U.S. patent application number 14/454884 was filed with the patent office on 2015-07-16 for humanized anti-beta7 antagonists and uses therefor.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Mark S. Dennis, Sherman Fong.
Application Number | 20150197560 14/454884 |
Document ID | / |
Family ID | 36000753 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197560 |
Kind Code |
A1 |
Fong; Sherman ; et
al. |
July 16, 2015 |
HUMANIZED ANTI-BETA7 ANTAGONISTS AND USES THEREFOR
Abstract
The invention provides therapeutic anti-beta7 antibodies,
compositions comprising, and methods of using these antibodies.
Inventors: |
Fong; Sherman; (Kalaheo,
HI) ; Dennis; Mark S.; (San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
36000753 |
Appl. No.: |
14/454884 |
Filed: |
August 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13788246 |
Mar 7, 2013 |
8835133 |
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14454884 |
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13348709 |
Jan 12, 2012 |
8779100 |
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13788246 |
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12390730 |
Feb 23, 2009 |
8124082 |
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13348709 |
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11219121 |
Sep 2, 2005 |
7528236 |
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12390730 |
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60607377 |
Sep 3, 2004 |
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Current U.S.
Class: |
424/133.1 ;
435/366; 530/387.3 |
Current CPC
Class: |
C07K 2317/55 20130101;
C07K 16/18 20130101; A61P 3/10 20180101; C07K 2317/565 20130101;
C07K 2317/14 20130101; C07K 2317/76 20130101; A61P 41/00 20180101;
A61P 37/06 20180101; A61P 1/04 20180101; C07K 2317/41 20130101;
A61P 29/00 20180101; A61P 11/06 20180101; A61P 1/00 20180101; A61P
43/00 20180101; C07K 16/2827 20130101; A61P 37/00 20180101; C07K
2317/24 20130101; C07K 16/2839 20130101; A61P 5/00 20180101; C07K
2317/92 20130101; A61K 2039/505 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-53. (canceled)
54. An anti-beta7 antibody comprising: an HVR-L1, HVR-L2, HVR-L3,
HVR-H1, HVR-H2 and HVR-H3, wherein each, in order, comprises
RASESVDDLLH (SEQ ID NO:9), KYASQSIS (SEQ ID NO:2), QQGNSLPNT (SEQ
ID NO:3), GFFITNNYWG (SEQ ID NO:4), GYISYSGSTSYNPSLKS (SEQ ID
NO:5), and RTGSSGYFDF (SEQ ID NO:66).
55-60. (canceled)
61. A method of inhibiting the interaction of a human beta7
integrin subunit with a second integrin subunit and/or a ligand by
contacting a humanized anti-beta7 antibody with the beta7
integrin.
62. The method of claim 61, wherein the anti-beta7 antibody
comprises an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,
wherein each, in order, comprises RASESVDDLLH (SEQ ID NO:9),
KYASQSIS (SEQ ID NO:2), QQGNSLPNT (SEQ ID NO:3), GFFITNNYWG (SEQ ID
NO:4), GYISYSGSTSYNPSLKS (SEQ ID NO:5), and RTGSSGYFDF (SEQ ID
NO:66).
63. A method of modulating beta7 integrin-mediated cellular
adhesion and/or recruitment in a human experiencing a disorder by
administering to the mammal an effective amount of a composition
comprising a humanized anti-beta7 antibody and a pharmaceutical
carrier.
64. The method of claim 63, wherein the anti-beta7 antibody
comprises an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,
wherein each, in order, comprises RASESVDDLLH (SEQ ID NO:9),
KYASQSIS (SEQ ID NO:2), QQGNSLPNT (SEQ ID NO:3), GFFITNNYWG (SEQ ID
NO:4), GYISYSGSTSYNPSLKS (SEQ ID NO:5), and RTGSSGYFDF (SEQ ID
NO:66).
Description
[0001] This is a continuation application of U.S. patent
application Ser. No. 13/348,709 filed Jan. 12, 2012, which is
pending, which is a continuation application of U.S. patent
application Ser. No. 12/390,730, filed Feb. 23, 2009, now U.S. Pat.
No. 8,124,082, which is a divisional application of U.S. patent
application Ser. No. 11/219,121 filed Sep. 2, 2005, now U.S. Pat.
No. 7,528,236, which claims the benefit under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Ser. No. 60/607,377,
filed Sep. 3, 2004, the entire contents of which is hereby
incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates generally to the fields of
molecular biology and growth factor regulation. More specifically,
the invention concerns modulators of the biological activity of
integrins containing the beta7 subunit, and uses of said
modulators.
BACKGROUND
[0003] The integrins are .alpha./.beta. heterodimeric cell surface
receptors involved in numerous cellular processes from cell
adhesion to gene regulation (Hynes, R. O., Cell, 1992, 69:11-25;
and Hemler, M. E., Annu Rev. Immunol., 1990, 8:365-368). Several
integrins have been implicated in disease processes and have
generated widespread interest as potential targets for drug
discovery (Sharar, S. R. et al., Springer Semin. Immunopathol.,
1995, 16:359-378). In the immune system, integrins are involved in
leukocyte trafficking, adhesion and infiltration during
inflammatory processes (Nakajima, H. et al., J. Exp. Med., 1994,
179:1145-1154). Differential expression of integrins regulates the
adhesive properties of cells and different integrins are involved
in different inflammatory responses. Butcher, E. C. et al.,
Science, 1996, 272:60-66. The beta7 integrins (i.e. alpha4beta7
(.alpha.4.beta.7) and alphaEbeta7 (.alpha.E.beta.7)) are expressed
primarily on monocytes, lymphocytes, eosinophils, basophils, and
macrophages but not on neutrophils. Elices, M. J. et al., Cell,
1990, 60:577-584. The primary ligands for .alpha.4.beta.7 integrin
are the endothelial surface proteins mucosal addressin cell
adhesion molecule (MAdCAM) and vascular cell adhesion molecule
(VCAM-1) (Makarem, R. et al., J. Biol. Chem., 1994, 269:4005-4011).
The binding of the .alpha.4.beta.7 to MAdCAM and/or VCAM expressed
on high endothelial venules (HEVs) at sites of inflammation results
in firm adhesion of the leukocyte to the endothelium followed by
extravasation into the inflamed tissue (Chuluyan, H. E. et al.,
Springer Semin. Immunopathol., 1995, 16:391-404). A primary ligand
for .alpha.E.beta.7 integrin is the intra-epithelial lymphocyte
(IEL) surface protein, E-cadherein, which facilitates adherence of
the .alpha.E.beta.7-bearing cell to epithelial lymphocytes.
Monoclonal antibodies directed against .alpha.4.beta.7, MAdCAM or
VCAM have been shown to be effective modulators in animal models of
chronic inflammatory diseases such as asthma (Laberge, S. et al.,
Am. J. Respir. Crit. Care Med., 1995, 151:822-829.), rheumatoid
arthritis (RA; Barbadillo, C. et al., Springer Semin.
Immunopathol., 1995, 16:375-379), colitis (Viney et al, J.
Immunol., 1996, 157: 2488-2497) and inflammatory bowel diseases
(IBD; Podalski, D. K., N. Eng. J. Med., 1991, 325:928-937; Powrie,
F. et al., Ther. Immunol., 1995, 2:115-123). Monoclonal antibodies
directed against beta7 subunit have been shown to bind the integrin
subunit (Tidswell, M. et al. (1997) J. Immunol. 159:1497-1505) but
as non-human or non-humanized antibodies, they lack clinical
usefulness.
[0004] A need exists for highly specific compounds, such as
humanized antibodies or binding fragments thereof which inhibit the
interaction between the alpha4beta7 integrin and its ligands MAdCAM
and/or VCAM as well as the interaction between the alphaEbeta7
integrin and its ligand E-cadherin. These compounds are useful for
treatment of chronic inflammatory diseases such as asthma, Crohn's
disease, ulcerative colitis, diabetes, complications of organ
transplantation, and allograft-related disorders.
[0005] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
DISCLOSURE OF THE INVENTION
[0006] The invention is in part based on the identification of a
variety of antagonists of biological pathways involving
beta7-containing integrins, which are generally biological/cellular
processes that presents as an important and advantageous
therapeutic target. Such biological pathways include, without
limitation, inflammation, particularly chronic inflammation
disorders such as asthma, allergy, IBD, diabetes, transplantation
and grafts versus host disorders. The invention provides
compositions and methods based on interfering with beta7
integrin-mediated cellular adhesion and/or recruitment, including
but not limited to inferfering with MAdCAM and VCAM-1 binding to
the extracellular portion of alpha4beta7 integrin and E-cadherin
interaction with the alphaEbeta7 integrin intereaction. Antagonists
of the invention, as described herein, provide important
therapeutic and diagnostic agents for use in targeting pathological
conditions associated with abnormal or unwanted signaling via a
beta7 integrin. Accordingly, the invention provides methods,
compositions, kits and articles of manufacture related to
modulating beta7 integrin-mediated pathways, including modulation
of MAdCAM-alpha4beta7 binding and leukocyte recruitment to
gastrointestinal epithelium, binding and allergy, asthma, IBD (such
as Crohn's disease and ulcerative colitis), diabetes, inflammation
associated with transplantation, graft versus host disorder and/or
allograft disorders and other biological/physiological activities
mediated by beta7 integrin.
[0007] In one aspect, the invention provides anti-beta7 therapeutic
agents suitable for therapeutic use and capable of effecting
varying degrees of disruption of a beta7 integrin-mediated pathway.
For example, in one embodiment, the invention provides a humanized
anti-beta7 antibody wherein the antibody as a Fab fragment has
substantially the same binding affinity to human beta7 as a murine
Fab fragment comprising, consisting or consisting essentially of a
light chain and heavy chain variable domain sequence as depicted in
FIGS. 1A and 1B or FIGS. 9A and 9B. In another embodiment, the
invention provides a humanized anti-beta7 antibody wherein the
antibody as a Fab fragment has a binding affinity to human beta7
that is lower, for example at least 3, at least 5, at least 7 or at
least 10-fold lower, than that of a murine or rat Fab fragment
comprising, consisting or consisting essentially of a light chain
and heavy chain variable domain sequence as depicted in FIGS. 1A
and 1B or the variable domain sequences depicted in FIGS. 9A and
9B. Alternatively, a humanized anti-beta7 antibody, or beta7
binding fragment thereof, of the invention exhibits monovalent
affinity to human beta7, which affinity is substantially the same
as or greater than monovalent affinity to human beta7 of an
antibody comprising light chain and heavy chain variable sequences
as depicted in FIG. 1A (SEQ ID NO:10) and/or FIG. 1B (SEQ ID
NO:11), or FIG. 9A (SEQ ID NO:12) and/or FIG. 9B (SEQ ID NO:13).
The antibody or binding fragment thereof having great affinity to
human beta7 exhibits an affinity which is at least 2-fold, at least
5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at
least 500-fold, at least 1000-fold, at least 5000-fold, or at least
10,000-fold greater than antibody comprising the light chain and
heavy chain sequences depicted in FIG. 1A (SEQ ID NO:10) and/or
FIG. 1B (SEQ ID NO:11), or FIG. 9A (SEQ ID NO:12) and/or FIG. 9B
(SEQ ID NO:13).
[0008] In another embodiment, the invention provides an anti-beta7
humanized antibody wherein the antibody as a Fab fragment has a
binding affinity to human beta7 that is greater, for example at
least 3, at least 5, at least 7, at least 9, at least 10, at least
15, at least 20, or at least 100-fold greater than that of a rodent
(such as rat or murine) Fab fragment comprising, consisting or
consisting essentially of a light chain and heavy chain variable
domain sequence as depicted in FIG. 1A and FIG. 1B, respectively.
In one embodiment, said rodent Fab fragment has the binding
affinity of a Fab fragment comprising variable domain sequences of
a rat antibody designated FIB504.64 produced by hybridoma cell line
deposited under American Type Culture Collection Accession Number
ATCC HB-293. In a further embodiment, a humanized Fab fragment of
the invention has the binding affinity of a Fab fragment comprising
variable domain sequences of an antibody produced by anyone of the
humanized anti-beta7 antibodies of the invention. As is
well-established in the art, binding affinity of a ligand to its
receptor can be determined using any of a variety of assays, and
expressed in terms of a variety of quantitative values.
Accordingly, in one embodiment, the binding affinity is expressed
as Kd values and reflects intrinsic binding affinity (e.g., with
minimized avidity effects). Generally and preferably, binding
affinity is measured in vitro, whether in a cell-free or
cell-associated setting. As described in greater detail herein,
fold difference in binding affinity can be quantified in terms of
the ratio of the binding affinity value of a humanized antibody in
Fab form and the binding affinity value of a reference/comparator
Fab antibody (e.g., a murine antibody having donor hypervariable
region sequences), wherein the binding affinity values are
determined under similar assay conditions. Thus, in one embodiment,
the fold difference in binding affinity is determined as the ratio
of the Kd values of the humanized antibody in Fab form and said
reference/comparator Fab antibody. Any of a number of assays known
in the art, including those described herein, can be used to obtain
binding affinity measurements, including, for example, Biacore.RTM.
(Biacore International Ab, Uppsala, Sweden) and ELISA.
[0009] In its various aspects and embodiments, the beta7 antagonist
antibody of the invention is directed to the following set of
potential claims for this application: Antibody comprising an
anti-beta7 antibody or beta7 binding fragment thereof
comprising:
[0010] (a) at least one, two, three, four, or five or hypervariable
region (HVR) sequences selected from the group consisting of:
[0011] (i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is
RASESVDTYLH (SEQ ID NO:1) [0012] (ii) HVR-L2 comprising sequence
B1-B8, wherein B1-B8 is KYASQSIS (SEQ ID NO:2) [0013] (iii) HVR-L3
comprising sequence C1-C9, wherein C1-C9 is QQGNSLPNT (SEQ ID NO:3)
[0014] (iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is
GFFITNNYWG (SEQ ID NO:4) [0015] (v) HVR-H2 comprising sequence
E1-E17, wherein E1-E17 is GYISYSGSTSYNPSLKS (SEQ ID NO:5); and
[0016] (vi) HVR-H3 comprising sequence F2-F11, wherein F2-F11 is
MTGSSGYFDF (SEQ ID NO:6).
[0017] In an embodiment of the polypeptide or antibody of claim 1,
the polypeptide or antibody comprises at least one variant HVR,
wherein the variant HVR sequence comprises modification of at least
one residue of at least one of the sequences depicted in SEQ ID
NOs:1, 2, 3, 4, 5, 6, 7, 8, and 9. In another embodiment of claim 1
or claim 2, the invention comprises an anti-beta7 antibody or beta7
binding fragment thereof comprising one, two, three, four, five or
six hypervariable regions (HVRs) selected from the group consisting
of HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein:
[0018] (i) HVR-L1 comprises amino acid sequence RASESVDTYLH (SEQ ID
NO:1); RASESVDSLLH (SEQ ID NO:7), RASESVDTLLH (SEQ ID NO:8), or
RASESVDDLLH (SEQ ID NO:9); [0019] (ii) HVR-L2 comprises amino acid
sequence KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:67, or
XYASQSIS (SEQ ID NO:68, where X represents any amino acid), [0020]
(iii) HVR-L3 comprises QQGNSLPNT (SEQ ID NO:3), [0021] (iv) HVR-H1
comprises amino acid sequence GFFITNNYWG (SEQ ID NO:4), [0022] (v)
HVR-H2 comprises amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID
NO:5), and [0023] (vi) HVR-H3 comprises amino acid sequence
MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:66) for relative
positions F2-F11; or comprises amino acid sequence F1-F11, wherein
F1-F11 is AMTGSSGYFDF (SEQ ID NO:63), ARTGSSGYFDF (SEQ ID NO:64),
or AQTGSSGYFDF (SEQ ID NO:65).
[0024] In still another embodiment of claim 1 or any of the
embodiments, the invention comprises an anti-beta7 antibody or
beta7 binding fragment thereof comprising one, two, three, four,
five or six hypervariable regions (HVRs) selected from the group
consisting of HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3,
wherein: [0025] (i) HVR-L1 comprises amino acid sequence A1-A11,
wherein A1-A11 is RASESVDTYLH (SEQ ID NO:1); RASESVDSLLH (SEQ ID
NO:7), RASESVDTLLH (SEQ ID NO:8), or RASESVDDLLH (SEQ ID NO:9) or a
variant of SEQ ID NOs:1, 7, 8 or 9 wherein amino acid A2 is
selected from the group consisting of A, G, S, T, and V and/or
amino acid A3 is selected from the group consisting of S, G, I, K,
N, P, Q, R, and T, and/or A4 is selected from the group consisting
of E, V, Q, A, D, G, H, I, K, L, N, and R, and/or amino acid A5 is
selected from the group consisting of S, Y, A, D, G, H, I, K, N, P,
R, T, and V, and/or amino acid A6 is selected from the group
consisting of V, R, I, A, G, K, L, M, and Q, and/or amino acid A7
is selected from the group consisting of D, V, S, A, E, G, H, I, K,
L, N, P, S, and T, and/or amino acid A8 is selected from the group
consisting of D, G, N, E, T, P and S, and/or amino acid A9 is
selected from the group consisting of L, Y, I and M, and/or amino
acid A10 is selected from the group consisting of L, A, I, M, and V
and/or amino acid A11 is selected from the group consisting of H,
Y, F, and S; [0026] (ii) HVR-L2 comprises amino acid sequence
B1-B8, wherein B1-B8 is KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID
NO:67, or XYASQSIS (SEQ ID NO:68, where X represents any amino
acid) or a variant of SEQ ID NOs:2, 67 or 68 wherein amino acid B1
is selected from the group consisting of K, R, N, V, A, F, Q, H, P,
I, L, Y and X (where X represents any amino acid), and/or amino
acid B4 is selected from the group consisting of S and D, and/or
amino acid B5 is selected from the group consisting of Q and S,
and/or amino acid B6 is selected from the group consisting of S, D,
L, and R, and/or amino acid B7 is selected from the group
consisting of I, V, E, and K; [0027] (iii) HVR-L3 comprises amino
acid sequence C1-C9, wherein C1-C9 is QQGNSLPNT (SEQ ID NO:3) or a
variant of SEQ ID NO:3 wherein amino acid C8 is selected from the
group consisting of N, V, W, Y, R, S, T, A, F, H, I, L, M, and Y;
[0028] (iv) HVR-H1 comprises amino acid sequence D1-D10 wherein
D1-D10 is GFFITNNYWG (SEQ ID NO:4), [0029] (v) HVR-H2 comprises
amino acid sequence E1-E17 wherein E1-E17 is GYISYSGSTSYNPSLKS (SEQ
ID NO:5), or a variant of SEQ ID NO:5 wherein amino acid E2 is
selected from the group consisting of Y, F, V, and D, and/or amino
acid E6 is selected from the group consisting of S and G, and/or
amino acid E10 is selected from the group consisting of S and Y,
and/or amino acid E12 is selected from the group consisting of N,
T, A, and D, and/or amino acid 13 is selected from the group
consisting of P, H, D, and A, and/or amino acid E15 is selected
from the group consisting of L and V, and/or amino acid E17 is
selected from the group consisting of S and G, and [0030] (vi)
HVR-H3 comprises amino acid sequence F2-F11 wherein F2-F11 is
MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:66); or comprises
amino acid sequence F1-F11, wherein F1-F11 is AMTGSSGYFDF (SEQ ID
NO:63), ARTGSSGYFDF (SEQ ID NO:64), or AQTGSSGYFDF (SEQ ID NO:65),
or a variant of SEQ ID NOs:6, 63, 64, 65, or 66 wherein amino acid
F2 is R, M, A, E, G, Q, S, and/or amino acid F11 is selected from
the group consisting of F and Y.
