domain antibody) or in another form,
e.g., as described herein, such as a scFv.
[0237] For the purposes for the disclosure, the term "antibody"
includes a protein capable of specifically binding to one or a few
closely related antigens (e.g., TL1a) by virtue of an antigen
binding domain contained within a Fv. This term includes four chain
antibodies (e.g., two light chains and two heavy chains),
recombinant or modified antibodies (e.g., chimeric antibodies,
humanized antibodies, human antibodies, CDR-grafted antibodies,
primatized antibodies, de-immunized antibodies, synhumanized
antibodies, half-antibodies, bispecific antibodies). An antibody
generally comprises constant domains, which can be arranged into a
constant region or constant fragment or fragment crystallizable
(Fc). Exemplary forms of antibodies comprise a four-chain structure
as their basic unit. Full-length antibodies comprise two heavy
chains (.sup..about.50 to 70 kD) covalently linked and two light
chains (.sup..about.23 kDa each). A light chain generally comprises
a variable region (if present) and a constant domain and in mammals
is either a K light chain or a .lamda. light chain. A heavy chain
generally comprises a variable region and one or two constant
domain(s) linked by a hinge region to additional constant
domain(s). Heavy chains of mammals are of one of the following
types .alpha., .delta., .epsilon., .gamma., or .mu.. Each light
chain is also covalently linked to one of the heavy chains. For
example, the two heavy chains and the heavy and light chains are
held together by inter-chain disulfide bonds and by non-covalent
interactions. The number of inter-chain disulfide bonds can vary
among different types of antibodies. Each chain has an N-terminal
variable region (V.sub.H or V.sub.L wherein each are
.sup..about.110 amino acids in length) and one or more constant
domains at the C-terminus. The constant domain of the light chain
(C.sub.L which is .sup..about.110 amino acids in length) is aligned
with and disulfide bonded to the first constant domain of the heavy
chain (C.sub.H1 which is 330 to 440 amino acids in length). The
light chain variable region is aligned with the variable region of
the heavy chain. The antibody heavy chain can comprise 2 or more
additional C.sub.H domains (such as, C.sub.H2, C.sub.H3 and the
like) and can comprise a hinge region between the C.sub.H1 and
C.sub.H2 constant domains. Antibodies can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2) or
subclass. In one example, the antibody is a murine (mouse or rat)
antibody or a primate (such as, human) antibody. In one example the
antibody heavy chain is missing a C-terminal lysine residue. In one
example, the antibody is humanized, synhumanized, chimeric,
CDR-grafted or deimmunized.
[0238] As used herein, "variable region" refers to the portions of
the light and/or heavy chains of an antibody as defined herein that
is capable of specifically binding to an antigen and, includes
amino acid sequences of complementarity determining regions (CDRs);
i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For
example, the variable region comprises three or four FRs (e.g.,
FR1, FR2, FR3 and optionally FR4) together with three CDRs. V.sub.H
refers to the variable region of the heavy chain. V.sub.L refers to
the variable region of the light chain.
[0239] As used herein, the term "complementarity determining
regions" (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the
amino acid residues of an antibody variable region the presence of
which are major contributors to specific antigen binding. Each
variable region domain (V.sub.H or V.sub.L) typically has three CDR
regions identified as CDR1, CDR2 and CDR3. In one example, the
amino acid positions assigned to CDRs and FRs are defined according
to Kabat Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred
to herein as "the Kabat numbering system"). In another example, the
amino acid positions assigned to CDRs and FRs are defined according
to the Enhanced Chothia Numbering Scheme
(http://www.bioinfo.org.uk/mdex.html). According to the numbering
system of Kabat, V.sub.H FRs and CDRs are positioned as follows:
residues 1 to 30 (FR1), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65
(CDR2), 66 to 94 (FR3), 95 to 102 (CDR3) and 103 to 113 (FR4).
According to the numbering system of Kabat, V.sub.L FRs and CDRs
are positioned as follows: residues 1 to 23 (FR1), 24 to 34 (CDR1),
35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3)
and 98 to 107 (FR4). The disclosure is not limited to FRs and CDRs
as defined by the Kabat numbering system, but includes all
numbering systems, including the canonical numbering system or of
Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et al.,
Nature 342: 877-883, 1989; and/or Al-Lazikani et al., J. Mol. Biol.
273: 927-948, 1997; the numbering system of Honnegher and Plukthun
J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in
Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997. In one
example, the CDRs are defined according to the Kabat numbering
system. Optionally, heavy chain CDR2 according to the Kabat
numbering system does not comprise the five C-terminal amino acids
listed herein or any one or more of those amino acids are
substituted with another naturally-occurring amino acid. In an
additional, or alternative, option, light chain CDR1 does not
comprise the four N-terminal amino acids listed herein or any one
or more of those amino acids are substituted with another
naturally-occurring amino acid. In this regard, Padlan et al.,
FASEB J., 9: 133-139, 1995 established that the five C-terminal
amino acids of heavy chain CDR2 and/or the four N-terminal amino
acids of light chain CDR1 are not generally involved in antigen
binding.
[0240] "Framework regions" (FRs) are those variable region residues
other than the CDR residues.
[0241] As used herein, the term "Fv" shall be taken to mean any
protein, whether comprised of multiple polypeptides or a single
polypeptide, in which a V.sub.L and a V.sub.H associate and form a
complex having an antigen binding domain, i.e., capable of
specifically binding to an antigen. The V.sub.H and the V.sub.L
which form the antigen binding domain can be in a single
polypeptide chain or in different polypeptide chains. Furthermore,
an Fv of the disclosure (as well as any protein of the disclosure)
may have multiple antigen binding domains which may or may not bind
the same antigen. This term shall be understood to encompass
fragments directly derived from an antibody as well as proteins
corresponding to such a fragment produced using recombinant means.
In some examples, the V.sub.H is not linked to a heavy chain
constant domain (C.sub.H) 1 and/or the V.sub.L is not linked to a
light chain constant domain (C.sub.L). Exemplary Fv containing
polypeptides or proteins include a Fab fragment, a Fab' fragment, a
F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or
higher order complex, or any of the foregoing linked to a constant
region or domain thereof, e.g., C.sub.H2 or C.sub.H3 domain, e.g.,
a minibody. A "Fab fragment" consists of a monovalent
antigen-binding fragment of an antibody, and can be produced by
digestion of a whole antibody with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain or can be produced using recombinant means. A "Fab'
fragment" of an antibody can be obtained by treating a whole
antibody with pepsin, followed by reduction, to yield a molecule
consisting of an intact light chain and a portion of a heavy chain
comprising a V.sub.H and a single constant domain. Two Fab'
fragments are obtained per antibody treated in this manner. A Fab'
fragment can also be produced by recombinant means. A "F(ab')2
fragment" of an antibody consists of a dimer of two Fab' fragments
held together by two disulfide bonds, and is obtained by treating a
whole antibody molecule with the enzyme pepsin, without subsequent
reduction. A "Fab.sub.2" fragment is a recombinant fragment
comprising two Fab fragments linked using, for example a leucine
zipper or a C.sub.H3 domain. A "single chain Fv" or "scFv" is a
recombinant molecule containing the variable region fragment (Fv)
of an antibody in which the variable region of the light chain and
the variable region of the heavy chain are covalently linked by a
suitable, flexible polypeptide linker.
[0242] As used herein, the term "binds" in reference to the
interaction of a TL1a-binding protein or an antigen binding domain
thereof with an antigen means that the interaction is dependent
upon the presence of a particular structure (e.g., an antigenic
determinant or epitope) on the antigen. For example, an antibody
recognizes and binds to a specific protein structure rather than to
proteins generally. If an antibody binds to epitope "A", the
presence of a molecule containing epitope "A" (or free, unlabeled
"A"), in a reaction containing labeled "A" and the antibody, will
reduce the amount of labeled "A" bound to the antibody.
[0243] As used herein, the term "specifically binds" or "binds
specifically" shall be taken to mean that a protein of the
disclosure reacts or associates more frequently, more rapidly, with
greater duration and/or with greater affinity with a particular
antigen or cell expressing same than it does with alternative
antigens or cells. For example, a protein that specifically binds
to an antigen binds that antigen with greater affinity, avidity,
more readily, and/or with greater duration than it binds to other
antigens. For example, a protein binds to TL1a (e.g., human TL1a)
with materially greater affinity than it does to other TNF
superfamily ligands or to antigens commonly recognized by
polyreactive natural antibodies (i.e., by naturally occurring
antibodies known to bind a variety of antigens naturally found in
humans). It is also understood by reading this definition that, for
example, a protein that specifically binds to a first antigen may
or may not specifically bind to a second antigen. As such,
"specific binding" does not necessarily require exclusive binding
or non-detectable binding of another antigen, this is meant by the
term "selective binding". In one example, "specific binding" of a
TL1a-binding protein of the disclosure to an antigen, means that
the protein binds to the antigen with an equilibrium constant
(K.sub.D) of 100 nM or less, such as 50 nM or less, for example 20
nM or less, such as, 15 nM or less or 10 nM or less or 5 nM or
less.
[0244] As used herein, the term "does not detectably bind" shall be
understood to mean that a TL1a-binding protein, e.g., an antibody,
binds to a candidate antigen at a level less than 20%, or 10% or 6%
or 5% above background. The background can be the level of binding
signal detected in the absence of the TL1a-binding protein and/or
in the presence of a negative control protein (e.g., an isotype
control antibody) and/or the level of binding detected in the
presence of a negative control antigen. The level of binding is
detected, for example, using ELISA in which the antigen is
immobilized and contacted with a TL1a-binding protein.
[0245] As used herein, the term "epitope" (syn. "antigenic
determinant") shall be understood to mean a region of TL1a to which
a protein comprising an antigen binding domain of an antibody
binds. This term is not necessarily limited to the specific
residues or structure to which the protein makes contact. For
example, this term includes the region spanning amino acids
contacted by the protein and/or at least 5 to 10 or 2 to 5 or 1 to
3 amino acids outside of this region. In some examples, the epitope
is a linear series amino acids. An epitope may also comprise a
series of discontinuous amino acids that are positioned close to
one another when TL1a is folded, i.e., a "conformational epitope".
The skilled artisan will also be aware that the term "epitope" is
not limited to peptides or polypeptides. For example, the term
"epitope" includes chemically active surface groupings of molecules
such as sugar side chains, phosphoryl side chains, or sulfonyl side
chains, and, in certain examples, may have specific three
dimensional structural characteristics, and/or specific charge
characteristics. An epitope or peptide or polypeptide comprising
same can be administered to an animal to generate antibodies
against the epitope.
[0246] As used herein, the term "inhibits interaction of TL1a and
DR3" shall be understood to mean that in an assay to measure
binding of TL1a and DR3, a protein is capable of inhibiting 50% of
binding (i.e., has an EC.sub.50) of less than about 13 nM, for
example, less than about 10 nM, such as less than about 7 nM, e.g.,
less than about 5 nM, for example, less than about 3 nM.
[0247] As used herein, the term "does not inhibit interaction of
TL1a and DcR3" will be understood to mean that a TL1a-binding
protein described herein does not inhibit interaction of TL1a and
DcR3 (i.e., such that interaction is no longer detectable, e.g.,
using an ELISA assay described herein). For example, at a
concentration of 100 .mu.g/ml, the protein does not completely
inhibit interaction of TL1a and DcR3. In some examples, the protein
reduces the interaction of TL1a and DcR3 by less than about 20% or
15% or 10%, e.g., when tested at a concentration of 10 .mu.g/ml or
100 .mu.g/ml.
[0248] As used herein, the term "does not detectably reduce" shall
be understood to mean that a protein as described herein reduces
binding of TL1a (or a biotinylated form thereof) to DcR3 by no more
than 20% or 8% or 6% or 5% or 4% or 3% or 2% above the level of
interaction in the absence of the protein or above the level of
background interaction, when tested at a concentration of 10
.mu.g/ml or 100 .mu.g/ml. The background can be the level of
binding signal detected in the absence of the protein and/or in the
presence of a negative control protein (e.g., an isotype control
antibody).
[0249] As used herein, the term "neutralize" shall be taken to mean
that a TL1a-binding protein is capable of reducing or preventing
TL1a-mediated activity in a cell. Methods for determining
neutralization are known in the art and/or described herein. For
example, TF-1 cells are contacted with TL1a, such as human TL1a
(e.g., expressed by a mammalian cell) and cycloheximide in the
presence or absence of the TL1a-binding protein. A TL1a-binding
protein that reduces the level of apoptosis of the cells compared
to the level in the absence of the protein is considered to
"neutralize" TL1a activity.
[0250] As used herein, the term "condition" refers to a disruption
of or interference with normal function, and is not to be limited
to any specific condition, and will include diseases or
disorders.
[0251] As used herein, a "TL1a-associated condition" refers to any
condition that is caused by or associated with TL1a or a cell
expressing TL1a. The skilled artisan will be readily able to
determine such conditions based on the disclosure herein and/or by
performing an assay to diagnose a TL1a-associated condition as
described herein. In this regard, in some examples the condition is
an inflammatory condition, an autoimmune condition and a condition
that can be treated by enhancing angiogenesis. A description of
exemplary conditions is included herein.
[0252] As used herein, the terms "preventing", "prevent" or
"prevention" include administering a protein of the disclosure to
thereby stop or hinder the development of at least one symptom of a
condition. This term also encompasses treatment of a subject in
remission to prevent or hinder relapse. For example, a subject
suffering from relapsing-remitting multiple sclerosis is treated
during remission to thereby prevent a relapse.
[0253] As used herein, the terms "treating", "treat" or "treatment"
include administering a protein described herein to thereby reduce
or eliminate at least one symptom of a specified disease or
condition.
[0254] As used herein, the term "subject" shall be taken to mean
any animal, such as, a mammal. In one example, the mammal is a
human or non-human primate. In one example, the mammal is a
human.
[0255] Reference herein to a "sample" should be understood as a
reference to any sample derived from a subject such as, but not
limited to, a body fluid (e.g., blood or blood fraction such as
serum or plasma, tears, urine, synovial fluid or cerebrospinal
fluid), cellular material (e.g. tissue aspirate), tissue biopsy
specimens or surgical specimens. In some examples, the "sample" is
any one or more of serum, plasma, PBMCs, or a buffy coat
fraction.
[0256] As used herein, the term "diagnosis", and variants thereof
such as, but not limited to, "diagnose", "diagnosed" or
"diagnosing" includes any primary diagnosis of a clinical state or
diagnosis of recurrent disease.
[0257] "Prognosis", "prognosing" and variants thereof as used
herein refer to the likely outcome or course of a disease,
including the chance of recovery or recurrence or the outcome of
treatment.
[0258] The term "expression construct" is to be taken in its
broadest context and includes a nucleic acid comprising one or more
promoter sequences operably linked with one or more nucleic acids
as described herein.
[0259] The term "expression vector" refers to a nucleic acid
comprising an expression construct that is additionally capable of
maintaining and or replicating nucleic acid in an expressible
format. For example, an expression vector may comprise a plasmid,
bacteriophage, phagemid, cosmid, virus sub-genomic or genomic
fragment. Selection of appropriate vectors is within the knowledge
of those having skill in the art.
[0260] As used herein, the term "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a genomic gene, including the TATA box or initiator
element, which is required for accurate transcription initiation,
with or without additional regulatory elements (e.g., upstream
activating sequences, transcription factor binding sites, enhancers
and silencers) that alter expression of a nucleic acid, e.g., in
response to a developmental and/or external stimulus, or in a
tissue specific manner. In the present context, the term "promoter"
is also used to describe a recombinant, synthetic or fusion nucleic
acid, or derivative which confers, activates or enhances the
expression of a nucleic acid to which it is operably linked. An
exemplary promoter can contain additional copies of one or more
specific regulatory elements to further enhance expression and/or
alter the spatial expression and/or temporal expression of said
nucleic acid.
[0261] As used herein, the term "operably linked to" means
positioning a promoter relative to a nucleic acid such that
expression of the nucleic acid is controlled by the promoter. A
promoter can be operably linked to numerous nucleic acids, e.g.,
through an internal ribosome entry site.
Antibodies
Immunization-Based Methods
[0262] To generate antibodies, TL1a or an epitope bearing fragment
or portion thereof or a modified form thereof (e.g., a fusion
protein comprising a human epitope within a mouse TL1a protein) or
nucleic acid encoding same, optionally formulated with any suitable
or desired adjuvant and/or pharmaceutically acceptable carrier, is
administered to a subject (for example, a non-human animal subject,
such as, a mouse, a rat, a chicken etc.) in the form of an
injectable composition. Exemplary non-human animals are mammals,
such as murine animals (e.g., rats or mice). Injection may be
intranasal, intramuscular, sub-cutaneous, intravenous, intradermal,
intraperitoneal, or by other known route. Optionally, the TL1a or
epitope bearing fragment or portion thereof or a nucleic acid
encoding same is administered numerous times. Means for preparing
and characterizing antibodies are known in the art (See, e.g.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988).
[0263] The production of polyclonal antibodies may be monitored by
sampling blood of the immunized animal at various points following
immunization. A second, booster injection, may be given, if
required to achieve a desired antibody titer. The process of
boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the
immunized animal is bled and the serum isolated and stored, and/or
the animal is used to generate monoclonal antibodies (mAbs).
[0264] Monoclonal antibodies are exemplary antibodies contemplated
by the disclosure. Generally, production of monoclonal antibodies
involves, immunizing a subject (e.g., a rodent, e.g., mouse or rat)
with TL1a or an epitope bearing fragment or portion thereof or a
nucleic acid encoding same under conditions sufficient to stimulate
antibody producing cells. In some examples, a mouse
genetically-engineered to express human immunoglobulin proteins and
not express murine immunoglobulin proteins, is immunized to produce
an antibody (e.g., as described in PCT/US2007/008231 and/or Lonberg
et al., Nature 368: 856-859, 1994). Following immunization,
antibody producing somatic cells (e.g., B lymphocytes) are fused
with immortal cells, e.g., immortal myeloma cells. Various methods
for producing such fused cells (hybridomas) are known in the art
and described, for example, in Kohler and Milstein, Nature 256:
495-497, 1975. The hybridoma cells can then be cultured under
conditions sufficient for antibody production.
[0265] The disclosure contemplates other methods for producing
antibodies, e.g., ABL-MYC technology (as described, for example in
Largaespada et al., Curr. Top. Microbiol. Immunol, 166: 91-96,
1990). Suitable antibodies are then selected based on methods
described herein.
Library-Based Methods
[0266] The disclosure also encompasses screening of libraries of
antibodies or proteins comprising antigen binding domains thereof
(e.g., comprising variable regions thereof) to identify a
TL1a-binding protein of the disclosure.
[0267] Examples of libraries contemplated by this disclosure
include naive libraries (from unchallenged subjects), immunized
libraries (from subjects immunized with an antigen) or synthetic
libraries. Nucleic acid encoding antibodies or regions thereof
(e.g., variable regions) are cloned by conventional techniques
(e.g., as disclosed in Sambrook and Russell, eds, Molecular
Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor
Laboratory Press, 2001) and used to encode and display proteins
using a method known in the art. Other techniques for producing
libraries of proteins are described in, for example in U.S. Pat.
No. 6,300,064 (e.g., a HuCAL library of Morphosys AG); U.S. Pat.
No. 5,885,793; U.S. Pat. No. 6,204,023; U.S. Pat. No. 6,291,158; or
U.S. Pat. No. 6,248,516.
[0268] The TL1a-binding proteins according to the disclosure may be
soluble secreted proteins or may be presented as a fusion protein
on the surface of a cell, or particle (e.g., a phage or other
virus, a ribosome or a spore). Various display library formats are
known in the art. For example, the library is an in vitro display
library (e.g., a ribosome display library, a covalent display
library or a mRNA display library, e.g., as described in U.S. Pat.
No. 7,270,969). In yet another example, the display library is a
phage display library wherein proteins comprising antigen binding
domains of antibodies are expressed on phage, e.g., as described in
U.S. Pat. No. 6,300,064; U.S. Pat. No. 5,885,793; U.S. Pat. No.
6,204,023; U.S. Pat. No. 6,291,158; or U.S. Pat. No. 6,248,516.
Other phage display methods are known in the art and are
contemplated by the disclosure. Similarly, methods of cell display
are contemplated by the disclosure, e.g., bacterial display
libraries, e.g., as described in U.S. Pat. No. 5,516,637; yeast
display libraries, e.g., as described in U.S. Pat. No. 6,423,538 or
a mammalian display library.
[0269] Methods for screening display libraries are known in the
art. In one example, a display library of the disclosure is
screened using affinity purification, e.g., as described in Scopes
(In: Protein purification: principles and practice, Third Edition,
Springer Verlag, 1994). Methods of affinity purification typically
involve contacting proteins comprising antigen binding domains
displayed by the library with a target antigen (e.g., TL1a) and,
following washing, eluting those domains that remain bound to the
antigen.
[0270] Any variable regions or scFvs identified by screening are
readily modified into a complete antibody, if desired. Exemplary
methods for modifying or reformatting variable regions or scFvs
into a complete antibody are described, for example, in Jones et
al., J. Immunol. Methods 354: 85-90, 2010; or Jostock et al., J.
Immunol. Methods, 289: 65-80, 2004. Alternatively, or additionally,
standard cloning methods are used, e.g., as described in Ausubel et
al., (In: Current Protocols in Molecular Biology. Wiley
Interscience, ISBN 047 150338, 1987), and/or (Sambrook et al., (In:
Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratories, New York, Third Edition 2001).
Deimmunized, Chimeric, CDR Grafted, Humanized, Synhumanized,
Primatized, Human and Composite TL1a-Binding Proteins
[0271] The TL1a-binding proteins of the disclosure may be CDR
grafted proteins which include CDRs from an antibody from a
non-human species (e.g., mouse or rat or non-human primate) grafted
onto or inserted into FRs from a human antibody or which include
CDRs from an antibody from one type of antibody (e.g., one type of
human antibody) grafted onto or inserted into FRs from another type
of antibody (e.g., another type of human antibody). This term also
encompasses a composite protein comprising, for example, one or
more CDR grafted variable regions and one or more, e.g., human
variable regions, chimeric variable regions, synhumanized variable
regions or primatized variable regions. Such proteins are
exemplified herein by the antibodies designated C320-16 to
C320-33.
[0272] The TL1a-binding proteins of the disclosure may be a
humanized protein.
[0273] The term "humanized protein" shall be understood to refer to
a protein comprising a human-like variable region, which includes
CDRs from an antibody from a non-human species (e.g., mouse or rat
or non-human primate) grafted onto or inserted into FRs from a
human antibody (this type of antibody is also referred to as a
"CDR-grafted antibody"). Humanized proteins also include proteins
in which one or more residues of the human protein are modified by
one or more amino acid substitutions and/or one or more FR residues
of the human protein are replaced by corresponding non-human
residues. Humanized proteins may also comprise residues which are
found in neither the human antibody or in the non-human antibody.
Any additional regions of the protein (e.g., Fc region) are
generally human. Humanization can be performed using a method known
in the art, e.g., U.S. Pat. No. 5,225,539, U.S. Pat. No. 6,054,297,
U.S. Pat. No. 7,566,771 or U.S. Pat. No. 5,585,089. The term
"humanized protein" also encompasses a super-humanized protein,
e.g., as described in U.S. Pat. No. 7,732,578. This term also
encompasses a composite protein comprising, for example, one or
more humanized variable regions and one or more, e.g., human
variable regions, chimeric variable regions, synhumanized variable
regions or primatized variable regions.
[0274] In one example, a humanized TL1a-binding protein comprises
the regions between 27d and 34, 50 and 55, and 89 and 96 in a light
chain sequence disclosed herein; and 31 and 35b, 50 and 58, and 95
and 101 in a heavy chain sequence disclosed herein (numbering
according to the Kabat numbering system). In this regard, Padlan et
al., FASEB J., 9: 133-139, 1995 presents evidence that these
regions are those most likely to bind or contact antigen.
[0275] The TL1a-binding proteins of the disclosure may be human
proteins. The term "human protein" as used herein refers to
proteins having variable and, optionally, constant antibody regions
found in humans, e.g. in the human germline or somatic cells or
from libraries produced using such regions. The "human" antibodies
can include amino acid residues not encoded by human sequences,
e.g. mutations introduced by random or site directed mutations in
vitro (in particular mutations which involve conservative
substitutions or mutations in a small number of residues of the
protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein).
These "human antibodies" do not necessarily need to be generated as
a result of an immune response of a human, rather, they can be
generated using recombinant means (e.g., screening a phage display
library) and/or by a transgenic animal (e.g., a mouse) comprising
nucleic acid encoding human antibody constant and/or variable
regions and/or using guided selection (e.g., as described in U.S.
Pat. No. 5,565,332). This term also encompasses affinity matured
forms of such antibodies. For the purposes of the disclosure, a
human protein will also be considered to include a protein
comprising FRs from a human antibody or FRs comprising sequences
from a consensus sequence of human FRs and in which one or more of
the CDRs are random or semi-random, e.g., as described in U.S. Pat.
No. 6,300,064 and/or U.S. Pat. No. 6,248,516.
[0276] Exemplary human TL1a-binding proteins are antibodies
comprising the following pairs of variable regions:
(i) a V.sub.H comprising a sequence set forth in SEQ ID NO: 2 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 6; (ii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 10 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 14; (iii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 18 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 22; (iv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 26 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 30; (v) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 34 and a
V.sub.L comprising a sequence set forth in SEQ ID NO:38; (vi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (vii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 50 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 54; (viii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (ix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (x) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 66 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 70 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 74 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 78 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 82; (xv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 86 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 90 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xvii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 58 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 62; (xviii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 163; (xx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165; (xxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 166; (xxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 167; (xxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 154 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 155 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 156 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxx) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 157 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 158 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 159 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 160 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxiv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 161 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 234 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 46; (xxxvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 164; (xxxvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 165;
(xxxviii) a V.sub.H comprising a sequence set forth in SEQ ID NO:
42 and a V.sub.L comprising a sequence set forth in SEQ ID NO: 166;
(xxxix) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 167;
(xl) a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 168; (xli)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 169; (xlii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 170; (xliii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 171; (xliv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 42 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 172; (xlv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 175 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 188; (xlvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 176 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 189; (xlvii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 177 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 190; (xlviii)
a V.sub.H comprising a sequence set forth in SEQ ID NO: 178 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 191; (xlix) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 179 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 192; (l) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 180 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 193; (li) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 181 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 194; (lii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 182 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 195; (liii) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 183 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 196; (liv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 184 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 197; (lv) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 185 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 198; (lvi) a
V.sub.H comprising a sequence set forth in SEQ ID NO: 186 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 199; or
(lvii) a V.sub.H comprising a sequence set forth in SEQ ID NO: 187
and a V.sub.L comprising a sequence set forth in SEQ ID NO:
200.