[0031] In one embodiment of claim 1 or any of the antibodies of the
invention, the amino acid at heavy chain framework position 71
(according to the Kabat numbering system) is selected from the
group consisting of R, A, and T, and/or the amino acid at heavy
chain framework position 73 (Kabat numbering system) is selected
from the group consisting of N and T, and/or the amino acid at
heavy chain framework position 78 (Kabat numbering system) is
selected from the group consisting of F, A, and L.
[0032] In one embodiment of claim 1 or any of the antibodies of the
invention, HVR-L1 of an antibody of the invention comprises the
sequence of SEQ ID NO:1. In one embodiment, HVR-L2 of an antibody
of the invention comprises the sequence of SEQ ID NO:2. In one
embodiment, HVR-L3 of an antibody of the invention comprises the
sequence of SEQ ID NO:3. In one embodiment, HVR-H1 of an antibody
of the invention comprises the sequence of SEQ ID NO:4. In one
embodiment, HVR-H2 of an antibody of the invention comprises the
sequence of SEQ ID NO:5. In one embodiment, HVR-H3 of an antibody
of the invention comprises the sequence of SEQ ID NOs:6 or 66 for
relative positions F2-F11 or SEQ ID NOs:63, 64, or 65 for relative
positions F1-F11. In one embodiment, HVR-L1 comprises RASESVDSLLH
(SEQ ID NO: 7). In one embodiment, HVR-L1 comprises RASESVDTLLH
(SEQ ID NO: 8). In one embodiment, HVR-L1 comprises RASESVDDLLH
(SEQ ID NO:9). In one embodiment, an antibody of the invention
comprising these sequences (in combinations as described herein) is
humanized or human.
[0033] In one aspect, the invention provides an antibody comprising
one, two, three, four, five or six HVRs, wherein each HVR
comprises, consists or consists essentially of a sequence selected
from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8 and
9, and wherein SEQ ID NO:1, 7, 8 or 9 corresponds to an HVR-L1, SEQ
ID NO:2 corresponds to an HVR-L2, SEQ ID NO:3 corresponds to an
HVR-L3, SEQ ID NO:4 corresponds to an HVR-H1, SEQ ID NO:5
corresponds to an HVR-H2, and SEQ ID NOs:6 corresponds to an
HVR-H3. In one embodiment, an antibody of the invention comprises
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each,
in order, comprises SEQ ID NO:1, 2, 3, 4, 5 and 6. In one
embodiment, an antibody of the invention comprises HVR-L1, HVR-L2,
HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,
comprises SEQ ID NO:7, 2, 3, 4, 5 and 6. In one embodiment, an
antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,
HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:8,
2, 3, 4, 5 and 6. In one embodiment, an antibody of the invention
comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3,
wherein each, in order, comprises SEQ ID NO:9, 2, 3, 4, 5 and 6. In
one embodiment, an antibody of the invention comprises HVR-L1,
HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,
comprises SEQ ID NO:9, 2, 3, 4, 5 and 66, or SEQ ID NO:9, 2, 3, 4,
5, 63 or SEQ ID NO:9, 2, 3, 4, 5, 64 or SEQ ID NO:9, 2, 3, 4, 5,
and 65 or SEQ ID NO:9, 67, 3, 4, 5, 64 or SEQ ID NO:9, 68, 3, 4, 5,
64.
[0034] Variant HVRs in an antibody of the invention can have
modifications of one or more residues within the HVR and the HVRs
and/or framework regions may be humanized. Embodiments of the
invention in which there is an HVR and/or framework modification
include, without limitation, the following potential claims for
this application: [0035] 2. The antibody of claim 1 or any of its
embodiments, wherein A8 in a variant HVR-L1 is S, D or T and A9 is
L. [0036] 3. The antibody of claim 1 or any of its embodiments,
wherein the antibody is humanized. [0037] 4. The antibody of claim
1 or any of its embodiments, wherein at least a portion of the
framework sequence is a human consensus framework sequence. [0038]
5. The antibody of claim 1 or any of its embodiments, wherein said
modification is substitution, insertion or deletion. [0039] 6. The
antibody of claim 1 or any of its embodiments, wherein a HVR-L1
variant comprises 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)
substitutions in any combination of the following positions: A2 (G,
S, T, or V); A3 (G, I, K, N, P, Q, R, or T), A4 (A, D, G, H, I, K,
L, N, Q, R, or V), A5 (A, D, G, H, I, K, N, P, R, T, V, or Y), A6
(A, G, I, K, L, M, Q, or R), A7 (A, E, G, H, I, K, L, N, P, S, T,
or V), A8 (S, D, E, G, P, or N) and A9 (L, I, or M), A10 (A, I, M,
or V), and A11 (F, S, or Y). [0040] 7. The antibody of claim 1 or
any of its embodiments, wherein a HVR-L2 variant comprises 1-4 (1,
2, 3, or 4) substitutions in any combination of the following
positions: B1 (N), 135 (S), B6 (R or L), and B7 (T, E, K, or V).
[0041] 8. The antibody of claim 1 or any of its embodiments,
wherein a HVR-L3 variant comprises at least one substitution at
position C8 (W, Y, R, S, A, F, H, I, L, M, N, T, or V). [0042] 9.
The antibody of claim 1 or any of its embodiments, wherein a HVR-H2
variant comprises 1-7 (1, 2, 3, 4, 5, 6, or 7) substitutions in any
combination of the following positions: E2 (V, D, or F), E6 (G),
E10 (Y), E12 (A, D, or T), E13 (D, A, or H), E15 (V), E17 (G).
[0043] 10. The antibody of claim 1 or any of its embodiments,
wherein a HVR-H3 variant comprises at 1 or 2 substitutions in any
combination of the following positions: F2 (A, s E, G, Q, R, or S),
and F11 (Y). [0044] 11. The antibody of claim 1 or any of its
embodiments, comprising an HVR-L1 having the sequence of SEQ ID
NO:7. [0045] 12. The antibody of claim 1 or any of its embodiments,
comprising an HVR-L1 having the sequence of SEQ ID NO:8. [0046] 13.
The antibody of claim 1 or any of its embodiments, comprising an
HVR-L1 having the sequence of SEQ ID NO:9. [0047] 14. The antibody
of any one of claims 11-13 comprising a heavy chain human subgroup
III heavy chain consensus framework sequence comprising a
substitution at position 71, 73 and/or 78. [0048] 15. The antibody
of claim 14, wherein the substitution is R71A, N73T and/or N78A.
[0049] 16. The antibody of claim 1 or any of its embodiments,
comprising an HVR-L3 having the sequence of SEQ ID NO:3. [0050] 17.
The antibody of claim 1 or any of its embodiments, wherein A8 in a
variant HVR-L1 is S. [0051] 18. The antibody of claim 1 or any of
its embodiments, wherein A8 in a variant HVR-L1 is D. [0052] 19.
The antibody of claim 1 or any of its embodiments, wherein A9 in a
variant HVR-L1 is L. [0053] 20. The antibody of claim 1 or any of
its embodiments, wherein a framework sequence between sequence
E1-E17 and F1-F11 is HFR3-1-HFR3-31 and wherein HFR3-6 is A or R,
HFR3-8 is N or T, and HFR3-13 is L or A or F. [0054] 21. A
humanized anti-beta7 antibody wherein monovalent affinity of the
antibody to human s beta7 is substantially the same as monovalent
affinity of a rat antibody comprising a light chain and heavy chain
variable sequence as depicted in FIG. 9. [0055] 22. A humanized
anti-beta7 antibody wherein monovalent affinity of the antibody to
human beta7 is at least 3-fold greater than monovalent affinity of
a rat antibody comprising a light chain and heavy chain variable
sequence as depicted in FIG. 9. [0056] 23. The humanized antibody
of claim 21 or 22 wherein the rat antibody is produced by hybridoma
cell line deposited under American Type Culture Collection
Accession Number ATCC with designation HB-293. [0057] 24. The
antibody of any of claims 21-23 wherein the binding affinity is
expressed as a Kd value. [0058] 25. The antibody of any of claim
21-24 wherein the binding affinity is measured by Biacore.TM. or
radioimmunoassay. [0059] 26. The antibody of claim 1 comprising
human .kappa. subgroup 1 light chain consensus framework sequence.
[0060] 27. The antibody of claim 1 comprising heavy chain human
subgroup III heavy chain consensus framework sequence. [0061] 28.
The antibody of claim 27 wherein the framework sequence comprises a
substitution at position 71, 73 and/or 78. [0062] 29. The antibody
of claim 28 wherein said substitution is R71A, N73T and/or N78A or
wherein the substituted amino acid at position 71 is R or A, and/or
the amino acid substitution at position 78 is N or T, and/or the
amino acid substitution at position 78 is L or A or F. [0063] 30.
The antibody of claim 28 wherein said substitution is L78F or A78F
or A78L or L78A. [0064] 31. A method of inhibiting the interaction
of a human beta7 integrin subunit with a second integrin subunit
and/or a ligand by contacting the antibody of any one of claims
1-30 with the second integrin subunit and/or the ligand. [0065] 32.
The method of claim 31, wherein the second integrin subunit is
alpha4 integrin subunit, and wherein the ligand is MAdCAM, VCAM or
fibronectin. [0066] 33. The method of claim 32, wherein the alpha4
integrin subunit is human. [0067] 34. The method of claim 33,
wherein the ligand is human. [0068] 35. The method of claim 32,
wherein the second integrin subunit is alphaE integrin subunit, and
wherein the ligand is E-cadherein. [0069] 36. The method of claim
35, wherein the alphaE integrin subunit is human. [0070] 37. The
method of claim 36, wherein the ligand is human. [0071] 38. The
method of claim 31, wherein the inhibiting reduces or alleviates
symptoms of a disorder selected from inflammation, asthma,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
diabetes, inflammation resulting of organ transplantation, graft
versus host disorder, and inflammation associated with allograft
disorders. Further embodiments of the invention include without
limitation the following:
[0072] In one embodiment, a HVR-L1 is SEQ ID NO:1, 7, 8, or 9 or a
HVR-L1 variant of SEQ ID NO:1, 7, 8, or 9 which comprises 1-10 (1,
2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions at relative positions
A1-A11, in any combination of the following positions: A2 (A, G, S,
T, or V); A3 (S, G, I, K, N, P, Q, R, or T), A4 (E, A, D, G, H, I,
K, L, N, Q, R, or V), A5 (S, A, D, G, H, I, K, N, P, R, T, V, or
Y), A6 (V, A, G, I, K, L, M, Q, or R), A7 (D, A, E, G, H, I, K, L,
N, P, S, T, or V), A8 (T, S, D, E, G, P, or N) and A9 (Y, L, I, or
M), A10 (L, A, I, M, or V), and A11 (H, F, S, or Y). In one
embodiment, a HVR-L2 is SEQ ID NO:2, 67, or 68 or a HVR-L2 variant
of SEQ ID NO:2, 67, or 68 which HVR-L2 variant comprises 1-4 (1, 2,
3, 4, 4 or 5) substitutions at relative positions B1-B8, in any
combination of the following positions: B1 (K, R, N, V, A, F, Q, H,
P, I, L, Y or X (where X represents any amino acid), B4 (S), B5 (Q
or S), B6 (S, R or L), and B7 (I, T, E, K, or V). In one
embodiment, a HVR-L3 is SEQ ID NO:3 or a HVR-L3 variant of SEQ ID
NO:3 which comprises at least one substitution at relative
positions C1-C8, such as at position C8 (W, Y, R, S, A, F, H, I, L,
M, N, T, or V). In one embodiment, a HVR-H1 is SEQ ID NO:4. In one
embodiment, a HVR-H2 is SEQ ID NO:5 or a HVR-H2 variant of SEQ ID
NO:5 which HVR-H2 variant comprises 1-7 (1, 2, 3, 4, 5, 6, or 7)
substitutions at relative positions E1-E11 in any combination of
the following positions: E2 (Y, V, D, or F), E6 (S or G), E10 (S or
Y), E12 (N, A, D, or T), E13 (P, D, A, or H), E15 (L or V), E17 (S
or G). In one embodiment, a HVR-H3 is SEQ ID NOs:6, 63, 64, 65, or
66 or a HVR-H3 variant of SEQ ID NOs:6, 63, 64, 65, or 66 which
comprises at relative positions F1-F11 for SEQ ID NOs:63, 64, and
65 or at relative positions F2-F11 for SEQ ID NOs:6 and 66, 1 or 2
substitutions in any combination of the following positions: F2 (M,
A, E, G, Q, R, or S), and F11 (F or Y). Letter(s) in parenthesis
following each position indicates an illustrative substitution
(i.e., replacement) amino acid for a consensus or other amino acid
as would be evident to one skilled in the art, suitability of other
amino acids as substitution amino acids in the context described
herein can be routinely assessed using techniques known in the art
and/or described herein.
[0073] In one embodiment, a HVR-L1 comprises the sequence of SEQ ID
NO:1. In one embodiment, A8 in a variant HVR-L1 is D. In one
embodiment, A8 in a variant HVR-L1 is S. In one embodiment, A9 in a
variant HVR-L1 is L. In one embodiment, A8 in a variant HVR-L1 is D
and A9 in a variant HVR-L1 is L. In one embodiment, A8 in a variant
HVR-L1 is S and A9 in a variant HVR-L1 is L. In some embodiments of
the invention comprises these variations in the HVR-L1, the HVR-L2,
HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprises or consists of or
consists essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6. In
some embodiments, HVR-H3 comprises or consists or consists
essentially of SEQ ID NO:6 or 66 (for relative positions F2-F11) or
SEQ ID NO:63 or 64 or 65 (for relative positions F1-F11).
[0074] In one embodiment, A8 in a variant HVR-L1 is I and the and
A9 in a variant HVR-L1 is L, which variant further comprises the
HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR comprising,
consisting of, or consist essentially of, in order, SEQ ID NO:2, 3,
4, 5, and 6.
[0075] In one embodiment, A8, A9, and A10 in a variant HVR-L1 are
D, L, and V, respectively, which variant further comprises the
HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR comprising,
consisting of, or consist essentially of, in order, SEQ ID NO:2, 3,
4, 5, and 6.
[0076] In one embodiment, A8 and A9 in a variant HVR-L1 are N and
L, respectively, which variant further comprises the HVR-L2,
HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting
of, or consist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and
6.
[0077] In one embodiment, A8 and A9 in a variant HVR-L1 are P and
L, respectively, and B6 and B7 in a variant HVR-L2 are R and T,
respectively, which variant further comprises the HVR-L2, HVR-L3,
HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, or
consist essentially of, in order, SEQ ID NO:3, 4, 5, and 6.
[0078] In one embodiment, A2, A4, A8, A9, and A10 in a variant
HVR-L1 are S, D, S, L, and V, respectively, which variant further
comprises the HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR
comprising, consisting of, or consist essentially of, in order, SEQ
ID NO:2, 3, 4, 5, and 6.
[0079] In one embodiment, A5 and A9 in a variant HVR-L1 are D and
T, respectively, which variant further comprises the HVR-L2,
HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting
of, or consist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and
6.
[0080] In one embodiment, A5 and A9 in a variant HVR-L1 are N and
L, respectively, which variant further comprises the HVR-L2,
HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting
of, or consist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and
6.
[0081] In one embodiment, A9 in a variant HVR-L1 is L, which
variant further comprises the HVR-L2, HVR-L3, HVR-H1, HVR-H2, and
HVR-H3, each HVR comprising, consisting of, or consist essentially
of, in order, SEQ ID NO:2, 3, 4, 5, and 6.
[0082] In one embodiment, the antibody or anti-beta7 binding
polypeptide of the invention comprises an HVR-L1, HVR-L2, HVR-L3,
HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, or
consisting essentially of, in order, SEQ ID NO:9, 2, 3, 4, 5, and
64. In another embodiment, each HVR comprises, consists of, or
consists essentially of, in order, SEQ ID NO:9, 67, 3, 4, 5, and
64. In another embodiment, each HVR comprises, consists of, or
consists essentially of, in order, SEQ ID NO:9, 68, 3, 4, 5, and
64. In another embodiment, each HVR comprises, consists of, or
consists essentially of, in order, SEQ ID NO:9, 2 or 67 or 68, 3,
4, 5, and 66.
[0083] In some embodiments, said variant HVR-L1 antibody variants
further comprises HVR-L2, HVR-L3, HVR-L3, HVR-H1, HVR-H2, and
HVR-H3, wherein each comprises, in order, the sequence depicted in
SEQ ID NOs:2 3, 4, 5, and 6. Where the antibody variant comprises
HVR-L1 A8(P) and A9(L) and HVR-L2 B6(R) and B7(T), in some
embodiments said HVR-L1, HVR-L2 variant further comprises HVR-L3,
HVR-H1, HVR-H2, and HVR-H3, wherein each comprises, in order, the
sequence depicted in SEQ ID NOs:3, 4, 5, and 6.
[0084] In some embodiments, these antibodies further comprise a
human subgroup III heavy chain framework consensus sequence. In one
embodiment of these antibodies, the framework consensus sequence
comprises substitution at position 71, 73 and/or 78. In some
embodiments of these antibodies, position 71 is A, 73 is T and/or
78 is A. In one embodiment of these antibodies, these antibodies
further comprise a human .kappa.I light chain framework consensus
sequence.
[0085] In one embodiment, an antibody of the invention comprises a
HVR-L1 comprising SEQ ID NO:1. In one embodiment, a variant
antibody of the invention comprises a variant HVR-L1 wherein A10 is
V. In one embodiment, said variant antibody further comprises
HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises,
in order, the sequence depicted in SEQ ID NOs:2, 3, 4, 5 and 6. In
some embodiments, these antibodies further comprise a human
subgroup III heavy chain framework consensus sequence. In one
embodiment of these antibodies, the framework consensus sequence
comprises substitution at position 71, 73 and/or 78. In some
embodiments of these antibodies, position 71 is A, 73 is T and/or
78 is A. In one embodiment of these antibodies, these antibodies
further comprise a human .kappa.I light chain framework consensus
sequence.
[0086] In one embodiment, an antibody of the invention comprises a
HVR-L3 comprising SEQ ID NO:3. In one embodiment, a variant
antibody of the invention comprises a variant HVR-L3 wherein C8 is
L. In one embodiment, said variant antibody further comprises
HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises,
in order, the sequence depicted in SEQ ID NOs:1, 2, 4, 5 and 6. In
one embodiment, an antibody of the invention comprises a variant
HVR-L3 wherein C8 is W. In one embodiment, said variant antibody
further comprises HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3,
wherein each comprises, in order, the sequence depicted in SEQ ID
NOs:1, 2, 4, 5 and 6. In some embodiment, HVR-L1 comprises SEQ ID
NO:7, 8, or 9. In some embodiments, these antibodies further
comprise a human subgroup III heavy chain framework consensus
sequence. In one embodiment of these antibodies, the framework
consensus sequence comprises substitution at position 71, 73 and/or
78. In some embodiments of these antibodies, position 71 is A, 73
is T and/or 78 is A. In one embodiment of these antibodies, these
antibodies further comprise a human .kappa.I light chain framework
consensus sequence.
[0087] In one embodiment, an antibody of the invention comprises a
HVR-H3 comprising SEQ ID NO:6. In one embodiment, a variant of said
antibody comprises a variant HVR-H3 wherein F1 is Q. In one
embodiment, said variant antibody further comprises HVR-L1, HVR-L2,
HVR-L3, HVR-H1 and HVR-H2, wherein each comprises, in order, the
sequence depicted in SEQ ID NOs:1, 2, 3, 4, and 5. In one
embodiment, an antibody of the invention comprises a variant HVR-H3
wherein F1 is R. In one embodiment, said variant antibody further
comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H2, wherein each
comprises, in order, the sequence depicted in SEQ ID NOs:1, 2, 3,
4, and 5. In one embodiment, HVR-L1 comprises SEQ ID NO:7, 8, or 9.
In some embodiments, these antibodies further comprise a human
subgroup III heavy chain framework consensus sequence. In one
embodiment of these antibodies, the framework consensus sequence
comprises substitution at position 71, 73 and/or 78. In some
embodiments of these antibodies, position 71 is A, 73 is T and/or
78 is A. In one embodiment of these antibodies, these antibodies
further comprise a human .kappa.I light chain framework consensus
sequence.
[0088] In one embodiment, an antibody of the invention comprises a
HVR-L1 comprising SEQ ID NO:1. In one embodiment, the antibody
comprises a variant HVR-L1 wherein A4 is Q. In one embodiment, said
variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2
and HVR-H3, wherein each comprises, in order, the sequence depicted
in SEQ ID NOs:2, 3, 4, 5, and 6. In one embodiment, an antibody of
the invention comprises a variant HVR-L1 wherein A6 is I. In one
embodiment, said variant antibody further comprises HVR-L2, HVR-L3,
HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, the
sequence depicted in SEQ ID NOs:2, 3, 4, 5, and 6. In one
embodiment, an antibody of the invention comprises a variant HVR-L1
wherein A7 is S. In one embodiment, said variant antibody further
comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each
comprises, in order, the sequence depicted in SEQ ID NOs:2, 3, 4,
5, and 6. In one embodiment, an antibody of the invention comprises
a variant HVR-L1 wherein A8 is D or N. In one embodiment, said
variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2
and HVR-H3, wherein each comprises, in order, the sequence depicted
in SEQ ID NOs:2, 3, 4, 5, and 6. In some embodiments, these
antibodies further comprise a human subgroup III heavy chain
framework consensus sequence. In one embodiment of these
antibodies, the framework consensus sequence comprises substitution
at position 71, 73 and/or 78. In some embodiments of these
antibodies, position 71 is A, 73 is T and/or 78 is A. In one
embodiment of these antibodies, these antibodies further comprise a
human .kappa.I light chain framework consensus sequence.
[0089] In one embodiment, an antibody of the invention comprises a
HVR-L2 comprising SEQ ID NO:2. In one embodiment, an antibody of
the invention comprises a variant HVR-L2 wherein B1 is N. In one
embodiment, an antibody of the invention comprises a variant HVR-L2
wherein B5 is S. In one embodiment, an antibody of the invention
comprises a variant HVR-L2 wherein B6 is L. In one embodiment, an
antibody of the invention comprises a variant HVR-L2 wherein B7 is
V. In one embodiment, an antibody of the invention comprises a
variant HVR-L2 wherein B7 is E or K. In some embodiments, said
variant antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2
and HVR-H3, wherein each comprises, in order, the sequence depicted
in SEQ ID NOs:1, 3, 4, 5, and 6. In some embodiments, HVR-L1
comprises SEQ ID NO:7, 8, or 9. In some embodiments, these
antibodies further comprise a human subgroup III heavy chain
framework consensus sequence. In one embodiment of these
antibodies, the framework consensus sequence comprises substitution
at position 71, 73 and/or 78. In some embodiments of these
antibodies, position 71 is A, 73 is T and/or 78 is A. In one
embodiment of these antibodies, these antibodies further comprise a
human .kappa.I light chain framework consensus sequence.
[0090] In one embodiment, an antibody of the invention comprises a
HVR-L3 comprising SEQ ID NO:3. In one embodiment, an antibody of
the invention comprises a variant HVR-L3 wherein C8 is W, Y, R, or
S. In some embodiments, said variant antibody further comprises
HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises,
in order, the sequence depicted in SEQ ID NOs:1, 2, 4, 5, and 6. In
some embodiments, HVR-L1 comprises SEQ ID NO:7, 8, or 9. In some
embodiments, these antibodies further comprise a human subgroup III
heavy chain framework consensus sequence. In one embodiment of
these antibodies, the framework consensus sequence comprises
substitution at position 71, 73 and/or 78. In some embodiments of
these antibodies, position 71 is A, 73 is T and/or 78 is A. In one
embodiment of these antibodies, these antibodies further comprise a
human .kappa.I light chain framework consensus sequence.
[0091] In one embodiment, an antibody of the invention comprises a
HVR-H2 comprising SEQ ID NO:5. In one embodiment, an antibody of
the invention comprises a variant HVR-H2 wherein E2 is F. In one
embodiment, an antibody of the invention comprises a variant HVR-H2
wherein E2 is V or D. In one embodiment, an antibody of the
invention comprises a variant HVR-H2 wherein E6 is G. In one
embodiment, an antibody of the invention comprises a variant HVR-H2
wherein E10 is Y. In one embodiment, an antibody of the invention
comprises a variant HVR-H2 wherein E12 is A, D, or T. In one
embodiment, an antibody of the invention comprises a variant HVR-H2
wherein E13 is D, A, or N. In one embodiment, an antibody of the
invention comprises a variant HVR-H2 wherein E15 is V. In one
embodiment, an antibody of the invention comprises a variant HVR-H2
wherein E11 is G. In some embodiments, said variant antibody
further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H3,
wherein each comprises, in order, the sequence depicted in SEQ ID
NOs:1, 2, 3, 4, and 6. In some embodiments, HVR-L1 comprises SEQ ID
NO:7, 8, or 9. In some embodiments, these antibodies further
comprise a human subgroup III heavy chain framework consensus
sequence. In one embodiment of these antibodies, the framework
consensus sequence comprises substitution at position 71, 73 and/or
78. In some embodiments of these antibodies, position 71 is A, 73
is T and/or 78 is A. In one embodiment of these antibodies, these
antibodies further comprise a human .kappa.I light chain framework
consensus sequence.
[0092] In one embodiment, an antibody of the invention comprises a
HVR-H3 comprising SEQ ID NO:6. In one embodiment, an antibody of
the invention comprises a variant HVR-H3 wherein F11 is Y. In some
embodiments, said variant antibody further comprises HVR-L1,
HVR-L2, HVR-L3, HVR-H1 and HVR-H3, wherein each comprises, in
order, the sequence depicted in SEQ ID NOs:1, 2, 3, 4, and 6. In
some embodiments, HVR-L1 comprises SEQ ID NO:7, 8, or 9. In some
embodiments, these antibodies further comprise a human subgroup III
heavy chain framework consensus sequence. In one embodiment of
these antibodies, the framework consensus sequence comprises
substitution at position 71, 73 and/or 78. In some embodiments of
these antibodies, position 71 is A, 73 is T and/or 78 is A. In one
embodiment of these antibodies, these antibodies further comprise a
human .kappa.I light chain framework consensus sequence.
[0093] In some embodiments, these antibodies further comprise a
human subgroup III heavy chain framework consensus sequence. In one
embodiment of these antibodies, the framework consensus sequence
comprises substitution at position 71, 73 and/or 78. In some
embodiments of these antibodies, position 71 is A, 73 is T and/or
78 is A. In one embodiment of these antibodies, these antibodies
further comprise a human .kappa.I light chain framework consensus
sequence.
[0094] A therapeutic agent for use in a host subject preferably
elicits little to no immunogenic response against the agent in said
subject. In one embodiment, the invention provides such an agent.
For example, in one embodiment, the invention provides a humanized
antibody that elicits and/or is expected to elicit a human
anti-rodent antibody response (such as anti-mouse or anti-rat
response) or a human anti-human response at a substantially reduced
level compared to an antibody comprising the sequence comprising
SEQ ID NOs:10 and/or 11 (FIGS. 1A and 1B) or SEQ ID NOs: 12 and/or
13 (FIGS. 9A and 9B depicting rat anti-mouse Fib504 amino acid
sequences) in a host subject. In another example, the invention
provides a humanized antibody that elicits and/or is expected to
elicit no human anti-rodent (such as human anti-mouse (HAMA) or
human anti-mouse) or human anti-human antibody response (HAHA).
[0095] A humanized antibody of the invention may comprise one or
more human and/or human consensus non-hypervariable region (e.g.,
framework) sequences in its heavy and/or light chain variable
domain. In some embodiments, one or more additional modifications
are present within the human and/or human consensus
non-hypervariable region sequences. In one embodiment, the heavy
chain variable domain of an antibody of the invention comprises a
human consensus framework sequence, which in one embodiment is the
subgroup III consensus framework sequence. In one embodiment, an
antibody of the invention comprises a variant subgroup III
consensus framework sequence modified at at least one amino acid
position. For example, in one embodiment, a variant subgroup III
consensus framework sequence may comprise a substitution at one or
more of positions 71, 73, 78 and/or 94. In one embodiment, said
substitution is R71A, N73T, L78A, and/or R94M, in any combination
thereof.
[0096] As is known in the art, and as described in greater detail
hereinbelow, the amino acid position/boundary delineating a
hypervariable region of an antibody can vary, depending on the
context and the various definitions known in the art (as described
below). Some positions within a variable domain may be viewed as
hybrid hypervariable positions in that these positions can be
deemed to be within a hypervariable region under one set of
criteria while being deemed to be outside a hypervariable region
under a different set of criteria. One or more of these positions
can also be found in extended hypervariable regions (as further
defined below). The invention provides antibodies comprising
modifications in these hybrid hypervariable positions. In one
embodiment, these hybrid hypervariable positions include one or
more of positions 26-30, 33-35B, 47-49, 49, 57-65, 93, 94 and 102
in a heavy chain variable domain. In one embodiment, these hybrid
hypervariable positions include one or more of positions 24-29,
35-36, 46-49, 49, 56 and 97 in a light chain variable domain. In
one embodiment, an antibody of the invention comprises a variant
human subgroup consensus framework sequence modified at one or more
hybrid hypervariable positions. In one embodiment, an antibody of
the invention comprises a heavy chain variable domain comprising a
variant human subgroup III consensus framework sequence modified at
one or more of positions 28-35, 49, 50, 52a, 53, 54, 58-61, 63, 65,
94 and 102. In one embodiment, the antibody comprises a, T28F,
F29I, S30T, S31N, Y32N, A33Y, M34W, and S35G substitution. In one
embodiment, the antibody comprises a S49G substitution. In one
embodiment, the antibody comprises a V50F or V50D or V50Y
substitution. In one embodiment, the antibody comprises a G53Y
substitution. In one embodiment, the antibody comprises a G54S
substitution. In one embodiment, the antibody comprises a Y58S
substitution. In one embodiment, the antibody comprises a A60N or
A60Dor A60T substitution. In one embodiment, the antibody comprises
a D61P or D61A or D61H substitution. In one embodiment, the
antibody comprises a V63L substitution. In one embodiment, the
antibody comprises a G65S substitution. In one embodiment, the
antibody comprises a R94M substitution. In one embodiment, the
antibody comprises a R94A or R94E or R94G or R94Q or R94S
substitution. In one embodiment, the antibody comprises a G95T
substitution. In one embodiment, the antibody comprises one or more
of the substitutions at positions 28-35, 49, 50, 52a, 53, 54,
58-61, 63, 65, 94 and 102 and further comprises one or more of the
substitutions at positions R71A or N73T or L78A or L78F. In one
embodiment, the antibody comprises a Y102F substitution. It can be
seen by reference to FIG. 1B that these substitutions are in the
HVR-H1, HVR-H2, and/or HVR-H3 of the heavy chain.
[0097] In one embodiment, an antibody of the invention comprises a
light chain variable domain comprising a variant human subgroup I
consensus framework sequence modified at one or more of positions
27, 29-31, 33, 34, 49, 50, 53-55, 91 and 96. In one embodiment, the
antibody comprises a Q27E substitution. In one embodiment, the
antibody comprises a I29V substitution. In one embodiment, the
antibody comprises a S30D substitution. In one embodiment, the
antibody comprises a N31T or N31S or N31D substitution. In one
embodiment, the antibody comprises a Y32L. In one embodiment, the
antibody comprises a A34H substitution. In one embodiment, the
antibody comprises a Y49K substitution. In one embodiment, the
antibody comprises a A50Y substitution. In one embodiment, the
antibody comprises a S53Q substitution. In one embodiment, the
antibody comprises a L54S substitution. In one embodiment, the
antibody comprises a E55I or E55V substitution. In one embodiment,
the antibody comprises a Y91G substitution. In one embodiment, the
antibody comprises a W96N or W96L substitution. In one embodiment,
the antibody comprises a A25S substitution. In one embodiment, the
antibody comprises a A25 to G, S, T, or V substitution. In one
embodiment, the antibody comprises a modification selected from one
or more of the following groups of substitutions. For example, in
one embodiment, the antibody comprises a S26 to G, I, K, N, P, Q,
or T substitution. In one embodiment, the antibody comprises a Q27
to E, A, D, G, H, I, K, L, N, Q, R, or V substitution. In one
embodiment, the antibody comprises a S28 to A, D, G, H, I, K, N, P,
R, T, V, or Y substitution. In one embodiment, the antibody
comprises a 129 to V, A, G, K, L, M, Q or R substitution. In one
embodiment, the antibody comprises a S30 to D, A, E, G, H, I, K, L,
N, P, S, T or V substitution. In one embodiment, the antibody
comprises a N31 to D, T, E, or G substitution. In one embodiment,
the antibody comprises a Y32 to L, I or M substitution. In one
embodiment, the antibody comprises a L33 to A, I, M or V
substitution. In one embodiment, the antibody comprises a A34 to H,
F, Y or S substitution. In one embodiment, the antibody comprises a
Y49 to K or N substitution. In one embodiment, the antibody
comprises a A50Y substitution. In one embodiment, the antibody
comprises S53Q substitution. In one embodiment, the antibody
comprises a L54S substitution. In one embodiment, the antibody
comprises a E55 to V, I or K substitution. In one embodiment, the
antibody comprises a Y91G substitution. In one embodiment, the
antibody comprises a W96 to N, L, W, Y, R, S, A, F, H, I, M, N, R,
S, T, V or Y substitution. It can be seen by reference to FIG. 1A
that these substitutions are in the HVR-L1, HVR-L2, and/or HVR-L3
of the light chain.
[0098] An antibody of the invention can comprise any suitable human
or human consensus light chain framework sequences, provided the
antibody exhibits the desired biological characteristics (e.g., a
desired binding affinity). In one embodiment, an antibody of the
invention comprises at least a portion (or all) of the framework
sequence of human .kappa. light chain. In one embodiment, an
antibody of the invention comprises at least a portion (or all) of
human .kappa. subgroup I framework consensus sequence.
[0099] In one embodiment, an antibody of the invention comprises a
heavy and/or light chain variable domain comprising framework
sequences depicted SEQ ID NOS:34-41 and in FIGS. 1, 7 and 8,
provided positions 49 of the light chain and 94 of the heavy chain
are included in the extended HVRs, and provided said position 49 is
K and said position 94 is preferably but not necessarily M and may
be R.
[0100] Antagonists of the invention can be used to modulate one or
more aspects of beta7 associated effects, including but not limited
to association with alpha4 integrin subunit, association with
alphaE integrin subunit, binding of alpha4beta7 integrin to MAdCAM,
VCAM-1 or fibronectin and binding of alphaEbeta7 integrin to
E-caderin. These effects can be modulated by any biologically
relevant mechanism, including disruption of ligand binding to beta7
subunit or to the alpha4beta7 or alphaEbeta dimeric integrin,
and/or by disrupting association between the alpha and beta
integrin subunits such that formation of the dimeric integrin is
inhibited. Accordingly, in one embodiment, the invention provides a
beta7 antagonist antibody that inhibits binding of alpha4 to beta7.