[0277] Additional exemplary human TL1a-binding proteins are
antibodies comprising a V.sub.H comprising a sequence set forth in
SEQ ID NO: 42 and a V.sub.L comprising a sequence set forth in SEQ
ID NO: 46, wherein the V.sub.H and/or V.sub.L comprise one or more
of the following substitutions or groups of substitutions:
(i) the V.sub.H comprises an alanine at position 16 of SEQ ID NO:
42; (ii) the V.sub.H comprises an alanine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (iii) the V.sub.H comprises a serine at position 100 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (iv) the V.sub.H comprises a histidine at
position 100 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (v) the V.sub.H comprises a
leucine at position 100 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (vi) the V.sub.H
comprises an aspartic acid at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(vii) the V.sub.H comprises a tyrosine at position 100 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (viii) the V.sub.H comprises a proline at position 100
of SEQ ID NO: 42 and the V.sub.L comprises a (ix) the V.sub.H
comprises a glutamine at position 100 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46; (x)
the V.sub.H comprises a lysine at position 100 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xi) the V.sub.H comprises an alanine at position 101 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xii) the V.sub.H comprises a serine at position 101 of SEQ
ID NO: 42 and the V.sub.L comprises a threonine at position 76 of
SEQ ID NO: 46; (xiii) the V.sub.H comprises a histidine at position
101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xiv) the V.sub.H comprises a leucine
at position 101 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xv) the V.sub.H
comprises an aspartic acid at position 101 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xvi) the V.sub.H comprises a tyrosine at position 101 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xvii) the V.sub.H comprises a glutamine at position 101
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xviii) the V.sub.H comprises a lysine at
position 101 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xix) the V.sub.H comprises an
alanine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xx) the V.sub.H
comprises a serine at position 102 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xxi) the
V.sub.H comprises a histidine at position 102 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxii) the V.sub.H comprises a leucine at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a (xxiii) the V.sub.H comprises a
tyrosine at position 102 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxiv) the V.sub.H
comprises a proline at position 102 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxv) the V.sub.H comprises a glutamine at position 102 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxvi) the V.sub.H comprises a lysine at position 102 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxvii) the V.sub.H comprises an alanine at
position 103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xxviii) the V.sub.H comprises a
serine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (xxix) the V.sub.H
comprises a histidine at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxx) the V.sub.H comprises a leucine at position 103 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (xxxi) the V.sub.H comprises an aspartic acid at position
103 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (xxxii) the V.sub.H comprises a
tyrosine at position 103 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xxxiii) the V.sub.H
comprises a proline at position 103 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xxxiv) the V.sub.H comprises a glutamine at position 103 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xxxv) the V.sub.H comprises a lysine at position 103 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxxvi) the V.sub.H comprises a serine at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a (xxxvii)
the V.sub.H comprises a histidine at position 104 of SEQ ID NO: 42
and the V.sub.L comprises a threonine at position 76 of SEQ ID NO:
46; (xxxviii) the V.sub.H comprises a leucine at position 104 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xxxix) the V.sub.H comprises an aspartic acid at
position 104 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xl) the V.sub.H comprises a
tyrosine at position 104 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (xli) the V.sub.H
comprises a proline at position 104 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlii) the V.sub.H comprises a glutamine at position 104 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (xliii) the V.sub.H comprises a lysine at position 104
of SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
76 of SEQ ID NO: 46; (xliv) the V.sub.H comprises an alanine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlv) the V.sub.H comprises a
histidine at position 105 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (xlvi) the
V.sub.H comprises a leucine at position 105 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(xlvii) the V.sub.H comprises an aspartic acid at position 105 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (xlviii) the V.sub.H comprises a tyrosine at
position 105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine
at position 76 of SEQ ID NO: 46; (xlix) the V.sub.H comprises a
proline at position 105 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (l) the V.sub.H
comprises a glutamine at position 105 of SEQ ID NO: 42 and the
V.sub.L comprises a (li) the V.sub.H comprises a lysine at position
105 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lii) the V.sub.H comprises an
alanine at position 107 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (liii) the V.sub.H
comprises a serine at position 107 of SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (liv) the
V.sub.H comprises a histidine at position 107 of SEQ ID NO: 42 and
the V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lv) the V.sub.H comprises a leucine at position 107 of SEQ ID NO:
42 and the V.sub.L comprises a threonine at position 76 of SEQ ID
NO: 46; (lvi) the V.sub.H comprises an aspartic acid at position
107 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lvii) the V.sub.H comprises a
tyrosine at position 107 of SEQ ID NO: 42 and the V.sub.L comprises
a threonine at position 76 of SEQ ID NO: 46; (lviii) the V.sub.H
comprises a proline at position 107 of SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lix) the V.sub.H comprises a glutamine at position 107 of SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 76 of SEQ
ID NO: 46; (lx) the V.sub.H comprises a lysine at position 107 of
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 76
of SEQ ID NO: 46; (lxi) the V.sub.H comprises a threonine at
position 41 of SEQ ID NO: 42 and the V.sub.L comprises an alanine
at position 23 of SEQ ID NO: 46; (lxii) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
an aspartic acid at position 28 of SEQ ID NO: 46; (lxiii) the
V.sub.H comprises a threonine at position 41 of SEQ ID NO: 42 and
the V.sub.L comprises a tyrosine at position 33 of SEQ ID NO: 46;
(lxiv) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises an aspartic acid at position 34 of
SEQ ID NO: 46; (lxv) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises an asparagine at
position 53 of SEQ ID NO: 46; (lxvi) the V.sub.H comprises a
threonine at position 41 of SEQ ID NO: 42 and the V.sub.L comprises
a serine at position 54 of SEQ ID NO: 46; (lxvii) the V.sub.H
comprises a threonine at position 41 of SEQ ID NO: 42 and the
V.sub.L comprises an alanine at position 82 of SEQ ID NO: 46;
(lxviii) the V.sub.H comprises a threonine at position 41 of SEQ ID
NO: 42 and the V.sub.L comprises a serine at position 95 of SEQ ID
NO: 46; (lxix) the V.sub.H comprises a threonine at position 41 of
SEQ ID NO: 42 and the V.sub.L comprises a serine at position 96 of
SEQ ID NO: 46; (lxx) the V.sub.H comprises a threonine at position
41 of SEQ ID NO: 42 and the V.sub.L comprises a threonine at
position 76 of SEQ ID NO: 46; (lxxi) the V.sub.H comprises a serine
at position 47 of SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23 of SEQ ID NO: 46; (lxxii) the V.sub.H
comprises a proline at position 41, an alanine at position 72, an
aspartic acid at position 73, an arginine at position 74 and a
threonine at position 76 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 76 of SEQ ID NO: 46;
(lxxiii) the V.sub.H comprises a proline at position 41, a leucine
at position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a serine at position 24 and
a threonine at position 76 each relative to SEQ ID NO: 46; (lxxiv)
the V.sub.H comprises a proline at position 41, a leucine at
position 51 and a glutamic acid at position 102 each relative to
SEQ ID NO: 42 and the V.sub.L comprises a threonine at position 23,
a serine at position 24 and a threonine at position 76 each
relative to SEQ ID NO: 46; (lxxv) the V.sub.H comprises a proline
at position 41, a leucine at position 51 and a glutamic acid at
position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 23 and a threonine at position 76
each relative to SEQ ID NO: 46; (lxxvi) the V.sub.H comprises a
proline at position 41, a leucine at position 51 and a glutamic
acid at position 102 each relative to SEQ ID NO: 42 and the V.sub.L
comprises a threonine at position 76 of SEQ ID NO: 46; (lxxvii) the
V.sub.H comprises a proline at position 41, a leucine at position
51, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24 and a threonine
at position 76 each relative to SEQ ID NO: 46; (lxxviii) the
V.sub.H comprises a proline at position 41, a leucine at position
51, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 76 of SEQ ID NO: 46; (lxxix) the V.sub.H
comprises a proline at position 41, an alanine at position 72, an
aspartic acid at position 73, an arginine at position 74 and a
threonine at position 76 each relative to SEQ ID NO: 42 and the
V.sub.L comprises a threonine at position 23, a serine at position
24 and a threonine at position 76 each relative to SEQ ID NO: 46;
(lxxx) the V.sub.H comprises a proline at position 41, an alanine
at position 72, an aspartic acid at position 73, an arginine at
position 74 and a threonine at position 76 each relative to SEQ ID
NO: 42 and the V.sub.L comprises a threonine at position 23, a
serine at position 24, a threonine at position 76 and a glutamic
acid at position 51 each relative to SEQ ID NO: 46; (lxxxi) the
V.sub.H comprises a proline at position 41, a leucine at position
51, an alanine at position 72, an aspartic acid at position 73, an
arginine at position 74, a threonine at position 76, a glutamic
acid at position 102 and an alanine at position 105 each relative
to SEQ ID NO: 42 and the V.sub.L comprises a threonine at position
23, a serine at position 24, a threonine at position 76 and a
glutamic acid at position 51 each relative to SEQ ID NO: 46; and
(i) the V.sub.H comprises a proline at position 41, a leucine at
position 51, an alanine at position 72, an aspartic acid at
position 73, an arginine at position 74, a threonine at position
76, a glutamic acid at position 102 and an alanine at position 105
each relative to SEQ ID NO: 42 and the V.sub.L comprises a
threonine at position 23, a serine at position 24, a threonine at
position 76 and a glycine at position 51 each relative to SEQ ID
NO: 46.
[0278] Optionally, the V.sub.H is linked to a heavy chain constant
region, e.g., an IgG1 heavy chain constant region. In one example,
the heavy chain constant region lacks the c-terminal lysine
residue.
[0279] Optionally, the V.sub.L is linked to a light chain constant
region.
[0280] The TL1a-binding proteins of the disclosure may be
synhumanized proteins. The term "synhumanized protein" refers to a
protein prepared by a method described in WO2007/019620. A
synhumanized TL1a-binding protein includes a variable region of an
antibody, wherein the variable region comprises FRs from a New
World primate antibody variable region and CDRs from a non-New
World primate antibody variable region. For example, a synhumanized
TL1a-binding protein includes a variable region of an antibody,
wherein the variable region comprises FRs from a New World primate
antibody variable region and CDRs from a mouse or rat antibody. In
one example, the synhumanized TL1a-binding protein is a
TL1a-binding antibody in which one or both of the variable regions
are synhumanized. This term also encompasses a composite protein
comprising, for example, one or more synhumanized variable regions
and one or more, e.g., human variable regions or humanized variable
regions or chimeric variable regions.
[0281] The TL1a-binding proteins of the disclosure may be
primatized proteins. A "primatized protein" comprises variable
region(s) from an antibody generated following immunization of a
non-human primate (e.g., a cynomolgus macaque). Optionally, the
variable regions of the non-human primate antibody are linked to
human constant regions to produce a primatized antibody. Exemplary
methods for producing primatized antibodies are described in U.S.
Pat. No. 6,113,898. This term also encompasses a composite protein
comprising, for example, one or more primatized variable regions
and one or more, e.g., human variable regions or humanized variable
regions or chimeric variable regions.
[0282] In one example a TL1a-binding protein of the disclosure is a
chimeric protein. The term "chimeric proteins" refers to proteins
in which an antigen binding domain is from a particular species
(e.g., murine, such as mouse or rat) or belonging to a particular
antibody class or subclass, while the remainder of the protein is
from a protein derived from another species (such as, for example,
human or non-human primate) or belonging to another antibody class
or subclass. In one example, a chimeric protein is a chimeric
antibody comprising a V.sub.H and/or a V.sub.L from a non-human
antibody (e.g., a murine antibody) and the remaining regions of the
antibody are from a human antibody. The production of such chimeric
proteins is known in the art, and may be achieved by standard means
(as described, e.g., in U.S. Pat. No. 6,331,415; U.S. Pat. No.
5,807,715; U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397).
This term also encompasses a composite protein comprising, for
example, one or more chimeric variable regions and one or more,
e.g., human variable regions or humanized variable regions or
chimeric variable regions.
[0283] The disclosure also contemplates a deimmunized TL1a-binding
protein, e.g., as described in WO2000/34317 and WO2004/108158.
De-immunized antibodies and proteins have one or more epitopes,
e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to
thereby reduce the likelihood that a subject will raise an immune
response against the antibody or protein. For example, a
TL1a-binding protein of the disclosure is analyzed to identify one
or more B or T cell epitopes and one or more amino acid residues
within the epitope is mutated to thereby reduce the immunogenicity
of the TL1a-binding protein. The present inventors have used such
techniques to identify epitopes that are predicted to bind to MHC
Class II molecules and identify TL1a binding proteins less likely
to induce an immune response in a subject.
[0284] It will be apparent to the skilled artisan from the
foregoing disclosure that a "composite" protein comprises one form
of V.sub.H (e.g., human) and another form of V.sub.L (e.g.,
humanized). The disclosure explicitly encompasses all combinations
of forms of V.sub.H and V.sub.L.
Other TL1a-Binding Proteins Comprising an Antigen Binding
Domain
[0285] The disclosure also contemplates other TL1a-binding proteins
comprising a variable region or antigen binding domain of an
antibody, such as:
(i) a single-domain antibody, which is a single polypeptide chain
comprising all or a portion of the V.sub.H or a V.sub.L of an
antibody (see, e.g., U.S. Pat. No. 6,248,516); (ii) diabodies,
triabodies and tetrabodies, e.g., as described in U.S. Pat. No.
5,844,094 and/or US2008152586; (iii) scFvs, e.g., as described in
U.S. Pat. No. 5,260,203; (iv) minibodies, e.g., as described in
U.S. Pat. No. 5,837,821; (v) "key and hole" bispecific proteins as
described in U.S. Pat. No. 5,731,168; (vi) heteroconjugate
proteins, e.g., as described in U.S. Pat. No. 4,676,980; (vii)
heteroconjugate proteins produced using a chemical cross-linker,
e.g., as described in U.S. Pat. No. 4,676,980; (viii) Fab'-SH
fragments, e.g., as described in Shalaby et al., J. Exp. Med., 175:
217-225, 1992; or (ix) Fab.sub.3 (e.g., as described in
EP19930302894).
Constant Domain Fusions
[0286] The disclosure encompasses a TL1a-binding protein comprising
an antigen binding domain of an antibody and a constant region or
Fc or a domain thereof, e.g., C.sub.H2 and/or C.sub.H3 domain.
Suitable constant regions and/or domains will be apparent to the
skilled artisan and/or the sequences of such polypeptides are
readily available from publicly available databases. Kabat et al
also provide description of some suitable constant
regions/domains.
[0287] Constant regions and/or domains thereof are useful for
providing biological activities such as, dimerization, extended
serum half life (e.g., by binding to FcRn), antibody-dependent cell
cytotoxicity (ADCC), complement dependent cytotoxicity (CDC),
antibody-dependent cell phagocytosis (ADCP).
[0288] The disclosure also contemplates TL1a-binding proteins
comprising mutant constant regions or domains, e.g., as described
in U.S. Pat. No. 7,217,797; U.S. Pat. No. 7,217,798; or
US20090041770 (having increased half-life) or US2005037000
(increased ADCC).
[0289] The C-terminal lysine of the heavy chain constant region of
an antibody of the disclosure or TL1a-binding protein of the
disclosure comprising a constant region or Fc may be removed, for
example, during production or purification of the antibody, or by
recombinantly engineering the nucleic acid encoding a heavy chain
of the antibody or protein. Accordingly, whole antibodies or
proteins may comprise antibody or protein populations with all
C-terminal lysine residues removed, antibody or protein populations
with no C-terminal lysine residues removed, or antibody or protein
populations having a mixture of antibodies with and without the
C-terminal lysine residue. In some examples, the antibody or
protein populations may additionally comprise antibodies or
proteins in which the C-terminal lysine residue is removed in one
of the heavy chain constant regions. Similarly, a composition of
antibodies or proteins may comprise the same or a similar mix of
antibody or protein populations with or without the C-terminal
lysine residue.
Enhancing Effector Function
[0290] In one example, a TL1a-binding protein of the disclosure may
induce effector function or enhanced effector function.
[0291] In the context of the disclosure, "effector functions" refer
to those biological activities mediated by cells or proteins that
bind to the Fc region (a native sequence Fc region or amino acid
sequence variant Fc region) of an antibody that result in killing
of a cell. Examples of effector functions induced by antibodies
include: complement dependent cytotoxicity;
antibody-dependent-cell-mediated cytotoxicity (ADCC);
antibody-dependent-cell-phagocytosis (ADCP); and B-cell
activation.
[0292] In one example, a TL1a-binding protein of the disclosure
binds to TL1a on the surface of a cell in such a manner that it is
capable of inducing an effector function, such as, ADCC or CDC.
[0293] For example, the TL1a-binding protein remains bound to the
TL1a on the surface of the cell for a time sufficient to induce an
effector function, such as ADCC and/or CDC.
[0294] In one example, a TL1a-binding protein of the disclosure is
capable of inducing enhanced effector function, e.g., by virtue of
a modified Fc region or by virtue of comprising a region capable of
binding to an immune effector cell. For example, the level of
effector function is increased compared to the level induced by a
human IgG1 or IgG3 Fc region. Enhancing effector function induced
by a TL1a-binding protein of the disclosure may result in enhanced
therapeutic or prophylactic effects, e.g., by not only blocking the
action of TL1a but also by killing or depleting cells causing a
condition, e.g., by killing auto-reactive T cells.
[0295] In one example, the Fc region of a TL1a-binding protein of
the disclosure is modified to increase the level of effector
function it is capable of inducing compared to the Fc region
without the modification. Such modifications can be at the amino
acid level and/or the secondary structural level and/or the
tertiary structural level and/or to the glycosylation of the Fc
region.
[0296] The skilled addressee will appreciate that greater effector
function may be manifested in any of a number of ways, for example
as a greater level of effect, a more sustained effect or a faster
rate of effect.
[0297] In one example, the Fc region comprises one or more amino
acid modifications that increase its ability to induce enhanced
effector function. In one example, the Fc region binds with greater
affinity to one or more Fc.gamma.Rs, such as Fc.gamma.RIII. In one
example, the Fc region comprise at least one amino acid
substitution at a position selected from the group consisting of:
230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276,
278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, numbered
according to the EU index of Kabat. In one example, the Fc region
comprises the following amino acid substitutions S239D/I332E,
numbered according to the EU index of Kabat. This Fc region has
about 14 fold increase in affinity for Fc.gamma.RIIIa compared to a
wild-type Fc region and about 3.3 increased ability to induce ADCC
compared to a wild-type Fc region. In one example, the Fc region
comprises the following amino acid substitutions S239D/A330L/1332E,
numbered according to the EU index of Kabat. This Fc region has
about 138 fold increase in affinity for Fc.gamma.RIIIa compared to
a wild-type Fc region and about 323 fold increased ability to
induce ADCC compared to a wild-type Fc region.
[0298] Additional amino acid substitutions that increase ability of
a Fc region to induce effector function are known in the art and/or
described, for example, in U.S. Pat. No. 6,737,056 or U.S. Pat. No.
7,317,091.
[0299] In one example, the glycosylation of the Fc region is
altered to increase its ability to induce enhanced effector
function. In this regard, native antibodies produced by mammalian
cells typically comprise a branched, biantennary oligosaccharide
that is generally attached by an N-linkage to Asn297 of the
C.sub.H2 domain of the Fc region. The oligosaccharide may include
various carbohydrates, e.g., mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached
to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some examples, Fc regions according to the disclosure
comprise a carbohydrate structure that lacks fucose attached
(directly or indirectly) to an Fc region, i.e., the Fc region is
"afucosylated". Such variants may have an improved ability to
induce ADCC. Methods for producing afucosylated antibodies include,
expressing the antibody or antigen binding fragment thereof in a
cell line incapable of expressing .alpha.-1,6-fucosyltransferase
(FUT8) (e.g., as described in Yumane-Ohnuki et al., Biotechnol.
Bioengineer. 87: 614-622, 2004), expressing the antibody or antigen
binding fragment thereof in cells expressing a small interfering
RNA against FUT8 (e.g., as described in Mori et al., Biotechnol.
Bioengineer., 88: 901-908, 2004), expressing the antibody or
antigen binding fragment thereof in cells incapable of expressing
guanosine diphosphate (GDP)-mannose 4,6-dehydratase (GMD) (e.g., as
described in Kanda et al., J. Biotechnol., 130: 300-310, 2007). The
disclosure also contemplates the use of proteins having a reduced
level of fucosylation, e.g., produced using a cell line modified to
express .beta.-(1,4)-N-acetylglucosaminyltransferase III (GnT-III)
(e.g., as described in Um na et al., Nat. Biotechnol. 17: 176-180,
1999).
[0300] Other methods include the use of cell lines which inherently
produce antibodies capable of inducing enhanced Fc-mediated
effector function (e.g. duck embryonic derived stem cells for the
production of viral vaccines, WO2008/129058; Recombinant protein
production in avian EBX.RTM. cells, WO 2008/142124).
[0301] TL1a-binding proteins of the disclosure also include those
with bisected oligosaccharides, e.g., in which a biantennary
oligosaccharide attached to the Fc region is bisected by GlcNAc.
Such proteins may have reduced fucosylation and/or improved ADCC
function. Examples of such proteins are described, e.g., in U.S.
Pat. No. 6,602,684 and US20050123546.
[0302] TL1a-binding proteins with at least one galactose residue in
the oligosaccharide attached to the Fc region are also
contemplated. Such proteins may have improved CDC function. Such
proteins are described, e.g., in WO1997/30087 and WO1999/22764.
[0303] TL1a-binding proteins can also comprise a Fc region capable
of inducing enhanced levels of CDC. For example, hybrids of IgG1
and IgG3 produce antibodies having enhanced CDC activity (Natsume
et al., Cancer Res. 68: 3863-3872, 2008).
[0304] TL1a-binding proteins can also or alternatively be fused to
or conjugated to proteins (e.g., antibody variable regions) that
bind to immune effector cells, e.g., by virtue of binding to CD3 or
CD16.
[0305] Methods for determining effector function are known in the
art. In one example, the level of ADCC activity is assessed using a
.sup.51Cr release assay, an europium release assay or a .sup.35S
release assay. In each of these assays, cells expressing TL1a are
cultured with one or more of the recited compounds for a time and
under conditions sufficient for the compound to be taken up by the
cell. In the case of a .sup.35S release assay, the cells can be
cultured with .sup.35S-labeled methionine and/or cysteine for a
time sufficient for the labeled amino acids to be incorporated into
newly synthesized proteins. Cells are then cultured in the presence
or absence of the protein and in the presence of immune effector
cells, e.g., PBMCs and/or NK cells. The amount of .sup.51Cr,
europium and/or .sup.35S in cell culture medium is then detected,
and an increase in the presence of the protein compared to in the
absence of protein indicates that the binding molecule/agent has
effector function. Exemplary publications disclosing assays for
assessing the level of ADCC induced by a protein include Hellstrom
et al. Proc. Natl Acad. Sci. USA 83: 7059-7063, 1986 and Bruggemann
et al., J. Exp. Med. 166: 1351-1361, 1987.
[0306] Other assays for assessing the level of ADCC induced by a
protein include ACTI.TM. nonradioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. CA, USA) or CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Wis., USA).
[0307] Alternatively, or additionally, effector function of a
TL1a-binding protein is assessed by determining its affinity for
one or more Fc.gamma.Rs, e.g., as described in U.S. Pat. No.
7,317,091.
[0308] C1q binding assays may also be carried out to confirm that
the TL1a-binding protein is able to bind C1q and may induce CDC. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163,
1996).
[0309] In another example, the TL1a-binding protein comprises one
or more amino acid substitutions that increase the half-life of the
protein. For example, the TL1a-binding protein comprises a constant
region comprising one or more amino acid substitutions that
increase the affinity of the constant region for the neonatal Fc
region (FcRn). For example, the constant region has increased
affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate
Fc/FcRn binding in an endosome. In one example, the constant region
has increased affinity for FcRn at about pH 6 compared to its
affinity at about pH 7.4, which facilitates the re-release of Fc
into blood following cellular recycling. These amino acid
substitutions are useful for extending the half life of a
TL1a-binding protein, by reducing clearance from the blood.
[0310] Exemplary amino acid substitutions include T250Q and/or
M428L or T252A, T254S and T266F or M252Y, 5254T and T256E or H433K
and N434F according to the EU numbering system. Additional or
alternative amino acid substitutions are described, for example, in
US20070135620 or U.S. Pat. No. 7,083,784.
Stabilized TL1a-Binding Proteins
[0311] Neutralizing TL1a-binding proteins of the disclosure can
comprise an IgG4 constant region or a stabilized IgG4 constant
region. The term "stabilized IgG4 constant region" will be
understood to mean an IgG4 constant region that has been modified
to reduce Fab arm exchange or the propensity to undergo Fab arm
exchange or formation of a half-antibody or a propensity to form a
half-antibody. "Fab arm exchange" refers to a type of protein
modification for human IgG4, in which an IgG4 heavy chain and
attached light chain (half-molecule) is swapped for a heavy-light
chain pair from another IgG4 molecule. Thus, IgG4 molecules may
acquire two distinct Fab arms recognizing two distinct antigens
(resulting in bispecific molecules). Fab arm exchange occurs
naturally in vivo and can be induced in vitro by purified blood
cells or reducing agents such as reduced glutathione. A
"half-antibody" forms when an IgG4 antibody dissociates to form two
molecules each containing a single heavy chain and a single light
chain.
[0312] In one example, a stabilized IgG4 constant region comprises
a proline at position 241 of the hinge region according to the
system of Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest Washington D.C. United States Department of
Health and Human Services, 1987 and/or 1991). This position
corresponds to position 228 of the hinge region according to the EU
numbering system (Kabat et al., Sequences of Proteins of
Immunological Interest Washington D.C. United States Department of
Health and Human Services, 2001 and Edelman et al., Proc. Natl.
Acad. USA, 63: 78-85, 1969). In human IgG4, this residue is
generally a serine. Following substitution of the serine for
proline, the IgG4 hinge region comprises a sequence CPPC. In this
regard, the skilled person will be aware that the "hinge region" is
a proline-rich portion of an antibody heavy chain constant region
that links the Fc and Fab regions that confers mobility on the two
Fab arms of an antibody. The hinge region includes cysteine
residues which are involved in inter-heavy chain disulfide bonds.
It is generally defined as stretching from Glu226 to Pro243 of
human IgG1 according to the numbering system of Kabat. Hinge
regions of other IgG isotypes may be aligned with the IgG1 sequence
by placing the first and last cysteine residues forming inter-heavy
chain disulphide (S--S) bonds in the same positions (see for
example WO2010/080538).
Mutant TL1a-Binding Proteins
[0313] The disclosure also provides a TL1-binding protein or a
nucleic acid encoding same having at least 80% identity to a
sequence disclosed herein. In one example, a TL1a-binding protein
or nucleic acid of the disclosure comprises sequence at least about
85% or 90% or 95% or 97% or 98% or 99% identical to a sequence
disclosed herein, wherein the protein specifically binds to TL1a
and inhibits interaction of TL1a and DR3 and does not inhibit
interaction of TL1a and DcR3.
[0314] Alternatively, or additionally, the TL1a-binding protein
comprises a CDR (e.g., three CDRs) at least about 80% or 85% or 90%
or 95% or 97% or 98% or 99% identical to CDR(s) of a V.sub.H or
V.sub.L as described herein according to any example, wherein the
protein is capable of specifically binding to TL1a and inhibiting
interaction of TL1a and DR3, wherein the protein does not inhibit
interaction of TL1a and DcR3. In this regard, the inventors have
produced numerous antibodies having diverse sequences within their
CDRs. Methods for determining binding of a protein TL1a and
determining the interaction of TL1a and DR3 or TL1a and DcR3 are
described herein.
[0315] For example, the inventors have identified a group of
TL1a-binding proteins sharing 60% identity in their HCDR1 according
to the Kabat numbering system and another subgroup of proteins
sharing 80% identity in their HCDR1 according to the Kabat
numbering system.
[0316] The inventors have also identified a subclass of
TL1a-binding proteins sharing 40% identity or 47% identity in their
HCDR2 according to the Kabat numbering system.
[0317] As discussed herein, it is also known in the art that the
five C-terminal residues of heavy chain CDR2 can be mutated to
conservative or non-conservative amino acid substitutions (31% of
residues) (Padlan et al., FASEB J. 9: 133-139, 1995). Thus, a
protein can comprise a CDR2 having at least about 69% identity to a
heavy chain CDR2 sequence disclosed herein.
[0318] The inventors have also identified a class of proteins
comprising variants of the variable regions of antibody C320
described herein. These variants permit identification of sites
within the variable regions that can be substituted without loss of
function.
[0319] For example, the inventors have identified several residues
in a V.sub.H comprising a sequence set forth in SEQ ID NO: 42 that
can be substituted without loss of function. Accordingly, the
disclosure encompasses proteins comprising a V.sub.H with at least
about 86% identity to a sequence set forth in SEQ ID NO: 42. In
this regard, the inventors have produced a modified form of SEQ ID
NO: 42 having about 17 amino acid substitutions (i.e., about 86%
identity thereto) that retains a function recited herein. In this
regard, the inventors have also produced a modified forms of SEQ ID
NO: 42 having about 90 or 94% identity thereto that retains a
function recited herein. In one example, the sequence has at least
about 95% or 96% or 97% or 98% identity to a sequence set forth in
SEQ ID NO: 42. In this regard, the inventors have produced proteins
having about 97% or 98% or 99% identity to a sequence set forth in
SEQ ID NO: 42. In one example, the sequence has at least about 99%
identity to a sequence set forth in SEQ ID NO: 42. In one example,
the sequence has at least about 99.2% identity to a sequence set
forth in SEQ ID NO: 42.
[0320] In one example, the TL1a-binding protein comprises between 1
and 17 amino acid substitutions compared to SEQ ID NO: 42. For
example, the TL1a-binding protein comprises 1 or 2 or 3 or 4 or 5
or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17
amino acid substitutions compared to SEQ ID NO: 42.
[0321] In one example, the TL1a-binding protein comprises between 1
and 17 amino acid substitutions in the FRs compared to SEQ ID NO:
42. For example, the TL1a-binding protein comprises 1 or 2 or 3 or
4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or
16 or 17 amino acid substitutions in the FRs compared to SEQ ID NO:
42. In one example, the substitution is not at position 73 of SEQ
ID NO: 42.
[0322] In one example, the TL1a-binding protein comprises between 1
and 3 amino acid substitutions in CDR3 compared to SEQ ID NO: 42.
For example, the TL1a-binding protein comprises 1 or 2 or 3 amino
acid substitutions in the CDR3 compared to SEQ ID NO: 42. In one
example, the substitution is not at one or more of positions 99 or
101 or 104 or 108 of SEQ ID NO: 42.
[0323] In one example, TL1a-binding protein comprises one or more
substitutions to prevent or reduce glycosylation of the protein,
wherein the substitution(s) are between amino acids 72 to 76 of SEQ
ID NO: 42. For example, the protein comprises an alanine in place
of the arginine at position 71 and/or an aspartic acid in place of
the asparagine at position 72 and/or an arginine in place of the
threonine at position 73 and/or a threonine in place of the
isoleucine at position 75 of SEQ ID NO: 42.
[0324] In one example, the V.sub.H of the protein is not
glycosylated and/or does not comprise a consensus site for N-linked
glycosylation.
[0325] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a serine at position
16 of SEQ ID NO: 42.
[0326] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a proline at
position 41.
[0327] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a arginine at
position 74.
[0328] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a glutamic acid or
glycine at position 49 of SEQ ID NO: 42.
[0329] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a proline at
position 41 and a leucine at position 51 and a glutamic acid at
position 102 and an alanine at position 105 each relative to SEQ ID
NO: 42.
[0330] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a proline at
position 41 and a leucine at position 51 and an alanine at position
72 and an aspartic acid at position 73 and an arginine at position
74 and a glutamic acid at position 102 and an alanine at position
105 each relative to SEQ ID NO: 42.
[0331] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 42,
wherein the mutant sequence at least comprises a proline at
position 41 and a leucine at position 51 and an alanine at position
72 and an aspartic acid at position 73 and an arginine at position
74 and a threonine at position 76 and a glutamic acid at position
102 and an alanine at position 105 each relative to SEQ ID NO:
42.
[0332] The inventors have identified a site within HCDR2 that can
be substituted. Coupled with the observations of Padlan et al.,
supra, the disclosure thus provides a protein comprising a V.sub.H
comprising a CDR2 having a sequence at least about 65% identical to
a sequence set forth in SEQ ID NO: 44. In one example, the
percentage identity is at least about 70% or 75% or 80% or 90%. In
one example, the percentage identity is at least about 94%.
[0333] The inventors have identified numerous sites within HCDR3
that can be substituted. The disclosure thus provides a protein
comprising a V.sub.H comprising a CDR3 having a sequence at least
about 60% identical to a sequence set forth in SEQ ID NO: 45. In
one example, the percentage identity is at least about 70%
identical to a sequence set forth in SEQ ID NO: 45, e.g., as
exemplified herein. In one example, the percentage identity is at
least about 75% or 80% or 90%. In one example, the percentage
identity is at least about 90%.
[0334] In one example, a mutant form of SEQ ID NO: 45 comprises a
glutamic acid at position 1 of SEQ ID NO: 45 and/or a proline at
position 3 of SEQ ID NO: 45 and/or an alanine at position 6 of SEQ
ID NO: 45. Optionally, the mutant form additionally comprises a
phenylalanine at position 8 of SEQ ID NO: 45 and/or a tyrosine at
position 10 of SEQ ID NO: 45.
[0335] Additional residues that can be mutated are set out in FIGS.
1C-1E and in SEQ ID NOs: 94, 137, 152, 162 and 173.
[0336] In one example, the inventors have identified several
residues in a V.sub.L comprising a sequence set forth in SEQ ID NO:
46 that can be substituted without loss of function. Accordingly,
the disclosure encompasses proteins comprising a V.sub.L with at
least about 95% identity to a sequence set forth in SEQ ID NO: 46.
For example, the inventors have mutated 32 residues in the V.sub.L
without loss of function, meaning that the disclosure provides a
protein having at least about 71% identity to a sequence set forth
in SEQ ID NO: 46. In one example, the percentage identity is at
least about 75% or 80% or 90% or 95% or 97%. In one example, the
percentage identity is at least about 98%. In one example, the
percentage identity is at least about 99%. In one example, the
percentage identity is at least about 99.1%.
[0337] In one example, the TL1a-binding protein comprises between 1
and 17 amino acid substitutions compared to SEQ ID NO: 42. For
example, the TL1a-binding protein comprises 1 or 2 or 3 or 4 or 5
or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17
or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28
or 29 or 30 or 31 or 32 amino acid substitutions compared to SEQ ID
NO: 46.
[0338] In one example, the TL1a-binding protein comprises between 1
and 32 amino acid substitutions in the FRs compared to SEQ ID NO:
46. For example, the TL1a-binding protein comprises 1 or 2 or 3 or
4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or
16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or
27 or 28 or 29 or 30 or 31 or 32 amino acid substitutions in the
FRs compared to SEQ ID NO: 46.