In one embodiment, a beta7 antagonist antibody of the invention
disrupts binding of alpha4beta7 to MAdCAM. In one embodiment, a
beta7 antagonist antibody of the invention disrupts binding of
alpha4beta7 to VCAM-1. In one embodiment, a beta7 antagonist
antibody of the invention disrupts binding of alpha4beta7 to
fibronectin. In one embodiment, a beta7 antagonist antibody of the
invention disrupts binding of beta7 to alphaE. In one embodiment, a
beta7 antagonist antibody of the invention disrupts binding
alphaEbeta7 integrin to E-cadherin. Interference can be direct or
indirect. For example, a beta7 antagonist antibody may bind to
beta7 within a sequence of the alpha4beta7 or alphaEbeta7
dimerization region, and thereby inhibit interaction of the
integrin subunits and formation of an integrin dimer. In a further
example, a beta7 antagonist antibody may bind to a sequence within
the ligand binding domain of beta7 subunit and thereby inhibit
interaction of said bound domain with its binding partner (such as
fibronectin, VCAM, and/or MAdCAM for the alpha4beta7 integrin; or
E-cadherin for the alphaEbeta7 integrin). In another example, a
beta7 antagonist antibody may bind to a sequence that is not within
the integrin subunit dimerization domain or a ligand binding
domain, but wherein said beta7 antagonist antibody binding results
in disruption of the ability of the beta7 domain to interact with
its binding partner (such as an alpha4 or alphaE integrin subunit
and/or a ligand such as fibronectin, VCAM, MAdCAM, or E-cadherein).
In one embodiment, an antagonist antibody of the invention binds to
beta7 (for example, the extracellular domain) such that beta7
dimerization with the alpha4 or alphaE subunit is disrupted. In one
embodiment, an antagonist antibody of the invention binds to beta7
such that ability of beta7 and/or an alpha4beta7 and/or an
alphaEbeta7 integrin to bind to its respective ligand or ligands is
disrupted. For example, in one embodiment, the invention provides
an antagonist antibody which upon binding to a beta7 molecule
inhibits dimerization of said molecule. In one embodiment, a beta7
antagonist antibody of the invention specifically binds a sequence
in the ligand binding domain of beta7. In one embodiment, a beta7
antagonist antibody of the invention specifically binds a sequence
in the ligand binding domain of beta7 such that ligand binding
(i.e., fibronectin, VCAM, and/or MAdCAM) to the alpha4beta7
integrin is disrupted. In one embodiment, a beta7 antagonist
antibody of the invention specifically binds a sequence in the
ligand binding domain of beta7 such that ligand binding (i.e.,
E-cadherin) to the alphaEbeta7 integrin is disrupted.
[0101] In one embodiment, an antagonist antibody of the invention
disrupts beta7 dimerization comprising heterodimerization (i.e.,
beta7 dimerization with an alpha4 or alphaE integrin subunit
molecule).
[0102] In one embodiment, an antagonist antibody of the invention
binds to an epitope on the beta7 integrin subunit that maps to
amino acids 176-237. In another embodiment, an antagonist antibody
of the invention binds to the same epitope on the beta7 integrin
that is the substantially the same epitope as Fib504.64 (ATCC
HB-293). Determination of epitope binding is by standard techniques
including without limitation competition binding analysis.
[0103] In one aspect, the invention provides an antibody comprising
a combination of one, two, three, four, five or all of the HVR
sequences depicted in the table of amino acid substitutions in FIG.
13.
[0104] A therapeutic agent for use in a host subject preferably
elicits little to no immunogenic response against the agent in said
subject. In one embodiment, the invention provides such an agent.
For example, in one embodiment, the invention provides a humanized
antibody that elicits and/or is expected to elicit a human anti-rat
or human anti-mouse or human anti-human antibody response at a
substantially reduced level compared to an antibody comprising the
sequence of SEQ ID NOS:10, 11, 12 and/or SEQ ID NO:13 (rat
anti-mouse Fib504 (ATCC HB-293), FIGS. 1 and 9) in a host subject.
In another example, the invention provides a humanized antibody
that elicits and/or is expected to elicit no human anti-mouse,
human anti-rat, or human anti-human antibody response.
[0105] A humanized antibody of the invention may comprise one or
more human and/or human consensus non-hypervariable region (e.g.,
framework) sequences in its heavy and/or light chain variable
domain. In some embodiments, one or more additional modifications
are present within the human and/or human consensus
non-hypervariable region sequences. In one embodiment, the heavy
chain variable domain of an antibody of the invention comprises a
human consensus framework sequence, which in one embodiment is the
subgroup III consensus framework sequence. In one embodiment, an
antibody of the invention comprises a variant subgroup III
consensus framework sequence modified at at least one amino acid
position. For example, in one embodiment, a variant subgroup III
consensus framework sequence may comprise a substitution at one or
more of positions 71, 73, 78 and/or 94, although position 94 is
part of an extended heavy chain hypervariable region-H3 of the
present invention. In one embodiment, said substitution is R71A,
N73T, N78A, and/or R94M, in any combination thereof.
[0106] An antibody of the invention can comprise any suitable human
or human consensus light chain framework sequences, provided the
antibody exhibits the desired biological characteristics (e.g., a
desired binding affinity). In one embodiment, an antibody of the
invention comprises at least a portion (or all) of the framework
sequence of human .kappa. light chain. In one embodiment, an
antibody of the invention comprises at least a portion (or all) of
human .kappa. subgroup I framework consensus sequence.
[0107] Antagonists of the invention can be used to modulate one or
more aspects of beta7 associated effects. For example, a beta7
antagonist antibody may bind to beta7 within a sequence of the
alpha4beta7 or alphaEbeta7 dimerization region, and thereby inhibit
interaction of the integrin subunits and formation of an integrin
dimer. In a further example, a beta7 antagonist antibody may bind
to a sequence within the ligand binding domain of beta7 subunit and
thereby inhibit interaction of said bound domain with its binding
partner (such as fibronectin, VCAM, and/or MAdCAM for the
alpha4beta7 integrin; or E-cadherin for the alphaEbeta7 integrin).
In another example, a beta7 antagonist antibody may bind to a
sequence that is not within the integrin subunit dimerization
domain or a ligand binding domain, but wherein said beta7
antagonist antibody binding results in disruption of the ability of
the beta7 domain to interact with its binding partner (such as an
alpha4 or alphaE integrin subunit and/or a ligand such as
fibronectin, VCAM, MAdCAM, or E-cadherein). In one embodiment, an
antagonist antibody of the invention binds to beta7 (for example,
the extracellular domain) such that beta7 dimerization with the
alpha4 or alphaE subunit is disrupted. In one embodiment, an
antagonist antibody of the invention binds to beta7 such that
ability of beta7 and/or an alpha4beta7 and/or an alphaEbeta7
integrin to bind to its respective ligand or ligands is disrupted.
For example, in one embodiment, the invention provides an
antagonist antibody which upon binding to a beta7 molecule inhibits
dimerization of said molecule. In one embodiment, a beta7
antagonist antibody of the invention specifically binds a sequence
in the ligand binding domain of beta7. In one embodiment, a beta7
antagonist antibody of the invention specifically binds a sequence
in the ligand binding domain of beta7 such that ligand binding
(i.e., fibronectin, VCAM, and/or MAdCAM) to the alpha4beta7
integrin is disrupted. In one embodiment, a beta7 antagonist
antibody of the invention specifically binds a sequence in the
ligand binding domain of beta7 such that ligand binding (i.e.,
E-cadherin) to the alphaEbeta7 integrin is disrupted.
[0108] In one embodiment, an antagonist antibody of the invention
disrupts beta7 dimerization comprising heterodimerization (i.e.,
beta7 dimerization with an alpha4 or alphaE integrin subunit
molecule.
[0109] In some instances, it may be advantageous to have a beta7
antagonist antibody that does not interfere with binding of a
ligand (such as fibronectin, VCAM, MAdCAM, or alphaE) to beta7
subunit as part of an integrin or to an alpha4beta7 integrin or an
alphaEbeta7 integrin as a dimer. Accordingly, in one embodiment,
the invention provides an antibody that does not bind a
fibronectin, VCAM, MAdCAM, or E-cadherin binding site on beta7 but,
instead, inhibits interaction between beta7 subunit and an alpha
subunit (such as alpha4 or alphaE integrin subunit) such that a
biologically active integrin is prevented from forming. In one
example, an antagonist antibody of the invention can be used in
conjunction with one or more other antagonists, wherein the
antagonists are targeted at different processes and/or functions
within the beta7 integrin axis. Thus, in one embodiment, a beta7
antagonist antibody of the invention binds to an epitope on beta7
distinct from an epitope bound by another beta7 or an alpha/beta
integrin antagonist (such as an alpha4beta7 antibody, including
monoclonal antibody or an antibody, such as a humanized antibody or
monoclonal antibody derived from and/or having the same or
effectively the same binding characteristics or specificity as an
antibody derived from a murine antibody.
[0110] In one embodiment, the invention provides a beta7 antagonist
antibody that disrupts beta7-alpha4 or -alphaE multimerization into
the respective integrin as well as ligand binding. For example, an
antagonist antibody of the invention that inhibits beta7
dimerization with alpha4 or alphaE integrin subunit may further
comprise an ability to compete with ligand for binding to beta7 or
the integrin dimer (e.g., it may interfere with the binding of
fibronectin, VCAM, and/or MAdCAM to beta7 and/or alpha4beta7; or it
may interfere with the binding of E-cadherin to beta7 or
alphaEbeta7.)
[0111] In one embodiment of a beta7 antagonist antibody of the
invention, binding of the antagonist to beta7 inhibits ligand
binding activated cellular adhesion. In another embodiment of a
beta7 antagonist antibody of the invention, binding of the
antagonist to beta7 in a cell inhibits recruitment of the cell to
the cells and/or tissue in which the beta7-containing integrin is
expressed.
[0112] In one embodiment, a beta7 antagonist antibody of the
invention specifically binds at least a portion of amino acids
176-250 (optionally amino acids 176-237) of the beta7 extracellular
domain (see Tidswell, M. et al. (1997) J. Immunol. 159:1497-1505)
or variant thereof, and reduces or blocks binding of ligands
MAdCAM, VCAM-1, fibronectin, and/or E-cadherin. In one embodiment,
such blocking of ligand binding disrupts, reduces and/or prevents
adhesion of a cell expressing the ligand to a cell expressing the
beta7-containing ligand. In one embodiment, an antagonist antibody
of the invention specifically binds an amino acid sequence of beta7
comprising residues 176-237. In one embodiment, an antagonist
antibody of the invention specifically binds a conformational
epitope formed by part or all of at least one of the sequences
selected from the group consisting of residues 176-237 of beta7. In
one embodiment, an antagonist antibody of the invention
specifically binds an amino acid sequence having at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 98%, at least 99% sequence identity or similarity with the
amino acid sequence of residues 176-237 or residues 176-250 of
human beta7. In one embodiment, the antagonist anti-beta7 antibody
of the invention binds the same epitope as the anti-beta7 antibody
Fib504 produced by hybridoma ATCC HB-293.
[0113] In one aspect, the invention provides compositions
comprising one or more antagonist antibodies of the invention and a
carrier. In one embodiment, the carrier is pharmaceutically
acceptable.
[0114] In one aspect, the invention provides nucleic acids encoding
a beta7 antagonist antibody of the invention.
[0115] In one aspect, the invention provides vectors comprising a
nucleic acid of the invention.
[0116] In one aspect, the invention provides host cells comprising
a nucleic acid or a vector of the invention. A vector can be of any
type, for example a recombinant vector such as an expression
vector. Any of a variety of host cells can be used. In one
embodiment, a host cell is a prokaryotic cell, for example, E.
coli. In one embodiment, a host cell is a eukaryotic cell, for
example a mammalian cell such as Chinese Hamster Ovary (CHO)
cell.
[0117] In one aspect, the invention provides methods for making an
antagonist of the invention. For example, the invention provides a
method of making a beta7 antagonist antibody (which, as defined
herein includes full length and fragments thereof), said method
comprising expressing in a suitable host cell a recombinant vector
of the invention encoding said antibody (or fragment thereof), and
recovering said antibody.
[0118] In one aspect, the invention provides an article of
manufacture comprising a container; and a composition contained
within the container, wherein the composition comprises one or more
beta7 antagonist antibodies of the invention. In one embodiment,
the composition comprises a nucleic acid of the invention. In one
embodiment, a composition comprising an antagonist antibody further
comprises a carrier, which in some embodiments is pharmaceutically
acceptable. In one embodiment, an article of manufacture of the
invention further comprises instructions for administering the
composition (for example, the antagonist antibody) to a
subject.
[0119] In one aspect, the invention provides a kit comprising a
first container comprising a composition comprising one or more
beta7 antagonist antibodies of the invention; and a second
container comprising a buffer. In one embodiment, the buffer is
pharmaceutically acceptable. In one embodiment, a composition
comprising an antagonist antibody further comprises a carrier,
which in some embodiments is pharmaceutically acceptable. In one
embodiment, a kit further comprises instructions for administering
the composition (for example, the antagonist antibody) to a
subject.
[0120] Beta7 integrins and their ligands are variously expressed in
disease states. [The expression of MAdCAM-1 on gut endothelium is
increased in sites of mucosal inflammation in patients with
inflammatory bowel disease (UC and CD) and colonic lamina propria
of UC and CD patients also show increased CD3+ and a4b7+ cells
compared to IBS controls (see Souza H., et al., Gut 45:856 (1999)).
MAdCAM-1 expression was observed to be associated with portal tract
inflammation in liver diseases and may be important in recruitment
of alpha4beta7+ lymphocytes to the liver during inflammation.
(Hillan, K., et al., Liver. 19(6):509-18 (1999)) MAdCAM-1 on
hepatic vessels supports adhesion of a4b7+ lymphocytes from
patients with IBD and primary sclerosing cholangitis. The adhesion
was inhibited by anti-MAdCAM-1, anti-alpha4beta7, or anti-alpha4
antibodies. (Grant A J. et al., Hepatology. 33(5):1065-72 (2001)).
MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial
cells and/or on microvessels in the inflamed central nervous
system. Beta7 integrins contribute to demyelinating disease of the
CNS (Kanwar et al., J. Neuroimmunology 103, 146 (2000)). Expression
of alpha4beta7 was significantly higher in the LPL of CD than in
controls and patients with UC (Oshitani, N. et al., International
Journal of Molecule Medicine 12, 715-719 (2003)). IELs from CD
patients may be chronically stimulated and recruited from the
periphery (Meresse, B., et al., Human Immunology, 62, 694-700
(2001)). In human liver disease, alphaEbeta7 T cells (CD4+ and
CD8+) are preferentially accumulated in human livers where
E-cadherin is expressed on hepatocytes and bile duct epithelium
(Shimizu, Y., et al., Journal of Hepatology 39, 918-924 (2003)). In
chronic pancreatitis, CD8+CD103+ T cells, analogous to intestinal
intraepithelial lymphocytes, infiltrate the pancreas in chronic
pancreatitis (Matthias, P., et al., Am J Gastroenterol 93:2141-2147
(1998)). Upregulation of alphaEbeta7 is found in systemic lupus
erythematosus patients with specific epithelial involvement (Pang
et al., Arthritis & Rheumatism 41:1456-1463 (1998)). In
Sjogren's Syndrome, CD8+ alphaEbeta7+T cells adhere and kill acinar
epithelial cells by inducing apoptosis (Kroneld et al., Scand J
Rheumatol 27:215-218, 1998) Integrin alpha4beta7 and alphaEbeta7
play a role in T cell epidermotropism during skin inflammation and
contribute to skin allograft rejection (Sun et al., Transplantation
74, 1202, 2002). Teraki and Shiohara showed preferential expression
of aEb7 integrin on CD8+T cells in psoriatic epidermis (Teraki and
Shiohara, Br. J. Dermatology 147, 1118, 2002). Sputum T lymphocytes
are activated IELs (CD69+ CD103+) in asthma, COPD, and normal
subjects (Leckie et. al., Thorax 58, 23, 2003). CD103+(aEb7+) CTL
accumulate with graft epithelium during clinical renal allograft
rejection (Hadley et al., Transplantation 72, 1548, 2001)] Thus, in
one aspect, the invention provides use of a beta7 antagonist
antibody of the invention to inhibit beta7 integrin-ligand
interaction to reduce or alleviate disease, such as one or more of
the above described disease states. In one embodiment, the antibody
of the invention is used in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as an
inflammatory disease including without limitation inflammatory
bowel disease (such as Crohn's disease and ulcerative colitis),
inflammatory liver disease, inflammation of the CNS, chronic
pancreatitis, systemic lupus erythematosus, Sjogren's syndrome,
psoriasis and skin inflammation, asthma, chronic obstructive
pulmonary disease (COPD), interstitial lung disease, allergy,
autoimmune disease, transplantation rejection, renal graft
rejection, graft versus host disease, diabetes, and cancer.
[0121] In one aspect, the invention provides use of a nucleic acid
of the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as an
immune (such as autoimmune or inflammatory) disorder including
without limitation, inflammatory bowel disease (such as Crohn's
disease or ulcerative colitis) and allergic reaction (such as
disorders of the respiratory system, skin, joints, allergic asthma
and other organs affected by allergic reaction mediated by a
beta7-containing integrin).
[0122] In one aspect, the invention provides use of an expression
vector of the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as an
immune (such as autoimmune or inflammatory) disorder including
without limitation, inflammatory bowel disease (such as Crohn's
disease or ulcerative colitis) and allergic reaction (such as
disorders of the respiratory system, skin, joints, and other organs
affected by allergic reaction mediated by a beta7-containing
integrin).
[0123] In one aspect, the invention provides use of a host cell of
the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disease, such as an
immune (such as autoimmune or inflammatory) disorder including
without limitation, inflammatory bowel disease (such as Crohn's
disease or ulcerative colitis) and allergic reaction (such as
disorders of the respiratory system, skin, joints, and other organs
affected by allergic reaction mediated by a beta7-containing
integrin).
[0124] In one aspect, the invention provides use of an article of
manufacture of the invention in the preparation of a medicament for
the therapeutic and/or prophylactic treatment of a disease, such as
an immune (such as autoimmune or inflammatory) disorder including
without limitation, inflammatory bowel disease (such as Crohn's
disease or ulcerative colitis) and allergic reaction (such as
disorders of the respiratory system, skin, joints, and other organs
affected by allergic reaction mediated by a beta7-containing
integrin).
[0125] In one aspect, the invention provides use of a kit of the
invention in the preparation of a medicament for the therapeutic
and/or prophylactic treatment of a disease, such as an immune (such
as autoimmune or inflammatory) disorder including without
limitation, inflammatory bowel disease (such as Crohn's disease or
ulcerative colitis) and allergic reaction (such as disorders of the
respiratory system, skin, joints, and other organs affected by
allergic reaction mediated by a beta7-containing integrin).
[0126] The invention provides methods and compositions useful for
modulating disease states associated with dysregulation of the
beta7 integrin mediated cell-cell interaction process. The beta7
integrins are involved in multiple biological and physiological
functions, including, for example, inflammatory disorders and
allergic reactions. Thus, in one aspect, the invention provides a
method comprising administering to a subject an antibody of the
invention.