[0339] In one example, the TL1a-binding protein comprises between 1
and 3 amino acid substitutions in a CDR compared to SEQ ID NO: 46.
For example, the TL1a-binding protein comprises 1 or 2 or 3 amino
acid substitutions in a CDR compared to SEQ ID NO: 46. In one
example, the substitution is not at one or more of positions 34,
54, 94 of SEQ ID NO: 46.
[0340] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 46,
wherein the mutant sequence at least comprises a serine at position
24 of SEQ ID NO: 46.
[0341] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 46,
wherein the mutant sequence at least comprises a threonine at
position 76 of SEQ ID NO: 46.
[0342] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 46,
wherein the mutant sequence at least comprises a glutamine at
position 81 of SEQ ID NO: 46.
[0343] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 46,
wherein the mutant sequence at least comprises a threonine at
position 23 and a serine at position 24 and a threonine at position
76 each relative to SEQ ID NO: 46.
[0344] In one example, a TL1a-binding protein of the disclosure
comprises a mutant of a sequence set forth in SEQ ID NO: 46,
wherein the mutant sequence at least comprises a threonine at
position 23 and a serine at position 24 and a threonine at position
76 and a glutamic acid at position 51 each relative to SEQ ID NO:
46.
[0345] The inventors have identified a plurality of sites within
LCDR1 that can be substituted. The disclosure thus provides a
protein comprising a V.sub.L comprising a CDR1 having a sequence at
least about 60% identical to a sequence set forth in SEQ ID NO: 47.
In one example, the percentage identity is at least about 70% or
75% or 80% or 90%. For example, the inventors have mutated two
sites within LCDR1 comprising a sequence set forth in SEQ ID NO:
47, thus producing a protein having at least about 86 identity to
the recited sequence. In one example, the percentage identity is at
least about 90%.
[0346] In one example, a TL1a binding protein of the disclosure
comprises a V.sub.H comprising a sequence set forth in SEQ ID NO:
42 and a V.sub.L comprising a sequence set forth in SEQ ID NO: 46,
wherein the V.sub.H and/or V.sub.L comprise one or more of the
following substitutions or groups of substitutions:
(i) the V.sub.H comprises at least a serine at position 16 of SEQ
ID NO: 42; (ii) the V.sub.L comprises at least a threonine at
position 76 of SEQ ID NO: 46; (iii) the V.sub.L comprises at least
a glutamine at position 81 of SEQ ID NO: 46; (iv) the V.sub.H
comprises at least a proline at position 41 and a leucine at
position 51 and a glutamic acid at position 102 and an alanine at
position 105 each relative to SEQ ID NO: 42 and the V.sub.L
comprises at least a threonine at position 23 and a serine at
position 24 and a threonine at position 76 each relative to SEQ ID
NO: 46; or (v) the VH comprises at least a proline at position 41
and a leucine at position 51 and an alanine at position 72 and an
aspartic acid at position 73 and an arginine at position 74 and a
glutamic acid at position 102 and an alanine at position 105 each
relative to SEQ ID NO: 42 and a VL comprises at least a threonine
at position 23 and a serine at position 24 and a threonine at
position 76 and a glutamic acid at position 51 each relative to SEQ
ID NO: 46.
[0347] In another example, a nucleic acid of the disclosure
comprises a sequence at least about 80% or 85% or 90% or 95% or 97%
or 98% or 99% identical to a sequence set forth herein and encoding
a TL1a-binding protein which is capable of specifically binding to
TL1a and inhibiting interaction of TL1a and DR3, wherein the
protein does not inhibit interaction of TL1a and DcR3. The
disclosure also encompasses nucleic acids encoding a TL1a-binding
protein of the disclosure, which differs from a sequence
exemplified herein as a result of degeneracy of the genetic
code.
[0348] The % identity of a nucleic acid or polypeptide is
determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453,
1970) analysis (GCG program) with a gap creation penalty=5, and a
gap extension penalty=0.3. The query sequence is at least 50
residues in length, and the GAP analysis aligns the two sequences
over a region of at least 50 residues. For example, the query
sequence is at least 100 residues in length and the GAP analysis
aligns the two sequences over a region of at least 100 residues.
For example, the two sequences are aligned over their entire
length.
[0349] As discussed above, the disclosure also contemplates a
nucleic acid that hybridizes under stringent hybridization
conditions to a nucleic acid encoding a TL1a-binding protein
described herein, e.g., nucleic acid encoding a V.sub.H or V.sub.L
of antibody C319, C320, C321, C323, C333, C334, C336, C320-3,
C320-5, C320-90, C320-103, C320-114, C320-115, C320-120, C320-129,
C320-130, C320-135, C320-162, C320-163, C320-164, C320-165,
C320-166, C320-167, C320-168, C320-169, C320-170, C320-171,
C320-172, C320-179 or, C320-183. A "moderate stringency" is defined
herein as being a hybridization and/or washing carried out in
2.times.SSC buffer, 0.1% (w/v) SDS at a temperature in the range
45.degree. C. to 65.degree. C., or equivalent conditions. A "high
stringency" is defined herein as being a hybridization and/or wash
carried out in 0.1.times.SSC buffer, 0.1% (w/v) SDS, or lower salt
concentration, and at a temperature of at least 65.degree. C., or
equivalent conditions. Reference herein to a particular level of
stringency encompasses equivalent conditions using
wash/hybridization solutions other than SSC known to those skilled
in the art. For example, methods for calculating the temperature at
which the strands of a double stranded nucleic acid will dissociate
(also known as melting temperature, or Tm) are known in the art. A
temperature that is similar to (e.g., within 5.degree. C. or within
10.degree. C.) or equal to the Tm of a nucleic acid is considered
to be high stringency. Medium stringency is to be considered to be
within 10.degree. C. to 20.degree. C. or 10.degree. C. to
15.degree. C. of the calculated Tm of the nucleic acid.
[0350] The disclosure also contemplates mutant forms of a
TL1a-binding protein of the disclosure comprising one or more
conservative amino acid substitutions compared to a sequence set
forth herein. In some examples, the TL1a-binding protein comprises
10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1
conservative amino acid substitutions. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain and/or
hydropathicity and/or hydrophilicity.
[0351] Families of amino acid residues having similar side chains
have been defined in the art, including basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), .beta.-branched
side chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Hydropathic indices are described, for example in Kyte and
Doolittle J. Mol. Biol., 157: 105-132, 1982 and hydrophylic indices
are described in, e.g., U.S. Pat. No. 4,554,101.
[0352] The disclosure also contemplates non-conservative amino acid
changes. For example, of particular interest are substitutions of
charged amino acids with another charged amino acid and with
neutral or positively charged amino acids. In some examples, the
TL1a-binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6
or 5 or 4 or 3 or 2 or 1 non-conservative amino acid
substitutions.
[0353] In one example, the mutation(s) occur within a FR of an
antigen binding domain of a TL1a-binding protein of the disclosure.
In another example, the mutation(s) occur within a CDR of a
TL1a-binding protein of the disclosure.
Exemplary methods for producing mutant forms of a TL1a-binding
protein include: [0354] mutagenesis of DNA (Thie et al., Methods
Mol. Biol. 525: 309-322, 2009) or RNA (Kopsidas et al., Immunol.
Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18,
2007; and WO1999/058661); [0355] introducing a nucleic acid
encoding the polypeptide into a mutator cell, e.g., XL-1Red,
XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene); [0356] DNA
shuffling, e.g., as disclosed in Stemmer, Nature 370: 389-91, 1994;
and [0357] site directed mutagenesis, e.g., as described in
Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory
Manual, Cold Spring Harbor Laboratories, NY, 1995).
[0358] Exemplary methods for determining biological activity of the
mutant TL1a-binding proteins of the disclosure will be apparent to
the skilled artisan and/or described herein, e.g., antigen binding.
For example, methods for determining antigen binding, competitive
inhibition of binding, affinity, association, dissociation and
therapeutic efficacy are described herein.
Exemplary TL1a-Binding Proteins
[0359] Exemplary variable region containing TL1a-binding proteins
produced by the inventors and their encoding nucleic acids are
described in Tables 1 and 2.
TABLE-US-00001 TABLE 1 Sequences of exemplary TL1a-binding proteins
and encoding nucleic acids V.sub.H V.sub.H V.sub.L V.sub.L amino
chain amino chain acid nucleotide acid nucleotide Antibody SEQ SEQ
SEQ SEQ Name ID NO ID NO ID NO ID NO 1 C336 2 96 6 97 2 C334 10 98
14 99 3 C333 18 100 22 101 4 C323 26 102 30 103 5 C321 34 104 38
105 6 C320 42 106 46 107 7 C319 50 108 54 109 8 C320-3 58 110 46
107 9 C320-5 42 106 62 111 10 C320-90 66 112 62 111 11 C320-103 70
113 62 111 12 C320-114 74 114 62 111 13 C320-115 78 115 62 111 14
C320-120 58 110 82 116 15 C320-129 86 117 46 107 16 C320-130 90 118
46 107 17 C320-135 58 110 62 111 18 C320-7 154 164 19 C320-8 155
163 20 C320-9 156 165 21 C320-10 157 166 22 C320-11 158 167 23
C320-12 159 168 24 C320-13 234 169 25 C320-14 160 170 26 C320-15
161 171 27 C320-16 154 46 28 C320-17 155 46 29 C320-18 156 46 30
C320-19 157 46 31 C320-20 158 46 32 C320-21 159 46 33 C320-22 234
46 34 C320-23 160 46 35 C320-24 161 46 36 C320-25 42 164 37 C320-26
42 163 38 C320-27 42 165 39 C320-28 42 166 40 C320-29 42 167 41
C320-30 42 168 42 C320-31 42 169 43 C320-32 42 170 44 C320-33 42
171 45 C320-162 175 222 188 228 46 C320-163 176 223 189 228 47
C320-164 177 224 190 229 48 C320-165 178 224 191 230 49 C320-166
179 224 192 231 50 C320-167 180 224 193 228 51 C320-168 181 225 194
230 52 C320-169 182 225 195 228 53 C320-170 183 226 196 230 54
C320-171 184 197 55 C320-172 185 226 198 232 56 C320-179 186 227
199 232 57 C320-183 187 227 200 233
TABLE-US-00002 TABLE 2 Amino acid substitutions in V.sub.H
(relative to SEQ ID NO: 42) and V.sub.L (relative to SEQ. ID NO:
46) of exemplary TL1a-binding proteins. Antibody V.sub.H V.sub.L
name substitution.sup.1 substitution.sup.1 1 C320-2 A16S none 2
C320-53 E99S A76T 3 C320-54 E99H A76T 4 C320-55 E99L A76T 5 C320-56
E99D A76T 6 C320-57 E99Y A76T 7 C320-58 E99P A76T 8 C320-59 E99Q
A76T 9 C320-60 E99K A76T 10 C320-61 V100A A76T 11 C320-62 V100S
A76T 12 C320-63 V100H A76T 13 C320-64 V100L A76T 14 C320-65 V100D
A76T 15 C320-66 V100Y A76T 16 C320-67 V100P A76T 17 C320-68 V100Q
A76T 18 C320-69 V100K A76T 19 C320-70 P101A A76T 20 C320-71 P101S
A76T 21 C320-72 P101H A76T 22 C320-73 P101L A76T 23 C320-74 P101D
A76T 24 C320-75 P101Y A76T 25 C320-76 P101Q A76T 26 C320-77 P101K
A76T 27 C320-78 D102A A76T 28 C320-79 D102S A76T 29 C320-80 D102H
A76T 30 C320-81 D102L A76T 31 C320-82 D102Y A76T 32 C320-83 D102P
A76T 33 C320-84 D102Q A76T 34 C320-85 D102K A76T 35 C320-86 T103A
A76T 36 C320-87 T103S A76T 37 C320-88 T103H A76T 38 C320-89 T103L
A76T 39 C320-90 T103D A76T 40 C320-91 T103Y A76T 41 C320-92 T103P
A76T 42 C320-93 T103Q A76T 43 C320-94 T103K A76T 44 C320-95 A104S
A76T 45 C320-96 A104H A76T 46 C320-97 A104L A76T 47 C320-98 A104D
A76T 48 C320-99 A104Y A76T 49 C320-100 A104P A76T 50 C320-101 A104Q
A76T 51 C320-102 A104K A76T 52 C320-103 S105A A76T 53 C320-104
S105H A76T 54 C320-105 S105L A76T 55 C320-106 S105D A76T 56
C320-107 S105Y A76T 57 C320-108 S105P A76T 58 C320-109 S105Q A76T
59 C320-110 S105K A76T 60 C320-111 E107A A76T 61 C320-112 E107S
A76T 62 C320-113 E107H A76T 63 C320-114 E107L A76T 64 C320-115
E107D A76T 65 C320-116 E107Y A76T 66 C320-117 E107P A76T 67
C320-118 E107Q A76T 68 C320-119 E107K A76T 69 C320-120 T41P A23T 70
C320-121 T41P D28N 71 C320-122 T41P L33Y 72 C320-123 T41P G34D 73
C320-124 T41P Y53N 74 C320-125 T41P Y54S 75 C320-126 T41P P82A 76
C320-127 T41P G95S 77 C320-128 T41P T96S 78 C320-129 D102E None 79
C320-130 M51L None 80 C320-131 None D49E 81 C320-135 T41P A76T 82
C320-162 T41P + M51L + S75A + A76T D102E 83 C320-163 T41P + R72A +
N73D + A76T T74R + I76T 84 C320-164 T41P + M51L + D102E G24S + A76T
85 C320-165 T41P + M51L + D102E A23T + G24S + A76T 86 C320-166 T41P
+ M51L + D102E A23T + A76T 87 C320-167 T41P + M51L + D102E A76T 98
C320-168 T41P + M51L + D102E + A23T + G24S + A76T S105A 89 C320-169
T41P + M51L + D102E + A76T S105A 90 C320-170 T41P + R72A + N73D +
A23T + G24S + A76T T74R + I76T 91 C320-171 T41P + R72A + N73D +
A23T + G24S + A76T + T74R + I76T Y51P 92 C320-172 T41P + R72A +
N73D + A23T + G24S + A76T + T74R + I76T Y51E 93 C320-179 T41P +
M51L + R72A + A23T + G24S + A76T + N73D + T74R + I76T + Y51E D102E
+ S105A 94 C320-183 T41P + M51L + R72A + A23T + G24S + A76T + N73D
+ T74R + I76T + Y51G D102E + S105A .sup.1substitutions are listed
as: residue in SEQ ID NO: 42 or 46; position in SEQ ID NO: 42 or
46; substituted amino acid, i.e., A16S in V.sub.H, means that at
position 16 of SEQ ID NO: 42 there is an alanine that has been
substituted with a serine in the TL1a-binding protein.
Recombinant Expression
[0360] As discussed herein, a nucleic acid encoding a TL1a-binding
protein of the disclosure (and/or polypeptides included in such a
TL1a-binding protein) is introduced into an expression construct,
such that it is operably linked to a promoter to thereby facilitate
its expression. Methods for producing expression constructs, e.g.,
cloning into expression constructs/vectors are known in the art
and/or described in Ausubel et al., (In: Current Protocols in
Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), and
(Sambrook et al., (In: Molecular Cloning: Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third
Edition 2001) and U.S. Pat. No. 7,270,969.
[0361] In one example, the TL1a-binding protein of the disclosure
is expressed in a bacterial cell. Typical promoters suitable for
expression in bacterial cells such as for example a bacterial cell
selected from the group comprising E. coli, Staphylococcus sp,
Corynebacterium sp., Salmonella sp., Bacillus sp., and Pseudomonas
sp., include, but are not limited to a promoter such as lacz, Ipp,
a temperature-sensitive (.sub.L or (.sub.R promoters, T7, T3, SP6
or semi-artificial promoters such as the IPTG-inducible tac
promoter or lacUV5 promoter.
[0362] In another example, the TL1a-binding protein is expressed in
a yeast cell. Typical promoters suitable for expression in yeast
cells such as, Pichia pastoris, Saccharomyces cerevisiae and S.
pombe, include, but are not limited to promoters from the following
genes ADH1, GAL1, GAL4, CUP1, PHO5, nmt, RPR1, or TEF1.
[0363] In a further example, the TL1a-binding protein is expressed
in an insect cell. Typical promoters suitable for expression in
insect cells, or in insects, include, but are not limited to, the
OPEI2 promoter, the insect actin promoter isolated from Bombyx
muri, the Drosophila sp. dsh promoter (Marsh et al., Hum. Mol.
Genet. 9: 13-25, 2000).
[0364] A TL1a-binding protein of the disclosure can also be
expressed in plant cells. Promoters for expressing peptides in
plant cells are known in the art, and include, but are not limited
to, the Hordeum vulgare amylase gene promoter, the cauliflower
mosaic virus 35S promoter, the nopaline synthase (NOS) gene
promoter, and the auxin inducible plant promoters P1 and P2.
[0365] In one example, a TL1a-binding protein of the disclosure is
expressed in a mammalian cell or in a mammal. Typical promoters
suitable for expression in a mammalian cell include, for example a
promoter selected from the group consisting of, retroviral LTR
elements, the SV40 early promoter, the SV40 late promoter, the CMV
IE (cytomegalovirus immediate early) promoter, the EF.sub.1
promoter (from human elongation factor 1), the EM7 promoter, the
UbC promoter (from human ubiquitin C). Examples of useful mammalian
host cell lines include monkey kidney CVI line transformed by SV40
(COS-7); human embryonic kidney line (HEK-293 cells); baby hamster
kidney cells (BHK); Chinese hamster ovary cells (CHO); African
green monkey kidney cells (VERO-76); or myeloma cells (e.g., NS/O
cells).
[0366] Other elements of expression constructs/vectors are known in
the art and include, for example, enhancers, transcriptional
terminators, polyadenylation sequences, nucleic acids encoding
selectable or detectable markers and origins of replication.
[0367] In one example, an expression construct is a bicistronic
expression construct. By "bicistronic" is meant a single nucleic
acid molecule that is capable of encoding two distinct polypeptides
from different regions of the nucleic acid, for example, a single
nucleic acid capable of encoding a V.sub.H containing polypeptide
and a V.sub.L containing polypeptide as distinct polypeptides.
Generally, the regions encoding each distinct polypeptide are
separated by an internal ribosome entry site (IRES) and the region
5' of the IRES does not comprise a transcription termination
sequence. Exemplary IRESs are described, for example, in
US20090247455.
[0368] Following production of a suitable expression construct, it
is introduced into a suitable cell using any method known in the
art. Exemplary methods include microinjection, transfection
mediated by DEAE-dextran, transfection mediated by liposomes such
as using commercially available reagents, PEG-mediated DNA uptake,
electroporation and microparticle bombardment such as by using
DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA)
amongst others.
[0369] The cells used to produce the TL1a-binding protein of this
disclosure are then cultured under conditions known in the art to
produce a TL1a-binding protein of the disclosure.
[0370] Cell free expression systems are also contemplated by the
disclosure, e.g., the TNT T7 and TNT T3 systems (Promega), the
pEXP1-DEST and pEXP2-DEST vectors (Invitrogen).
Protein Purification
[0371] Following production/expression, a TL1a-binding protein of
the disclosure is purified using a method known in the art. Such
purification provides the protein of the disclosure substantially
free of nonspecific protein, acids, lipids, carbohydrates, and the
like. In one example, the protein will be in a preparation wherein
more than about 90% (e.g. 95%, 98% or 99%) of the protein in the
preparation is a TL1a-binding protein of the disclosure.
[0372] Standard methods of peptide purification are employed to
obtain an isolated TL1a-binding protein of the disclosure,
including but not limited to various high-pressure (or performance)
liquid chromatography (HPLC) and non-HPLC polypeptide isolation
protocols, such as size exclusion chromatography, ion exchange
chromatography, hydrophobic interaction chromatography, mixed mode
chromatography, phase separation methods, electrophoretic
separations, precipitation methods, salting in/out methods,
immunochromatography, and/or other methods.
[0373] In one example, affinity purification is useful for
isolating a fusion protein comprising a label. Methods for
isolating a protein using affinity chromatography are known in the
art and described, for example, in Scopes (In: Protein
purification: principles and practice, Third Edition, Springer
Verlag, 1994). For example, an antibody or compound that binds to
the label (in the case of a polyhistidine tag this may be, for
example, nickel-NTA) is immobilized on a solid support. A sample
comprising a protein is then contacted to the immobilized antibody
or compound for a time and under conditions sufficient for binding
to occur. Following washing to remove any unbound or
non-specifically bound protein, the protein is eluted.
[0374] In the case of a TL1a-binding protein comprising a Fc region
of an antibody, protein A or protein G or modified forms thereof
can be used for affinity purification. Protein A is useful for
isolating purified proteins comprising a human .gamma.1, .gamma.2,
or .gamma.4 heavy chain Fc region. Protein G is recommended for all
mouse Fc isotypes and for human .gamma.3.
[0375] In one example, a TL1a-binding protein of the disclosure is
conjugated to a compound. For example, the compound is selected
from the group consisting of a radioisotope, a detectable label, a
therapeutic compound, a colloid, a toxin, a nucleic acid, a
peptide, a protein, a compound that increases the half life of the
TL1a-binding protein in a subject and mixtures thereof.
[0376] The other compound can be directly or indirectly bound to
the TL1a-binding protein (e.g., can comprise a linker in the case
of indirect binding). Examples of compounds include, a radioisotope
(e.g., iodine-131, yttrium-90 or indium-111), a detectable label
(e.g., a fluorophore or a fluorescent nanocrystal or quantum dot),
a therapeutic compound (e.g., a chemotherapeutic or an
anti-inflammatory), a colloid (e.g., gold), a toxin (e.g., ricin or
tetanus toxoid), a nucleic acid, a peptide (e.g., a serum albumin
binding peptide), a protein (e.g., a protein comprising an antigen
binding domain of an antibody or serum albumin), a compound that
increases the half life of the TL1a-binding protein in a subject
(e.g., polyethylene glycol or other water soluble polymer having
this activity) and mixtures thereof. Exemplary compounds that can
be conjugated to a TL1a-binding protein of the disclosure and
methods for such conjugation are known in the art and described,
for example, in WO2010/059821.
[0377] The TL1a-binding protein may be conjugated to nanoparticles
(for example as reviewed in Kogan et al., Nanomedicine (Lond). 2:
287-306, 2007). The nanoparticles may be metallic
nanoparticles.
[0378] The TL1a-binding protein may be comprised in
antibody-targeted bacterial minicells (for example as described in
PCT/IB2005/000204).
[0379] Some exemplary compounds that can be conjugated to a
TL1a-binding protein of the disclosure are listed in Table 3.
TABLE-US-00003 TABLE 3 Compounds useful in conjugation. Group
Detail Radioisotopes .sup.123I, .sup.125I, .sup.130I, .sup.133I,
.sup.135I, .sup.47Sc, .sup.72As, .sup.72Sc, .sup.90Y, .sup.88Y,
(either directly .sup.97Ru, .sup.100Pd, .sup.101mRh, .sup.101mRh,
.sup.119Sb, .sup.128Ba, .sup.197Hg, .sup.211At, or indirectly)
.sup.212Bi, .sup.153Sm, .sup.169Eu, .sup.212Pb, .sup.109Pd,
.sup.111In, .sup.67Gu, .sup.68Gu, .sup.67Cu, .sup.75Br, .sup.76Br,
.sup.77Br, .sup.99mTc, .sup.11C, .sup.13N, .sup.15O, .sup.18I,
.sup.188Rc, .sup.203Pb, .sup.64Cu, .sup.105Rh, .sup.198Au,
.sup.199Ag or .sup.177Lu Half life Polyethylene glycol extenders
Glycerol Glucose Fluorescent Phycoerythrin (PE) probes
Allophycocyanin (APC) Alexa Fluor 488 Cy5.5 Biologics fluorescent
proteins such as Renilla luciferase, GFP immune modulators or
proteins, such as cytokines, e.g., an interferon toxins an
immunoglobulin or antibody or antibody variable region half life
extenders such as albumin or antibody variable regions or peptides
that bind to albumin Chemo- Taxol therapeutics 5-FU Doxorubicin
Idarubicin
[0380] TL1a-binding proteins comprising antibody binding domains of
the disclosure are readily screened for biological activity, e.g.,
as described below.
Binding Assays
[0381] One form of assay is an antigen binding assay, e.g., as
described in Scopes (In: Protein purification: principles and
practice, Third Edition, Springer Verlag, 1994). Such a method
generally involves labeling the TL1a-binding protein and contacting
it with immobilized antigen. Following washing to remove
non-specific bound protein, the amount of label and, as a
consequence, bound protein is detected. Of course, the TL1a-binding
protein can be immobilized and the antigen labeled. Panning-type
assays, e.g., as described or exemplified herein can also be used.
Alternatively, or additionally, surface plasmon resonance assays
can be used.
[0382] In one example, a binding assay is performed with peptide
comprising an epitope of TL1a. In this way, TL1a-binding proteins
that bind to a specific region of TL1a are selected.
Inhibition of Interaction of TL1a and DR3
[0383] Methods for identifying TL1a-binding proteins that inhibit
interaction of TL1a and DR3 will be apparent to the skilled artisan
based on the description herein.
[0384] For example, DR3 (e.g., 2 .mu.g/ml of DR3) is immobilized on
a surface and contacted with TL1a (e.g., 1 .mu.g/ml TL1a) and with
a TL1a-binding protein to be tested (in the case of controls, an
isotype matched control antibody is added). A reduced level of TL1a
bound to the DR3 in the presence of the TL1a-binding protein
compared to in the absence of the TL1a-binding protein indicates
that the TL1a-binding protein inhibits binding of TL1a to DR3. The
assay can also be performed with immobilized TL1a to which DR3 is
contacted. The assay can also be performed with labeled TL1a and/or
DR3 to assist with detection.
[0385] In some examples, various concentrations of the TL1a-binding
protein are tested and the concentration at which 50% of the
maximum inhibition of binding of TL1a to DR3 by the TL1a-binding
protein is determined (this concentration is known as EC.sub.50).
In one example, the EC.sub.50 of a TL1a-binding protein of the
disclosure is less than about 5 nM or 4 nM or 3.5 nM or 3 nM or 2.5
nM or 2.3 nM or 1 nM or 0.5 nM. In one example, the EC.sub.50 is
less than 3 nM. In one example, the EC.sub.50 is less than 2.5 nM.
In one example, the EC.sub.50 is less than 1 nM. In one example,
the EC.sub.50 is less than 0.5 nM.
[0386] In some examples, the maximal inhibition of interaction of
TL1a and DR3 is assessed by determining the level of interaction of
TL1a and DR3 in the presence and absence of a TL1a-binding protein.
The level of inhibition of interaction of TL1a and DR3 in the
presence of the protein is then expressed as a percentage of the
level of interaction in the absence of the protein. In one example,
the TL1a-binding protein inhibits at least about 80% of interaction
between TL1a and DR3. For example, the percentage inhibition is at
least about 84% or 85% or 90% or 93% or 94% or 95%. In one example,
the percentage inhibition is at least about 93%. In one example,
the percentage inhibition is at least about 94%. In one example,
the percentage inhibition is assessed using a polypeptide
comprising DR3 fused to an antibody Fc region. In one example, the
TL1a-binding protein is used at a concentration of about 10
.mu.g/ml.
Selective Inhibition of Interaction of TL1a and DR3
[0387] Methods for identifying TL1a-binding proteins that inhibit
interaction of TL1a and DR3 but not TL1a and DcR3 will be apparent
to the skilled artisan and/or described herein.
[0388] For example, DcR3 (e.g., 2 .mu.g/ml DcR3) is immobilized on
a surface and contacted with TL1a (e.g., 1 .mu.g/ml TL1a) and a
TL1a-binding protein to be tested (in the case of negative
controls, an isotype-matched control antibody is used). The level
of TL1a bound to DcR3 is then determined. A reduced level of TL1a
bound to the DcR3 in the presence of the TL1a-binding protein
compared to in the absence of the TL1a-binding protein indicates
that the TL1a-binding protein inhibits binding of TL1a to DcR3. A
similar level of bound TL1a in the presence or absence of the
TL1a-binding protein indicates that the TL1a-binding protein does
not inhibit interaction of TL1a and DcR3. The assay can also be
performed with immobilized TL1a to which DcR3 is contacted.
[0389] In some examples, various concentrations of the TL1a-binding
protein are tested to determine the level of inhibition of TL1a
interaction with DcR3 at different concentrations.
[0390] In some examples, the maximal inhibition of interaction of
TL1a and DcR3 is assessed by determining the level of interaction
of TL1a and DcR3 in the presence and absence of a TL1a-binding
protein. The level of inhibition of interaction of TL1a and DcR3 in
the presence of the protein is then expressed as a percentage of
the level of interaction in the absence of the protein. In one
example, the TL1a-binding protein at a concentration of 10 .mu.g/ml
inhibits 25% or less of the interaction between TL1a and DcR3. For
example, the percentage inhibition is 20% or less or 18% or less or
15% or less or 12% or less or 10% or less or 8% or less or 5% or
less. In one example, the percentage inhibition is about 18% or
less. In one example, the percentage inhibition is about 7% or
less. In one example, the percentage inhibition is about 5% or
less. In one example, the percentage inhibition is assessed using a
polypeptide comprising DcR3 fused to an antibody Fc region. In one
example, the TL1a-binding protein is used at a concentration of
about 10 .mu.g/ml.
Neutralization Assays
[0391] Methods for identifying TL1a-binding proteins that
neutralize TL1a activity through DR3 will also be apparent to the
skilled artisan, e.g., based on the description herein.
[0392] For example, DR3-expressing cells (e.g., TF-1 cells) (e.g.,
about 7.times.10.sup.4 cells to 8.times.10.sup.4 cells (e.g.,
7.5.times.10.sup.4 cells) are contacted with TL1a and a protein
synthesis inhibitor (e.g. cycloheximide) in the presence or absence
of a TL1a-binding protein to be tested. The level of apoptosis of
the cells is then assessed, e.g., by detecting activation of
caspases or propidium iodide uptake or other known assays. A
TL1a-binding protein that reduces the level of apoptosis compared
to the level of apoptosis in the absence of the TL1a-binding
protein is considered to inhibit TL1a activity or neutralize TL1a
activity through DR3.
[0393] In some examples, various concentrations of the TL1a-binding
protein are tested to determine the level of neutralization at
different concentrations. In some examples, the concentration at
which 50% of the maximum inhibition of apoptosis by the
TL1a-binding protein is determined (this concentration is known as
EC.sub.50). In one example, the EC.sub.50 of a TL1a-binding protein
of the disclosure is 3 nM or less, for example, about 2.5 nM or
less, such as about 2.4 nM or less, for example, about 2 nM or
less, such as about 1.5 nM or less, such as about 1 nM or less. In
one example, the EC.sub.50 of a TL1a-binding protein of the
disclosure about 0.99 nM or less. In one example, the EC.sub.50 of
a TL1a-binding protein of the disclosure is about 0.6 nM or less.