[0127] In one aspect, the invention provides a method of inhibiting
beta7 integrin mediated inflammation, said method comprising
contacting a cell or tissue with an effective amount of a antibody
of the invention, whereby lymphocyte or B-cell interaction and
binding to a beta7 integrin-expressing cell is inhibited.
[0128] In one aspect, the invention provides a method of treating a
pathological condition associated with dysregulation of beta7
integrin binding in a subject, said method comprising administering
to the subject an effective amount of an antibody of the invention,
whereby said condition is treated.
[0129] In one aspect, the invention provides a method of inhibiting
the binding of a lymphocyte expressing a beta7 integrin ligand
(such as a cell expressing MAdCAM, VCAM, E-cadherein or
fibronectin) to a cell that expresses beta7 integrin (such as
alpha4beta7 or alphaEbeta7 integrins), said method comprising
contacting said cell with an antibody of the invention thereby
inhibiting or preventing adhesion of the cells and causing a
reduction of inflammatory reaction.
[0130] In one aspect, the invention provides a method for treating
or preventing an inflammatory disorder associated with increased
expression or activity of beta7 integrin or increased interaction
between a beta7 integrin on one cell and a beta7 integrin receptor
on another cell, said method comprising administering to a subject
in need of such treatment an effective amount of an antibody of the
invention, thereby effectively treating or preventing said
inflammatory disorder. In one embodiment, said inflammatory
disorder is inflammatory bowel disease (IBD). In another
embodiment, said inflammatory disorder is an allergic reaction.
[0131] Methods of the invention can be used to affect any suitable
pathological state, for example, cells and/or tissues associated
with dysregulation of the beta7 integrin binding pathway. Beta7
integrins are expressed primarily on leukocytes (Tidswell, M. et
al. (1997) supra). In one embodiment, a leukocyte is targeted in a
method of the invention and is prevented from binding to a cell
expressing a ligand of the beta7 integrin. For example, an an
intra-epithelial lymphocyte expressing E-cadherin is prevented,
according to the invention, from binding to an
alphaEbeta7-expressing cell by an antagonist anti-beta7 antibody.
Cells expressing MAdCAM, VCAM-1 or fibronectin are prevented by an
antagonist anti-beta7 antibody of the invention from binding to a
leukocyte expressing alpha4beta7.
[0132] Methods of the invention can further comprise additional
treatment steps. For example, in one embodiment, a method further
comprises a step wherein a targeted cell and/or tissue (for
example, an endothelial cell of the intestinal lining) is exposed
to an anti-TNF antibody or a small molecule therapeutic agent
including without limitation 5-ASA compounds (including without
limitation
[0133] As described herein, beta7 integrins mediate important
biological processes the dysregulation of which leads to numerous
pathological conditions. Accordingly, in one embodiment of methods
of the invention, a cell that is targeted (for example, an
endothelial cell) is one in which adhesion of a cell expressing a
beta7 integrin ligand of a beta7 integrin (where the cell may be,
without limitation, a lymphocyte, and the ligand may be MAdCAM,
VCAM or E-cadherin) is disrupted, inhibited, or prevented as
compared to the cells in the absence of the anti-beta7 antagonist
antibody of the invention. In one embodiment, a method of the
invention inhibits lymphocyte homing, thereby inhibiting
inflammation at the site of beta7 integrin expression. For example,
contact with an antagonist of the invention may result in a cell's
inability to adhere to a cell expressing a ligand of a beta7
integrin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0134] FIGS. 1A and 1B depict alignment of sequences of the
variable light and heavy chains for the following: light chain
human subgroup kappa I consensus sequence (FIG. 1A, SEQ ID NO:23),
heavy chain human subgroup III consensus sequence (FIG. 1B, SEQ ID
NO:24), rat anti-mouse beta7 antibody (Fib504) variable light chain
(FIG. 1A, SEQ ID NO:10), rat anti-mouse beta7 antibody (Fib504)
variable heavy chain (FIG. 1B, SEQ ID NO:11), and humanized
antibody variants: Humanized hu504Kgraft variable light chain (FIG.
1A, SEQ ID NO:25), humanized hu504K graft variable heavy chain
(FIG. 1B, SEQ ID NO:26), variant hu504.5 (amino acid variations
from humanized hu504K graft are indicated in FIG. 1A (light chain)
and FIG. 1B (heavy chain) for variants hu504.5, hu504.16, and
hu504.32. Additional amino acid substitutions in the HVR-H1 and
HVR-H2 of the hu504K graft which resulted in beta7 binding
antibodies are indicated in FIG. 1C.
[0135] FIGS. 2A and 2B depict the full length sequence of the human
consensus subgroup III sequence light chain (FIG. 2A, SEQ ID NO:27)
and heavy chain (FIG. 2B, SEQ ID NO:28). HVRs are underlined.
[0136] FIGS. 3A and 3B depict the full length sequence of the
humanized 504 graft containing rat Fib504 hypervariable regions (as
described herein) grafted into the human kappa I consensus sequence
light chain (FIG. 3A, SEQ ID NO:29) and into the human subgroup III
consensus sequence heavy chain (FIG. 3B, SEQ ID NO:30). HVRs are
underlined.
[0137] FIGS. 4A and 4B depict the full length sequence of the
humanized 504Kgraft in which position 49 of the light chain of the
hu504 graft is a Y49K substitution. The hu504Kgraft light chain is
depicted by SEQ ID NO:31 and the hu504Kgraft heavy chain is
depicted by SEQ ID NO:30. HVRs are underlined.
[0138] FIGS. 5A and 5B depict the full length sequence of the
hu504K-RF graft in which positions 71 and 78 of the heavy chain of
the hu504 graft are an A71R substitution and a A78F substitution
from the hu504Kgraft sequence. The hu504K-RF graft light chain is
depicted by SEQ ID NO:31 and the hu504K-RF graft heavy chain is
depicted by SEQ ID NO:32. HVRs are underlined.
[0139] FIGS. 6A and 6B depict the full length sequence of the
hu504.32 variant comprising the heavy chain of the hu504K-RF graft
(SEQ ID NO:32) and T31D and Y32L substitutions in the light chain
of the hu504Kgraft (SEQ ID NO:33). HVRs are underlined.
[0140] FIG. 7A-FIG. 7B and FIG. 8A-FIG. 8B depict exemplary
acceptor human consensus framework sequences for use in practicing
the instant invention with sequence identifiers as follows:
Variable Light (VL) Consensus Frameworks (FIG. 7A,B)
[0141] human VL kappa subgroup I consensus framework (SEQ ID NO:14)
human VL kappa subgroup I consensus framework minus extended HVR-L2
(SEQ ID NO:15) human VL kappa subgroup II consensus framework (SEQ
ID NO:16) human VL kappa subgroup III consensus framework (SEQ ID
NO:17) human VL kappa subgroup IV consensus framework (SEQ ID
NO:18) Shaded regions represent light chain HVRs (indicated as L1,
L2, and L3).
Variable Heavy (VH) Consensus Frameworks (FIG. 8A, B)
[0142] human VH subgroup I consensus framework minus Kabat CDRs
(SEQ ID NO:19) human VH subgroup I consensus framework minus
extended hypervariable regions (SEQ ID NOs:20-22) human VH subgroup
II consensus framework minus Kabat CDRs (SEQ ID NO:48) human VH
subgroup II consensus framework minus extended hypervariable
regions (SEQ ID NOs:49-51) human VH subgroup III consensus
framework minus Kabat CDRs (SEQ ID NO:52) human VH subgroup III
consensus framework minus extended hypervariable regions (SEQ ID
NOs:53-55) human VH acceptor framework minus Kabat CDRs (SEQ ID
NO:56) human VH acceptor framework minus extended hypervariable
regions (SEQ ID NOs:57-58) human VH acceptor 2 framework minus
Kabat CDRs (SEQ ID NO:59) human VH acceptor 2 framework minus
extended hypervariable regions (SEQ ID NOs:60-62)
[0143] FIGS. 9A and 9B depict an amino acid sequence of the
variable chains of rat anti-mouse integrin beta? Fib504 antibody
produced by the hybridoma ATCC HB-293. HVRs are underlined.
Variable light chain is depicted in FIG. 9A (SEQ ID NO:12) and
variable heavy chain is depicted in FIG. 9B (SEQ ID NO:13).
[0144] FIG. 10A depicts amino acid positions in the heavy chain of
various consensus sequences (hu subgroups I-III). The consensus
sequence used for development of the Herceptin.RTM. anti-HER2
antibody, rat Fib504, and hu504-RL and hu504-RF frameworks are
described in the Examples herein. FIG. 10B is a bar graph showing
the relative binding of alpha4beta7 to hu504graft antibody and
hu504Kgraft antibody as a function of "RL" or "RF" framework
modifications as described in Example 1.
[0145] FIG. 11A-11C. FIG. 11A tabulates the HVR changes resulting
from affinity-maturation performed by offering a limited range of
amino acid substitutions in the hu504.16 variant. The results are
from libraries with individually modified HVRs in the hu504.16
variant as described in Example 2 herein. Amino acid abbreviations
in boxes are amino acids found more frequently in the beta7-binding
antibodies (phage-selected antibodies). FIGS. 11B and 11C are bar
graphs of the results in FIG. 11A indicating the number and type of
amino acid substitutions in the hu504.16 variant (light chain, FIG.
11B; heavy chain, FIG. 11C) detectable by the mutagenesis and
selection methods of Example 2.
[0146] FIG. 12 tabulates the results of affinity maturation
performed by offering a broad range of possible amino acid
substitutions in the HVRs of hu504.32 variant as described in
Example 2. The boxes indicate the amino acid that was detected most
frequently in antibodies detected as beta7-binding antibodies by
the mutagenesis and selection methods of Example 2.
[0147] FIGS. 13A and 13B depict HVR sequences of rat anti-mouse
Fib504 (ATCC-293), and the human consensus (left columns). Examples
of amino acid substitutions observed for each HVR position (not
meant to be limiting) by the assays described in the Examples
(amino acid substitutions observed by soft amino acid
randomization, broad amino acid substitution scan, and limited
amino acid substitution scan) are shown to the right, (a useful
method of modifying HVRs for humanization, applicable to variants
of the present invention, is found in U.S. Application Ser. No.
60/545,840, filed Feb. 19, 2004).
[0148] FIG. 14 is an exemplary graphical representation of Fib504
and variant antibody binding to MAdCAM as a function of antibody
concentration as described in Example 3. IC.sub.50 and IC.sub.90
values for the antibodies were determined.
[0149] FIGS. 15A and 15B depict the light and heavy chain HVR amino
acid sequences for the 504.32R anti-beta7 antibody with respect to
position according to the Kabat numbering system and a relative
numbering system (A-F) for the six HVRs of the antibody. Amino
acids at positions 71, 73, and 78 of the heavy chain FR3 region are
also depicted. Useful amino acid substitutions are also listed for
many of the positions in the HVRs or the heavy chain FR3
region.
[0150] FIG. 16 shows bar graphs of the relative ability of the
504.32M and 504.32R antibodies to block homing of radiolabelled T
cells to the colon of mice experiencing inflammatory bowel
disease.
MODES FOR CARRYING OUT THE INVENTION
[0151] The invention provides methods, compositions, kits and
articles of manufacture for identifying and/or using inhibitors of
the beta7 signaling pathway.
[0152] Details of these methods, compositions, kits and articles of
manufacture are provided herein.
GENERAL TECHNIQUES
[0153] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature, such
as, "Molecular Cloning: A Laboratory Manual", second edition
(Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait,
ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987);
"Methods in Enzymology" (Academic Press, Inc.); "Current Protocols
in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and
periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et
al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal
Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et
al., 2001).
DEFINITIONS
[0154] By "beta7 subunit" or ".beta.7 subunit" is meant the human
.beta.7 integrin subunit (Erle et al., (1991) J. Biol. Chem.
266:11009-11016). The beta7 subunit associates with alpha4 intetrin
subunit, such as the human .alpha.4 subunit (Kilger and Holzmann
(1995) J. Mol. Biol. 73:347-354). The alpha4beta7 integrin is
expressed on a majority of mature lymphocytes, as well as a small
population of thymocytes, bone marrow cells and mast cells.
(Kilshaw and Murant (1991) Eur. J. Immunol. 21:2591-2597; Gurish et
al., (1992) 149: 1964-1972; and Shaw, S. K. and Brenner, M. B.
(1995) Semin. Immunol. 7:335). The beta7 subunit also associates
with the alphaE subunit, such as the human alphaE integrin subunit
(Cepek, K. L, et al. (1993) J. Immunol. 150:3459). The alphaEbeta7
integrin is expressed on intra-intestinal epithelial lymphocytes
(iIELs) (Cepek, K. L. (1993) supra). The beta7 subunit that binds
to the humanized anti-beta7 antibody of the invention may be
naturally occurring and may be soluble or localized to the surface
of a cell.
[0155] By "alphaE subunit" or "alphaE integrin subunit" or "aE
subunit" or "aE integrin subunit" or "CD103" is meant an integrin
subunit found to be associated with beta7 integrin on
intra-epithelial lymphocytes, which alphaEbeta7 integrin mediates
binding of the iELs to intestinal epithelium expressing E-caderin
(Cepek, K. L. et al. (1993) J. Immunol. 150:3459; Shaw, S. K. and
Brenner, M. B. (1995) Semin. Immunol. 7:335).
[0156] "MAdCAM" or "MAdCAM-1" are used interchangeably in the
context of the present invention and refer to the protein mucosal
addressin cell adhesion molecule-1, which is a single chain
polypeptide comprising a short cytoplasmic tail, a transmembrane
region and an extracellular sequence composed of three
immunoglobulin-like domains. The cDNAs for murine, human and
macaque MAdCAM-1 have been cloned (Briskin, et al, (1993) Nature,
363:461-464; Shyjan et al., (1996) J. Immunol. 156:2851-2857).
[0157] "VCAM-1" or "vascular cell adhesion molecule-1" "CD106"
refers to a ligand of alpha4beta7 and alpha4beta1, expressed on
activated endothelium and important in endothelial-leukocyte
interactions such as binding and transmigration of leukocytes
during inflammation.
[0158] "E-cadherin" refers to a member of the family of cadherins,
where E-cadherin is expressed on epithelial cells. E-cadherin is a
ligand of the alphaEbeta7 integrin and mediates binding of
iEL-expressed alphaEbeta7 to intestinal epithelium, although its
function in lymphocyte homing is unclear. E-cadherin expression is
upregulated by TGF-beta1.
[0159] "Fibronectin" refers to Fibronectin is involved in tissue
repair, embryogenesis, blood clotting, and cell migration/adhesion.
It serves as a linker in the ECM (extracellular matrix), and as
dimer found in the plasma (plasma fibronectin). The plasma form is
synthesized by hepatocytes, while the ECM form is made by
fibroblasts, chondrocytes, endothelial cells, macrophages, as well
as certain epithelial cells. In this context, it interacts with the
alpha4beta7 integrin to mediate aspects of lymphocyte homing or
adhesion. The ECM form of fibronectin serves as a general cell
adhesion molecule by anchoring cells to collagen or proteoglycan
substrates. Fibronectin also can serve to organize cellular
interaction with the ECM by binding to different components of the
extracellular matrix and to membrane-bound fibronectin receptors on
cell surfaces. Finally, fibronectin is important in cell migration
events during embryogenesis.
[0160] "Gastrointestinal inflammatory disorders" are a group of
chronic disorders that cause inflammation and/or ulceration in the
mucous membrane. These disorders include, for example, inflammatory
bowel disease (e.g., Crohn's disease, ulcerative colitis,
indeterminate colitis and infectious colitis), mucositis (e.g.,
oral mucositis, gastrointestinal mucositis, nasal mucositis and
proctitis), necrotizing enterocolitis and esophagitis.
[0161] "Inflammatory Bowel Disease" or "IBD" is used
interchangeably herein to refer to diseases of the bowel that cause
inflammation and/or ulceration and includes without limitation
Crohn's disease and ulcerative colitis.
[0162] "Crohn's disease (CD)" or "ulcerative colitis (UC)" are
chronic inflammatory bowel diseases of unknown etiology. Crohn's
disease, unlike ulcerative colitis, can affect any part of the
bowel. The most prominent feature Crohn's disease is the granular,
reddish-purple edmatous thickening of the bowel wall. With the
development of inflammation, these granulomas often lose their
circumscribed borders and integrate with the surrounding tissue.
Diarrhea and obstruction of the bowel are the predominant clinical
features. As with ulcerative colitis, the course of Crohn's disease
may be continuous or relapsing, mild or severe, but unlike
ulcerative colitis, Crohn's disease is not curable by resection of
the involved segment of bowel. Most patients with Crohn's disease
require surgery at some point, but subsequent relapse is common and
continuous medical treatment is usual.
[0163] Crohn's disease may involve any part of the alimentary tract
from the mouth to the anus, although typically it appears in the
ileocolic, small-intestinal or colonic-anorectal regions.
Histopathologically, the disease manifests by discontinuous
granulomatomas, crypt abscesses, fissures and aphthous ulcers. The
inflammatory infiltrate is mixed, consisting of lymphocytes (both T
and B cells), plasma cells, macrophages, and neutrophils. There is
a disproportionate increase in IgM- and IgG-secreting plasma cells,
macrophages and neutrophils.
[0164] Anti-inflammatory drugs sulfasalazine and 5-aminosalisylic
acid (5-ASA) are useful for treating mildly active colonic Crohn's
disease and is commonly prescribed to maintain remission of the
disease. Metroidazole and ciprofloxacin are similar in efficacy to
sulfasalazine and appear to be particularly useful for treating
perianal disease. In more severe cases, corticosteroids are
effective in treating active exacerbations and can even maintain
remission. Azathioprine and 6-mercaptopurine have also shown
success in patients who require chronic administration of cortico
steroids. It is also possible that these drugs may play a role in
the long-term prophylaxis. Unfortunately, there can be a very long
delay (up to six months) before onset of action in some
patients.
[0165] Antidiarrheal drugs can also provide symptomatic relief in
some patients. Nutritional therapy or elemental diet can improve
the nutritional status of patients and induce symtomatic
improvement of acute disease, but it does not induce sustained
clinical remissions. Antibiotics are used in treating secondary
small bowel bacterial overgrowth and in treatment of pyogenic
complications.
[0166] "Ulcerative colitis (UC)" afflicts the large intestine. The
course of the disease may be continuous or relapsing, mild or
severe. The earliest lesion is an inflammatory infiltration with
abscess formation at the base of the crypts of Lieberkuhn.
Coalescence of these distended and ruptured crypts tends to
separate the overlying mucosa from its blood supply, leading to
ulceration. Symptoms of the disease include cramping, lower
abdominal pain, rectal bleeding, and frequent, loose discharges
consisting mainly of blood, pus and mucus with scanty fecal
particles. A total colectomy may be required for acute, severe or
chronic, unremitting ulcerative colitis.
[0167] The clinical features of UC are highly variable, and the
onset may be insidious or abrupt, and may include diarrhea,
tenesmus and relapsing rectal bleeding. With fulminant involvement
of the entire colon, toxic megacolon, a life-threatening emergency,
may occur. Extraintestinal manifestations include arthritis,
pyoderma gangrenoum, uveitis, and erythema nodosum.
[0168] Treatment for UC includes sulfasalazine and related
salicylate-containing drugs for mild cases and corticosteroid drugs
in severe cases. Topical adminstration of either salicylates or
corticosteroids is sometimes effective, particularly when the
disease is limited to the distal bowel, and is associated with
decreased side effects compared with systemic use. Supportive
measures such as administration of iron and antidiarrheal agents
are sometimes indicated. Azathioprine, 6-mercaptopurine and
methotrexate are sometimes also prescribed for use in refractory
corticosteroid-dependent cases.
[0169] A "modification" of an amino acid residue/position, as used
herein, refers to a change of a primary amino acid sequence as
compared to a starting amino acid sequence, wherein the change
results from a sequence alteration involving said amino acid
residue/positions. For example, typical modifications include
substitution of the residue (or at said position) with another
amino acid (e.g., a conservative or non-conservative substitution),
insertion of one or more (generally fewer than 5 or 3) amino acids
adjacent to said residue/position, and deletion of said
residue/position. An "amino acid substitution," or variation
thereof, refers to the replacement of an existing amino acid
residue in a predetermined (starting) amino acid sequence with a
different amino acid residue. Generally and preferably, the
modification results in alteration in at least one
physicobiochemical activity of the variant polypeptide compared to
a polypeptide comprising the starting (or "wild type") amino acid
sequence. For example, in the case of an antibody, a
physicobiochemical activity that is altered can be binding
affinity, binding capability and/or binding effect upon a target
molecule.
[0170] The term "amino acid" within the scope of the present
invention is used in its broadest sense and is meant to include the
naturally occurring L .alpha.-amino acids or residues. The commonly
used one- and three-letter abbreviations for naturally occurring
amino acids are used herein (Lehninger, A. L., Biochemistry, 2d
ed., pp. 71-92, (Worth Publishers, New York, N.Y., 1975). The term
includes D-amino acids as well as chemically modified amino acids
such as amino acid analogs, naturally occurring amino acids that
are not usually incorporated into proteins such as norleucine, and
chemically synthesized compounds having properties known in the art
to be characteristic of an amino acid. For example, analogs or
mimetics of phenylalanine or proline, which allow the same
conformational restriction of the peptide compounds as natural Phe
or Pro are included within the definition of amino acid. Such
analogs and mimetics are referred to herein as "functional
equivalents" of an amino acid. Other examples of amino acids are
listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis,
Biology, Gross and Meiehofer, Eds., Vol. 5, p. 341 (Academic Press,
Inc., New York, N.Y., 1983), which is incorporated herein by
reference. Where a single letter is used to designate one of the
naturally occurring amino acid, the designations are as commonly
found in the relevant literature (see, for example, Alberts, B. et
al. Molecular Biology of the Cell, 3rd ed., Garland Publishing,
Inc. 1994, page 57).
[0171] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0172] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat", and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0173] The phrase "substantially similar," or "substantially the
same", as used herein, denotes a sufficiently high degree of
similarity between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is
preferably less than about 50%, preferably less than about 40%,
preferably less than about 30%, preferably less than about 20%,
preferably less than about 10% as a function of the value for the
reference/comparator antibody.
[0174] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0175] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay that measures solution
binding affinity of Fabs for antigen by equilibrating Fab with a
minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5 ug/ml
of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23.degree. C.). In a non-adsorbant plate (Nunc
#269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed with serial
dilutions of a Fab of interest (e.g., consistent with assessment of
an anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res.
57:4593-4599). The Fab of interest is then incubated overnight;
however, the incubation may continue for a longer period (e.g., 65
hours) to insure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature (e.g., for one hour). The solution is then removed
and the plate washed eight times with 0.1% Tween-20 in PBS. When
the plates have dried, 150 ul/well of scintillant (MicroScint-20;
Packard) is added, and the plates are counted on a Topcount gamma
counter (Packard) for ten minutes. Concentrations of each Fab that
give less than or equal to 20% of maximal binding are chosen for
use in competitive binding assays. According to another embodiment
the Kd or Kd value is measured by using surface plasmon resonance
assays using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore,
Inc., Piscataway, N.J.) at 25 C with immobilized antigen CM5 chips
at .about.10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 ug/ml (.about.0.2 uM) before injection at a flow rate of 5
ul/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen, Y.,
et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stir red cuvette.
[0176] An "on-rate" or "rate of association" or "association rate"
or "k.sub.on" according to this invention can also be determined
with the same surface plasmon resonance technique described above
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25 C with immobilized antigen CM5 chips at
.about.10 response units (RU). Briefly, carboxymethylated dextran
biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 ug/ml (.about.0.2 uM) before injection at a flow rate of 5
ul/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of 1M ethanolamine to
block unreacted groups. For kinetics measurements, two-fold serial
dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%
Tween 20 (PBST) at 25.degree. C. at a flow rate of approximately 25
ul/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIAcore Evaluation Software version 3.2) by
simultaneous fitting the association and dissociation sensorgram.
The equilibrium dissociation constant (Kd) was calculated as the
ratio k.sub.off/k.sub.on. See, e.g., Chen, Y., et al., (1999) J.
Mol Biol 293:865-881. However, if the on-rate exceeds 10.sup.6
M.sup.-1 S.sup.-1 by the surface plasmon resonance assay above,
then the on-rate is preferably determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stirred cuvette. The "Kd" or "Kd value" according to this invention
is in one embodiment measured by a radiolabeled antigen binding
assay (RIA) performed with the Fab version of the antibody and
antigen molecule as described by the following assay that measures
solution binding affinity of Fabs for antigen by equilibrating Fab
with a minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5 ug/ml
of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23.degree. C.). In a non-adsorbant plate (Nunc
#269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed with serial
dilutions of a Fab of interest (consistent with assessment of an
anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res.
57:4593-4599). The Fab of interest is then incubated overnight;
however, the incubation may continue for a longer period (e.g., 65
hours) to insure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature for one hour. The solution is then removed and the
plate washed eight times with 0.1% Tween-20 in PBS. When the plates
have dried, 150 ul/well of scintillant (MicroScint-20; Packard) is
added, and the plates are counted on a Topcount gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays. According to another embodiment, the Kd
or Kd value is measured by using surface plasmon resonance assays
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25 C with immobilized antigen CM5 chips at
.about.10 response units (RU). Briefly, carboxymethylated dextran
biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 ug/ml (.about.0.2 uM) before injection at a flow rate of 5
ul/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen, Y.,
et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stir red cuvette.
[0177] In one embodiment, an "on-rate" or "rate of association" or
"association rate" or "k.sub.on" according to this invention is
determined with the same surface plasmon resonance technique
described above using a BIAcore.TM.-2000 or a BIAcore.TM.-3000
(BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilized antigen
CM5 chips at .about.10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium
acetate, pH 4.8, into 5 ug/ml (.about.0.2 uM) before injection at a
flow rate of 5 ul/minute to achieve approximately 10 response units
(RU) of coupled protein. Following the injection of 1M ethanolamine
to block unreacted groups. For kinetics measurements, two-fold
serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS
with 0.05% Tween 20 (PBST) at 25.degree. C. at a flow rate of
approximately 25 ul/min. Association rates (k.sub.on) and
dissociation rates (k.sub.off) are calculated using a simple
one-to-one Langmuir binding model (BIAcore Evaluation Software
version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
was calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen,
Y., et al., (1999) J. Mol Biol 293:865-881. However, if the on-rate
exceeds 10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance
assay above, then the on-rate is preferably determined by using a
fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or
a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with
a stirred cuvette.
[0178] The phrase "substantially reduced," or "substantially
different", as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values, HAMA
response). The difference between said two values is preferably
greater than about 10%, preferably greater than about 20%,
preferably greater than about 30%, preferably greater than about
40%, preferably greater than about 50% as a function of the value
for the reference/comparator antibody.
[0179] "Percent (%) amino acid sequence identity" with respect to a
peptide or polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the specific peptide or polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2, wherein the complete
source code for the ALIGN-2 program is provided in Table A below.
The ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc. and the source code shown in Table A below has been
filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech, Inc., South San Francisco, Calif. or
may be compiled from the source code provided in FIG. 8 below. The
ALIGN-2 program should be compiled for use on a UNIX operating
system, preferably digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
[0180] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A.
[0181] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program.
[0182] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
phage vector. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "recombinant vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector.
[0183] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label. Other types of modifications include, for example, "caps",
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
ply-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C.) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0184] "Oligonucleotide," as used herein, generally refers to
short, generally single stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not mutually exclusive. The description above
for polynucleotides is equally and fully applicable to
oligonucleotides.
[0185] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (for example, full length or intact monoclonal
antibodies), polyclonal antibodies, multivalent antibodies,
multispecific antibodies (e.g., bispecific antibodies so long as
they exhibit the desired biological activity) and may also include
certain antibody fragments (as described in greater detail herein).
An antibody can be human, humanized and/or affinity matured.
[0186] "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion preferably retains at least one,
preferably most or all, of the functions normally associated with
that portion when present in an intact antibody. In one embodiment,
an antibody fragment comprises an antigen binding site of the
intact antibody and thus retains the ability to bind antigen. In
another embodiment, an antibody fragment, for example one that
comprises the Fc region, retains at least one of the biological
functions normally associated with the Fc region when present in an
intact antibody, such as FcRn binding, antibody half life
modulation, ADCC function and complement binding. In one
embodiment, an antibody fragment is a monovalent antibody that has
an in vivo half life substantially similar to an intact antibody.
For example, such an antibody fragment may comprise on antigen
binding arm linked to an Fc sequence capable of conferring in vivo
stability to the fragment.
[0187] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen.
[0188] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)).
[0189] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the
following review articles and references cited therein: Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994).
[0190] An "antigen" is a predetermined antigen to which an antibody
can selectively bind. The target antigen may be polypeptide,
carbohydrate, nucleic acid, lipid, hapten or other naturally
occurring or synthetic compound. Preferably, the target antigen is
a polypeptide. An "acceptor human framework" for the purposes
herein is a framework comprising the amino acid sequence of a VL or
VH framework derived from a human immunoglobulin framework, or from
a human consensus framework. An acceptor human framework "derived
from" a human immunoglobulin framework or human consensus framework
may comprise the same amino acid sequence thereof, or may contain
pre-existing amino acid sequence changes. Where pre-existing amino
acid changes are present, preferably no more than 5 and preferably
4 or less, or 3 or less, pre-existing amino acid changes are
present. Where pre-existing amino acid changes are present in a VH,
preferably those changes are only at three, two or one of positions
71H, 73H and 78H; for instance, the amino acid residues at those
positions may be 71A, 73T and/or 78A. In one embodiment, the VL
acceptor human framework is identical in sequence to the VL human
immunoglobulin framework sequence or human consensus framework
sequence.
[0191] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residue in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al.
[0192] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al. In one embodiment,
the VL subgroup I consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00001 (SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITC- L1- (SEQ ID
NO: 35) WYQQKPGKAPKLLI- L2- (SEQ ID NO: 36)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC- L3- (SEQ ID NO: 37)
FGQGTKVEIKR.
[0193] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al. In one embodiment, the
VH subgroup III consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00002 (SEQ ID NO: 38) EVQLVESGGGLVQPGGSLRLSCAAS- H1- (SEQ
ID NO: 39) WVRQAPGKGLEWV- H2- (SEQ ID NO: 40)
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA- H3- (SEQ ID NO: 41)
WGQGTLVTVSS.
[0194] An "unmodified human framework" is a human framework which
has the same amino acid sequence as the acceptor human framework,
e.g. lacking human to non-human amino acid substitution(s) in the
acceptor human framework.
[0195] An "altered hypervariable region" for the purposes herein is
a hypervariable region comprising one or more (e.g. one to about
16) amino acid substitution(s) therein.
[0196] An "un-modified hypervariable region" for the purposes
herein is a hypervariable region having the same amino acid
sequence as a non-human antibody from which it was derived, i.e.
one which lacks one or more amino acid substitutions therein.
[0197] The term "hypervariable region", "HVR", or "HV", when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six hypervariable regions;
three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A
number of hypervariable region delineations are in use and are
encompassed herein. The Kabat Complementarity Determining Regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). Chothia refers instead to the
location of the structural loops (Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)). The AbM hypervariable regions represent a
compromise between the Kabat CDRs and Chothia structural loops, and
are used by Oxford Molecular's AbM antibody modeling software. The
"contact" hypervariable regions are based on an analysis of the
available complex crystal structures. The residues from each of
these hypervariable regions are noted below.
TABLE-US-00003 TABLE 1 Loop Kabat AbM Chothia Contact L1 L24-L34
L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3
L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32
H30-H35B Kabat numbering H1 H31-H35 H26-H35 H26-H32 H30-H35 Chothia
numbering H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0198] Hypervariable regions may comprise "extended hypervariable
regions" as follows: 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56
or 52-56 (L2) and 89-97 (L3) in the VL and 26-35 (H1), 50-65 or
49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The
variable domain residues are numbered according to Kabat et al.,
supra for each of these definitions.
[0199] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0200] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
[0201] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al. Proc Nat. Acad.
Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol.
Biol. 226:889-896 (1992).
[0202] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
bind. Preferred blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0203] An "agonist antibody", as used herein, is an antibody which
mimics at least one of the functional activities of a polypeptide
of interest.
[0204] A "disorder" is any condition that would benefit from
treatment with a substance/molecule or method of the invention.
This includes chronic and acute disorders or diseases including
those pathological conditions which predispose the mammal to the
disorder in question. Non-limiting examples of disorders to be
treated herein include malignant and benign tumors; non-leukemias
and lymphoid malignancies; neuronal, glial, astrocytal,
hypothalamic and other glandular, macrophagal, epithelial, stromal
and blastocoelic disorders; and inflammatory, immunologic and other
angiogenesis-related disorders.
[0205] The term "immune related disease" means a disease in which a
component of the immune system of a mammal causes, mediates or
otherwise contributes to a morbidity in the mammal. Also included
are diseases in which stimulation or intervention of the immune
response has an ameliorative effect on progression of the disease.
Included within this term are immune-mediated inflammatory
diseases, non-immune-mediated inflammatory diseases, infectious
diseases, immunodeficiency diseases, neoplasia, etc.
[0206] Examples of immune-related and inflammatory diseases, some
of which are immune or T cell mediated, which can be treated
according to the invention include systemic lupus erythematosis,
rheumatoid arthritis, juvenile chronic arthritis,
spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic
inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's
syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria),
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia), thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis), diabetes mellitus, immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis), demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy, hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis, inflammatory
bowel disease (ulcerative colitis: Crohn's disease),
gluten-sensitive enteropathy, and Whipple's disease, autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis, allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria, immunologic diseases of the lung
such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis, transplantation associated diseases
including graft rejection and graft-versus-host-disease. Infectious
diseases including viral diseases such as AIDS (HIV infection),
hepatitis A, B, C, D, and E, herpes, etc., bacterial infections,
fungal infections, protozoal infections and parasitic
infections.
[0207] An "autoimmune disorder" or "autoimmune disease" as used
interchangeably herein is a non-malignant disease or disorder
arising from and directed against an individual's own tissues. The
autoimmune diseases described herein specifically exclude malignant
or cancerous diseases or conditions, particularly excluding B cell
lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic
leukemia (CLL), Hairy cell leukemia, and chronic myeloblastic
leukemia. Examples of autoimmune diseases or disorders include, but
are not limited to, inflammatory responses such as inflammatory
skin diseases including psoriasis and dermatitis (for example,
atopic dermatitis); systemic scleroderma and sclerosis; responses
associated with inflammatory bowel disease (such as Crohn's disease
and ulcerative colitis); respiratory distress syndrome (including
adult respiratory distress syndrome; ARDS); dermatitis; meningitis;
encephalitis; uveitis; colitis; glomerulonephritis; allergic
conditions such as eczema and asthma and other conditions involving
infiltration of T cells and chronic inflammatory responses;
atherosclerosis; leukocyte adhesion deficiency; rheumatoid
arthritis; systemic lupus erythematosus (SLE); diabetes mellitus
(e.g. Type I diabetes mellitus or insulin dependent diabetes
mellitis); multiple sclerosis; Reynaud's syndrome; autoimmune
thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome;
juvenile onset diabetes; and immune responses associated with acute
and delayed hypersensitivity mediated by cytokines and
T-lymphocytes typically found in tuberculosis, sarcoidosis,
polymyositis, granulomatosis and vasculitis; pernicious anemia
(Addison's disease); diseases involving leukocyte diapedesis;
central nervous system (CNS) inflammatory disorder; multiple organ
injury syndrome; hemolytic anemia (including, but not limited to
cryoglobinemia or Coombs positive anemia); myasthenia gravis;
antigen-antibody complex mediated diseases; anti-glomerular
basement membrane disease;
[0208] antiphospholipid syndrome; allergic neuritis; Graves'
disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;
pemphigus; autoimmune polyendocrinopathies; Reiter's disease;
stiff-man syndrome; Behcet disease; giant cell arteritis; immune
complex nephritis; IgA nephropathy; IgM polyneuropathies; immune
thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia
etc.
[0209] The term "gastrointestinal inflammatory disorders" is a
group of chronic disorders that cause inflammation and/or
ulceration in the mucous membrane. As such, the term includes
inflammatory bowel disease (e.g., Crohn's disease, ulcerative
colitis, indeterminate colitis and infectious colitis), mucositis
(e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis
and proctitis), necrotizing enterocolitis and esophagitis.
[0210] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0211] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0212] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers
include squamous cell cancer, small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of
the lung, cancer of the peritoneum, hepatocellular cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney cancer, liver
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma and various types of head and neck cancer.