In one example, the EC.sub.50 of a TL1a-binding protein of the
disclosure is about 0.4 nM or less.
[0394] The ability of a TL1a-binding protein of the disclosure to
neutralize TL1a-activity can also be assessed by determining their
ability to reduce cytokine secretion by immune cells. For example,
PBMCs are contacted with Concanavalin A in the presence or absence
of a TL1a-binding protein. The level of secretion of a TL1a-induced
cytokine (e.g., interferon .gamma. or IL-13) is then assessed,
e.g., using an ELISA. Reduction of cytokine secretion in the
presence of the TL1a-binding protein compared to in the absence of
the protein indicates that the TL1a-binding protein neutralizes
TL1a activity.
[0395] In some examples, various concentrations of the TL1a-binding
protein are tested to determine the level of reduction in cytokine
secretion at different concentrations. In some examples, the
concentration at which 50% of the maximum inhibition of cytokine
secretion by the TL1a-binding protein is determined (this
concentration is known as EC.sub.50). For example, the EC.sub.50
for inhibiting secretion of interferon-.gamma. is 4 nM or less,
such as 3 nM or less or 2.5 nm or less or 2 nM or less. In another
example, the EC.sub.50 for inhibition of secretion of IL-13 is 15
nM or less, such as 10 nM or less, for example, 5 nM or less, such
as 1 nM or less. For example, the EC.sub.50 for inhibition of
secretion of IL-13 is 0.5 nM or less.
[0396] Other assays for determining neutralization of TL1a activity
include determining the level of proliferation, migration and tube
formation of endothelial cells in the presence and absence of the
TL1a-binding protein. For example, endothelial cells are cultured
in an extracellular matrix, e.g., Matrigel.TM., and the level of
migration and/or tube formation is determined, e.g., using
microscopy. An increase in migration and/or tube formation in the
presence of the TL1a-binding protein compared to in the absence of
the TL1a-binding protein indicates that the TL1a-binding protein
neutralizes TL1a activity through DR3.
[0397] In vivo Matrigel.TM. plug assays can also be performed,
e.g., essentially as described in Bagley et al., Cancer Res 63:
5866, 2003.
[0398] Additional assays include assessing the ability of a
TL1a-binding protein to reduce or prevent interferon .gamma.
secretion from peripheral blood T cells and/or NK cells stimulated
with IL-12 and/or IL-18 or in Fc.gamma.R activated monocytes.
In Vivo Assays
[0399] TL1a-binding proteins of the disclosure can also be assessed
for therapeutic efficacy in an animal model of a condition, e.g., a
TL1a-mediated condition. For example, the TL1a-binding protein is
administered to a model of inflammatory bowel disease or colitis
(e.g., dextran sodium sulphate (DSS)-induced colitis or CD45Rb
adoptive transfer model of colitis (e.g., Kanai et al., Inflamm.
Bowel Dis. 12: 89-99, 2006). In another example, a TL1a-binding
protein is administered to a model of multiple sclerosis, e.g., EAE
models in which a mouse or rat is immunized with a myelin sheath
protein or peptide derived therefrom (e.g., MOG, MBP or PLP) and an
immune response is generated against the protein thereby inducing a
model of multiple sclerosis. Exemplary EAE models are reviewed in,
for example Tsunoda and Fujinami, J. Neuropathol. Exp. Neurol. 55:
673-686, 1996. The TL1a-binding protein can also or alternatively
be tested in a model of arthritis e.g., a SKG strain of mouse
(Sakaguchi et al., Nature 426: 454-460, 1995), rat type II collagen
arthritis model, mouse type II collagen arthritis model or antigen
induced arthritis models (Bendele J. Musculoskel. Neuron. Interact.
1: 377-385, 2001) and/or a model of inflammatory airway disease
(for example, OVA challenge or cockroach antigen challenge), or in
a model of inflammatory uveitis for example interphotoreceptor
retinoid binding protein immunization-induced uveoretinitis (Caspi,
Curr Protoc Immunol Chapter 15: unit 15.6, 2003).
[0400] The ability of a TL1a-binding protein of the disclosure to
neutralize TL1a activity can also or alternatively be assessed in a
model of graft-versus-host-response, e.g., in which splenocytes
from one animal are injected into an allogeneic animal (e.g., a MHC
or HLA unmatched animal).
Epitope Mapping Assays
[0401] In another example, the epitope bound by a protein described
herein is mapped. Epitope mapping methods will be apparent to the
skilled artisan. For example, a series of overlapping peptides
spanning the TL1a sequence or a region thereof comprising an
epitope of interest, e.g., peptides comprising 10 to 15 amino acids
are produced. The TL1a-binding protein is then contacted to each
peptide or a combination thereof and the peptide(s) to which it
binds determined. This permits determination of peptide(s)
comprising the epitope to which the TL1a-binding protein binds. If
multiple non-contiguous peptides are bound by the protein, the
protein may bind a conformational epitope.
[0402] Alternatively, or in addition, amino acid residues within
TL1a are mutated, e.g., by alanine scanning mutagenesis, and
mutations that reduce or prevent protein binding are determined.
Any mutation that reduces or prevents binding of the TL1a-binding
protein is likely to be within the epitope bound by the protein. A
form of this method is exemplified herein.
[0403] A further method involves binding TL1a or a region thereof
to an immobilized TL1a-binding protein of the disclosure and
digesting the resulting complex with proteases. Peptide that
remains bound to the immobilized protein are then isolated and
analyzed, e.g., using mass spectrometry, to determine their
sequence.
Affinity Assays
[0404] Optionally, the dissociation constant (Kd) or association
constant (Ka) or equilibrium constant (K.sub.D) of a TL1a-binding
protein for TL1a or a peptide comprising an epitope thereof is
determined. These constants for a TL1a-binding protein are in one
example measured by a radiolabeled or fluorescently-labeled TL1a
binding assay. This assay equilibrates the protein with a minimal
concentration of labeled TL1a in the presence of a titration series
of unlabeled TL1a. Following washing to remove unbound TL1a, the
amount of label is determined.
[0405] Affinity measurements can be determined by standard
methodology for antibody reactions, for example, immunoassays,
surface plasmon resonance (SPR) (Rich and Myszka Curr. Opin.
Biotechnol 11::54, 2000; Englebienne Analyst. 123: 1599, 1998),
isothermal titration calorimetry (ITC) or other kinetic interaction
assays known in the art.
[0406] In one example, the constants are measured by using surface
plasmon resonance assays, e.g., using BIAcore surface plasmon
resonance (BIAcore, Inc., Piscataway, N.J.) with immobilized TL1a
or a region thereof. Exemplary SPR methods are described in U.S.
Pat. No. 7,229,619.
[0407] In one example, a TL1a-binding protein as described herein
according to any example has a K.sub.D for TL1a of 100 nM or less,
such as 50 nM or less, for example, 20 nM or less, for example, 10
nM or less or 6 nM or less. For example, a TL1a-binding protein has
a K.sub.D of 5.5 nM or less. For example, a TL1a-binding protein
has a K.sub.D of 5 nM or less. For example, a TL1a-binding protein
has a K.sub.D of 4 nM or less.
Half Life Assays
[0408] Some TL1a-binding proteins encompassed by the disclosure
have an improved half-life, e.g., are modified to extend their
half-life compared to TL1a-binding proteins that are unmodified.
Methods for determining a TL1a-binding protein with an improved
half-life will be apparent to the skilled person. For example, the
ability of a TL1a-binding protein to bind to a neonatal Fc receptor
(FcRn) is assessed. In this regard, increased binding affinity for
FcRn increased the serum half-life of the TL1a-binding protein (see
for example, Kim et al., Eur. J. Immunol. 24: 2429, 1994).
[0409] The half-life of a TL1a-binding protein of the disclosure
can also be measured by pharmacokinetic studies, e.g., according to
the method described by Kim et al, Eur. J. of Immunol. 24: 542,
1994. According to this method radiolabeled TL1a-binding protein is
injected intravenously into mice and its plasma concentration is
periodically measured as a function of time, for example at 3
minutes to 72 hours after the injection. The clearance curve thus
obtained should be biphasic, that is, an alpha phase and beta
phase. For the determination of the in vivo half-life of the
TL1a-binding protein, the clearance rate in beta-phase is
calculated and compared with that of the wild type or unmodified
TL1a-binding protein.
Stability Assays
[0410] Stability of a TL1a-binding protein of the disclosure can be
assessed by any of a variety of assays. For example, the
TL1a-binding protein is exposed to a condition, e.g., heat or acid
or stored for a period of time (e.g., 1 month) at room temperature.
Aggregation of the TL1a-binding protein can then be assessed by
determining turbidity (with an increase in turbidity following
exposure to the condition indicating instability), size exclusion
chromatography, non-reducing gel electrophoresis or a binding or
neutralization study described herein.
[0411] The TL1a-binding protein of the disclosure or nucleic acid
encoding same or cell expressing same (syn. active ingredient) is
useful for parenteral, topical, oral, or local administration,
aerosol administration, or transdermal administration, for
prophylactic or for therapeutic treatment.
[0412] Formulation of a TL1a-binding protein or nucleic acid
encoding same or cell expressing same to be administered will vary
according to the route of administration and formulation (e.g.,
solution, emulsion, capsule) selected. An appropriate
pharmaceutical composition comprising TL1a-binding protein or
nucleic acid encoding same or cell expressing same to be
administered can be prepared in a physiologically acceptable
carrier. A mixture of TL1a-binding proteins can also be used. For
solutions or emulsions, suitable carriers include, for example,
aqueous or alcoholic/aqueous solutions, emulsions or suspensions,
including saline and buffered media. Parenteral vehicles can
include sodium chloride solution, Ringer's dextrose, dextrose and
sodium chloride, lactated Ringer's or fixed oils. A variety of
appropriate aqueous carriers are known to the skilled artisan,
including water, buffered saline, polyols (e.g., glycerol,
propylene glycol, liquid polyethylene glycol), dextrose solution
and glycine. Intravenous vehicles can include various additives,
preservatives, or fluid, nutrient or electrolyte replenishers (See,
generally, Remington's Pharmaceutical Science, 16th Edition, Mack,
Ed. 1980). The compositions can optionally contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering agents
and toxicity adjusting agents, for example, sodium acetate, sodium
chloride, potassium chloride, calcium chloride and sodium lactate.
The TL1a-binding protein of this disclosure can be lyophilized for
storage and reconstituted in a suitable carrier prior to use
according to art-known lyophilization and reconstitution
techniques.
[0413] The optimum concentration of the active ingredient(s) in the
chosen medium can be determined empirically, according to
procedures well known to the skilled artisan, and will depend on
the ultimate pharmaceutical formulation desired.
[0414] The dosage ranges for the administration of the TL1a-binding
protein of the disclosure are those large enough to produce the
desired effect. For example, the composition comprises a
therapeutically or prophylactically effective amount of the
TL1a-binding protein or nucleic acid encoding same or cell
expressing same.
[0415] As used herein, the term "effective amount" shall be taken
to mean a sufficient quantity of the TL1a-binding protein, nucleic
acid or cells to induce/increase or inhibit/reduce/prevent
signaling of TL1a in a subject. The skilled artisan will be aware
that such an amount will vary depending on, for example, the
TL1a-binding protein, nucleic acid or cells and/or the particular
subject and/or the type or severity of a condition being treated.
Accordingly, this term is not to be construed to limit the
disclosure to a specific quantity, e.g., weight or number of
TL1a-binding proteins, nucleic acids or cells.
[0416] As used herein, the term "therapeutically effective amount"
shall be taken to mean a sufficient quantity of TL1a-binding
protein, nucleic acid or cells to reduce or inhibit one or more
symptoms of a condition.
[0417] As used herein, the term "prophylactically effective amount"
shall be taken to mean a sufficient quantity of TL1a-binding
protein, nucleic acid or cells to prevent or inhibit or delay the
onset of one or more detectable symptoms of a condition.
[0418] The dosage should not be so large as to cause adverse side
effects, such as hyper viscosity syndromes, pulmonary edema,
congestive heart failure, and the like. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the
patient and can be determined by one of skill in the art. The
dosage can be adjusted by the individual physician in the event of
any complication. Dosage can vary from about 0.1 mg/kg to about 300
mg/kg, e.g., from about 0.2 mg/kg to about 200 mg/kg, such as, from
about 0.5 mg/kg to about 20 mg/kg, in one or more dose
administrations daily, for one or several days.
[0419] In one example, the TL1a-binding protein is administered at
a dosage of between about 1 mg/kg to about 15 mg/kg. In one
example, the TL1a-binding protein is administered at a dosage of
between about 2 mg/kg to about 10 mg/kg. In one example, the
TL1a-binding protein is administered subcutaneously or
intravenously.
[0420] In some examples, the TL1a-binding protein or other active
ingredient is administered at an initial (or loading) dose which is
higher than subsequent (maintenance doses). For example, the
binding molecule is administered at an initial dose of between
about 1 mg/kg to about 30 mg/kg. The binding molecule is then
administered at a maintenance dose of between about 0.0001 mg/kg to
about 1 mg/kg. The maintenance doses may be administered every 7 to
35 days, such as, every 14 or 21 or 28 days.
[0421] In some examples, a dose escalation regime is used, in which
a TL1a-binding protein or other active ingredient is initially
administered at a lower dose than used in subsequent doses. This
dosage regime is useful in the case of subject's initially
suffering adverse events
[0422] In the case of a subject that is not adequately responding
to treatment, multiple doses in a week may be administered.
Alternatively, or in addition, increasing doses may be
administered.
[0423] One or more TL1a-binding proteins of the disclosure can be
administered to an individual by an appropriate route, either alone
or in combination with (before, simultaneous with, or after)
another drug or agent. For example, the TL1a-binding protein of the
disclosure can also be used in combination with proteins, e.g., a
TNF antagonist, an anti-IL-12/23 antibody, an anti-inflammatory, a
corticosteroid, methotrexate or a painkiller. The TL1a-binding
protein of the disclosure can be used as separately administered
compositions given in conjunction with antibiotics and/or
antimicrobial agents.
[0424] It will be appreciated by those skilled in the art that the
TL1a-binding proteins of the disclosure may be introduced into a
subject by administering an expression construct of the disclosure
or a cell expressing a TL1a-binding protein of the disclosure. A
variety of methods can be used for introducing a nucleic acid
encoding the antibody into a target cell in vivo. For example, the
naked nucleic acid may be injected at the target site, may be
encapsulated into liposomes, or may be introduced by way of a viral
vector.
[0425] The following assays can be performed with a TL1a-binding
protein of the disclosure, e.g., a TL1a-binding protein conjugated
to a detectable label as discussed herein. Detection of TL1a with
an assay described herein is useful for diagnosing or prognosing a
condition.
[0426] An immunoassay is an exemplary assay format for diagnosing a
condition in a subject or detecting TL1a in a sample. The
disclosure contemplates any form of immunoassay, including Western
blotting, enzyme-linked immunosorbent assay (ELISA),
fluorescence-linked immunosorbent assay (FLISA), competition assay,
radioimmunoassay, lateral flow immunoassay, flow-through
immunoassay, electrochemiluminescent assay, nephelometric-based
assays, turbidometric-based assay, and fluorescence activated cell
sorting (FACS)-based assays.
[0427] One form of a suitable immunoassay is, for example, an ELISA
or FLISA.
[0428] In one form such an assay involves immobilizing a
TL1a-binding protein of the disclosure onto a solid matrix, such
as, for example a polystyrene or polycarbonate microwell or
dipstick, a membrane, or a glass support (e.g. a glass slide). A
test sample is then brought into direct contact with the
TL1a-binding protein and TL1a in the sample is bound or captured.
Following washing to remove any unbound protein in the sample, a
protein that binds to TL1a at a distinct epitope is brought into
direct contact with the captured TL1a. This detector protein is
generally labeled with a detectable reporter molecule, such as for
example, an enzyme (e.g. horseradish peroxidase (HRP), alkaline
phosphatase (AP) or .beta.-galactosidase) in the case of an ELISA
or a fluorophore in the case of a FLISA. Alternatively, a second
labeled protein can be used that binds to the detector protein.
Following washing to remove any unbound protein the detectable
reporter molecule is detected by the addition of a substrate in the
case of an ELISA, such as for example hydrogen peroxide, TMB, or
toluidine, or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside
(x-gal). Of course, the immobilized (capture) protein and the
detector protein may be used in the opposite manner.
[0429] The level of the antigen in the sample is then determined
using a standard curve that has been produced using known
quantities of the marker or by comparison to a control sample.
[0430] The assays described above are readily modified to use
chemiluminescence or electrochemiluminescence as the basis for
detection.
[0431] As will be apparent to the skilled artisan, other detection
methods based on an immunosorbent assay are useful in the
performance of the disclosure. For example, an immunosorbent method
based on the description supra using a radiolabel for detection, or
a gold label (e.g. colloidal gold) for detection, or a liposome,
for example, encapsulating NAD+ for detection or an acridinium
linked immunosorbent assay.
[0432] In some examples of the disclosure, the level of TL1a is
determined using a surface plasmon resonance detector (e.g.,
BIAcore.TM., GE Healthcare, Piscataway, N.J.), a flow through
device, for example, as described in U.S. Pat. No. 7,205,159; a
micro- or nano-immunoassay device (e.g., as described in
US20030124619); a lateral flow devices (e.g., as described in
US20040228761 or US20040265926); a fluorescence polarization
immunoassay (FPIA e.g., as described in U.S. Pat. No. 4,593,089 or
U.S. Pat. No. 4,751,190); or an immunoturbidimetric assay (e.g., as
described in U.S. Pat. No. 5,571,728 or U.S. Pat. No.
6,248,597).
Samples and Control Samples
[0433] As will be apparent to the skilled artisan, some of the
examples described herein require some degree of quantification to
determine the level of TL1a. Such quantification may be determined
by the inclusion of a suitable control sample in an assay of the
disclosure.
[0434] In one example, a suitable control sample is a sample that
is derived from a healthy subject or a normal subject.
[0435] In the present context, the term "healthy subject" shall be
taken to mean an individual who is known not to suffer from a
condition associated with TL1a, e.g., an inflammatory
condition.
[0436] The term "normal subject" shall be taken to mean an
individual having a normal level of TL1a in a sample compared to a
population of individuals.
[0437] The disclosure also contemplates the control sample as being
a data set obtained from a normal and/or healthy subject or a
population of normal and/or healthy subjects.
[0438] In one example, a method of the disclosure additionally
comprises determining the level of TL1a in a control sample, e.g.,
using a method described herein.
[0439] In one example, a sample from the subject and a control
sample are assayed at approximately or substantially the same
time.
[0440] In one example, the sample from the subject and the control
sample are assayed using the same method of the disclosure as
described herein in any one or more examples to allow for
comparison of results.
[0441] Exemplary conditions that can be
treated/prevented/diagnosed/prognosed by performing a method of the
disclosure include autoimmune diseases, inflammatory conditions,
and conditions characterized by insufficient angiogenesis.
[0442] In one example, the condition is an autoimmune disease.
[0443] Exemplary conditions include inflammatory bowel disease
(IBD), irritable bowel syndrome (IBS), Crohn's disease, ulcerative
colitis, diverticular disease, systemic lupus erythematosus,
rheumatoid arthritis, juvenile chronic arthritis,
spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic
inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's
syndrome, systemic vasculitis, sarcoidosis, demyelinating diseases
of the central and peripheral nervous systems such as multiple
sclerosis, idiopathic polyneuropathy, autoimmune or immune-mediated
eye disease such as autoimmune uveitis and uveitis associated with
various vasculitides, autoimmune or immune-mediated skin diseases
including bullous skin diseases, erythema multiforme and contact
dermatitis, psoriasis, allergic diseases of the lung such as
asthma, airway hypersensitivity, eosinophilic pneumonia, chronic
obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis
and hypersensitivity pneumonitis, atherosclerosis or graft versus
host disease.
[0444] In one example, the condition is arthritis, e.g., rheumatoid
arthritis, polyarthritis, osteoarthritis or a spondyloarthropathy.
In this regard, Bull et al., J. Exp. Med. 205: 2457, 2008 have
shown that antibodies that antagonize TL1a are useful for the
treatment of rheumatoid arthritis. The selective nature of the
TL1a-binding proteins of the disclosure make them useful for
treating rheumatoid arthritis.
[0445] In one example, the condition is multiple sclerosis. In this
regard, US20090317388 shows that mice deficient in TL1a do not
develop experimental autoimmune encephalomyelitis (EAE), which is
an accepted model of multiple sclerosis.
[0446] In one example, the inflammatory condition is an
inflammatory mucosal condition, e.g., an inflammatory disease of
the bowel (e.g., inflammatory bowel disease, Crohn's disease or
ulcerative colitis), or an inflammatory disease of the lung (e.g.,
airway hyperreactivity or asthma).
[0447] In one example, the condition is inflammatory bowel disease
and/or colitis. In this regard, Takedatsu et al., Gastroenterology
135: 552, 2008 have shown that antibodies that antagonize TL1a are
useful for the treatment of colitis.
[0448] In one example, the condition is asthma or airway
hypersensitivity or chronic obstructive pulmonary disease
(COPD).
[0449] In another example, the condition is an inflammatory skin
disease (e.g., an autoimmune or immune-mediated skin disease),
e.g., a bullous skin diseases, erythema multiforme, contact
dermatitis. Alternatively, the skin disease is psoriasis.
[0450] Exemplary conditions characterized by insufficient
angiogenesis include cardiovascular disease, autoimmune conditions
(e.g., rheumatoid arthritis, psoriatic arthritis, systemic lupus
erythematosus (SLE) and systemic sclerosis), antineutrophil
cytoplasmic antibodies (ANCA)-associated vasculitis, ischemia
(including ischemia resulting from a transplant) or necrosis.
[0451] The disclosure additionally comprises a kit comprising one
or more of the following:
(i) a TL1a-binding protein of the disclosure or expression
construct(s) encoding same; (ii) a cell of the disclosure; or (iii)
a pharmaceutical composition of the disclosure.
[0452] In the case of a kit for detecting TL1a, the kit can
additionally comprise a detection means, e.g., linked to a
TL1a-binding protein of the disclosure.
[0453] In the case of a kit for therapeutic/prophylactic use, the
kit can additionally comprise a pharmaceutically acceptable
carrier.
[0454] Optionally a kit of the disclosure is packaged with
instructions for use in a method described herein according to any
example.
[0455] The disclosure includes the following non-limiting
Examples.
Example 1
Materials and Methods
[0456] In the following examples, reference to a position of a
residue is a reference to the position in the relevant sequence as
set forth herein, unless otherwise indicated.
1.1 HEK293/pTT5 Expression System
[0457] For all transfections involving the HEK293E/pTT5 expression
system (Durocher et al., Nucl. Acids Res., 30: E9, 2002), HEK293E
cells were cultured in complete cell growth media (1 L of F17
medium (Invitrogen), 9 ml of Pluronic F68 (Invitrogen), 2 mM
Glutamine containing 20% (w/v) Tryptone NI (Organotechnie) with
Geneticin.TM. (50 mg/ml, Invitrogen) at 50 .mu.l/100 ml culture).
At the day before transfection, the cells were harvested by
centrifugation and resuspended in fresh media without
Geneticin.TM.. The next day, DNA was mixed with a commercial
transfection reagent and the DNA transfection mix added to the
culture drop-wise. The culture was incubated overnight at
37.degree. C., 5% CO.sub.2 and 120 rpm without Geneticin.TM.. The
next day 12.5 ml of Tryptone and 250 .mu.l of Geneticin.TM. were
added per 500 ml culture. The culture was incubated at 37.degree.
C., 5% CO.sub.2 and 120 rpm for seven days, then the supernatants
were harvested and purified.
1.2 TL1a Protein
[0458] Human TL1a was purchased (Peprotech and Genscript: both E.
coli-expressed) or produced in the mammalian HEK293E/pTT5
expression system, using a DNA expression construct coding for the
extracellular domain (ECD) of human TL1a with an N-terminally
located HIS and FLAG tag (SEQ ID NO: 1). Culture supernatant
containing the secreted TL1a protein was harvested by
centrifugation at 2000 g for 10 mins to remove the cells. The TL1a
protein was purified from the supernatant using a HisTrap.TM. HP
column (GE Healthcare). The eluted protein was buffer-exchanged
into PBS using a HiLoad 16/60 Superdex 200 prep grade column (GE
Healthcare) and .sup..about.70 kDa fraction was separated by gel
filtration on a HiLoad 26/60 Superdex 200 prep grade column (GE
Healthcare).
[0459] For phage display experiments, recombinant human TL1a
(Peprotech, Genscript HEK293E-derived) was biotinylated using an
EZ-link Sulfo-NHS-LC-biotin kit (Pierce) at a 3:1 ratio of
biotin:TL1a. Free biotin was removed from the protein preparation
by dialysis against PBS using a Slide-A-Lyzer dialysis cassette
with a 3.5 kDa molecular weight cut-off.
[0460] TL1a was also produced with a tag that allowed for single
site biotinylation. This TL1a protein was expressed in HEK-293
cells and purified as described previously. It was then
biotinylated using a enzyme that selectively incorporates a biotin
on the TL1a protein.
1.3 Phage Display
[0461] Antibodies that bind specifically to TL1a were isolated from
a naive phagemid library comprising more than 10.times.10.sup.10
individual human FAb fragments.
[0462] Anti-TL1a antibodies were isolated from the phage display
library over the course of several panning `campaigns` (i.e.
discrete phage display experiments with different reagents or
panning conditions). The general protocol followed the method
outlined by Marks and Bradbury (Methods Mol. Biol. 248: 161-176,
2004)
[0463] Each phage display campaign involved three rounds of
panning. For each round, .sup..about.1.times.10.sup.13 phage
particles were blocked by mixing 1:1 with blocking buffer (5% skim
milk in phosphate buffered saline (PBS) pH 7.4) and incubating for
1 hr at room temperature. The blocked phage library was then
pre-depleted for streptavidin binders by incubation for 45 mins
with 1004 of streptavidin-coupled Dynabeads (Invitrogen), which
were blocked as described for the library. The beads (and
streptavidin binders attached to them) were discarded after the
incubation step.
[0464] Recombinant human TL1a antigen was prepared for panning by
capture onto the surface of streptavidin-coupled Dynabeads
(Invitrogen). To achieve this, 10 to 100 pmol of biotinlyated TL1a
was incubated with 100 .mu.L of beads for 45 mins at room
temperature. The resulting TL1a-bead complexes were washed with PBS
to remove free TL1a and then used in the subsequent panning
reaction.
[0465] Library panning was conducted by mixing the blocked and
pre-depleted library with the TL1a-bead complexes in a 1.5 mL
microcentrifuge tube and rotating for 2 hrs at room temperature.
Non-specifically bound phage were removed using a series of washes.
Each wash involved pulling the bead complexes from the solution
onto the tube wall using a magnetic rack, aspirating the
supernatant and then re-suspending the beads in fresh wash buffer.
This was repeated a number of times with either PBS wash buffer
(PBS with 0.5% skim milk) or PBS-T wash buffer (PBS with 0.05%
TWEEN-20 (Sigma) and 0.5% skim milk). Phage that remained bound
after the washing process were eluted from the TL1a-bead complexes
by incubation with 0.5 mL of 100 mM triethylamine (TEA) (Merck) for
20 mins at room temperature. The eluted `output` phage were
neutralized by adding 0.25 mL of 1 M Tris-HCl pH 7.4 (Sigma).
[0466] At the end of the first and second rounds of panning, the
output phage were added to a 10 mL culture of exponentially growing
TG1 E. coli (yeast-tryptone (YT) growth media) and allowed to
infect the cells by incubating for 30 mins at 37.degree. C. without
shaking, then with shaking at 250 rpm for 30 mins. The phagemids
encoding the phage display output were then rescued as phage
particles following a standard protocol (Marks and Bradbury,
supra).
[0467] At the end of the third panning round TG1 cells were
infected with output phage, but the cells were plated on solid YT
growth media (supplemented with 2% glucose and 100 .mu.g/mL
carbenicillin) at a sufficient dilution to produce discrete E. coli
colonies. These colonies were used to inoculate 1 mL liquid
cultures to allow expression of FAb fragments for use in screening
experiments.
1.4 SPR-Based Screening of FAbs for TL1a Binding
[0468] Each individual E. coli colony was used to express a FAb
that could be screened for TL1a binding activity. Colonies were
inoculated into 1 mL YT starter cultures (supplemented with 100
.mu.g/mL carbenicillin and 2% glucose) in 96-well deepwell plates
(Costar) and incubated overnight at 30.degree. C. with shaking at
650 rpm. These starter cultures were diluted 1:50 into a 1 mL
expression culture (YT supplemented with 100 .mu.g/mL carbenicillin
only) and grown to an optical density of 0.8 to 1.0 at 600 nm. FAb
expression was induced by adding
isopropyl-beta-D-thiogalactopyranoside to a final concentration of
1 mM. Cultures were incubated at 20.degree. C. for 16 hrs.
[0469] FAb samples were prepared by harvesting cells by
centrifugation (2500 g, 10 mins) and performing a periplasmic
extraction. The cell pellet was resuspended in 75 .mu.L of
extraction buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA, 20% Sucrose)
and shaken at 1000 rpm for 10 mins at 4.degree. C. Extract
preparation was completed by adding 225 .mu.L of H.sub.2O, shaking
at 1000 rpm for 1 hr and clearing the extract by centrifugation at
2500 g for 10 mins. The supernatants were recovered, filtered
through Acroprep 100 kDa molecular-weight cutoff plates (Pall
Corporation) and stored at 4.degree. C. until required for further
experiments.
[0470] The FAb samples were screened for TL1a-binding activity
using a surface plasmon resonance (SPR) assay. High-throughput SPR
screening was conducted using a BIAcore 4000 Biosensor (GE
Healthcare) in a single concentration analyte pass assay.
Approximately 10,000 RU of antiV5 antibody (Invitrogen cat#R960CUS)
was immobilized on a CM5 Series S Sensor chip, using standard amine
coupling chemistry at pH 5.5 on spots 1, 2, 4 & 5 of each of
the four flow cells leaving spot 3 unmodified. The running buffer
used was HBS-EP+ (GE Healthcare) and all interactions measured at
25.degree. C. and data collection rate set to 10 Hz. Crude
periplasmic preparations of V5-tagged FAbs, were diluted two-fold
in running buffer before capturing at a flow rate of 10 ul/min for
100 sec (typically around 200RU of FAb was captured) on spot 1 or 5
of each flow cell. Following a short stabilization period, human
TL1a trimer was passed over all spots of all four flow cells
simultaneously at a flow rate of 30 .mu.l/min for 100 sec.
Dissociation of the interaction was measured for 100 sec prior to
regeneration back to the antiV5 antibody using a 30 sec pulse of
100 mM phosphoric acid. Generated sensorgrams were referenced
against an adjacent antiV5 antibody spot for each flow cell, and
fitted using a 1:1 Langmuir equation to determine k.sub.a, k.sub.d
and K.sub.D.