[0213] Dysregulation of angiogenesis can lead to many disorders
that can be treated by compositions and methods of the invention.
These disorders include both non-neoplastic and neoplastic
conditions. Neoplastics include but are not limited those described
above. Non-neoplastic disorders include but are not limited to
undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis
(RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis,
atherosclerotic plaques, diabetic and other proliferative
retinopathies including retinopathy of prematurity, retrolental
fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
angle (rubeosis), ocular neovascular disease, vascular restenosis,
arteriovenous malformations (AVM), meningioma, hemangioma,
angiofibroma, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, chronic inflammation,
lung inflammation, acute lung injury/ARDS, sepsis, primary
pulmonary hypertension, malignant pulmonary effusions, cerebral
edema (e.g., associated with acute stroke/closed head
injury/trauma), synovial inflammation, pannus formation in RA,
myositis ossificans, hypertropic bone formation, osteoarthritis
(OA), refractory ascites, polycystic ovarian disease,
endometriosis, 3rd spacing of fluid diseases (pancreatitis,
compartment syndrome, burns, bowel disease), uterine fibroids,
premature labor, chronic inflammation such as IBD (Crohn's disease
and ulcerative colitis), renal allograft rejection, inflammatory
bowel disease, nephrotic syndrome, undesired or aberrant tissue
mass growth (non-cancer), hemophilic joints, hypertrophic scars,
inhibition of hair growth, Osler-Weber syndrome, pyogenic granuloma
retrolental fibroplasias, scleroderma, trachoma, vascular
adhesions, synovitis, dermatitis, preeclampsia, ascites,
pericardial effusion (such as that associated with pericarditis),
and pleural effusion.
[0214] As used herein, "treatment" refers to clinical intervention
in an attempt to alter the natural course of the individual or cell
being treated, and can be performed either for prophylaxis or
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing metastasis,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis. In some embodiments, antibodies of the invention are
used to delay development of a disease or disorder.
[0215] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0216] A "therapeutically effective amount" of a substance/molecule
of the invention, agonist or antagonist may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the substance/molecule, agonist or
antagonist to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the substance/molecule, agonist or
antagonist are outweighed by the therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0217] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0218] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; an esperamicin; as well as neocarzinostatin
chromophore and related chromoprotein enediyne antiobiotic
chromophores), aclacinomysins, actinomycin, authramycin, azaserine,
bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMF.RTM.); retinoids
such as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0219] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0220] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell whose
growth is dependent upon beta7 activation either in vitro or in
vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of beta7-dependent cells in S
phase. Examples of growth inhibitory agents include agents that
block cell cycle progression (at a place other than S phase), such
as agents that induce G1 arrest and M-phase arrest. Classical
M-phase blockers include the vincas (vincristine and vinblastine),
taxanes, and topoisomerase II inhibitors such as doxorubicin,
epirubicin, daunorubicin, etoposide, and bleomycin. Those agents
that arrest G1 also spill over into S-phase arrest, for example,
DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and
ara-C. Further information can be found in The Molecular Basis of
Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell
cycle regulation, oncogenes, and antineoplastic drugs" by Murakami
et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The
taxanes (paclitaxel and docetaxel) are anticancer drugs both
derived from the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc
Rorer), derived from the European yew, is a semisynthetic analogue
of paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0221] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0222] Compounds useful in combination therapy with an antagonist
anti-beta7 antibody of the invention include antibodies (including
without limitation other anti-beta7 antagonist antibodies (Fib 21,
22, 27, 30 (Tidswell, M. (1997) supra) or humanized derivatives
thereof), anti-alpha4 antibodies (such as ANTEGEN.RTM.), anti-TNF
(REMICADE.RTM.)) or non-protein compounds including without
limitation 5-ASA compounds ASACOL.RTM., PENTASA.TM., ROWASA.TM.,
COLAZAL.TM., and other compounds such as Purinethol and steroids
such as prednisone. In an embodiment, the invention encompasses a
method of treating a patient, such as a human patient, with the
antagonist anti-beta7 antibody of the invention alone or in
combination with a second compound that is also useful in treating
inflammation. In one embodiment the second compound is selected
from the group consisting of Fib 21, 22, 27, 30, or humanized
derivatives thereof), anti-alpha4 antibodies, ANTEGEN.RTM.,
anti-TNF, REMICADE.RTM., 5-ASA compounds, ASACOL.RTM., PENTASA.TM.,
ROWASA.TM., COLAZAL.TM., Purinethol, steroids, and prednisone. In
one embodiment of the invention, administration of the antagonist
anti-beta7 antibody of the invention substantially reduces the dose
of the second compound. In one embodiment, said reduction in the
dose of the second compound is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%. In one embodiment of the invention, the combination of
the antibody of the invention and the reduced dose of the second
compound relieves symptoms in the patient to substantially the same
degree or better than administration of the second compound
alone.
Generating Variant Antibodies Exhibiting Reduced or Absence of HAMA
Response
[0223] Reduction or elimination of a HAMA (human anti-mouse (also
applicable to human anti-rat or human anti-human) response is a
significant aspect of clinical development of suitable therapeutic
agents. See, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988),
80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et
al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response
Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et
al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988),
332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described
herein, the invention provides antibodies that are humanized such
that HAMA response is reduced or eliminated. Variants of these
antibodies can further be obtained using routine methods known in
the art, some of which are further described below.
[0224] For example, an amino acid sequence from an antibody as
described herein can serve as a starting (parent) sequence for
diversification of the framework and/or hypervariable sequence(s).
A selected framework sequence to which a starting hypervariable
sequence is linked is referred to herein as an acceptor human
framework. While the acceptor human frameworks may be from, or
derived from, a human immunoglobulin (the VL and/or VH regions
thereof), preferably the acceptor human frameworks are from, or
derived from, a human consensus framework sequence as such
frameworks have been demonstrated to have minimal, or no,
immunogenicity in human patients.
[0225] Where the acceptor is derived from a human immunoglobulin,
one may optionally select a human framework sequence that is
selected based on its homology to the donor framework sequence by
aligning the donor framework sequence with various human framework
sequences in a collection of human framework sequences, and select
the most homologous framework sequence as the acceptor.
[0226] In one embodiment, human consensus frameworks herein are
from, or derived from, VH subgroup III and/or VL kappa subgroup I
consensus framework sequences.
[0227] Thus, the VH acceptor human framework may comprise one, two,
three or all of the following framework sequences:
FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:38), FR2
comprising WVRQAPGKGLEWV (SEQ ID NO:39), FR3 comprising FR3
comprises RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYCA (SEQ ID NO:42),
wherein X1 is A or R, X2 is T or N, and X3 is A, L, or F, FR4
comprising WGQGTLVTVSS (SEQ ID NO:41). Examples of VH consensus
frameworks include: human VH subgroup I consensus framework minus
Kabat CDRs (SEQ ID NO:19); human VH subgroup I consensus framework
minus extended hypervariable regions (SEQ ID NOs:20-22); human VH
subgroup II consensus framework minus Kabat CDRs (SEQ ID NO:48);
human VH subgroup II consensus framework minus extended
hypervariable regions (SEQ ID NOs:49-51); human VH subgroup III
consensus framework minus Kabat CDRs (SEQ ID NO:52); human VH
subgroup III consensus framework minus extended hypervariable
regions (SEQ ID NO:53-55); human VH acceptor framework minus Kabat
CDRs (SEQ ID NO:56); human VH acceptor framework minus extended
hypervariable regions (SEQ ID NOs:57-58); human VH acceptor 2
framework minus Kabat CDRs (SEQ ID NO:59); or human VH acceptor 2
framework minus extended hypervariable regions (SEQ ID
NOs:60-62).
[0228] In one embodiment, the VH acceptor human framework comprises
one, two, three or all of the following framework sequences:
TABLE-US-00004 FR1 comprising (SEQ ID NO: 38)
EVQLVESGGGLVQPGGSLRLSCAAS, FR2 comprising (SEQ ID NO: 39)
WVRQAPGKGLEWV, FR3 comprising (SEQ ID NO: 43)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCA, FR3 comprises (SEQ ID NO: 44)
RFTISRDTSKNTAYLQMNSLRAEDTAVYYCA, (SEQ ID NO: 45)
RFTISRDTSKNTFYLQMNSLRAEDTAVYYCA, (SEQ ID NO: 46)
RFTISADTSKNTFYLQMNSLRAEDTAVYYCA, FR4 comprising (SEQ ID NO: 41)
WGQGTLVTVSS.
[0229] The VL acceptor human framework may comprise one, two, three
or all of the following framework sequences:
TABLE-US-00005 FR1 comprising (SEQ ID NO: 34)
DIQMTQSPSSLSASVGDRVTITC, FR2 comprising (SEQ ID NO: 35)
WYQQKPGKAPKLLI, FR3 comprising (SEQ ID NO: 36)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, FR4 comprising (SEQ ID NO: 37)
FGQGTKVEIKR.
[0230] Examples of VL consensus frameworks include:
human VL kappa subgroup I consensus framework (SEQ ID NO:14); human
VL kappa subgroup I consensus framework (extended HVR-L2) (SEQ ID
NO:15); human VL kappa subgroup II consensus framework (SEQ ID
NO:16); human VL kappa subgroup III consensus framework (SEQ ID
NO:17); or human VL kappa subgroup IV consensus framework (SEQ ID
NO:18)
[0231] While the acceptor may be identical in sequence to the human
framework sequence selected, whether that is from a human
immunoglobulin or a human consensus framework, the present
invention contemplates that the acceptor sequence may comprise
pre-existing amino acid substitutions relative to the human
immunoglobulin sequence or human consensus framework sequence.
These pre-existing substitutions are preferably minimal; usually
four, three, two or one amino acid differences only relative to the
human immunoglobulin sequence or consensus framework sequence.
[0232] Hypervariable region residues of the non-human antibody are
incorporated into the VL and/or VH acceptor human frameworks. For
example, one may incorporate residues corresponding to the Kabat
CDR residues, the Chothia hypervariable loop residues, the Abm
residues, and/or contact residues. Optionally, the extended
hypervariable region residues as follows are incorporated: 24-34
(L1), 49-56 (L2) and 89-97 (L3), 26-35 (H1), 50-65 or 49-65 (H2)
and 93-102, 94-102, or 95-102 (H3).
[0233] While "incorporation" of hypervariable region residues is
discussed herein, it will be appreciated that this can be achieved
in various ways, for example, nucleic acid encoding the desired
amino acid sequence can be generated by mutating nucleic acid
encoding the mouse variable domain sequence so that the framework
residues thereof are changed to acceptor human framework residues,
or by mutating nucleic acid encoding the human variable domain
sequence so that the hypervariable domain residues are changed to
non-human residues, or by synthesizing nucleic acid encoding the
desired sequence, etc.
[0234] In the examples herein, hypervariable region-grafted
variants were generated by Kunkel mutagenesis of nucleic acid
encoding the human acceptor sequences, using a separate
oligonucleotide for each hypervariable region. Kunkel et al.,
Methods Enzymol. 154:367-382 (1987). Appropriate changes can be
introduced within the framework and/or hypervariable region, using
routine techniques, to correct and re-establish proper
hypervariable region-antigen interactions.
[0235] Phage(mid) display (also referred to herein as phage display
in some contexts) can be used as a convenient and fast method for
generating and screening many different potential variant
antibodies in a library generated by sequence randomization.
However, other methods for making and screening altered antibodies
are available to the skilled person.
[0236] Phage(mid) display technology has provided a powerful tool
for generating and selecting novel proteins which bind to a ligand,
such as an antigen. Using the techniques of phage(mid) display
allows the generation of large libraries of protein variants which
can be rapidly sorted for those sequences that bind to a target
molecule with high affinity. Nucleic acids encoding variant
polypeptides are generally fused to a nucleic acid sequence
encoding a viral coat protein, such as the gene III protein or the
gene VIII protein. Monovalent phagemid display systems where the
nucleic acid sequence encoding the protein or polypeptide is fused
to a nucleic acid sequence encoding a portion of the gene III
protein have been developed. (Bass, S., Proteins, 8:309 (1990);
Lowman and Wells, Methods: A Companion to Methods in Enzymology,
3:205 (1991)). In a monovalent phagemid display system, the gene
fusion is expressed at low levels and wild type gene III proteins
are also expressed so that infectivity of the particles is
retained. Methods of generating peptide libraries and screening
those libraries have been disclosed in many patents (e.g. U.S. Pat.
No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717,
U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,498,530).
[0237] Libraries of antibodies or antigen binding polypeptides have
been prepared in a number of ways including by altering a single
gene by inserting random DNA sequences or by cloning a family of
related genes. Methods for displaying antibodies or antigen binding
fragments using phage(mid) display have been described in U.S. Pat.
Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197,
5,580,717, and 5,658,727. The library is then screened for
expression of antibodies or antigen binding proteins with the
desired characteristics.
[0238] Methods of substituting an amino acid of choice into a
template nucleic acid are well established in the art, some of
which are described herein. For example, hypervariable region
residues can be substituted using the Kunkel method. See, for
example, Kunkel et al., Methods Enzymol. 154:367-382 (1987).
[0239] The sequence of oligonucleotides includes one or more of the
designed codon sets for the hypervariable region residues to be
altered. A codon set is a set of different nucleotide triplet
sequences used to encode desired variant amino acids. Codon sets
can be represented using symbols to designate particular
nucleotides or equimolar mixtures of nucleotides as shown in below
according to the IUB code.
[0240] IUB Codes
[0241] G Guanine
[0242] A Adenine
[0243] T Thymine
[0244] C Cytosine
[0245] R (A or G)
[0246] Y (C or T)
[0247] M (A or C)
[0248] K (G or T)
[0249] S (C or G)
[0250] W (A or T)
[0251] H (A or C or T)
[0252] B (C or G or T)
[0253] V (A or C or G)
[0254] D (A or G or T) H
[0255] N (A or C or G or T)
[0256] For example, in the codon set DVK, D can be nucleotides A or
G or T; V can be A or G or C; and K can be G or T. This codon set
can present 18 different codons and can encode amino acids Ala,
Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.
[0257] Oligonucleotide or primer sets can be synthesized using
standard methods. A set of oligonucleotides can be synthesized, for
example, by solid phase synthesis, containing sequences that
represent all possible combinations of nucleotide triplets provided
by the codon set and that will encode the desired group of amino
acids. Synthesis of oligonucleotides with selected nucleotide
"degeneracy" at certain positions is well known in that art. Such
sets of nucleotides having certain codon sets can be synthesized
using commercial nucleic acid synthesizers (available from, for
example, Applied Biosystems, Foster City, Calif.), or can be
obtained commercially (for example, from Life Technologies,
Rockville, Md.). Therefore, a set of oligonucleotides synthesized
having a particular codon set will typically include a plurality of
oligonucleotides with different sequences, the differences
established by the codon set within the overall sequence.
Oligonucleotides, as used according to the invention, have
sequences that allow for hybridization to a variable domain nucleic
acid template and also can include restriction enzyme sites for
cloning purposes.
[0258] In one method, nucleic acid sequences encoding variant amino
acids can be created by oligonucleotide-mediated mutagenesis. This
technique is well known in the art as described by Zoller et al.
Nucleic Acids Res. 10:6487-6504(1987). Briefly, nucleic acid
sequences encoding variant amino acids are created by hybridizing
an oligonucleotide set encoding the desired codon sets to a DNA
template, where the template is the single-stranded form of the
plasmid containing a variable region nucleic acid template
sequence. After hybridization, DNA polymerase is used to synthesize
an entire second complementary strand of the template that will
thus incorporate the oligonucleotide primer, and will contain the
codon sets as provided by the oligonucleotide set.
[0259] Generally, oligonucleotides of at least 25 nucleotides in
length are used. An optimal oligonucleotide will have 12 to 15
nucleotides that are completely complementary to the template on
either side of the nucleotide(s) coding for the mutation(s). This
ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule. The oligonucleotides are
readily synthesized using techniques known in the art such as that
described by Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765
(1978).
[0260] The DNA template is generated by those vectors that are
either derived from bacteriophage M13 vectors (the commercially
available M13mp18 and M13mp19 vectors are suitable), or those
vectors that contain a single-stranded phage origin of replication
as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus,
the DNA that is to be mutated can be inserted into one of these
vectors in order to generate single-stranded template. Production
of the single-stranded template is described in sections 4.21-4.41
of Sambrook et al., above.
[0261] To alter the native DNA sequence, the oligonucleotide is
hybridized to the single stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA
polymerase or the Klenow fragment of DNA polymerase I, is then
added to synthesize the complementary strand of the template using
the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such that one strand of DNA encodes the
mutated form of gene 1, and the other strand (the original
template) encodes the native, unaltered sequence of gene 1. This
heteroduplex molecule is then transformed into a suitable host
cell, usually a prokaryote such as E. coli JM101. After growing the
cells, they are plated onto agarose plates and screened using the
oligonucleotide primer radiolabelled with a 32-Phosphate to
identify the bacterial colonies that contain the mutated DNA.
[0262] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutation(s). The modifications are as follows:
The single stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTT), is combined with a modified
thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide
complex. Upon addition of DNA polymerase to this mixture, a strand
of DNA identical to the template except for the mutated bases is
generated. In addition, this new strand of DNA will contain
dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of
the double-stranded heteroduplex is nicked with an appropriate
restriction enzyme, the template strand can be digested with ExoIII
nuclease or another appropriate nuclease past the region that
contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded.
A complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can
then be transformed into a suitable host cell.
[0263] As indicated previously the sequence of the oligonucleotide
set is of sufficient length to hybridize to the template nucleic
acid and may also, but does not necessarily, contain restriction
sites. The DNA template can be generated by those vectors that are
either derived from bacteriophage M13 vectors or vectors that
contain a single-stranded phage origin of replication as described
by Viera et al. Meth. Enzymol., 153:3 (1987). Thus, the DNA that is
to be mutated must be inserted into one of these vectors in order
to generate single-stranded template. Production of the
single-stranded template is described in sections 4.21-4.41 of
Sambrook et al., supra.
[0264] According to another method, a library can be generated by
providing upstream and downstream oligonucleotide sets, each set
having a plurality of oligonucleotides with different sequences,
the different sequences established by the codon sets provided
within the sequence of the oligonucleotides. The upstream and
downstream oligonucleotide sets, along with a variable domain
template nucleic acid sequence, can be used in a polymerase chain
reaction to generate a "library" of PCR products. The PCR products
can be referred to as "nucleic acid cassettes", as they can be
fused with other related or unrelated nucleic acid sequences, for
example, viral coat proteins and dimerization domains, using
established molecular biology techniques.