[0471] The data from the SPR screening process was used to select
potential TL1a-binders. FAb k.sub.d values were ranked and up to
200 of the strongest binders were submitted for DNA sequence
analysis. FAb with unique sequences were selected for conversion to
full-length human IgG.sub.1 antibodies.
1.5 Variable Region Sequencing
[0472] DNA sequencing was conducted by the Applied Genetic
Diagnostics group at the Melbourne University Department of
Pathology (Melbourne, Australia). Phagemid DNA (.sup..about.500 ng)
was mixed with 5 pmols of the appropriate primer for sequencing
either the Fab V.sub.H or V.sub.L chain. Sequencing reactions were
conducted using a BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems) according to manufacturer's instructions.
Samples were analyzed by capillary separation on 3130xl Genetic
Analyzers (Applied Biosystems). Sequence chromatogram data was
analyzed using the Chromas Lite software package (Technelysium,
Brisbane, QLD, Australia). Sequence text files were translated to
amino acid sequences and analyzed using the SeqAgent software
package (Xoma, Berkely, Calif., USA).
1.6 Construction of Vectors Expressing Antibodies
[0473] V.sub.H amino acid chains were expressed with a human
constant region (human IgG1 heavy chain C.sub.H1, hinge, C.sub.H2
and C.sub.H3 domains (e.g., SwissProt No. P01857)). This was
achieved by back-translation of amino acid sequences into DNA
sequences which were synthesized de novo by assembly of synthetic
oligonucleotides. Following gene synthesis the whole sequence was
subcloned into the multiple cloning site of the pTT5 heavy chain
vector (Durocher et al., Nucl. Acids Res. 30: E9, 2002). V.sub.L
amino acid chains were expressed with a human kappa light chain
constant region (SwissProt No. P01834.1) or a human lambda light
chain constant region (SwissProt No. P0CG05.1) by subcloning the
sequence into the multiple cloning site of the pTT5 light chain
vector.
[0474] Alternatively, FAb V.sub.H and V.sub.L sequences were
amplified directly from phagemid DNA by PCR and subcloned into IgG
expression vectors. PCR reactions were carried out using a Platinum
PCR Supermix kit (Invitrogen) as per manufacturer's instructions.
Approximately 50 ng of template phagemid DNA encoding a FAb was
mixed with 10 pmols of the appropriate forward and reverse PCR
primers and the PCR Supermix reagent to a final volume of 50 .mu.L.
Each reaction used a sequence-specific forward primer combined with
a generic V.sub.H or V.sub.L specific reverse primer. To facilitate
cloning into IgG expression vectors, primers for V.sub.H
amplification were designed to add 5' BsiWI and 3' NheI restriction
enzyme sites, primers for V.sub.L-kappa amplification added 5'
BssHII and 3' BsiWI sites and primers for V.sub.L-lambda
amplification added 5' BssHII and 3' AvrII sites.
[0475] PCRs were carried out in thin-walled PCR (96-well
GENEAMP.RTM. PCR system 9700, Applied Biosystems, Scoresby,
Victoria, Australia). Cycling conditions comprised an initial five
minute denaturation step at 94.degree. C., followed by 30
amplification cycles (denaturation at 94.degree. C. for 30 seconds,
followed by primer annealing at 55.degree. C. for 30 seconds, then
extension at 68.degree. C. for 30 to 90 seconds), and a final
extension step at 68.degree. C. for seven minutes. The amplified
V.sub.H genes were purified using a Minelute PCR purification kit
(Qiagen), digested with BsiWI and NheI enzymes (New England
Biolabs) and re-purified using a Minelute Reaction Clean-up kit
(Qiagen). Amplified V.sub.L-kappa genes were prepared similarly,
but digestion was carried out using BssHII and BsiWI. Amplified
V.sub.L-lambda genes were also prepared in the same way, but
digested with BssHII and AvrII. The resulting V.sub.H and V.sub.L
gene fragments were cloned into the multicloning site of the
appropriate pTT5 heavy or light chain vector (either kappa or
lambda light chain), as described above.
1.7 Expression and Purification of Antibodies
[0476] Heavy and light chain DNA were co-transfected into the
HEK293/pTT5 expression system and cultured for seven days. The
supernatants derived from these transfections were adjusted to pH
7.4 before being loaded onto a HiTrap Protein A column (5 ml, GE
Healthcare). The column was washed with 50 ml of PBS (pH 7.4).
Elution was performed using 0.1M citric acid pH 2.5. The eluted
antibody was desalted using Zeba Desalting columns (Pierce) into
PBS (pH 7.4). The antibody was analyzed using SDS-PAGE. The
concentration of the antibody was determined using a BCA assay kit
(Pierce). For conversion between antibody concentrations in
.mu.g/ml and molar concentrations an assumed molecular weight of
150 kDa was used for all antibodies.
1.8 TF-1 Cell Line Potency Assay
[0477] To determine which anti-TL1a antibodies functionally
neutralize the biological activity of TL1a, antibodies were tested
for their ability to neutralize TL1a-induced apoptosis in a TF-1
cell line. The TF-1 human erythroleukemic cell line (ATCC:
CRL-2003) was maintained in culture under standard conditions. TF-1
cells (7.5.times.10.sup.4/well) were incubated in black-sided
96-well plates (Greiner) with recombinant human TL1a 100 ng/ml and
cycloheximide 10 .mu.m/ml to induce apoptosis. Test antibodies at a
concentration of 10 .mu.g/mL (66.7 nM) or less were added to the
plates and incubated for 4 to 5 hours. Induction of apoptosis was
then assessed using the Homogeneous Caspases Kit (Roche) according
to manufacturer's instructions. In experiments to test the ability
of antibodies to neutralize function of TL1a from cynomolgus and
rhesus macaque, mouse, rat, guinea pig, pig or rabbit, the
appropriate species TL1a was substituted for human TL1a in this
protocol.
[0478] Data were normalized by expression as a percentage of
maximum apoptosis (apoptosis levels achieved by recombinant human
TL1a plus cycloheximide in the absence of anti-TL1a antibody).
1.9 Receptor Selectivity of Lead Antibodies
[0479] TL1a binds both to its cognate signaling receptor, DR3, and
to a decoy receptor, DcR3, which also serves as a decoy receptor
for TNF family members Fas-L and LIGHT.
[0480] Antibodies were assessed for their ability to inhibit
binding of TL1a to its receptors in a competition ELISA. DR3/Fc
Chimera (R&D Systems) or DcR3/Fc Chimera (R&D Systems) was
coated onto a 96-well plate (Maxisorp, Nunc) at a concentration of
2 .mu.g/ml. Serially diluted test antibodies were pre-incubated
with single-site biotinylated recombinant human TL1a 1 .mu.g/ml for
30 minutes then added to the DR3/Fc or DcR3/Fc coated wells. Bound
TL1a was detected using streptavidin-horseradish peroxidase 1:2000
(BD Pharmingen). Data were normalized by expression as a percentage
of maximum binding of TL1a to receptor in the absence of anti-TL1a
antibody.
[0481] An antibody described to inhibit TL1a activity (1B4; which
has a V.sub.H comprising a sequence set forth in SEQ ID NO: 119 and
a V.sub.L comprising a sequence set forth in SEQ ID NO: 120, which
is described in WO 2009/064854 and US patent application
publication US 200900280116) was included in assays for
comparison
1.10 Epitope Mapping
[0482] Epitope mapping was performed using alanine scanning
experiments. Modeling analysis was carried out to determine
probable exposed residues on TL1a. TL1a constructs were then
designed in which each of these theoretically exposed residues was
substituted with an alanine. These constructs were then expressed
and supernatant from the expression cultures tested for protein
expression and binding to anti-TL1a antibody C320 using SPR. A
Ni-NTA sensor chip (Biacore) was used to capture the HIS tagged
TL1a mutein from the supernatant of transfected HEK-293E cells. The
mutein was captured on flow cell 2 (or 4) and then C320 was passed
over flow cell 1+2 (or 3+4). The mutein expression (RU) was
determined as the change in RU at the end of injection of the
mutein. The C320 binding (RU) was determined as the change in RU
determined at the end of the C320 injection.
[0483] TL1a constructs that expressed but had apparently lost
binding to C320 were re-transfected and purified for testing by
ELISA. ELISA plates were coated with polyclonal rabbit anti-human
TL1a (Peprotech) (1 .mu.g/ml), DR3/Fc chimera (R&D systems) (2
.mu.g/ml) or DcR3/Fc chimera (R&D systems) (1 .mu.g/ml). TL1a
muteins or unsubstituted TL1a were added to the plates at a
concentration of 1 .mu.g/ml. Bound TL1a was then detected with
biotinylated polyclonal rabbit anti-human TL1a (Peprotech) (250
ng/ml) and streptavidin-horseradish peroxidase (BD Pharmingen)
1:2000. Constructs that could be detected by polyclonal anti-TL1a
were deemed to have expressed and folded appropriately.
Appropriately expressed and folded constructs that did not bind to
one or either of the receptors were considered to be important for
TL1a binding to that receptor. Binding of test antibodies to TL1a
muteins was tested by direct ELISA. ELISA plates were coated with
different TL1a muteins (1 .mu.g/ml). Serially diluted test
antibodies were then added, and bound antibodies were detected with
anti-human IgG-horseradish peroxidase (Invitrogen) 1:2000. Binding
of test antibodies to different TL1a isoforms was detected using
the same method.
1.11 Creation of Membrane Bound TL1a (mbTL1a)-Expressing Cell
Line
[0484] HEK 293 cells were electroporated with a vector containing a
sequence encoding full length TL1a (SEQ ID NO: 123) and maintained
in selective media (media supplemented with blasticidin 6
.mu.g/ml). After multiple passages, the cells were tested for cell
surface expression of TL1a by flow cytometry. 2.5.times.10.sup.5
cells per well were plated into 96-well round bottom plates
(Corning) and incubated with biotinylated polyclonal rabbit
anti-human TL1a (Peprotech) on ice for 30 minutes. Samples were
washed then incubated with streptavidin-FITC for a further 30
minutes on ice. Samples were then washed, resuspended and data
acquired on a flow cytometer.
1.12 Flow Cytometry Detection
[0485] After stable cell surface expression of TL1a had been
confirmed (as described in Example 1.10), antibodies C320, C321 and
C323 were screened using both transfected and untransfected cells
(2.5.times.10.sup.5 cells per well in 96-well round bottom plates
(Corning)) at decreasing concentrations starting from 10 .mu.g/ml.
Polyclonal goat anti-human IgG-FITC (Sigma) at a dilution of 1:200
was used as detection antibody. Anti-TL1a antibody 1B4 was included
for comparison.
1.13 Inhibition of Cytokine Production
[0486] To further characterize antibodies described herein, their
function on primary human cells was tested. To confirm endogenous
TL1a production, PBMCs were isolated from buffy coats over a
lymphoprep (Nycomed) gradient and cultured in 96-well tissue
culture plates (Corning) with Concanavalin A at decreasing
concentrations starting from 2 .mu.g/ml. Plates were incubated
overnight then supernatants were harvested and assayed for TL1a
using the human TL1a ELISA kit (Peprotech).
[0487] Additionally, adherent cells were harvested and assessed for
TL1a expression by flow cytometry using biotinylated anti-human
TL1a (Peprotech) 1:100 and streptavidin-FITC (Zymed) 1:200.
1.14 Isoelectric Focusing Gel Experiments
[0488] Isoelectric focusing gels were performed using the
NOVEX.RTM. Xcell Surelock.TM. system (Life Technologies) according
to manufacturer's instructions.
1.15 Protein A HPLC
[0489] Supernatants from HEK-293E cells transfected to transiently
express antibodies were analysed by Protein A HPLC using a
POROS.RTM. A/20 2.1.times.30 mm Id column (Applied Biosystems)
connected to an Agilent 1100 chromatography system. The column was
equilibrated with phosphate buffered saline (PBS) pH7.4, 0.2 ml of
HEK-293E supernatant containing protein was loaded and the protein
eluted with PBS adjusted to pH 2.2. The chromatograms at the
wavelengths of 215 nm or 280 nm were integrated using the
manufacturer's software and the area under the curve (AUC)
reported.
1.16 Antibody Expression and Antigen Binding as Determined by
SPR
[0490] Using a CM5 sensor chip (Biacore.TM.) Protein A was coupled
to the chip surface using amine coupling. Protein A was coupled on
flow cell 1 and 2 (or alternatively 3 and 4) using a Biacore 3000.
Cell supernatant for HEK-293 cells containing antibody were passed
over the surface of flow cell 2, while buffer (HBS-EP) was passed
over flow cell 1. At the end of injection of the supernatant the
change in response units was measured. This value is reported as
Protein A capture (SPR). The % expression is the Protein A capture
(SPR) of the tested antibody as a percentage of the Protein A
capture (SPR) of C320 in the same experiment. To determine if the
antibody binds TL1a, the TL1a was then passed over the flow cell 1
and 2. The sensorgrams were double referenced (Flow cell 2 is
subtracted from flow cell 1 and a buffer blank).
Example 2
Results of Phage Display
[0491] Phage display campaigns were conducted against recombinant
human TL1a. Six discreet campaigns were conducted using recombinant
bacterial-expressed recombinant TL1a. A low number of FAbs were
isolated that bound to TL1a, with no neutralizing antibodies
isolated.
[0492] Subsequent campaigns were conducted with mammalian-expressed
TL1a. The percentage of FAbs isolated that bound TL1a was
substantially higher using mammalian derived TL1a than bacterially
derived TL1a. The number of FAbs that were shown to be positive for
TL1a binding across the total phage display campaigns was in excess
of 200. From these, 55 FAbs with unique sequences were identified,
of which 29 were selected for conversion to full length IgG.sub.1
antibody.
Example 3
Neutralization of TL1a Activity
[0493] The ability of full length IgG1 antibodies comprising FAbs
isolated using phage display to inhibit or reduce TL1a-induced
apoptosis was assessed as described above (Example 1.8). Under
these conditions, 15/29 of the antibodies tested showed better than
50% inhibition of TL1a-induced apoptosis (FIG. 2). These antibodies
included: C319, C320, C321, C323, C333, C334, C335, C336.
[0494] These data demonstrate that while antibodies can be isolated
that bind to TL1a, only a limited subset of these antibodies have
the specificity required to functionally inhibit TL1a activity.
[0495] Within the group of antibodies that demonstrated inhibition
of recombinant human TL1a, the relative inhibition profile of each
antibody was assessed using the EC.sub.50 value (Table 4 and FIG.
3). Only antibodies C320, C321 and C323 had an inhibitory EC.sub.50
value of 1 nM or below.
TABLE-US-00004 TABLE 4 Antibody EC.sub.50 values for inhibition of
TL1a-induced apoptosis Variable Variable Antibody Heavy Chain Light
Chain EC.sub.50 Designation (SEQ ID NO) (SEQ ID NO) (nM) C336 2 6
4.05 C334 10 14 22.14 C333 18 22 2.34 C323 26 30 0.99 C321 34 38
0.56 C320 42 46 0.31 C319 50 54 11.17 1B4 74 75 3.37
Example 4
Receptor Selectivity
[0496] Antibodies C320, C321 and C323 inhibit interaction of TL1a
with DR3 (FIG. 4A) but do not inhibit interaction of TL1a with DcR3
(FIG. 4B). Using the same assay, antibodies C319, C333, C334 and
C336 were shown to demonstrate the same selective
neutralization.
[0497] Using a similar assay the binding of antibodies C320 and
derivatives thereof described below, particularly C320-168 and
C320-179, were shown to inhibit interaction of TL1a with DR3 (FIG.
4C) with an EC.sub.50 1 nM or less. These antibodies also did not
inhibit interaction of TL1a with DcR3 (FIG. 4D) when tested at
concentrations ranging from 0.1 .mu.g/mL to 100 .mu.g/mL. These
results contrast with those of antibodies C300-25 and 1B4. In this
regard, antibody C300-25 is a rat monoclonal antibody produced by
the inventors.
TABLE-US-00005 TABLE 5 Antibody EC.sub.50 values for inhibition of
TL1a interaction with receptors DR3 and DcR3 EC.sub.50 (nM)
Antibody DR3 DcR3 C320 0.97 DNI C320-179 0.72 DNI C320-168 0.97 DNI
C300-25 13.5 9.84 1B4 17.8 26.5 DNI--did not inhibit
[0498] DR3 and DcR3 have been shown to compete for binding to TL1a.
Therefore it was unexpected that antibodies have been isolated with
specificity, targeting an epitope on TL1a that is neutralizing for
DR3, but not DcR3. These results indicate that antibodies described
herein are capable of preventing TL1a activity through its cognate
receptor DR3 without disturbing either the natural antagonistic
function of DcR3 or a homeostatic balance of DcR3 binding. Without
being bound by any theory or mode of action, such selective
inhibition may be biologically relevant because DcR3 regulates the
amount of free Fas-L and LIGHT available for binding to their
cognate signaling receptors (Fas and H-VEM, respectively).
Consequently, inhibiting the interaction between TL1a and DcR3
could increase the amount of Fas-L and LIGHT bound by DcR3 thus
decreasing the amount available for signaling through their cognate
receptors. As Fas-mediated killing plays a role in cancer
surveillance, potential downstream consequences of increasing the
amount of DcR3 to bind to Fas-L could include increased
susceptibility to cancer. Again, without being bound by theory or
mode of action, isolating antibodies that specifically inhibit
interaction of TL1a and DR3, but not DcR3, could be advantageous in
treating disease but without compromising safety.
Example 5
Epitope Mapping
[0499] Amino acid substitutions were introduced into the sequence
of soluble TL1a (SEQ ID NO: 202) to generate a series of TL1a
muteins.
[0500] Using the SPR-based mutein analysis assay described in
Example 1.10, TL1a muteins in supernatants from transfected
HEK-293E cells were tested for expression levels and binding to
C320. The results are presented in Table 6.
TABLE-US-00006 TABLE 6 Expression and binding of soluble human TL1a
and muteins thereof to antibody C320 Amino Mutein C320 Amino Mutein
C320 acid Expres- Bind- acid Expres- Bind- substi- sion ing substi-
sion ing tution (RU) (RU) tution (RU) (RU) Wild Type 3244.9 125.6
A55S 2933.1 33.2 L1A 3264.3 115 A55L 2746.5 -7.6 K2A 3094.4 152
A55R 2307.7 -12.3 Q4A 3366.2 120 A55G 1991.2 -5.8 E5A 3086.5 360.4
A55D 2386.5 -21.1 F6A 2948.3 240.2 T57A 3335.6 42.7 P8A 3455.9
124.3 K58A 3241.5 4.3 S9A 3594.2 94.5 N59A 3382.9 73.8 H10A 3372.5
122.7 R60A 3405.8 116.5 Q11A 3572.9 71.4 N62A 3264.9 117.4 Q12A
3098.5 193.8 T64A 3083.6 289.8 V13A 3213.9 167.8 N65A 3226.8 156.6
Y14A 2883.5 401 K66A 3183.3 27.1 P16A 2938 225 F67A 3216.1 304.7
L17A 2904.1 194.5 L69A 3389.8 212.6 R18A 3102.5 132.2 E72A 3171.5
136.2 D20A 3247.6 154.6 S73A 3129.3 146 G21S 2915.3 184.8 R85A
3047.1 -33.5 G21L 3424.4 72.8 M87A 3161.2 46.1 G21R 3197 116.2 S89A
3600.3 10.8 G21A 3483 85 E90A 3129.7 1089.1 G21D 3032 158.6 E93A
3181.6 343.9 D22A 3185.6 169.8 I94A 3125.1 352.3 R32A 3103.7 -36.5
R95A 3634.8 28.8 T34A 3278.7 389.2 Q96A 3256.5 149.4 P35A 3385
-28.6 R99A 3351.3 105.2 T36A 2771.9 486.1 P100A 2898.5 199.5 Q37A
2789.3 319.2 K102A 3420.8 155.7 H38A 3133 147.4 D104A 2982.9 533.2
F39A 2509.8 445.5 S105A 3425.8 127.1 K40A 3213.5 55.5 D115A 3648.5
110.1 N41A 2967.5 248.5 S116A 2962.4 282.1 Q42A 3073 175.7 Y117A
3323.8 189.8 F43A 3245.8 109.3 P118A 3254.9 161.7 P44A 3180.1 200.4
E119A 3263.7 278.5 A45S 3407.3 61.5 P120A 3143 164.8 A45L 3087.7
35.6 Q122A 3189 148.5 A45R 3167 107.2 S135A 3109.4 95.4 A45G 3385.9
77.1 F138A 3496.7 21 A45D 3395.1 38.9 S148A 2883.9 228.7 H47A
3466.4 159.1 Q150A 3151.1 193.5 H50A 3249.3 132.4 E151A 3796.5 90.8
E51A 3136.5 33.8 K154A 3610.3 86.7 L52A 3105.5 52.7 S160A 1272.2
901.6 G53S 3238.5 19 D161A 3391.4 223.4 G53L 1299.7 -3.9 I162A
3139.2 72.4 G53R 3796.7 7.2 S163A 3054 481 G53A 3652.4 -4.8 Y167A
2942.2 70.7 G53D 2130.3 -6.1 T168A 3335.1 2.6 K169A 3005 69.7 E170A
3418.7 16
[0501] Muteins that expressed well (Mutein expression greater than
2000 RU) but failed to bind antibody C320 (TL1a binding less than
17 RU) were selected for further analysis.
[0502] Muteins with substitutions at amino acids G53 and A55
expressed poorly and had reduced binding to polyclonal anti-TL1a,
suggesting that these are important residues for TL1a structural
integrity. The other muteins all expressed adequately, bound
polyclonal anti-TL1a and ran equivalently to wild-type TL1a on both
reducing and non-reducing SDS-PAGE gels. These characteristics all
suggest that the muteins were appropriately expressed and folded
into TL1a proteins.
[0503] The muteins exhibited differential binding patterns to the
different monoclonal anti-TL1a antibodies described herein.
Antibodies C320, C320-168 and C320-179 did not detectably bind to
the R32A and R85A muteins or to a mutein containing both of these
substitutions (FIGS. 5A and B). C320 also showed negligible binding
to the P35A, T168A and E170A muteins while C320-168 and C320-179
had reduced binding to these muteins but not to the same extent as
C320. In contrast, TL1a-binding antibodies 1B4 and 16H2 showed
binding to the muteins R32A, R85A, P35A, T168A at greater than 50%
of the wild-type TL1a, indicating that amino acids at these
positions are not crucial to the binding of 1B4 and 16H2 to
TL1a.
[0504] Given that the antibodies C320, C320-168 and C320-179
inhibit the binding of TL1a to DR3 but not DcR3, the antibodies are
likely bind to amino acids involved in the binding of DR3 to TL1a
and disrupt this interaction. DR3, but not DcR3, had significantly
decreased binding to the R32A and R85A muteins, suggesting that
these residues are involved with TL1a binding to DR3 (FIG. 5C).
[0505] The binding site of the antibodies C320, C320-168 and
C320-179 on TL1a is illustrated in FIG. 5D. As can be seen from the
diagram, the residues R32 and R85, while being distant in primary
amino acid sequence are located adjacent to each other in the
protein's tertiary structure. Hence, these can be considered key
residues within the epitope on TL1a to which the antibodies C320,
C320-168 and C320-179 bind.
Example 6
Characterization of Antibodies Binding to Cell-Surface TL1a
[0506] TL1a, like most TNF-ligand superfamily members, is present
as a cell surface form that is cleaved to generate soluble TL1a and
as for other members the membrane form of TL1 (mbTL1a) has been
shown to have biological function. Therefore to neutralize the
activity of TL1a it would be advantageous to have antibodies that
bind strongly to both membrane and soluble TL1a. Antibodies that
displayed functional inhibition of TL1a were screened by flow
cytometry on a human cell line transfected stably with a membrane
anchored TL1a (produced as described in Example 1.11).
[0507] Antibodies C320, C321, C323 and 1B4 all bound to the TL1a
transfected cell line (FIG. 6) with EC.sub.50s of 0.5 to 2 nM, but
not normal untransfected cells.
[0508] To further characterize antibodies described herein, their
function on primary human cells was tested. PBMCs were isolated as
described in Example 1.12. Using the assay conditions described in
Example 1.13 these cells were shown to produce both mbTL1a (FIG.
7A) and soluble TL1a (FIG. 7B).
[0509] PBMCs were stimulated as described in Example 1.13 and
assayed for cytokine production in the presence of anti-TL1a
antibodies. Since no exogenous TL1a was added to the cell culture,
the effects of anti-TL1a antibodies were due to neutralization of
endogenous TL1a. Antibodies, C320 and C323 demonstrated good
inhibition of endogenous TL1a-induced cytokine production by PBMCs
(FIG. 8). This inhibition was apparent on cytokines typically
produced by Th1 cells (IFN-.gamma. FIG. 8A) and Th2 cells (IL-13;
FIG. 8B). These data demonstrate that these antibodies are likely
to be capable of inhibiting TL1a produced from primary human cells,
and that these antibodies could be used as diagnostic reagents to
detect TL1a on cells or in sera.
Example 7
Generating Improved Variants of C320
[0510] The C320 antibody was further optimized through alterations
to the antibody's sequence with the aim of yielding a positive
effect on the antibody's biophysical properties whilst having
minimal impact on the potency of C320.
[0511] For example, alterations that enhance the expression level
of the antibody with concomitantly increased production levels are
desirable. Removal of a N-linked glycosylation site in the V.sub.H
through amino acid substitution to reduce product heterogeneity may
further enhance C320. Substitution of amino acid residues with the
potential to impact the stability of the antibody through oxidation
or isomerization during purification or storage with amino acids
that do not undergo such transitions (Wang et al., Journal of
Pharmaceutical Sciences 96:1-26, 2006) may further improve C320.
Substitution of rare or non-germ-line C320 sequences which may
potentially contribute to immunogenicity with those of lower
predicted immunogenicity could further improve C320. Such changes
are described in more detail as follows.
Enhancing the Expression of C320
[0512] A database of human antibody germline amino acid sequences
was interrogated by Basic Local Alignment Search Tool searches
(BLAST: Altschul et al., J Mol Biol 215: 403-410, 1990) using the
C320 V.sub.H and V.sub.L amino acid sequences. This identified
twenty of the most homologous human antibody germline sequences to
those of the C320 V.sub.H and C320 V.sub.L, respectively. Aligning
the human germline sequences with those of C320 enabled the
identification of residues present in C320 but not common in the
majority of the germlines. The most common amino acid from the
germline sequences was substituted into the C320 amino acid
sequence. Table 7 lists the amino acid substitutions and the
resultant impact on the expression level of each antibody from a
transient transfection in HEK-293E cells.
TABLE-US-00007 TABLE 7 Effect of amino acid substitutions on
expression and potency Amino Acid % Expression Potency Antibody
Substitution Level relative TF-1 Designation (chain) to C320 EC-50
(pM) C320 N/A 100 233 C320-2 A16S (Heavy) 156 159 C320-3 T41P
(Heavy) 338 428 C320-4 N73D (Heavy) DNE N/A C320-5 A76T (Light) 231
89 C320-6 L81Q (Light) 98 67 C320-135 T41P (Heavy) & 288 462
A76T (Light) Amino acid substitutions are relative to SEQ ID NO: 42
for heavy chain and SEQ ID NO: 46 for light chain.
[0513] Two substitutions, present in C320-3 and C320-5 raised the
expression level of the antibody greater than two-fold and whilst
minimally impacting the potency of the antibody in a TF-1 potency
assay. A further antibody incorporating both substitutions,
C320-135, had largely equivalent potency and maintained the
improved expression level.
[0514] C320-4 contained a N73D (N72D according to the numbering
system of Kabat) substitution which aimed to improve expression
while also attempted to remove an N-linked glycosylation motif.
However, the N73D substitution abolished antibody expression,
suggesting N73 is desirable for expression of the antibody.
[0515] To further improve C320 expression the CDRs of C320 were
grafted onto other frameworks of antibodies possessing a known
crystal structure. This unusual approach was adopted as antibodies
of known crystal structure usually possess a matched
V.sub.H:V.sub.L pair. To select antibodies with suitable V.sub.H-
and V.sub.L frameworks BLAST searches of a database of crystal
structures were performed to identify antibodies of similar amino
acid sequence to C320. BLAST searches using the C320 V.sub.H were
used to identify the 100 most homologous antibody sequences with
known crystal structures. Similarly, the 100 most homologous
antibody sequences to the C320 V.sub.L with known crystal
structures were identified. Crystal structures appearing in both
heavy- and light chain lists were deemed suitable acceptor
frameworks for CDR grafting. These were 1TZG, 1RHH, 2DD8, 2JB5,
3FKU, 3GBM, 3IYW, 3LMJ, and 3P30. The CDRs of C320 were then used
to replace the CDR sequences present in each of the above antibody
sequences. An alignment of each of the sequence containing the C320
CDR regions is given in FIG. 1D (heavy chain) and FIG. 1G (light
chain). The antibody heavy and light chains were then paired as
follows and assessed for protein expression and TL1a binding and
the results listed in Table 8.
TABLE-US-00008 TABLE 8 Results of CDR grafting on expression and
TL1a binding AUC Protein A Protein A TL1a Antibody Heavy Light
capture HPLC binding Designation Chain Chain (RU) (215 nm) (RU)
C320 C320 C320 779 2764 282 mock N/A N/A 25 376 N/D C320-7 1TZG
1TZG 187 912 N/D C320-8 1RHH 1RHH 437 1718 29 C320-9 2DD8 2DD8 2235
8687 305 C320-10 2JB5 2JB5 2098 11406 368 C320-11 3FKU 3FKU 129 637
N/D C320-12 3GBM 3GBM 100 697 N/D C320-13 3IYW 3IYW 1566 4488 524
C320-14 3LMJ 3LMJ 570 1125 171 C320-15 3P30 3P30 1063 2192 318
C320-16 1TZG C320 2527 11604 653 C320-17 1RHH C320 2241 11980 683
C320-18 2DD8 C320 1528 5786 408 C320-19 2JB5 C320 1805 8018 465
C320-20 3FKU C320 986 1909 310 C320-21 3GBM C320 39 428 N/D C320-22
3IYW C320 2107 8622 665 C320-23 3LMJ C320 1119 2044 324 C320-24
3P30 C320 1023 2353 311 C320-25 C320 1TZG 376 1062 42 C320-26 C320
1RHH 438 1012 81 C320-27 C320 2DD8 2272 9795 578 C320-28 C320 2JB5
2176 11771 623 C320-29 C320 3FKU 1549 3195 355 C320-30 C320 3GBM
1288 3356 455 C320-31 C320 3IYW 1061 2806 381 C320-32 C320 3LMJ 892
1888 331 C320-33 C320 3P30 1527 4473 521 Note: The sequences of
heavy and light chains of these antibodies are listed in FIG. 1D
and 1G. RU is response units - a measure of binding to the surface
using SPR and AEC is area under the curve.
[0516] When the CDRs of C320 were grafted onto different antibody
frameworks a large number of the resultant antibodies expressed at
a level above that of the C320 antibody. The antibody C320-16, in
which the heavy chain C320 CDRs were grafted on the antibody 1TZG
framework and was paired with the C320 light chain, expressed
3-fold better than C320. This experiment also demonstrated that it
is possible to change the isotype of the antibody and retain
protein expression and binding to TL1a using this approach, as some
of the light chain antibody frameworks into which the C320 light
chain CDRs were grafted were of the kappa isotype as opposed to the
lambda isotype present in C320.
[0517] To further improve the expression of the antibody, amino
acid substitutions were introduced into the CDR3 region of the
variable heavy chain of C320 and the antibodies transfected into
HEK-293E cells and screened for expression levels using Protein A
HPLC and Protein A capture using surface plasmon resonance (SPR).
The results are described in Table 9.
TABLE-US-00009 TABLE 9 Effect of HCDR3 substitutions on expression
Heavy Chain Light Chain Substitution Substitution AUC (relative
(relative Protein A (Protein A Antibody to SEQ ID to SEQ ID capture
HPLC) Designation NO: 42) NO: 46) (RU) (280 nm) C320-0 N/A N/A 6413
487 mock N/A N/A 198 N/D C320-53 E99S A76T 6831 581 C320-54 E99H
A76T 6594 444 C320-55 E99L A76T 6797 540 C320-56 E99D A76T 5823 343
C320-57 E99Y A76T 7789 670 C320-58 E99P A76T 5679 335 C320-59 E99Q
A76T 8534 1149 C320-60 E99K A76T 8293 839 C320-61 V100A A76T 8381
863 C320-62 V100S A76T 8423 803 C320-63 V100H A76T 8828 751 C320-64
V100L A76T 8288 910 C320-65 V100D A76T 8816 1003 C320-66 V100Y A76T
7750 570 C320-67 V100P A76T 7831 660 C320-68 V100Q A76T 8170 715
C320-69 V100K A76T 8741 706 C320-70 P101A A76T 5022 234 C320-71
P101S A76T 5083 268 C320-72 P101H A76T 5403 230 C320-73 P101L A76T
5518 283 C320-74 P101D A76T 5756 297 C320-75 P101Y A76T 5084 212
C320-76 P101Q A76T 5036 263 C320-77 P101K A76T 5702 309 C320-78
D102A A76T 8249 684 C320-79 D102S A76T 8964 1102 C320-80 D102H A76T
8957 830 C320-81 D102L A76T 7969 515 C320-82 D102Y A76T 8135 558
C320-83 D102P A76T 7765 462 C320-84 D102Q A76T 7826 635 C320-85
D102K A76T 8487 890 C320-86 T103A A76T 10209 1882 C320-87 T103S
A76T 8236 567 C320-88 T103H A76T 4934 227 C320-89 T103L A76T 7580
822 C320-90 T103D A76T 8694 1106 C320-91 T103Y A76T 3838 130
C320-92 T103P A76T 4792 219 C320-93 T103Q A76T 4104 164 C320-94
T103K A76T 3592 149 C320-95 A104S A76T 4811 240 C320-96 A104H A76T
4882 251 C320-97 A104L A76T 4557 189 C320-98 A104D A76T 5371 279
C320-99 A104Y A76T 6305 410 C320-100 A104P A76T 5678 339 C320-101
A104Q A76T 6634 508 C320-102 A104K A76T 5826 438 C320-103 S105A
A76T 8159 825 C320-104 S105H A76T 6664 426 C320-105 S105L A76T 6193
357 C320-106 S105D A76T 7752 992 C320-107 S105Y A76T 8209 1072
C320-108 S105P A76T 6132 483 C320-109 S105Q A76T 6767 465 C320-110
S105L A76T 6999 452 C320-111 E105K A76T 6919 500 C320-112 E107S
A76T 7713 631 C320-113 E107H A76T 6723 459 C320-114 E107L A76T 7739
839 C320-115 E107D A76T 8505 1034 C320-116 E107Y A76T 6465 375
C320-117 E107P A76T 6699 400 C320-118 E107Q A76T 8109 704 C320-119
E107K A76T 8776 952 Note: Substitutions in this table refer to
those made into the C320 antibody
[0518] Several antibodies were generated that had significant
expression levels above that of C320 as determined by capture on a
Protein A surface. Several of the high expressing antibodies were
re-transfected, purified by Protein A chromatography and their
potency measured in a TF-1 assay. The results are listed in Table
10.
TABLE-US-00010 TABLE 10 Effect of HCDR3 substitutions on TL1a
inhibition Heavy Chain Light Chain Substitution Substitution
(relative (relative Potency Antibody to SEQ ID to SEQ ID TF-1
Designation NO: 42) NO: 46) EC-50 (pM) C320-0 N/A N/A 233 C320-61
V100A A76T 272 C320-63 V100H A76T 1067 C320-65 V100D A76T 546
C320-68 V100Q A76T 535 C320-86 T103A A76T 303 C320-87 T103S A76T
356 C320-90 T103D A76T 370 C320-103 S105A A76T 455 C320-104 S105H
A76T 349 C320-106 S105D A76T 1587 C320-112 E107S A76T 242 C320-114
E107L A76T 514 C320-115 E107D A76T 321 C320-117 E107P A76T 251
C320-119 E107K A76T 4142 Note: Substitutions in this table refer to
those made into the C320 antibody
[0519] The potency of the resulting antibodies ranged from reduced
compared to C320, such as C320-119, to levels equivalent to that of
C320.
[0520] The light chain sequence of C320 was aligned against the
germline sequence of highest homology, IGLV1-40*01. Differences in
amino acid sequences in the CDR regions were identified, as shown
in FIG. 9A. At a position in which the sequences differed a back
substitution of the amino acid from the germline sequence into C320
was performed. The antibodies were expressed in HEK-293 cells,
purified and the protein yield relative to C320 determined. A
potency assay using TF-1 cells was used to characterize the
inhibition profile of the antibodies. The results are displayed in
Table 11.
TABLE-US-00011 TABLE 11 Effect of germlining C320 on TF1a
inhibition Heavy Chain Light Chain Substitution Substitution
(relative (relative % Expression Potency Antibody to SEQ ID to SEQ
ID Level relative TF-1 Designation NO: 42) NO: 46) to C320 EC-50
(pM) C320 N/A N/A 100 233 C320-120 T41P A23T 279 343 C320-121 T41P
D28N 239 1433 C320-122 T41P L33Y 247 5480 C320-123 T41P G34D 207
DNI C320-124 T41P Y53N 128 1613 C320-125 T41P Y54S 259 13393
C320-126 T41P P82A 187 539 C320-127 T41P G95S 140 541 C320-128 T41P
T96S 134 328 Note: Substitutions in this table refer to those made
into the C320 antibody
[0521] The antibody C320-120 demonstrated a good level of
expression and the highest potency of all the antibodies tested in
this experiment. Some of the substitutions, such as those in
C320-123 and C320-125 resulted in antibodies that expressed but no
longer inhibited or weakly inhibited TL1a induced apoptosis on TF-1
cells. This suggested that the amino acids at the positions G34
(G32 according to the numbering system of Kabat) and Y54 (Y52
according to the numbering system of Kabat) of the variable light
chain may be important in enabling the antibody to inhibit the
activity of TL1a.
Removal of Putative Oxidation and Isomerization Sites from C320
[0522] Amino acid analysis of the variable heavy and light chain
sequences identified several amino acids that may undergo oxidation
or isomerization. Particular emphasis was placed on amino acids
present in the CDRs of the antibody. Changes to these amino acids
may, over time, alter the binding profile of the antibody. In the
variable heavy chain M51 (M51 according to the numbering system of
Kabat) was identified as a potential oxidation site along with the
potential aspartate isomerization site D102 (D98 according to the
numbering system of Kabat). In the light chain, D94 (D92 according
to the numbering system of Kabat) was identified as a potential
isomerization site. To reduce the potential impact of these
predicted issues variants of C320 were produced containing
conservative or semi-conservative amino acid substitutions in these
positions. These substitutions and their impact on the potency of
the resulting C320 variants are listed in Table 12.
TABLE-US-00012 TABLE 12 Effect of amino acid substitutions on
expression and TL1a inhibition Amino Acid Substitution % Expression
Potency Antibody from C320 Level relative TF-1 Designation (chain)
to C-320 EC-50 (pM) C320 N/A 100 233 C320-129 D102E (Heavy) 201 433
C320-130 M51L (Heavy) 185 528 C320-131 D94E (Light) 103 10680 Note:
Substitutions in this table refer to those made into the C320
antibody, with heavy chain substitutions relative to SEQ ID NO: 42
and light chain substitutions relative to SEQ ID NO: 46
[0523] The potential oxidation and isomerization site present in
the heavy chain were successfully substituted with conservative
amino acids which resulted in improved expression and retention of
potency. The substitution of D94E in the light chain CDR region
resulted in an antibody that expressed at similar levels to C320,
but had significantly reduced potency, indicating that D94 of the
light chain may be important in mediating the functional activity
of C320.
Removal of the N-Glycosylation Site from the C320 Heavy Chain
[0524] As an earlier attempt to remove the V.sub.H glycosylation
site NTS by substitution to DTS (N73D; N72D according to the
numbering system of Kabat) was not successful, further attempts to
disrupt the N-linked glycosylation motif, NX(S/T) (where X.noteq.P)
using a more comprehensive range of substitutions were made.
Firstly, further residues were tested in place of the asparagine at
position 73. Secondly the substitution T74P (T73P according to the
numbering system of Kabat) was tested, since proline in this
position is known to prevent N-linked glycosylation. A range of
amino acids were also tested in place of S75 (S74 according to the
numbering system of Kabat). The constructs tested in attempts to
remove this glycosylation site and their impact on antibody
expression levels are listed in Table 13.
TABLE-US-00013 TABLE 13 Effect of amino acid substitutions on
expression levels Heavy Chain Light Chain Substitution Substitution
AUC Antibody relative to relative to (Protein A Designation SEQ ID
NO: 42 SEQ ID NO: 46 HPLC) C320-0 N/A N/A 162 C320-120 T41P A23T
710 mock N/A N/A N/D C320-138 N73S A23T 87 C320-139 N73K A23T 58
C320-140 N73H A23T N/D C320-141 N73T A23T 66 C320-142 N73Q A23T 70
C320-143 N73G A23T N/D C320-144 N73P A23T N/D C320-145 N73L A23T 54
C320-146 N73Y A23T N/D C320-147 T74P A23T 110 C320-148 S75L A23T
115 C320-149 S75I A23T 96 C320-150 S75A A23T 194 C320-151 S75Y A23T
88 C320-152 S75K A23T 115 C320-153 S75E A23T 110 C320-154 S75F A23T
78 C320-155 S75H A23T 118 Note: Substitutions in this table refer
to those made into the C320 antibody
[0525] The majority of the C320 variants tested exhibited lower
levels of expression compared to C320, suggesting that substituting
individual amino acids present in the NTS motif, in C320, reduces
the expression of the antibody.
[0526] A result of earlier attempts to enhance the expression of
C320 by grafting the C320 CDRs onto different antibody frameworks
was the antibody designated C320-16, which used the frameworks of
1TZG. This antibody expressed well and bound TL1a (Table 7). The
frameworks of 1TZG and C320-16 were devoid of the N-linked
glycosylation sequence motif, as the RNTSI (SEQ ID NO: 203) at
positions 72 through 76 of SEQ ID NO: 42 (71 through 75 according
to the numbering system of Kabat) of C320 V.sub.H sequence is
replaced by ADRST (SEQ ID NO: 204) in the corresponding V.sub.H
sequence of 1TZG.
[0527] Variant C320-135 comprising expression enhancing
substitutions V.sub.H T41P and V.sub.L A73T was subjected to
further heavy chain engineering. C320 V.sub.H sequence RNTSI (SEQ
ID NO: 203) at positions 71 through 75 was replaced by ADRST (SEQ
ID NO: 204) from the corresponding V.sub.H sequence of 1TZG to
produce antibody C320-163. This antibody, now devoid of an N-linked
glycosylation site, expressed when transfected into HEK-293E cells
and, when purified, was capable of functional inhibition of TL1a
mediated apoptosis of TF-1 cells (Table 14).
Improved Variants of C320
[0528] The amino acid substitutions identified in previous
experiments as conferring advantageous properties over C320 were
subsequently combined and the resultant antibodies expressed,
purified and assayed for potency. The sequences of these are listed
in FIG. 9B (variable heavy) and FIG. 9C (variable light).
Comparative potency and expression data of each antibody is listed
in Table 14.
TABLE-US-00014 TABLE 14 Effect of amino acid substitutions on
expression and TL1a inhibition % expression Potency Antibody
compared TF-1 Designation to C320 EC-50 (pM) C320 100 233 C320-162
74 1053 C320-163 170 1160 C320-164 299 338 C320-165 380 240
C320-166 339 793 C320-167 344 239 C320-168 219 46 C320-169 N/A 477
C320-170 232 287 C320-171 102 DNI C320-172 140 269 C320-179 161 96
C320-183 129 439 * Note: DNI = did not inhibit
[0529] Comparing antibodies C320-168 (containing a N-linked glycan
motif) with C320-163 and C320-170 (lacking a N-linked glycan motif)
on an isoelectric focusing (IEF) gel demonstrated removal of charge
heterogeneity from the antibody profile on removing the N-linked
glycosylation site (FIG. 9D). C320-168 has 5 to 6 distinct charged
isoforms compared to 1-2 isoforms visualized for C320-163 and
C320-170. This reduction in charge heterogeneity is an advantageous
property providing improved batch to batch consistency in large
scale manufacturing processes (Wang et al., Journal of
Pharmaceutical Sciences 96:1-26, 2006).
[0530] Of the antibodies tested, two were more potent than the
parental C320 antibody-C320-168 and C320-179 (FIG. 9E). Both of
these C320 variants include the light chain variable region
substitution G24S (G25S according to the numbering system of Kabat)
that was found to improve potency in TF-1 assays in combination
with the other advantageous amino acids substitutions described
previously.
Removal of Predicted MHC Class II Binding Peptides from
Antibodies
[0531] The amino acid sequence of C320-168 variable heavy and light
chain regions were analysed in silico using the software package
Epibase.TM. (LONZA). This software predicts the propensity of
peptide sequences present in C320-168 to bind to MHC Class II
alleles. Such binding of peptides to MHC Class II alleles is an
important step in a host immune response against the administered
antibody.
[0532] A peptide sequence in the heavy chain of C320-168,
GLEWMGWLNPNSGNT (SEQ ID NO: 205), was predicted to strongly bind to
DRB1*0401. A peptide sequence in the light chain of C320-168,
LLIYGYYNRPSGVPD (SEQ ID NO: 206), was predicted to strongly bind to
DRB1*1101, DRB1*1104 and DRB1*1501.
[0533] The above listed peptide sequences and modified versions of
these peptide sequences were synthesized and incubated with the
corresponding MHC Class II protein and an ELISA performed to
determine if the peptide formed a complex with the MHC Class II
protein. The influenza peptide, PKYVKQNTLKLAT (SEQ ID NO: 207), was
used as positive control in these assays to indicated the formation
of a peptide:MHC Class II complex. The binding of each peptide to
the MHC Class II allele is listed in Table 14.
TABLE-US-00015 TABLE 14 Binding of peptides to MHC Class II Binding
MHC Class II (% of positive Peptide Allele control) GLEWMGWLNPNSGNT
(SEQ ID NO: 205) DRB1*0401 6 LLIYGYYNRPSGVPD (SEQ ID NO: 206)
DRB1*1501 123 LLIEGYYNRPSGVPD (SEQ ID NO: 208) DRB1*1501 41
LLIGGYYNRPSGVPD (SEQ ID NO: 209) DRB1*1501 42 LLIPGYYNRPSGVPD (SEQ
ID NO: 210) DRB1*1501 0 LLIKGYYNRPSGVPD (SEQ ID NO: 211) DRB1*1501
74 LLIYGYYNRPSGVPD (SEQ ID NO: 212) DRB1*1101 1 LLIEGYYNRPSGVPD
(SEQ ID NO: 213) DRB1*1101 0 LLIGGYYNRPSGVPD (SEQ ID NO: 214)
DRB1*1101 0 LLIPGYYNRPSGVPD (SEQ ID NO: 215) DRB1*1101 0
LLIKGYYNRPSGVPD (SEQ ID NO: 216) DRB1*1101 0 LLIYGYYNRPSGVPD (SEQ
ID NO: 217) DRB1*1104 1 LLIEGYYNRPSGVPD (SEQ ID NO: 218) DRB1*1104
0 LLIGGYYNRPSGVPD (SEQ ID NO: 219) DRB1*1104 1 LLIPGYYNRPSGVPD (SEQ
ID NO: 220) DRB1*1104 0 LLIKGYYNRPSGVPD (SEQ ID NO: 221) DRB1*1104
0
[0534] Peptides that may be immunologically significant or warrant
further investigation as good binders are considered to be those
peptides with scores 15% of the positive control. Therefore the
peptide GLEWMGWLNPNSGNT (SEQ ID NO: 205) has a low likelihood of
being immunogenic and no attempt was made to modify this motif in
C320 related antibodies.
[0535] The peptide LLIYGYYNRPSGVPD (SEQ ID NO: 206) was shown to
form a complex with MHC Class II protein DRB1*1501 (123% binding)
but not DRB1*1101 or DRB1*1104. An attempt to reduce the formation
of the peptide:MHC Class II complex was made by modifying the
tyrosine residue at the fourth position of the peptide
(corresponding to position 51 (position 49 according to the
numbering system of Kabat) in the C320-168 variable light chain
sequence) to one of either glutamic acid, glycine, proline, or
lysine. The introduction of a glutamic acid or a glycine residue at
this position reduced the complex formation by over 50% (Table 14).
Introducing proline at this position prevented complex formation.
Antibodies C320-172 and C320-179 incorporated substitution Y51E
(Y49E according to the numbering system of Kabat), in their V.sub.L
region, whilst C320-183 incorporated the change Y51G (Y49G
according to the numbering system of Kabat) in the V.sub.L region.
All three antibodies expressed and functionally inhibited TL1a, as
illustrated in Table 13. Expression of antibody C320-171
incorporating substitution Y51E (Y49P according to the numbering
system of Kabat) was detected, however, this antibody failed to
functionally inhibit TL1a, indicating that not all amino acids are
tolerated at position 49 in the light chain (Table 13).
Example 8
Generation of High Potency DR3-Selective Antibodies
[0536] New antibodies capable of neutralizing TL1a activity with
high potency and capable of selective inhibition of
TL1a-DR3-mediated activity are generated by focusing on the C320
antibody epitope on TL1a. For example, mutant variable regions
and/or individual substitutions showing improved expression and/or
TL1a-neutralization (e.g., as described in Example 7) are combined
and re-tested to determine if the improvement is additive.
[0537] In other approaches antibodies that bind to the epitope
bound by C320 are selected by any of a variety of techniques.
8.1 Selection from an Antibody Library Using the C320 Antibody
[0538] A phage display protocol is used where a first panning round
is conducted using an antigen density (i.e. biotinlyated TL1a) of
about 100 pmol and a TEA-based elution step as described
previously. The second and third rounds use a reduced antigen
density (e.g., about 50 pmol). Phage are eluted by adding the C320
IgG at a 10-fold molar excess and incubating the reactions at room
temperature for 2, 5, 10 or 20 mins. The IgG is expected to
specifically displace and elute phage expressing FAbs that bound to
the C320 epitope. Non-specific binders and phage bound to other
regions on the TL1a surface are less likely to elute under these
conditions.
[0539] The washing regimen comprises six washes with M-PBS for
round 1 and 2. For round 3 the washes are three washes with PBST
and then three washes with PBS.
[0540] Eluted phage are used to infect TG1 E. coli for phagemid
rescue or generation of colonies for screening as described for
other phage display experiments.
8.2 Selection/Production of Antibodies Using Mutant TL1a
[0541] Using mutated versions of TL1a as panning reagents for phage
display, antibodies that recognize an epitope similar to that of
C320 may be obtained. A phage display library may be depleted of
antibodies that recognize TL1a with an amino acid substitution of
R32A and/or R85A. The library may then be panned against TL1a. The
resultant isolated antibodies will likely bind to the residues R32
and/or R85.
Example 9
Affinity Maturation of C320
[0542] Antibody C320 is already a potent inhibitor of TL1a
activity. However, potency may be enhanced using affinity
maturation approaches. Enhanced potency frequently confers dosing
and efficacy advantages.
[0543] Numerous methods for affinity maturation of antibodies are
known in the art. Many of these are based on the general strategy
of generating panels or libraries of variant proteins by
mutagenesis followed by selection and/or screening for improved
affinity. Mutagenesis is often performed at the DNA level, for
example by error prone PCR (Thie et al., Methods Mol. Biol. 525:
309-322, xv, 2009), by gene shuffling (Kolkman and Stemmer, Nat.
Biotechnol. 19: 423-428, 2001), by use of mutagenic chemicals or
irradiation, by use of `mutator` strains with error prone
replication machinery (Greener et al., In Vitro Mutagenesis
Protocols. (Humana press, NJ), 1996) or by somatic hypermutation
approaches that harness natural affinity maturation machinery
(Peled et al., Annu. Rev. Immunol. 26: 481-511, 2008). Mutagenesis
is also performed at the RNA level, for example by use of Q.beta.
replicase (Kopsidas et al., Immunol. Lett. 107: 163-168, 2006).
Library-based methods allowing screening for improved variant
proteins are based on various display technologies such as phage,
yeast, ribosome, bacterial or mammalian cells, and are known in the
art (Benhar, Expert Opin. Biol. Ther. 7: 763-779, 2007). Affinity
maturation is also achieved by more directed/predictive methods for
example by site-directed mutagenesis or gene synthesis guided by
findings from 3D protein modeling (see for example U.S. Pat. No.
6,180,370 or U.S. Pat. No. 5,225,539).
[0544] Affinity maturation using ribosome display (Kopsidas et al.,
BMC Biotechnol. 7: 18, 2007) is performed using RNA encoding the
V.sub.L and V.sub.H domains of a C320-related antibody in scFv
format. A library of variants of this RNA is generated using
Q.beta. replicase (typically 1 to 3 changes per molecule) and this
library is displayed and selected on ribosomes for binding to TL1a.
Phenotype-genotype linkage is achieved because the RNA constructs
used remain attached to the ribosome translating the functional
scFv protein. C320-related scFv-RNA-ribosome complexes are panned
against TL1a and isolated. The RNA is converted into DNA and
incorporated into a bacterial expression system. Individual
bacteria encoding a single scFv are then isolated and induced to
express the scFv. Using a competition ELISA, scFvs with a higher
affinity for TL1a than a C320-related ScFv are identified. These
scFvs are converted into full length antibodies which are screened
in the TF-1 apoptosis assay as described in Example 1.8 to
determine improvement in inhibition of TL1a biological activity
over C320.
Example 11
Efficacy of Anti-TL1a Antibodies in Animal Models of Colitis
[0545] Rodent cross-reactive anti-TL1a antibodies described herein
were tested in rodent models of acute colitis induced by
intrarectal administration of di- or tri-nitrobenzenesulfonic acid
(D/TNBS) or oxazolone, and chronic colitis induced by
administration of DSS in drinking water (as described in Wirtz et
al., Nat. Protoc. 2: 541-546, 2007). DNBS and oxazolone induce
localized ulceration and inflammation. DSS administration induces
robust generalized inflammation of the intestinal tract
characterized by erosive lesions and inflammatory infiltrate.
Symptoms of all these models usually include diarrhoea, occult
blood, weight loss and occasionally rectal prolapse.
[0546] In a prophylactic model, antibody treatment was commenced at
the start of administration of the colitis-inducing compound. In a
therapeutic model, antibody treatment was commenced several days
after commencement of induction. The effect of the treatment on
weight, stool consistency and occult blood, as well as microscopic
effects on epithelial integrity and degree of inflammatory
infiltrate was determined. Daily clinical scoring was performed
based on stool consistency and presence of occult blood giving a
disease activity index (DAI) score.
[0547] Antibody C320-168 showed comparable efficacy to a standard
of care compound when dosed prophylactically in DNBS- or
oxazolone-induced colitis (FIGS. 10A-10C), and when dosed
therapeutically in DSS-induced colitis (FIGS. 11A and 11B).
Example 12
Efficacy of Anti-TL1a Antibodies in Animal Models of Disease
[0548] Antibodies are additionally tested in a rodent model of
multiple sclerosis (Racke, Curr. Protoc. Neurosci. Chapter 9, Unit
9 7, 2001). In this model either acute or chronic central
demyelination is induced by administration of spinal cord
homogenate or purified myelin peptides in adjuvant. This
administration causes autoimmune destruction of the myelin sheath
around the spinal cord neurons leading to lower limb weakness which
may develop to paralysis, and is characterized by significant
inflammatory infiltrate into the spinal cord. The acute form is
monophasic and animals recover spontaneously. The chronic form
resembles relapsing-remitting multiple sclerosis and consists of
two or more episodes of hind limb weakness/paralysis.
Sequence CWU 1
1
2351207PRTHomo sapiens 1Asp Asp Asp Asp Lys Gly Ser His His His His
His His His His Gly 1 5 10 15 Ser Gly Ser Leu Val Pro Arg Gly Ser
Gly Ser Leu Lys Gly Gln Glu 20 25 30 Phe Ala Pro Ser His Gln Gln
Val Tyr Ala Pro Leu Arg Ala Asp Gly 35 40 45 Asp Lys Pro Arg Ala
His Leu Thr Val Val Arg Gln Thr Pro Thr Gln 50 55 60 His Phe Lys
Asn Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly 65 70 75 80 Leu
Ala Phe Thr Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu 85 90
95 Ile Pro Glu Ser Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg
100 105 110 Gly Met Thr Ser Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg
Pro Asn 115 120 125 Lys Pro Asp Ser Ile Thr Val Val Ile Thr Lys Val
Thr Asp Ser Tyr 130 135 140 Pro Glu Pro Thr Gln Leu Leu Met Gly Thr
Lys Ser Val Cys Glu Val 145 150 155 160 Gly Ser Asn Trp Phe Gln Pro
Ile Tyr Leu Gly Ala Met Phe Ser Leu 165 170 175 Gln Glu Gly Asp Lys
Leu Met Val Asn Val Ser Asp Ile Ser Leu Val 180 185 190 Asp Tyr Thr
Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 195 200 205
2117PRTArtificialamino acid sequence of C336 VH 2Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Gly Gln Thr His Leu Asp
Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115
35PRTArtificialamino acid sequence of C336 HCDR1 3Gly Tyr Tyr Met
His 1 5 417PRTArtificialamino acid sequence of C336 HCDR2 4Trp Ile
Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15
Gly 58PRTArtificialamino acid sequence of C336 HCDR3 5Gly Gly Gln
Thr His Leu Asp Val 1 5 6108PRTArtificialamino acid sequence of
C336 VL 6Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
Ile Thr Asp Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Ile 85 90 95 Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105
711PRTArtificialamino acid sequence of C336 LCDR1 7Gln Ala Ser Gln
Asp Ile Thr Asp Tyr Leu Asn 1 5 10 87PRTArtificialamino acid
sequence of C336 LCDR2 8Asp Ala Ser Asn Leu Glu Thr 1 5
99PRTArtificialamino acid sequence of C336 LCDR3 9Gln Gln Tyr Asp
Asn Leu Pro Ile Thr 1 5 10122PRTArtificialamino acid sequence of
C334 VH 10Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe Ser Ser Tyr 20 25 30 Thr Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly
Thr Ala Asn His Ala Gln Ser Phe 50 55 60 Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr
Asn Ser Tyr Ser Ser Ser Trp Tyr Asp Ala Phe Asp Ile Trp 100 105 110
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
115PRTArtificialamino acid sequence of C334 HCDR1 11Ser Tyr Thr Ile
Ser 1 5 1217PRTArtificialamino acid sequence of C334 HCDR2 12Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn His Ala Gln Ser Phe Gln 1 5 10
15 Gly 1313PRTArtificialamino acid sequence of C334 HCDR3 13Asn Ser
Tyr Ser Ser Ser Trp Tyr Asp Ala Phe Asp Ile 1 5 10
14108PRTArtificialamino acid sequence of C334 VL 14Asp Val Val Met
Thr Gln Ser Pro Ala Phe Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30
Leu Ala Trp Tyr Gln Gln Met Pro Gly Gln Ala Pro Arg Leu Leu Leu 35
40 45 Tyr Asp Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Ser Glu Phe Thr Leu Thr Ile Ser Gly
Leu Gln Ser 65 70 75 80 Ala Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 105 1511PRTArtificialamino acid sequence of C334
LCDR1 15Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu Ala 1 5 10
167PRTArtificialamino acid sequence of C334 LCDR2 16Asp Ala Ser Thr
Arg Ala Thr 1 5 179PRTArtificialamino acid sequence of C334 LCDR3
17Gln Gln Tyr Asn Asn Trp Pro Leu Thr 1 5 18122PRTArtificialamino
acid sequence of C333 VH 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Thr Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile
Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85
90 95 Ser Thr Asn Ser Tyr Ser Ser Ser Trp Tyr Asp Ala Phe Asp Ile
Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
195PRTArtificialamino acid sequence of C333 HCDR1 19Ser Tyr Thr Ile
Ser 1 5 2017PRTArtificialamino acid sequence of C333 HCDR2 20Gly
Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe Gln 1 5 10
15 Gly 2113PRTArtificialamino acid sequence of C333 HCDR3 21Asn Ser
Tyr Ser Ser Ser Trp Tyr Asp Ala Phe Asp Ile 1 5 10
22108PRTArtificialamino acid sequence of C333 VL 22Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Asn Asn 20 25 30
Leu Ala Trp Tyr Gln Gln Leu Pro Gly Gln Ala Pro Arg Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser
Gly 50 55 60 Thr Gly Ser Gly Ser Glu Phe Thr Leu Thr Ile Ser Gly
Leu Gln Ser 65 70 75 80 Ala Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp
Ile Lys Arg 100 105 2311PRTArtificialamino acid sequence of C333
LCDR1 23Arg Ala Ser Gln Ser Ile Thr Asn Asn Leu Ala 1 5 10
247PRTArtificialamino acid sequence of C333 LCDR2 24Asp Ala Ser Thr
Arg Ala Thr 1 5 259PRTArtificialamino acid sequence of C333 LCDR3
25Gln Gln Tyr Asn Asn Trp Pro Leu Thr 1 5 26122PRTArtificialamino
acid sequence of C323 VH 26Gln Met Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Thr Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile
Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85
90 95 Ser Thr Asn Ser Tyr Ser Ser Ser Trp Tyr Asp Ala Phe Asp Ile
Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
275PRTArtificialamino acid sequence of C323 HCDR1 27Ser Tyr Thr Ile
Ser 1 5 2817PRTArtificialamino acid sequence of C323 HCDR2 28Gly
Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe Gln 1 5 10
15 Gly 2913PRTArtificialamino acid sequence of C323 HCDR3 29Asn Ser
Tyr Ser Ser Ser Trp Tyr Asp Ala Phe Asp Ile 1 5 10
30108PRTArtificialamino acid sequence of C323 VL 30Ala Ile Gln Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Ala 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Tyr Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Ser Tyr Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 105 3111PRTArtificialamino acid sequence of C323
LCDR1 31Arg Ala Ser Gln Gly Ile Gly Ser Ala Leu Ala 1 5 10
327PRTArtificialamino acid sequence of C323 LCDR2 32Asp Ala Ser Ser
Leu Gln Ser 1 5 339PRTArtificialamino acid sequence of C323 LCDR3
33Gln Gln Phe Ser Tyr Tyr Pro Leu Thr 1 5 34122PRTArtificialamino
acid sequence of C321 VH 34Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser
Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly
Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asp Ser His Ile Tyr Asp Ile Leu Thr Gly Tyr Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
355PRTArtificialamino acid sequence of C321 HCDR1 35Ser Tyr Gly Ile
Ser 1 5 3617PRTArtificialamino acid sequence of C321 HCDR2 36Trp
Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln 1 5 10
15 Gly 3713PRTArtificialamino acid sequence of C321 HCDR3 37Asp Ser
His Ile Tyr Asp Ile Leu Thr Gly Tyr Asp Tyr 1 5 10
38113PRTArtificialamino acid sequence of C321 VL 38Asp Val Val Met
Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys His Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35
40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Asp 85 90 95 Thr Gln Phe Pro Gln Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 3916PRTArtificialamino
acid sequence of C321 LCDR1 39His Ser Ser Gln Ser Leu Val His Ser
Asp Gly Asn Thr Tyr Leu Ser 1 5 10 15 407PRTArtificialamino acid
sequence of C321 LCDR2 40Lys Ile Ser Asn Arg Phe Ser 1 5
419PRTArtificialamino acid sequence of C321 LCDR3 41Met Gln Asp Thr
Gln Phe Pro Gln Thr 1 5 42119PRTArtificialamino acid sequence of
C320 VH 42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Thr Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 435PRTArtificialamino acid sequence
of C320 HCDR1 43Ser Tyr Asp Ile Asn 1 5 4417PRTArtificialamino acid
sequence of C320 HCDR2 44Trp Met Asn Pro Asn Ser Gly Asn Thr Gly
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 4510PRTArtificialamino acid
sequence of C320 HCDR3 45Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr 1
5 10 46111PRTArtificialamino acid sequence of C320 VL 46Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25
30 Leu Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu 65 70 75 80 Leu Pro Glu Asp Glu Gly Asp Tyr Tyr Cys
Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly 100 105 110 4714PRTArtificialamino acid
sequence of C320 LCDR1 47Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly
Leu Gly Val His 1 5 10 487PRTArtificialamino acid sequence of C320
LCDR2 48Gly Tyr Tyr Asn Arg Pro Ser 1 5 4910PRTArtificialamino acid
sequence of C320 LCDR3 49Gln Ser Tyr Asp Gly Thr Leu Ser Ala Leu 1
5 10
50123PRTArtificialamino acid sequence of C319 VH 50Gln Leu Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Arg 20 25 30
Asn Trp Trp Ser Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp 35
40 45 Ile Gly Glu Ile Tyr His Ser Asp Ile Thr Asn Tyr Asn Pro Ser
Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn
Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Glu Ala Gly Gly Thr
Tyr Ile Asp Ala Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 516PRTArtificialamino acid sequence of C319
HCDR1 51Ser Arg Asn Trp Trp Ser 1 5 5216PRTArtificialamino acid
sequence of C319 HCDR2 52Glu Ile Tyr His Ser Asp Ile Thr Asn Tyr
Asn Pro Ser Leu Lys Ser 1 5 10 15 5314PRTArtificialamino acid
sequence of C319 HCDR3 53Asp Gly Glu Ala Gly Gly Thr Tyr Ile Asp
Ala Phe Asp Val 1 5 10 54111PRTArtificialamino acid sequence of
C319 VL 54Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro
Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Ile Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Ile Ile Tyr Asp Val Ser Glu Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Asp
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Tyr Ser Tyr Ala Gly Thr 85 90 95 Tyr Thr
Ser Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110
5514PRTArtificialamino acid sequence of C319 LCDR1 55Thr Gly Thr
Ser Ser Asp Val Gly Ile Tyr Asn Tyr Val Ser 1 5 10
567PRTArtificialamino acid sequence of C319 LCDR2 56Asp Val Ser Glu
Arg Pro Ser 1 5 5710PRTArtificialamino acid sequence of C319 LCDR3
57Tyr Ser Tyr Ala Gly Thr Tyr Thr Ser Leu 1 5 10
58119PRTartificialamino acid sequence of C320-3 VH 58Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala
Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 595PRTartificialamino acid sequence of C320-3 HCDR1 59Ser
Tyr Asp Ile Asn 1 5 6017PRTartificialamino acid sequence of C320-3
HCDR2 60Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
Gln 1 5 10 15 Gly 6110PRTartificialamino acid sequence of C320-3
HCDR3 61Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr 1 5 10
62111PRTartificialamino acid sequence of C320-5 VL 62Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25
30 Leu Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Thr
Ile Thr Gly Leu 65 70 75 80 Leu Pro Glu Asp Glu Gly Asp Tyr Tyr Cys
Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly 100 105 110 6314PRTartificialamino acid
sequence of C320-5 LCDR1 63Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly
Leu Gly Val His 1 5 10 647PRTartificialamino acid sequence of
C320-5 LCDR2 64Gly Tyr Tyr Asn Arg Pro Ser 1 5
6510PRTartificialamino acid sequence of C320-5 LCDR3 65Gln Ser Tyr
Asp Gly Thr Leu Ser Ala Leu 1 5 10 66119PRTartificialamino acid
sequence of C320-90 VH 66Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg
Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn
Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Val Pro Asp Asp Ala Ser Phe Glu Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
675PRTartificialamino acid sequence of C320-90 HCDR1 67Ser Tyr Asp
Ile Asn 1 5 6817PRTartificialamino acid sequence of C320-90 HCDR2
68Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln 1
5 10 15 Gly 6910PRTartificialamino acid sequence of C320-90 HCDR3
69Glu Val Pro Asp Asp Ala Ser Phe Glu Tyr 1 5 10
70119PRTartificialamino acid sequence of C320-103 VH 70Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala
Ala Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 715PRTartificialamino acid sequence of C320-103 HCDR1 71Ser
Tyr Asp Ile Asn 1 5 7217PRTartificialamino acid sequence of
C320-103 HCDR2 72Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Lys Phe Gln 1 5 10 15 Gly 7310PRTartificialamino acid sequence
of C320-103 HCDR3 73Glu Val Pro Asp Thr Ala Ala Phe Glu Tyr 1 5 10
74119PRTartificialamino acid sequence of C320-114 VH 74Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala
Ser Phe Leu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 755PRTartificialamino acid sequence of C320-114 HCDR1 75Ser
Tyr Asp Ile Asn 1 5 7617PRTartificialamino acid sequence of
C320-114 HCDR2 76Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Lys Phe Gln 1 5 10 15 Gly 7710PRTartificialamino acid sequence
of C320-114 HCDR3 77Glu Val Pro Asp Thr Ala Ser Phe Leu Tyr 1 5 10
78119PRTartificialamino acid sequence of C320-115 VH 78Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala
Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 795PRTartificialamino acid sequence of C320-115 HCDR1 79Ser
Tyr Asp Ile Asn 1 5 8017PRTartificialamino acid sequence of
C320-115 HCDR2 80Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Lys Phe Gln 1 5 10 15 Gly 8110PRTartificialamino acid sequence
of C320-115 HCDR3 81Glu Val Pro Asp Thr Ala Ser Phe Asp Tyr 1 5 10
82111PRTartificialamino acid sequence of C320-120 VL 82Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25
30 Leu Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu 65 70 75 80 Leu Pro Glu Asp Glu Gly Asp Tyr Tyr Cys
Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly 100 105 110 8314PRTartificialamino acid
sequence of C320-120 LCDR1 83Thr Gly Ser Ser Ser Asp Ile Gly Ala
Gly Leu Gly Val His 1 5 10 847PRTartificialamino acid sequence of
C320-120 LCDR2 84Gly Tyr Tyr Asn Arg Pro Ser 1 5
8510PRTartificialamino acid sequence of C320-120 LCDR3 85Gln Ser
Tyr Asp Gly Thr Leu Ser Ala Leu 1 5 10 86119PRTartificialamino acid
sequence of C320-129 VH 86Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg
Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn
Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Val Pro Glu Thr Ala Ser Phe Glu Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
875PRTartificialamino acid sequence of C320-129 HCDR1 87Ser Tyr Asp
Ile Asn 1 5 8817PRTartificialamino acid sequence of C320-129 HCDR2
88Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln 1
5 10 15 Gly 8910PRTartificialamino acid sequence of C320-129 HCDR3
89Glu Val Pro Glu Thr Ala Ser Phe Glu Tyr 1 5 10
90119PRTartificialamino acid sequence of C320-130 VH 90Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala
Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 915PRTartificialamino acid sequence of C320-130 HCDR1 91Ser
Tyr Asp Ile Asn 1 5 9217PRTArtificialamino acid sequence of
C320-130 HCDR2 92Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Lys Phe Gln 1 5 10 15 Gly 9310PRTartificialamino acid sequence
of C320-130 HCDR3 93Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr 1 5 10
94119PRTartificialamino acid sequence of VH consensus sequence of
C320 and derivatives 94Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln
Ala Xaa Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Xaa Asn Pro
Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Val Pro Xaa Xaa Ala Xaa Phe Xaa Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 95111PRTartificialamino
acid sequence of VL consensus sequence of C320 and derivatives
95Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Xaa Gly Ser Ser Ser Asp Ile Gly Ala
Gly 20 25 30 Leu Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Xaa Ile Thr Gly Leu 65 70 75 80 Leu Pro Glu Asp Glu Gly Asp
Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
96351DNAArtificialnucleotide sequence encoding VH of C336
96gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg
cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg accagggaca
cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac
acggccgtgt attactgtgc gagtgggggc 300cagacccacc tggacgtctg
gggccaaggg accacggtca ccgtctcctc a 35197324DNAArtificialnucleotide
sequence encoding VL of C336 97gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtgggaga cagagtcacc 60atcacttgcc
aggcgagtca ggacattacc gactatttaa attggtatca gcagagacca
120gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg
ggttccatca 180aggttcagtg gaagtggatc tgggacatat tttactttca
ccatcagcag cctgcagcct 240gaagattttg cgacatatta ctgtcaacag
tatgataatc tcccgatcac cttcggccag 300gggacacgac tggagattaa acgt
32498366DNAArtificialnucleotide sequence encoding VH of C334
98caaatgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60tcctgcaagg cttctggagg caccttcagc agctatacta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac
agcaaaccac 180gcacagagct tccagggcag agtcacgatt accgcggacg
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtat attattgttc aaccaactcg 300tatagcagca gctggtatga
tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360tcttca
36699324DNAArtificialnucleotide sequence encoding VL of C334
99gatgttgtga tgacacagtc tccagctttc ctctctgtgt ctcctgggga aagagccacc
60ctctcctgca gggccagtca gagtattagc aacaacttag cctggtatca gcaaatgcct
120ggccaggctc ccaggctcct tctttatgat gcatccacca gggccactga
tatcccagcc 180aggttcagtg gcagtgggtc tgggtcagag ttcactctca
ccatcagcgg cctgcagtct 240gcagattttg cagtttatta ctgtcaacaa
tacaataact ggcctctcac tttcggcgga 300gggaccaagc tggagatcaa acgt
324100366DNAArtificialnucleotide sequence encoding VH of C333
100caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc agctatacta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggaggg atcatcccta
tctttggtac aacaaactac 180gcacagaggt tccagggcag agtcacgatt
accgcggacg aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccatat attactgttc aaccaactcc 300tatagcagca
gctggtatga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360tcttca
366101324DNAArtificialnucleotide sequence encoding VL of C333
101gaaattgtgt tgacgcagtc tccagccacc ctgtctgtgt ctcctgggga
aagagccacc 60ctctcctgca gggccagtca gagtattacc aacaacttag cctggtatca
acaactgcct 120ggccaggctc ccaggctcct tatttacgat gcatccacca
gggccactga tatcccagcc 180aggttcagtg gcactgggtc tgggtcagag
ttcactctca ccatcagcgg cctgcagtct 240gcggattttg cagtttatta
ctgtcaacaa tacaataact ggcctctcac tttcggcgga 300gggaccaaag
tggatatcaa acgt 324102366DNAArtificialnucleotide sequence encoding
VH of C323 102caaatgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc agctatacta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaggg
atcatcccta tctttggtac aacaaactac 180gcacagaggt tccagggcag
agtcacgatt accgcggacg aatccacgag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccatat attactgttc aaccaactcc
300tatagcagca gctggtatga tgcttttgat atctggggcc aagggacaat
ggtcaccgtc 360tcttca 366103324DNAArtificialnucleotide sequence
encoding VL of C323 103gccatccagt tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gcgcaagtca gggcattggc agtgctttag
cctggtatca gcagaaacca 120gggaaagctc ctaagctcct gatctatgat
gcctccagtt tgcaaagtgg ggtcccatca 180aggtacagcg gcagtggatc
tgggacagat ttcactctca ccatcagcgg cctgcagcct 240gaagattttg
caacttatta ctgtcaacag tttagttatt acccgctcac tttcggcgga
300gggaccaagc tggagatcaa acgt 324104366DNAArtificialnucleotide
sequence encoding VH of C321 104caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta cacctttacc
agctatggta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atcagcgctt acaatggtaa cacaaactat 180gcacagaagc
tccagggcag agtcaccatg accacagaca catccacgag cacagcctac
240atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc
gagagactcc 300catatttacg atattttgac tggttatgac tactggggcc
agggaaccct ggtcaccgtc 360tcctca 366105339DNAArtificialnucleotide
sequence encoding VL of C321 105gatgttgtga tgacacagac tccactctcc
tcacctgtca cccttggaca gccggcctcc 60atctcctgcc actctagtca aagcctcgta
cacagtgatg gaaacaccta cttgagttgg 120cttcagcaga ggcccggcca
gcctccaaga ctcctaattt ataagatttc taaccggttc 180tctggggtcc
cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc
240agcagggtgg aagctgagga tgtcggggtt tattactgca tgcaagatac
acaatttcct 300cagacgttcg gccaagggac caaggtggaa atcaaacgt
339106357DNAArtificialnucleotide sequence encoding VH of C320
106caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt
gcgacaggcc 120actggacaag ggcttgagtg gatgggatgg atgaacccta
acagtggtaa cacaggctat 180gcacagaagt tccagggcag agtcaccatg
accaggaaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagaagtg 300cctgatacgg
cctcctttga gtactggggc cagggaaccc tggtcaccgt ctcctca
357107333DNAArtificialnucleotide sequence encoding VL of C320
107cagtctgtgc tgacgcagcc gccctcagtg tctggggccc cagggcagag
ggtcaccatc 60tcctgcgctg ggagtagttc cgacatcggg gcaggtcttg gcgtgcactg
gtatcagcag 120cttccaggaa cagcccccaa actcctcatc tatggttact
acaatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc
acctcagcct ccctggccat cactgggctc 240ctgcctgagg atgagggtga
ttattactgc cagtcctatg acggcactct gagtgcccta 300ttcggcggag
ggaccaagct gaccgtccta ggt 333108369DNAArtificialnucleotide sequence
encoding VH of C319 108cagctgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcggggac cctgtccctc 60acctgcgctg tctctggtgg ctccatcagc agtagaaact
ggtggagttg ggtccgccag 120tccccaggga aggggctgga gtggattggg
gaaatctatc acagtgatat aaccaactat 180aacccgtccc tcaagagtcg
agtcaccatt tcagtagaca agtccaagaa ccagttctcc 240ctgaagctga
gctctgtgac cgccgcggac acggccgtgt attactgtgc gaaggacggg
300gaggcgggcg ggacctacat tgatgctttt gatgtctggg gccaagggac
aatggtcacc 360gtctcttca 369109333DNAArtificialnucleotide sequence
encoding VL of C319 109cagtctgccc tgactcagcc tcgctcagtg tccgggtctc
ctggacagtc agtcaccatc 60tcctgcactg gaaccagcag tgatgttggt atttataact
atgtctcctg gtaccaacaa 120cacccaggca aagcccccaa actcataatt
tatgatgtca gtgagcggcc ctcaggggtc 180cctgatcgct tctctggctc
caagtctgac aacacggcct ccctgaccat ctctgggctc 240caggctgagg
atgaggctga ttattactgc tactcatatg caggcaccta cacttcctta
300ttcggcggag ggaccaaggt caccgtccta ggt
333110357DNAartificialnucleotide sequence encoding VH of C320-3
110caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt
gcgacaggcc 120cccggacaag ggcttgagtg gatgggatgg atgaacccta
acagtggtaa cacaggctat 180gcacagaagt tccagggcag agtcaccatg
accaggaaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagaagtg 300cctgatacgg
cctcctttga gtactggggc cagggaaccc tggtcaccgt ctcctca
357111333DNAartificialnucleotide sequence encoding VL of C320-5
111cagtctgtgc tgacgcagcc gccctcagtg tctggggccc cagggcagag
ggtcaccatc 60tcctgcgctg ggagtagttc cgacatcggg gcaggtcttg gcgtgcactg
gtatcagcag 120cttccaggaa cagcccccaa actcctcatc tatggttact
acaatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc
acctcagcct ccctgaccat cactgggctc 240ctgcctgagg atgagggtga
ttattactgc cagtcctatg acggcactct gagtgcccta 300ttcggcggag
ggaccaagct gaccgtccta ggt 333112357DNAartificialnucleotide sequence
encoding VH of C320-90 112caggtgcagc tggtgcagtc tggcgccgaa
gtgaagaaac ctggcgccag cgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc
agctacgaca tcaactgggt ccgacaggcc 120accggccagg gcctggaatg
gatgggctgg atgaacccca acagcggcaa caccggctac 180gcccagaaat
tccagggcag agtgaccatg acccggaaca ccagcatcag caccgcctac
240atggaactga gcagcctgcg gagcgaggac accgccgtgt actactgcgc
cagagaggtg 300cccgacgacg ccagcttcga gtattggggc cagggcaccc
tggtcaccgt gtctagc 357113357DNAartificialnucleotide sequence
encoding VH of C320-103 113caggtgcagc tggtgcagtc tggcgccgaa
gtgaagaaac ctggcgccag cgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc
agctacgaca tcaactgggt ccgacaggcc 120accggccagg gcctggaatg
gatgggctgg atgaacccca acagcggcaa caccggctac 180gcccagaaat
tccagggcag agtgaccatg acccggaaca ccagcatcag caccgcctac
240atggaactga gcagcctgcg gagcgaggac accgccgtgt actactgcgc
cagagaggtg 300cccgacaccg ccgccttcga gtattggggc cagggcaccc
tggtcaccgt gtctagc 357114357DNAartificialnucleotide sequence
encoding VH of C320-114 114caggtgcagc tggtgcagtc tggcgccgaa
gtgaagaaac ctggcgccag cgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc
agctacgaca tcaactgggt ccgacaggcc 120accggccagg gcctggaatg
gatgggctgg atgaacccca acagcggcaa caccggctac 180gcccagaaat
tccagggcag agtgaccatg acccggaaca ccagcatcag caccgcctac
240atggaactga gcagcctgcg gagcgaggac accgccgtgt actactgcgc
cagagaggtg 300cccgacaccg ccagcttcct gtattggggc cagggcaccc
tggtcaccgt gtctagc 357115357DNAartificialnucleotide sequence
encoding VH of C320-115 115caggtgcagc tggtgcagtc tggcgccgaa
gtgaagaaac ctggcgccag cgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc
agctacgaca tcaactgggt ccgacaggcc 120accggccagg gcctggaatg
gatgggctgg atgaacccca acagcggcaa caccggctac 180gcccagaaat
tccagggcag agtgaccatg acccggaaca ccagcatcag caccgcctac
240atggaactga gcagcctgcg gagcgaggac accgccgtgt actactgcgc
cagagaggtg 300cccgacaccg ccagcttcga ctattggggc cagggcaccc
tggtcaccgt gtctagc 357116333DNAartificialnucleotide sequence
encoding VL of C320-120 116cagagcgtgc tgacacagcc ccctagcgtg
tcaggcgccc ctggccagag agtgaccatc 60tcttgcaccg gcagcagcag cgacatcgga
gctggactgg gcgtgcactg gtatcagcag 120ctgcctggca ccgcccccaa
gctgctgatc tacggctact acaaccggcc cagcggcgtg 180cccgacagat
tcagcggcag caagagcggc accagcgcca gcctggccat cactggactg
240ctgcccgagg acgagggcga ctactactgc cagagctacg acggcaccct
gagcgccctg 300tttggcggag gcaccaagct gaccgtccta ggt
333117357DNAartificialnucleotide sequence encoding VH of C320-129
117caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt
gcgacaggcc 120actggacaag ggcttgagtg gatgggatgg atgaacccta
acagtggtaa cacaggctat 180gcacagaagt tccagggcag agtcaccatg
accaggaaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagaagtg 300cctgagacgg
cctcctttga gtactggggc cagggaaccc tggtcaccgt ctcctca
357118357DNAartificialnucleotide sequence encoding VH of C320-130
118caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt
gcgacaggcc 120actggacaag ggcttgagtg gatgggatgg ctgaacccta
acagtggtaa cacaggctat 180gcacagaagt tccagggcag agtcaccatg
accaggaaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagaagtg 300cctgatacgg
cctcctttga gtactggggc cagggaaccc tggtcaccgt ctcctca
357119124PRTArtificialamino acid sequence of VH of humanized
antibody 1B4 119Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys
Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe
Ser Leu Ser Thr Ser 20 25 30 Asn Met Gly Val Val Trp Ile Arg Gln
Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Leu Trp
Asp Asp Arg Glu Tyr Ser Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu
Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr
Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys
Ala Arg Met Ser Arg Asn Tyr Tyr Gly Ser Ser Tyr Val Met Asp 100 105
110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
120106PRTArtificialamino acid sequence of VL of humanized antibody
1B4 120Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val
Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr
Tyr Cys His Gln Trp Asn Asn Tyr Gly Thr Phe 85 90 95 Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 121372DNAArtificialnucleotide
sequence encoding VH of humanized antibody 1B4 121caggtcacac
tgaaagagtc cggccctgcc ctggtcaagc ccacccagac cctgaccctg 60acatgcacct
tcagcggctt cagcctgagc accagcaaca tgggcgtcgt gtggatcaga
120cagccccctg gcaaggccct ggaatggctg gcccacatcc tgtgggacga
cagagagtac 180agcaaccccg ccctgaagtc ccggctgacc atcagcaagg
acaccagcaa gaaccaggtg 240gtgctgacca tgaccaacat ggaccccgtg
gacaccgcca cctactactg cgcccggatg 300agccggaact actacggcag
cagctacgtg atggactact ggggccaggg caccctggtc 360accgtgtcct ca
372122318DNAArtificialnucleotide sequence encoding VL of humanized
antibody 1B4 122gacatccagc tgacccagag ccccagcttc ctgagcgcca
gcgtgggcga cagagtgacc 60atcacctgta gcgccagcag cagcgtgaac tacatgcact
ggtatcagca gaagcccggc 120aaggccccca agctgctgat ctacagcacc
agcaacctgg ccagcggcgt gcccagcaga 180ttttctggca gcggcagcgg
caccgagttc accctgacca tcagcagcct gcagcccgag 240gacttcgcca
cctactactg ccaccagtgg aacaactacg gcaccttcgg ccagggcacc
300aaggtggaaa tcaagcgt 318123251PRTHomo sapiens 123Met Ala Glu Asp
Leu Gly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 Met Leu
Pro Glu His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30
Ala Arg Trp Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35
40 45 Gly Leu Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly
Glu 50 55 60 Ala Cys Val Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe
Ala Pro Ser 65 70 75 80 His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp
Gly Asp Lys Pro Arg 85 90 95 Ala His Leu Thr Val Val Arg Gln Thr
Pro Thr Gln His Phe Lys Asn 100 105 110 Gln Phe Pro Ala Leu His Trp
Glu His Glu Leu Gly Leu Ala Phe Thr 115 120 125 Lys Asn Arg Met Asn
Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140 Gly Asp Tyr
Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser 145 150 155 160
Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser 165
170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro
Thr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly
Ser Asn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser
Leu Gln Glu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile
Ser Leu Val Asp Tyr Thr Lys 225 230 235 240 Glu Asp Lys Thr Phe Phe
Gly Ala Phe Leu Leu 245 250 124177PRTMus musculus 124 Ile Thr Glu
Glu Arg Ser Glu Pro Ser Pro Gln Gln Val Tyr Ser Pro 1 5 10 15 Pro
Arg Gly Lys Pro Arg Ala His Leu Thr Ile Lys Lys Gln Thr Pro 20 25
30 Ala Pro His Leu Lys Asn Gln Leu Ser Ala Leu His Trp Glu His Asp
35 40 45 Leu Gly Met Ala Phe Thr Lys Asn Gly Met Lys Tyr Ile Asn
Lys Ser 50 55 60 Leu Val Ile Pro Glu Ser Gly Asp Tyr Phe Ile Tyr
Ser Gln Ile Thr 65 70 75 80 Phe Arg Gly Thr Thr Ser Val Cys Gly Asp
Ile Ser Arg Gly Arg Arg 85 90 95 Pro Asn Lys Pro Asp Ser Ile Thr
Met Val Ile Thr Lys Val Ala Asp 100 105 110 Ser Tyr Pro Glu Pro Ala
Arg Leu Leu Thr Gly Ser Lys Ser Val Cys 115 120 125 Glu Ile Ser Asn
Asn Trp Phe Gln Ser Leu Tyr Leu Gly Ala Thr Phe 130 135 140 Ser Leu
Glu Glu Gly Asp Arg Leu Met Val Asn Val Ser Asp Ile Ser 145 150 155
160 Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu
165 170 175 Leu 125180PRTMacaca mulatta 125Leu Lys Gly Gln Glu Phe
Ala Pro Ser His Gln Gln Val Tyr Ala Pro 1 5 10 15 Leu Arg Ala Asp
Gly Asp Lys Pro Arg Ala His Leu Thr Val Val Arg 20 25 30
Gln Thr Pro Thr Gln His Leu Lys Asn Gln Phe Pro Ala Leu His Trp 35
40 45 Glu His Glu Leu Gly Leu Ala Phe Thr Lys Asn Arg Met Asn Tyr
Thr 50 55 60 Asn Lys Phe Leu Leu Ile Pro Glu Ser Gly Asp Tyr Phe
Val Tyr Ser 65 70 75 80 Gln Val Thr Phe Arg Gly Met Thr Ser Glu Cys
Ser Glu Ile Arg Gln 85 90 95 Ala Gly Arg Pro Asn Lys Pro Asp Ser
Ile Thr Val Val Ile Thr Lys 100 105 110 Val Thr Asp Ser Tyr Pro Glu
Pro Thr Gln Leu Leu Met Gly Thr Lys 115 120 125 Ser Val Cys Glu Val
Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu Gly 130 135 140 Ala Met Phe
Ser Leu Gln Glu Gly Asp Lys Leu Met Val Asn Val Ser 145 150 155 160
Asp Ile Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly 165
170 175 Ala Phe Leu Leu 180 126177PRTRattus rattus 126Val Thr Glu
Glu Arg Ser Ala Pro Ser Ala Gln Pro Val Tyr Thr Pro 1 5 10 15 Ser
Arg Asp Lys Pro Lys Ala His Leu Thr Ile Met Arg Gln Thr Pro 20 25
30 Val Pro His Leu Lys Asn Glu Leu Ala Ala Leu His Trp Glu Asn Asn
35 40 45 Leu Gly Met Ala Phe Thr Lys Asn Arg Met Asn Tyr Thr Asn
Lys Phe 50 55 60 Leu Val Ile Pro Glu Ser Gly Asp Tyr Phe Ile Tyr
Ser Gln Ile Thr 65 70 75 80 Phe Arg Gly Thr Thr Ser Glu Cys Gly Asp
Ile Ser Arg Val Arg Arg 85 90 95 Pro Lys Lys Pro Asp Ser Ile Thr
Val Val Ile Thr Lys Val Ala Asp 100 105 110 Ser Tyr Pro Glu Pro Ala
His Leu Leu Thr Gly Thr Lys Ser Val Cys 115 120 125 Glu Ile Ser Ser
Asn Trp Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe 130 135 140 Ser Leu
Glu Glu Gly Asp Arg Leu Met Val Asn Val Ser Asp Ile Ser 145 150 155
160 Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu
165 170 175 Ile 127179PRTOryctolagus cuniculus 127Leu Lys Gly Arg
Glu Phe Gly Pro Ser Gln Gln Arg Ala Tyr Met Pro 1 5 10 15 Leu Arg
Ala Asp Gly Asn Lys Pro Arg Ala His Leu Thr Ala Val Lys 20 25 30
Gln Thr Pro Thr Gln Pro Leu Arg Asn His Phe Pro Ala Leu His Trp 35
40 45 Glu His Glu Leu Gly Leu Ala Phe Thr Lys Asn Arg Met Asn Tyr
Thr 50 55 60 Asn Lys Phe Leu Val Ile Pro Glu Ser Gly Asp Tyr Phe
Val Tyr Ser 65 70 75 80 Gln Val Thr Phe Arg Gly Thr Thr Ser Glu Cys
Gly Val Ile Asn Gln 85 90 95 Arg Arg Arg Gln Thr Lys Pro Asp Ser
Ile Val Val Val Ile Thr Lys 100 105 110 Val Thr Asp Asn Tyr Pro Glu
Pro Ala Gln Leu Leu Thr Gly Thr Lys 115 120 125 Ser Val Cys Glu Met
Gly Asn Trp Phe Gln Pro Ile Tyr Leu Gly Ala 130 135 140 Met Phe Ser
Leu Glu Glu Gly Asp Lys Leu Met Val Asn Val Ser Asp 145 150 155 160
Val Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala 165
170 175 Phe Leu Leu 128180PRTCavia porcellus 128Thr Ile Asn Glu Gln
Arg Phe Gly Pro Ser Tyr Gln Arg Val Tyr Thr 1 5 10 15 Pro Leu Arg
Asp Asp Arg Asp Lys Pro Arg Ala His Leu Thr Val Val 20 25 30 Arg
Gln Thr Pro Thr Gln His Leu Lys Asn Gln Phe Pro Ala Leu His 35 40
45 Trp Glu His Glu Leu Gly Leu Ala Phe Thr Lys Asn Arg Met Asn Tyr
50 55 60 Thr Asn Lys Phe Leu Val Ile Pro Glu Thr Gly Asp Tyr Phe
Val Tyr 65 70 75 80 Ser Gln Ile Thr Phe Arg Gly Thr Thr Ser Glu Cys
Gly Ile Ser Pro 85 90 95 Gly Arg Gln Gln Asn Lys Pro Asp Ser Ile
Phe Val Val Ile Thr Lys 100 105 110 Val Thr Asp Ser Tyr Pro Glu Pro
Ser Gln Leu Leu Thr Gly Thr Lys 115 120 125 Ser Val Cys Glu Ile Ser
Ser Asn Trp Phe Gln Pro Leu Tyr Leu Gly 130 135 140 Ala Met Phe Ser
Leu Gln Glu Gly Asp Lys Leu Met Val Asn Val Ser 145 150 155 160 Asp
Ile Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly 165 170
175 Ala Phe Leu Leu 180 129417PRTHomo sapiens 129Met Glu Gln Arg
Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu 1 5 10 15 Leu Val
Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg 20 25 30
Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys 35
40 45 Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu
Pro 50 55 60 Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr
Phe Leu Ala 65 70 75 80 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg
Cys Gln Ala Cys Asp 85 90 95 Glu Gln Ala Ser Gln Val Ala Leu Glu
Asn Cys Ser Ala Val Ala Asp 100 105 110 Thr Arg Cys Gly Cys Lys Pro
Gly Trp Phe Val Glu Cys Gln Val Ser 115 120 125 Gln Cys Val Ser Ser
Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys 130 135 140 Gly Ala Leu
His Arg His Thr Arg Leu Leu Cys Ser Arg Arg Gly Thr 145 150 155 160
Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys 165
170 175 Val Ser Cys Pro Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys
Ala 180 185 190 Ala Val Cys Gly Trp Arg Gln Met Phe Trp Val Gln Val
Leu Leu Ala 195 200 205 Gly Leu Val Val Pro Leu Leu Leu Gly Ala Thr
Leu Thr Tyr Thr Tyr 210 215 220 Arg His Cys Trp Pro His Lys Pro Leu
Val Thr Ala Asp Glu Ala Gly 225 230 235 240 Met Glu Ala Leu Thr Pro
Pro Pro Ala Thr His Leu Ser Pro Leu Asp 245 250 255 Ser Ala His Thr
Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys Ile Cys 260 265 270 Thr Val
Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr Pro Glu Thr 275 280 285
Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp Asp Gln Leu Pro 290
295 300 Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr Leu Ser Pro Glu
Ser 305 310 315 320 Pro Ala Gly Ser Pro Ala Met Met Leu Gln Pro Gly
Pro Gln Leu Tyr 325 330 335 Asp Val Met Asp Ala Val Pro Ala Arg Arg
Trp Lys Glu Phe Val Arg 340 345 350 Thr Leu Gly Leu Arg Glu Ala Glu
Ile Glu Ala Val Glu Val Glu Ile 355 360 365 Gly Arg Phe Arg Asp Gln
Gln Tyr Glu Met Leu Lys Arg Trp Arg Gln 370 375 380 Gln Gln Pro Ala
Gly Leu Gly Ala Val Tyr Ala Ala Leu Glu Arg Met 385 390 395 400 Gly
Leu Asp Gly Cys Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly 405 410
415 Pro 130300PRTHomo sapiens 130Met Arg Ala Leu Glu Gly Pro Gly
Leu Ser Leu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu
Pro Val Pro Ala Val Arg Gly Val Ala Glu 20 25 30 Thr Pro Thr Tyr
Pro Trp Arg Asp Ala Glu Thr Gly Glu Arg Leu Val 35 40 45 Cys Ala
Gln Cys Pro Pro Gly Thr Phe Val Gln Arg Pro Cys Arg Arg 50 55 60
Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr Gln 65
70 75 80 Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Cys Asn Val Leu
Cys Gly 85 90 95 Glu Arg Glu Glu Glu Ala Arg Ala Cys His Ala Thr
His Asn Arg Ala 100 105 110 Cys Arg Cys Arg Thr Gly Phe Phe Ala His
Ala Gly Phe Cys Leu Glu 115 120 125 His Ala Ser Cys Pro Pro Gly Ala
Gly Val Ile Ala Pro Gly Thr Pro 130 135 140 Ser Gln Asn Thr Gln Cys
Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala 145 150 155 160 Ser Ser Ser
Ser Ser Glu Gln Cys Gln Pro His Arg Asn Cys Thr Ala 165 170 175 Leu
Gly Leu Ala Leu Asn Val Pro Gly Ser Ser Ser His Asp Thr Leu 180 185
190 Cys Thr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val Pro Gly Ala
195 200 205 Glu Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe Gln
Asp Ile 210 215 220 Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu
Glu Ala Pro Glu 225 230 235 240 Gly Trp Gly Pro Thr Pro Arg Ala Gly
Arg Ala Ala Leu Gln Leu Lys 245 250 255 Leu Arg Arg Arg Leu Thr Glu
Leu Leu Gly Ala Gln Asp Gly Ala Leu 260 265 270 Leu Val Arg Leu Leu
Gln Ala Leu Arg Val Ala Arg Met Pro Gly Leu 275 280 285 Glu Arg Ser
Val Arg Glu Arg Phe Leu Pro Val His 290 295 300
1312PRTArtificialsequence from TL1a 131Leu Ala 1
1324PRTArtificialsequence from TL1a 132Gln Pro Ile Tyr 1
1337PRTArtificialsequence from TL1a 133Asn Val Ser Asp Ile Ser Leu
1 5 134330PRTHomo sapiens 134Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195
200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315
320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 135106PRTHomo
sapiens 135 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 105 136105PRTHomo sapiens
136Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser
Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp
Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala
Pro Thr Glu Cys Ser 100 105 137120PRTArtificial SequenceVH
consensus sequence of C320 and derivatives 137Xaa Val Gln Xaa Xaa
Xaa Xaa Ser Gly Ala Glu Val Xaa Xaa Pro Gly 1 5 10 15 Xaa Ser Val
Xaa Val Ser Cys Xaa Xaa Ser Gly Tyr Thr Phe Thr Ser 20 25 30 Tyr
Asp Ile Asn Trp Val Arg Gln Ala Xaa Gly Xaa Gly Xaa Xaa Trp 35 40
45 Xaa Gly Trp Xaa Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys
50 55 60 Phe Gln Gly Arg Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa
Thr Xaa 65 70 75 80 Tyr Xaa Xaa Leu Xaa Xaa Leu Arg Xaa Xaa Asp Thr
Ala Xaa Tyr Tyr 85 90 95 Cys Xaa Xaa Glu Xaa Pro Xaa Xaa Ala Xaa
Phe Xaa Tyr Trp Gly Xaa 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser
115 120 138112PRTArtificial Sequenceconsensus sequence of C320 and
derivatives 138Xaa Xaa Xaa Leu Thr Gln Xaa Xaa Xaa Xaa Xaa Ser Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Ser
Ser Asp Ile Gly Ala 20 25 30 Gly Leu Gly Val His Trp Xaa Gln Gln
Xaa Xaa Gly Xaa Xaa Pro Xaa 35 40 45 Leu Xaa Xaa Xaa Gly Xaa Xaa
Asn Arg Pro Ser Xaa Xaa Xaa Asp Arg 50 55 60 Phe Xaa Gly Xaa Lys
Ser Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Glu Xaa Xaa Xaa Asp Tyr Tyr Cys Gln Ser Tyr Asp Xaa 85 90 95 Xaa
Leu Ser Ala Leu Phe Xaa Gly Gly Thr Xaa Xaa Xaa Xaa Xaa Gly 100 105
110 13914PRTartificialLCDR1 consensus sequence of C320 and
derivatives 139Xaa Xaa Ser Ser Ser Asp Ile Gly Ala Gly Leu Gly Val
His 1 5 10 1407PRTartificialLCDR2 consensus sequence of C320 and
derivatives 140Gly Tyr Tyr Asn Arg Pro Ser 1 5
14110PRTartificialLCDR3 consensus sequence of C320 and
derivatives
141Gln Ser Tyr Asp Gly Thr Leu Ser Ala Leu 1 5 10
14217PRTartificialHCDR2 consensus sequence of C320 and derivatives
142Trp Xaa Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln
1 5 10 15 Gly 14310PRTartificialHCDR3 consensus sequence of C320
and derivatives 143Glu Val Pro Xaa Thr Ala Xaa Phe Glu Tyr 1 5 10
14430PRTArtificial Sequenceamino acid sequence of HFR1 consesnys
seq od C320 and der 144Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr 20 25 30 14514PRTArtificial SequenceHFR2
consensus sequence of C320 and derivatives 145Trp Val Arg Gln Ala
Xaa Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 14632PRTArtificial
SequenceHFR3 consensus sequence of C320 and derivatives 146Arg Val
Thr Met Thr Xaa Xaa Xaa Xaa Xaa Ser Thr Ala Tyr Met Glu 1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20
25 30 14711PRTArtificial SequenceHFR4 consensus sequence of C320
and derivatives 147Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5
10 14822PRTArtificial SequenceLFR1 consensus sequence of C320 and
derivatives 148Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala
Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys 20 14915PRTArtificial
SequenceLFR2 consensus sequence of C320 and derivatives 149Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Xaa 1 5 10 15
15032PRTArtificial SequenceLFR3 consensus sequence of C320 and
derivatives 150Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr
Ser Ala Ser 1 5 10 15 Leu Xaa Ile Thr Gly Leu Leu Pro Glu Asp Glu
Gly Asp Tyr Tyr Cys 20 25 30 15111PRTArtificial SequenceLFR4
consensus sequence of C320 and derivatives 151Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly 1 5 10 152119PRTArtificial SequenceVH
consensus sequence of C320 and derivatives 152Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp
Ile Asn Trp Val Arg Gln Ala Xaa Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Xaa Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Xaa Xaa Ala Xaa Phe
Xaa Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
153111PRTArtificial SequenceVl consensus sequence of C320 and
derivatives 153Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala
Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Xaa Gly Ser Ser Ser
Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr Tyr Asn
Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser
Gly Thr Ser Ala Ser Leu Xaa Ile Thr Gly Leu 65 70 75 80 Leu Pro Glu
Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90 95 Leu
Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
154119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 1TZG 154Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Arg Pro Gly Ser 1 5 10 15 Ser Val Thr Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Met 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Ile Thr Ile Thr Ala Asp Arg Ser Thr Ser Thr Ala Tyr 65 70
75 80 Leu Glu Leu Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
155120PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 1RHH 155Glu Val Gln Leu Leu Glu Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly 1 5 10 15 Ser Ser Val Gln Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser 20 25 30 Tyr Asp Ile Asn Trp
Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp 35 40 45 Met Gly Trp
Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys 50 55 60 Phe
Gln Gly Arg Val Thr Phe Thr Ala Asp Gln Ala Thr Ser Thr Ala 65 70
75 80 Tyr Met Glu Leu Thr Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr 85 90 95 Cys Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
156119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 2DD8 156Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
157119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 2JB5 157Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
158119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 3FKU 158Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr
Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Gln Ser Thr Arg Thr Ala Tyr 65 70
75 80 Met Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
159119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 3GBM 159Glu Val Gln Leu Val Glu Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Asp Phe Ala Gly Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Lys Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Lys Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
160119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 3LMJ 160Gln Val Gln Val Val Gln Ser Gly Ala
Glu Val Arg Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Val Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Lys Arg Val Ser Met Thr Glu Asp Thr Ser Thr Asn Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Thr Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
161119PRTArtificial SequenceVH comprising CDRs from C320 grafted
onto FRs of antibody 3P30 161Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Ala Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Pro Gln Trp Met 35 40 45 Gly Trp Met
Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Leu Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70
75 80 Leu Glu Leu Ser Ser Leu Arg Tyr Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Thr Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
162120PRTArtificial Sequenceamino acid sequence of VH consensus
sequence of C320 and derivatives 162Xaa Val Gln Xaa Xaa Xaa Xaa Ser
Gly Ala Glu Val Xaa Xaa Pro Gly 1 5 10 15 Xaa Ser Val Xaa Val Ser
Cys Xaa Xaa Ser Gly Tyr Thr Phe Thr Ser 20 25 30 Tyr Asp Ile Asn
Trp Val Arg Gln Ala Xaa Gly Xaa Gly Xaa Xaa Trp 35 40 45 Xaa Gly
Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys 50 55 60
Phe Gln Gly Arg Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa 65
70 75 80 Tyr Xaa Xaa Leu Xaa Xaa Leu Arg Xaa Xaa Asp Thr Ala Xaa
Tyr Tyr 85 90 95 Cys Xaa Xaa Glu Val Pro Asp Thr Ala Ser Phe Glu
Tyr Trp Gly Xaa 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser 115 120
163112PRTArtificial SequenceVL comprising CDRs from C320 grafted
onto FRs of antibody 1RHH 163Glu Leu Val Leu Thr Gln Ser Pro Gly
Thr Leu Ser Leu Ser Ala Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Ala Gly Ser Ser Ser Asp Ile Gly Ala 20 25 30 Gly Leu Gly Val His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 35 40 45 Leu Leu Ile
Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Ile Pro Asp Arg 50 55 60 Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Arg 65 70
75 80 Leu Glu Pro Glu Asp Leu Ala Val Tyr Tyr Cys Gln Ser Tyr Asp
Gly 85 90 95 Thr Leu Ser Ala Leu Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg 100 105 110 164112PRTArtificial SequenceVL comprising
CDRs from C320 grafted onto FRs of antibody 1TZGL 164Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Gln Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala 20 25
30 Gly Leu Gly Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg
35 40 45 Leu Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Val Ala
Asp Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Ser Tyr Asp Gly 85 90 95 Thr Leu Ser Ala Leu Phe Gly Gln
Gly Thr Lys Val Glu Val Lys Arg 100 105 110 165111PRTArtificial
SequenceVL comprising CDRs from C320 grafted onto FRs of antibody
2DD8 165Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly
Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Ala Gly Ser Ser Ser Asp Ile
Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Val Leu 35 40 45 Val Val Tyr Gly Tyr Tyr Asn Arg Pro
Ser Gly Ile Pro Glu Arg Phe 50 55 60 Ser Gly Ser Asn Ser Gly Asn
Thr Ala Thr Leu Thr Ile Ser Arg Val 65 70 75 80 Glu Ala Gly Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala
Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110
166111PRTArtificial SequenceVL comprising CDRs from C320 grafted
onto FRs of antibody 2JB5 166Asp Ile Ala Leu Thr Gln Pro Ala Ser
Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ala
Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp
Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70
75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly
Thr 85 90 95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly 100 105 110 167111PRTArtificial SequenceVL comprising CDRs
from C320 grafted onto FRs of antibody 3FKU 167Gln Pro Gly Leu Thr
Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr
Leu Thr Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu
Gly Val His Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu 35
40 45 Leu Ser Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Ile Ser Glu Arg
Phe 50 55 60 Ser Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile
Thr Gly Leu 65 70 75 80 Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly 100 105 110 168111PRTArtificial SequenceVL
comprising CDRs from C320 grafted onto FRs of antibody 3GBM 168Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10
15 Lys Val Thr Ile Ser Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly
20 25 30 Leu Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Ile
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr
Leu Gly Ile Thr Gly Leu 65 70 75 80 Gln Thr Gly Asp Glu Ala Asn Tyr
Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 169111PRTArtificial
SequenceVL comprising CDRs from C320 grafted onto FRs of antibody
3LMJ 169Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly
Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ala Gly Ser Ser Ser Asp Ile
Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln Gln Leu Pro Gly
Ala Ala Pro Lys Leu 35 40 45 Leu Ile Phe Gly Tyr Tyr Asn Arg Pro
Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Thr Gly Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90 95 Leu Ser Ala
Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
170111PRTArtificial SequenceVL comprising CDRs from C320 grafted
onto FRs of antibody 3P30 170Gln Ser Val Leu Thr Gln Pro Pro Ser
Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ala
Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70
75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly
Thr 85 90 95 Leu Ser Ala Leu Phe Gly Ala Gly Thr Lys Leu Thr Val
Leu Gly 100 105 110 171111PRTArtificial SequenceVL comprising CDRs
from C320 grafted onto FRs of antibody 3IYW 171Gln Ser Val Leu Thr
Gln Pro Ser Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu
Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40
45 Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Tyr Asp Gly Thr 85 90 95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 172112PRTArtificial SequenceVL
consensus sequence of C320 and derivatives 172Xaa Xaa Xaa Leu Thr
Gln Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Cys Ala Gly Ser Ser Ser Asp Ile Gly Ala 20 25 30 Gly
Leu Gly Val His Trp Xaa Gln Gln Xaa Xaa Gly Xaa Xaa Pro Xaa 35 40
45 Leu Xaa Xaa Tyr Gly Tyr Tyr Asn Arg Pro Ser Xaa Xaa Pro Asp Arg
50 55 60 Phe Xaa Gly Xaa Lys Ser Xaa Xaa Xaa Xaa Ser Xaa Ala Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Glu Xaa Xaa Xaa Asp Tyr Tyr Cys Gln
Ser Tyr Asp Gly 85 90 95 Thr Leu Ser Ala Leu Phe Xaa Gly Gly Thr
Xaa Xaa Xaa Xaa Xaa Gly 100 105 110 173119PRTArtificial SequenceVH
consensus sequence of C320 and derivatives 173Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp
Ile Asn Trp Val Arg Gln Ala Xaa Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Xaa Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Met Thr Xaa Xaa Xaa Xaa Xaa Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Xaa Thr Ala Xaa Phe
Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
174111PRTArtificial SequenceVL consensus sequence of C320 and
derivatives 174Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala
Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Xaa Xaa Ser Ser Ser
Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Xaa Gly Tyr Tyr Asn
Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser
Gly Thr Ser Ala Ser Leu Xaa Ile Thr Gly Leu 65 70 75 80 Leu Pro Glu
Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90 95 Leu
Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
175119PRTArtificial Sequenceamino acid sequence of VH of antibody
C320-162 175Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asn Thr Ala Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Pro Glu Thr Ala Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 176119PRTArtificial Sequenceamino
acid sequence of VH of antibody C320-163 176Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Ala Asp Arg Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
177119PRTArtificial Sequenceamino acid sequence of VH of antibody
C320-164 177Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Pro Glu Thr Ala Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 178119PRTArtificial Sequenceamino
acid sequence of VH of antibody C320-165 178Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Glu Thr Ala Ser Phe Glu
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
179119PRTArtificial Sequenceamino acid sequence of VH of antibody
C320-166 179Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Pro Glu Thr Ala Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 180119PRTArtificial Sequenceamino
acid sequence of VH of antibody C320-167 180Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Glu Thr Ala Ser Phe Glu
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
181119PRTArtificial Sequenceamino acid sequence of VH of antibody
C320-168 181Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Pro Glu Thr Ala Ala Phe Glu Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 182119PRTArtificial Sequenceamino
acid sequence of VH of antibody C320-169 182Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Glu Thr Ala Ala Phe Glu
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
183119PRTArtificial Sequenceamino acid sequence of VH of antibody
C320-170 183Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Ala Asp Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 184119PRTArtificial Sequenceamino
acid sequence of VH of antibody C320-171 184Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Ala Asp Arg Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser Phe Glu
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
185119PRTArtificial Sequenceamino acid sequence of VH of antibody
C320-172 185Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn Pro Asn Ser Gly
Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Ala Asp Arg Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Asp Thr Ala Ser
Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 186119PRTArtificial Sequenceamino acid sequence of VH of
antibody C320-179 186Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Asn Pro
Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Ala Asp Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Val Pro Glu Thr Ala Ala Phe Glu Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 187119PRTArtificial
Sequenceamino acid sequence of VH of antibody C320-183 187Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20
25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ala Asp Arg Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Pro Glu Thr
Ala Ala Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 188111PRTArtificial Sequenceamino acid sequence of VL
of antibody C320-162 188Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ala Gly Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 189111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-163 189Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ala Gly Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 190111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-164 190Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ala Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 191111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-165 191Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 192111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-166 192Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 193111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-167 193Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ala Gly Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 194111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-168 194Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 195111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-169 195Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ala Gly Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 196111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-170 196Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 197111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-171 197Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Pro Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 198111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-172 198Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Glu Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 199111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-179 199Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Glu Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 200111PRTArtificial Sequenceamino acid sequence of VL of
antibody C320-183 200Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Ser Ser
Ser Ser Asp Ile Gly Ala Gly 20 25 30 Leu Gly Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Gly Gly Tyr
Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Leu
Pro Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly Thr 85 90
95 Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 201111PRTArtificial Sequenceamino acid sequence of VL of
germline sequence IGLV1-40*1 201Gln Ser Val Leu Thr Gln Pro Pro Ser
Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr
Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr
Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70
75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
Ser 85 90 95 Leu Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 100 105 110 202180PRTArtificial Sequenceamino acid sequence
of soluble hTL1a 202Leu Lys Gly Gln Glu Phe Ala Pro Ser His Gln Gln
Val Tyr Ala Pro 1 5 10 15 Leu Arg Ala Asp Gly Asp Lys Pro Arg Ala
His Leu Thr Val Val Arg 20 25 30 Gln Thr Pro Thr Gln His Phe Lys
Asn Gln Phe Pro Ala Leu His Trp 35 40 45 Glu His Glu Leu Gly Leu
Ala Phe Thr Lys Asn Arg Met Asn Tyr Thr 50 55 60 Asn Lys Phe Leu
Leu Ile Pro Glu Ser Gly Asp Tyr Phe Ile Tyr Ser 65 70 75 80 Gln Val
Thr Phe Arg Gly Met Thr Ser Glu Cys Ser Glu Ile Arg Gln 85 90 95
Ala Gly Arg Pro Asn Lys Pro Asp Ser Ile Thr Val Val Ile Thr Lys 100
105 110 Val Thr Asp Ser Tyr Pro Glu Pro Thr Gln Leu Leu Met Gly Thr
Lys 115 120 125 Ser Val Cys Glu Val Gly Ser Asn Trp Phe Gln Pro Ile
Tyr Leu Gly 130 135 140 Ala Met Phe Ser Leu Gln Glu Gly Asp Lys Leu
Met Val Asn Val Ser 145 150 155 160 Asp Ile Ser Leu Val Asp Tyr Thr
Lys Glu Asp Lys Thr Phe Phe Gly 165 170 175 Ala Phe Leu Leu 180
2035PRTArtificial Sequenceamino acid sequence of N-linked
glycosylation site in VH of C320 203Arg Asn Thr Ser Ile 1 5
2045PRTArtificial Sequenceamino acid sequence from VH of 1TZG
corresponding to N-linked glycosylation site in VH of C320 204Ala
Asp Arg Ser Thr 1 5 20515PRTArtificial Sequenceamino acid sequence
of peptide from VH of C320-168 205Gly Leu Glu Trp Met Gly Trp Leu
Asn Pro Asn Ser Gly Asn Thr 1 5 10 15 20615PRTArtificial
Sequenceamino acid sequence of peptide from VL of C320-168 206Leu
Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10
15
20713PRTArtificial Sequenceamino acid sequence of influenza peptide
207Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr 1 5 10
20815PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 208Leu Leu Ile Glu Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 20915PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 209Leu Leu Ile Gly
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
21015PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 210Leu Leu Ile Pro Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 21115PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 211Leu Leu Ile Lys
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
21215PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 212Leu Leu Ile Tyr Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 21315PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 213Leu Leu Ile Glu
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
21415PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 214Leu Leu Ile Gly Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 21515PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 215Leu Leu Ile Pro
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
21615PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 216Leu Leu Ile Lys Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 21715PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 217Leu Leu Ile Tyr
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
21815PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 218Leu Leu Ile Glu Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 21915PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 219Leu Leu Ile Gly
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
22015PRTArtificial Sequenceamino acid sequence of mutant peptide
from VL of C320-168 220Leu Leu Ile Pro Gly Tyr Tyr Asn Arg Pro Ser
Gly Val Pro Asp 1 5 10 15 22115PRTArtificial Sequenceamino acid
sequence of mutant peptide from VL of C320-168 221Leu Leu Ile Lys
Gly Tyr Tyr Asn Arg Pro Ser Gly Val Pro Asp 1 5 10 15
222357DNAArtificial Sequencenucleotide sequence encoding VH of
antibody C320-162 222caggtgcagc tggtgcagtc tggggccgag gtgaagaagc
ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata
tcaactgggt gcgacaggcc 120cccggacaag ggcttgagtg gatgggatgg
ctgaacccta acagtggtaa cacaggctat 180gcacagaagt tccagggcag
agtcaccatg accaggaaca ccgccataag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaagtg
300cctgagacgg cctcctttga gtactggggc cagggaaccc tggtgacagt gtcctca
357223357DNAArtificial Sequencenucleotide sequence encoding VH of
antibody C320-163 223caggtgcagc tggtgcagtc tggggcggag gtgaagaagc
ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata
tcaactgggt gcgacaggcc 120cccggacaag ggcttgagtg gatgggatgg
atgaacccta acagtggtaa cacaggctat 180gcacagaagt tccagggcag
agtcaccatg accgcagatc gttccaccag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaagtg
300cctgatacgg cctcctttga gtactggggc cagggaaccc tggtcaccgt ctcctca
357224357DNAArtificial Sequencenucleotide sequence encoding VH of
antibodies C320-164, C320-165, C320-166 and C320-167 224caggtgcagc
tggtgcagtc tggggccgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc agttatgata tcaactgggt gcgacaggcc
120cccggacaag ggcttgagtg gatgggatgg ctgaacccta acagtggtaa
cacaggctat 180gcacagaagt tccagggcag agtcaccatg accaggaaca
cctccataag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagaagtg 300cctgagacgg cctcctttga
gtactggggc cagggaaccc tggtgacagt gtcctca 357225357DNAArtificial
Sequencenucleotide sequence encoding VH of antibodies C320-168 and
C320-169 225caggtgcagc tggtgcagtc tggggccgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt
gcgacaggcc 120cccggacaag ggcttgagtg gatgggatgg ctgaacccta
acagtggtaa cacaggctat 180gcacagaagt tccagggcag agtcaccatg
accaggaaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagaagtg 300cctgagacgg
ccgcctttga gtactggggc cagggaaccc tggtgacagt gtcctca
357226357DNAArtificial Sequencenucleotide sequence encoding VH of
antibodies C320-170 and C320-172 226caggtgcagc tggtgcagtc
tggggcggag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata
caccttcacc agttatgata tcaactgggt gcgacaggcc 120cccggacaag
ggcttgagtg gatgggatgg atgaacccta acagtggtaa cacaggctat
180gcacagaagt tccagggcag agtcaccatg accgcagatc gttccaccag
cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtgc gagagaagtg 300cctgatacgg cctcctttga gtactggggc
cagggaaccc tggtcaccgt ctcctca 357227357DNAArtificial
Sequencenucleotide sequence encoding VH of antibodies C320-179 and
C320-183 227caggtgcagc tggtgcagtc tggggcggag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt
gcgacaggcc 120cccggacaag ggcttgagtg gatgggatgg ctgaacccta
acagtggtaa cacaggctat 180gcacagaagt tccagggcag agtcaccatg
accgcagatc gttccaccag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagaagtg 300cctgagacgg
ccgcctttga gtactggggc cagggaaccc tggtcaccgt ctcctca
357228333DNAArtificial Sequencenucleotide sequence encoding VL of
antibodies C320-162, C320-163, C320-167 and C320-169 228cagtctgtgc
tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcgctg
ggagtagttc cgacatcggg gcaggtcttg gcgtgcactg gtatcagcag
120cttccaggaa cagcccccaa actcctcatc tatggttact acaatcggcc
ctcaggggtc 180cctgaccgat tctctggctc caagtctggc acctcagcct
ccctgaccat cactgggctc 240ctgcctgagg atgagggtga ttattactgc
cagtcctatg acggcactct gagtgcccta 300ttcggcggag ggaccaagct
gaccgtccta ggt 333229333DNAArtificial Sequencenucleotide sequence
encoding VL of antibody C320-164 229cagtctgtgc tgacgcagcc
gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcgcta gcagtagttc
cgacatcggg gcaggtcttg gcgtgcactg gtatcagcag 120cttccaggaa
cagcccccaa actcctcatc tatggttact acaatcggcc ctcaggggtc
180cctgaccgat tctctggctc caagtctggc acctcagcct ccctgaccat
cactgggctc 240ctgcctgagg atgagggtga ttattactgc cagtcctatg
acggcactct gagtgcccta 300ttcggcggag ggaccaagct gaccgtccta ggt
333230333DNAArtificial Sequencenucleotide sequence encoding VL of
antibodies C320-165, C320-168 and C320-170 230cagtctgtgc tgacgcagcc
gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcacta gcagtagttc
cgacatcggg gcaggtcttg gcgtgcactg gtatcagcag 120cttccaggaa
cagcccccaa actcctcatc tatggttact acaatcggcc ctcaggggtc
180cctgaccgat tctctggctc caagtctggc acctcagcct ccctgaccat
cactgggctc 240ctgcctgagg atgagggtga ttattactgc cagtcctatg
acggcactct gagtgcccta 300ttcggcggag ggaccaagct gaccgtccta ggt
333231333DNAArtificial Sequencenucleotide sequence encoding VL of
antibody C320-166 231cagtctgtgc tgacgcagcc gccctcagtg tctggggccc
cagggcagag ggtcaccatc 60tcctgcactg ggagtagttc cgacatcggg gcaggtcttg
gcgtgcactg gtatcagcag 120cttccaggaa cagcccccaa actcctcatc
tatggttact acaatcggcc ctcaggggtc 180cctgaccgat tctctggctc
caagtctggc acctcagcct ccctgaccat cactgggctc 240ctgcctgagg
atgagggtga ttattactgc cagtcctatg acggcactct gagtgcccta
300ttcggcggag ggaccaagct gaccgtccta ggt 333232333DNAArtificial
Sequencenucleotide sequence encoding VL of antibodies C320-172 and
C320-179 232cagtctgtgc tgacgcagcc gccctcagtg tctggggccc cagggcagag
ggtcaccatc 60tcctgcacta gcagtagttc cgacatcggg gcaggtcttg gcgtgcactg
gtatcagcag 120cttccaggaa cagcccccaa actcctcatc gaaggttact
acaatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc
acctcagcct ccctgaccat cactgggctc 240ctgcctgagg atgagggtga
ttattactgc cagtcctatg acggcactct gagtgcccta 300ttcggcggag
ggaccaagct gaccgtccta ggt 333233333DNAArtificial Sequencenucleotide
sequence encoding VL of antibody C320-183 233cagtctgtgc tgacgcagcc
gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcacta gcagtagttc
cgacatcggg gcaggtcttg gcgtgcactg gtatcagcag 120cttccaggaa
cagcccccaa actcctcatc ggaggttact acaatcggcc ctcaggggtc
180cctgaccgat tctctggctc caagtctggc acctcagcct ccctgaccat
cactgggctc 240ctgcctgagg atgagggtga ttattactgc cagtcctatg
acggcactct gagtgcccta 300ttcggcggag ggaccaagct gaccgtccta ggt
333234119PRTartificialamino acid sequence of VH of antibody C320-12
and C320-22 234Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys
Pro Gly Ala 1 5 10 15 Ser Thr Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Met Asn Pro Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Glu Val Pro Asp Thr Ala Ser Phe Glu Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 23510PRTartificialconsensus of
HCDR3 of C320 and derivatives 235Glu Xaa Pro Xaa Xaa Ala Xaa Phe
Xaa Tyr 1 5 10
* * * * *
References