[0265] The sequence of the PCR primers includes one or more of the
designed codon sets for the solvent accessible and highly diverse
positions in a hypervariable region. As described above, a codon
set is a set of different nucleotide triplet sequences used to
encode desired variant amino acids.
[0266] Antibody selectants that meet the desired criteria, as
selected through appropriate screening/selection steps can be
isolated and cloned using standard recombinant techniques.
Vectors, Host Cells and Recombinant Methods
[0267] For recombinant production of an antibody of the invention,
the nucleic acid encoding it is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The choice of vector depends in part on the
host cell to be used. Generally, preferred host cells are of either
prokaryotic or eukaryotic (generally mammalian) origin.
Generating Antibodies Using Prokaryotic Host Cells:
Vector Construction
[0268] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0269] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0270] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as XGEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0271] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g. the presence
or absence of a nutrient or a change in temperature.
[0272] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0273] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .beta.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the target light and heavy chains
(Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors
to supply any required restriction sites.
[0274] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0275] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB.sup.- strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).
[0276] The present invention provides an expression system in which
the quantitative ratio of expressed polypeptide components can be
modulated in order to maximize the yield of secreted and properly
assembled antibodies of the invention. Such modulation is
accomplished at least in part by simultaneously modulating
translational strengths for the polypeptide components.
[0277] One technique for modulating translational strength is
disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes
variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a series of amino acid or nucleic acid
sequence variants can be created with a range of translational
strengths, thereby providing a convenient means by which to adjust
this factor for the desired expression level of the specific chain.
TIR variants can be generated by conventional mutagenesis
techniques that result in codon changes which can alter the amino
acid sequence, although silent changes in the nucleotide sequence
are preferred. Alterations in the TIR can include, for example,
alterations in the number or spacing of Shine-Dalgarno sequences,
along with alterations in the signal sequence. One method for
generating mutant signal sequences is the generation of a "codon
bank" at the beginning of a coding sequence that does not change
the amino acid sequence of the signal sequence (i.e., the changes
are silent). This can be accomplished by changing the third
nucleotide position of each codon; additionally, some amino acids,
such as leucine, serine, and arginine, have multiple first and
second positions that can add complexity in making the bank. This
method of mutagenesis is described in detail in Yansura et al.
(1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.
[0278] Preferably, a set of vectors is generated with a range of
TIR strengths for each cistron therein. This limited set provides a
comparison of expression levels of each chain as well as the yield
of the desired antibody products under various TIR strength
combinations. TIR strengths can be determined by quantifying the
expression level of a reporter gene as described in detail in
Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational
strength comparison, the desired individual TIRs are selected to be
combined in the expression vector constructs of the invention.
[0279] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhuA (AtonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP41 kan.sup.R (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli.sub..lamda. 1776 (ATCC
31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These
examples are illustrative rather than limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria
having defined genotypes are known in the art and described in, for
example, Bass et al., Proteins, 8:309-314 (1990). It is generally
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon. Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may
desirably be incorporated in the cell culture.
Antibody Production
[0280] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0281] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0282] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0283] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0284] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the preferred
temperature ranges from about 20.degree. C. to about 39.degree. C.,
more preferably from about 25.degree. C. to about 37.degree. C.,
even more preferably at about 30.degree. C. The pH of the medium
may be any pH ranging from about 5 to about 9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8
to about 7.4, and more preferably about 7.0.
[0285] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. Preferably,
the phosphate-limiting medium is the C.R.A.P medium (see, for
example, Simmons et al., J. Immunol. Methods (2002), 263:133-147).
A variety of other inducers may be used, according to the vector
construct employed, as is known in the art.
[0286] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0287] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, preferably about 1,000 to 100,000
liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0288] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD.sub.550 of
about 180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0289] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
[0290] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0291] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
Antibody Purification
[0292] In one embodiment, the antibody protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed. The
following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
[0293] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the full length antibody
products of the invention. Protein A is a 41 kD cell wall protein
from Staphylococcus aureas which binds with a high affinity to the
Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
[0294] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
Finally the antibody of interest is recovered from the solid phase
by elution.
Generating Antibodies Using Eukaryotic Host Cells:
[0295] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0296] (i) Signal Sequence Component
[0297] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected preferably is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0298] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0299] (ii) Origin of Replication
[0300] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used only because it contains the early promoter.
[0301] (iii) Selection Gene Component
[0302] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0303] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0304] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0305] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity (e.g., ATCC CRL-9096).
[0306] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0307] (iv) Promoter Component
[0308] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody polypeptide nucleic acid. Promoter sequences are known
for eukaryotes. Virtually alleukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors.
[0309] Antibody polypeptide transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0310] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0311] (v) Enhancer Element Component
[0312] Transcription of DNA encoding the antibody polypeptide of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0313] (vi) Transcription Termination Component
[0314] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0315] (vii) Selection and Transformation of Host Cells
[0316] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0317] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0318] (viii) Culturing the Host Cells
[0319] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma-Aldrich, St. Louis, Mo., USA), Minimal
Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's
Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing
the host cells. In addition, any of the media described in Ham et
al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255
(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655;
or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may
be used as culture media for the host cells. Any of these media may
be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor),
salts (such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0320] (ix) Purification of Antibody
[0321] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0322] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM.resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0323] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
Activity Assays
[0324] The antibodies of the present invention can be characterized
for their physical/chemical properties and biological functions by
various assays known in the art.
[0325] The purified immunoglobulins can be further characterized by
a series of assays including, but not limited to, N-terminal
sequencing, amino acid analysis, non-denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange chromatography and papain digestion.
[0326] In certain embodiments of the invention, the immunoglobulins
produced herein are analyzed for their biological activity. In some
embodiments, the immunoglobulins of the present invention are
tested for their antigen binding activity. The antigen binding
assays that are known in the art and can be used herein include
without limitation any direct or competitive binding assays using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immnosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays. An illustrative antigen binding assay is provided
below in the Examples section.
[0327] In one embodiment, the present invention contemplates an
altered antibody that possesses some but not all effector
functions, which make it a desired candidate for many applications
in which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In certain embodiments, the Fc
activities of the produced immunoglobulin are measured to ensure
that only the desired properties are maintained. In vitro and/or in
vivo cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An
example of an in vitro assay to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
[0328] Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656
(1998). C1q binding assays may also be carried out to confirm that
the antibody is unable to bind C1q and hence lacks CDC activity. To
assess complement activation, a CDC assay, for example as described
in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may
be performed. FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the
art, for example those described in the Examples section.
Humanized Antibodies
[0329] The present invention encompasses humanized antibodies.
Various methods for humanizing non-human antibodies are known in
the art. For example, a humanized antibody can have one or more
amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al. (1986)
Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;
Verhoeyen et al. (1988) Science 239:1534-1536), by substituting
hypervariable region sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially
less than intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0330] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al.
(1987) J. Mol. Biol. 196:901. Another method uses a particular
framework derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized
antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA,
89:4285; Presta et al. (1993) J. Immunol., 151:2623.
[0331] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to one
method, humanized antibodies are prepared by a process of analysis
of the parental sequences and various conceptual humanized products
using three-dimensional models of the parental and humanized
sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer
programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and
combined from the recipient and import sequences so that the
desired antibody characteristic, such as increased affinity for the
target antigen(s), is achieved. In general, the hypervariable
region residues are directly and most substantially involved in
influencing antigen binding.
Antibody Variants
[0332] In one aspect, the invention provides antibody fragment
comprising modifications in the interface of Fc polypeptides
comprising the Fc region, wherein the modifications facilitate
and/or promote heterodimerization. These modifications comprise
introduction of a protuberance into a first Fc polypeptide and a
cavity into a second Fc polypeptide, wherein the protuberance is
positionable in the cavity so as to promote complexing of the first
and second Fc polypeptides. Methods of generating antibodies with
these modifications are known in the art, for example, as described
in U.S. Pat. No. 5,731,168.
[0333] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody nucleic acid, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of, residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0334] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0335] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g. for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0336] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative substitutions are shown in Table 2 under the heading
of "preferred substitutions". If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table 2, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00006 TABLE 2 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0337] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe
(F), Trp (W), Met (M) (2) uncharged polar: Gly (G), Ser (S), Thr
(T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) acidic: Asp (D), Glu
(E) (4) basic: Lys (K), Arg (R), His (H)
[0338] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0339] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0340] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0341] (3) acidic: Asp, Glu;
[0342] (4) basic: His, Lys, Arg;
[0343] (5) residues that influence chain orientation: Gly, Pro;
[0344] (6) aromatic: Trp, Tyr, Phe.
[0345] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0346] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the parent antibody from which they are
generated. A convenient way for generating such substitutional
variants involves affinity maturation using phage display. Briefly,
several hypervariable region sites (e.g. 6-7 sites) are mutated to
generate all possible amino acid substitutions at each site. The
antibodies thus generated are displayed from filamentous phage
particles as fusions to the gene III product of M13 packaged within
each particle. The phage-displayed variants are then screened for
their biological activity (e.g. binding affinity) as herein
disclosed. In order to identify candidate hypervariable region
sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0347] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0348] It may be desirable to introduce one or more amino acid
modifications in an Fc region of the immunoglobulin polypeptides of
the invention, thereby generating a Fc region variant. The Fc
region variant may comprise a human Fc region sequence (e.g., a
human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions including that of a hinge cysteine.
[0349] In accordance with this description and the teachings of the
art, it is contemplated that in some embodiments, an antibody used
in methods of the invention may comprise one or more alterations as
compared to the wild type counterpart antibody, for example in the
Fc region. These antibodies would nonetheless retain substantially
the same characteristics required for therapeutic utility as
compared to their wild type counterpart. For example, it is thought
that certain alterations can be made in the Fc region that would
result in altered (i.e., either improved or diminished) C1q binding
and/or Complement Dependent Cytotoxicity (CDC), for example, as
described in WO99/51642. See also Duncan & Winter Nature
322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No.
5,624,821; and WO94/29351 concerning other examples of Fc region
variants.
Immunoconjugates
[0350] The invention also pertains to immunoconjugates, or
antibody-drug conjugates (ADC), comprising an antibody conjugated
to a cytotoxic agent such as a chemotherapeutic agent, a drug, a
growth inhibitory agent, a toxin (e.g., an enzymatically active
toxin of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0351] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278)
theoretically allows targeted delivery of the drug moiety to
tumors, and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet
pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer
Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.
Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may effect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies or protein
receptor ligands.
[0352] ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al
(2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate
composed of a hu CD33 antibody linked to calicheamicin, was
approved in 2000 for the treatment of acute myeloid leukemia by
injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
4,970,198; 5,079,233; 5585089; 5606040; 5693762; 5739116; 5767285;
5773001). Cantuzumab mertansine (Immunogen, Inc.), an antibody drug
conjugate composed of the huC242 antibody linked via the disulfide
linker SPP to the maytansinoid drug moiety, DM1, is advancing into
Phase II trials for the treatment of cancers that express CanAg,
such as colon, pancreatic, gastric, and others. MLN-2704
(Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody
drug conjugate composed of an anti-prostate specific membrane
antigen (PSMA) monoclonal antibody linked to the maytansinoid drug
moiety, DM1, is under development for the protential treatment of
prostate tumors. The auristatin peptides, auristatin E (AE),
monomethylauristatin E (MMAE), and synthetic analogs of dolastatin,
were conjugated to chimeric monoclonal antibodies cBR96 (specific
to Lewis Y on carcinomas) and cAC 10 (specific to CD30 on
hematological malignancies) (Doronina et al. (2003) Nature
Biotechnology 21(7):778-784; and Francisco, et al. (2003) Blood,
102, 1458-1465) and are under therapeutic development. Other
compounds for use as drug conjugate cytotoxic agents include
without limitation auristatin E (AE), MMAF (a variant of auristatin
E (MMAE) with a phenylalanine at the C-terminus of the drug), and
AEVB (auristatin E valeryl benzylhydrazone, an acid labile linker
through the C-terminus of AE). Useful conjugate linkers for
attaching a drug to an antibody include without limitation MC
(maleimidocaproyl), Val Cit (valine-citrulline, dipeptide site in
protease cleavable linker), Citrulline (2-amino-5-ureido pentanoic
acid), PAB (p-aminobenzylcarbamoyl, a "self immolative" portion of
linker), Me (N-methyl-valine citrulline where the linker peptide
bond has been modified to prevent its cleavage by cathepsin B),
MC(PEG)6-OH (maleimidocaproyl-polyethylene glycol, attached to
antibody cysteines), SPP (N-Succinimidyl
4-(2-pyridylthio)pentanoate), and SMCC (N-Succinimidyl
4-(N-maleimidomethyl)cyclohexane-1 carboxylate). These and other
useful drug conjugates and their preparation are disclosed, for
example, in Doronina, S. O. et al., Nature Biotechnology 21:778-794
(2003), incorporated herein by reference in its entirety.
Particularly preferred linker molecules include, for example,
N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see, e.g.,
Carlsson et al., Biochem. J., 173, 723-737 (1978)), N-succinimidyl
4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.
4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP)
(see, e.g., CAS Registry number 341498-08-6), N-succinimidyl
4-(N-maleimidomethyl)cyclohe-xane-1-carboxylate (SMCC) (see, e.g.,
Yoshitake et al., Eur. J. Biochem., 101, 395-399 (1979)), and
N-succinimidyl 4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate
(SMNP) (see, e.g., U.S. Pat. No. 4,563,304).
[0353] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0354] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothecene,
and CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and Maytansinoids
[0355] In one embodiment, an antibody (full length or fragments) of
the invention is conjugated to one or more maytansinoid
molecules.
[0356] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
Maytansinoid-Antibody Conjugates
[0357] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Antibody-Maytansinoid Conjugates (Immunoconjugates)
[0358] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0359] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0360] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0361] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
Calicheamicin
[0362] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sup.I.sub.1 (Hinman et al., Cancer Research 53:3336-3342
(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug that the antibody can be conjugated is QFA which is
an antifolate. Both calicheamicin and QFA have intracellular sites
of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
Other Cytotoxic Agents
[0363] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0364] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0365] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0366] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0367] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0368] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0369] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
Preparation of Antibody Drug Conjugates
[0370] In the antibody drug conjugates (ADC) of the invention, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.
about 1 to about 20 drug moieties per antibody, through a linker
(L). The ADC of Formula I may be prepared by several routes,
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
nucleophilic group of an antibody with a bivalent linker reagent,
to form Ab-L, via a covalent bond, followed by reaction with a drug
moiety D; and (2) reaction of a nucleophilic group of a drug moiety
with a bivalent linker reagent, to form D-L, via a covalent bond,
followed by reaction with the nucleophilic group of an
antibody.
Ab-(L-D).sub.p I
[0371] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol.
[0372] Antibody drug conjugates of the invention may also be
produced by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic substituents on the
linker reagent or drug. The sugars of glycosylated antibodies may
be oxidized, e.g. with periodate oxidizing reagents, to form
aldehyde or ketone groups which may react with the amine group of
linker reagents or drug moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g. by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either glactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0373] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0374] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0375] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
Antibody Derivatives
[0376] The antibodies of the present invention can be further
modified to contain additional nonproteinaceous moieties that are
known in the art and readily available. Preferably, the moieties
suitable for derivatization of the antibody are water soluble
polymers. Non-limiting examples of water soluble polymers include,
but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymers are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
Pharmaceutical Formulations
[0377] Therapeutic formulations comprising an antibody of the
invention are prepared for storage by mixing the antibody having
the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of aqueous solutions, lyophilized or other dried formulations.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0378] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0379] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0380] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0381] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the
immunoglobulin of the invention, which matrices are in the form of
shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated immunoglobulins remain
in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
Uses
[0382] An antibody of the present invention may be used in, for
example, in vitro, ex vivo and in vivo therapeutic methods.
Antibodies of the invention can be used as an antagonist to
partially or fully block the specific antigen activity in vitro, ex
vivo and/or in vivo. Moreover, at least some of the antibodies of
the invention can neutralize antigen activity from other species.
Accordingly, the antibodies of the invention can be used to inhibit
a specific antigen activity, e.g., in a cell culture containing the
antigen, in human subjects or in other mammalian subjects having
the antigen with which an antibody of the invention cross-reacts
(e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or
mouse). In one embodiment, the antibody of the invention can be
used for inhibiting antigen activities by contacting the antibody
with the antigen such that antigen activity is inhibited.
Preferably, the antigen is a human protein molecule.
[0383] In one embodiment, an antibody of the invention can be used
in a method for inhibiting an antigen in a subject suffering from a
disorder in which the antigen activity is detrimental, comprising
administering to the subject an antibody of the invention such that
the antigen activity in the subject is inhibited. Preferably, the
antigen is a human protein molecule and the subject is a human
subject. Alternatively, the subject can be a mammal expressing the
antigen with which an antibody of the invention binds. Still
further the subject can be a mammal into which the antigen has been
introduced (e.g., by administration of the antigen or by expression
of an antigen transgene). An antibody of the invention can be
administered to a human subject for therapeutic purposes. Moreover,
an antibody of the invention can be administered to a non-human
mammal expressing an antigen with which the immunoglobulin
cross-reacts (e.g., a primate, pig or mouse) for veterinary
purposes or as an animal model of human disease. Regarding the
latter, such animal models may be useful for evaluating the
therapeutic efficacy of antibodies of the invention (e.g., testing
of dosages and time courses of administration). Blocking antibodies
of the invention that are therapeutically useful include, for
example but are not limited to, anti-HER2, anti-VEGF, anti-IgE,
anti-CD11, anti-interferon and anti-tissue factor antibodies. The
antibodies of the invention can be used to treat, inhibit, delay
progression of, prevent/delay recurrence of, ameliorate, or prevent
diseases, disorders or conditions associated with abnormal
expression and/or activity of one or more antigen molecules,
including but not limited to malignant and benign tumors;
non-leukemias and lymphoid malignancies; neuronal, glial,
astrocytal, hypothalamic and other glandular, macrophagal,
epithelial, stromal and blastocoelic disorders; and inflammatory,
angiogenic and immunologic disorders.
[0384] In one aspect, a blocking antibody of the invention is
specific to a ligand antigen, and inhibits the antigen activity by
blocking or interfering with the ligand-receptor interaction
involving the ligand antigen, thereby inhibiting the corresponding
signal pathway and other molecular or cellular events. The
invention also features receptor-specific antibodies which do not
necessarily prevent ligand binding but interfere with receptor
activation, thereby inhibiting any responses that would normally be
initiated by the ligand binding. The invention also encompasses
antibodies that either preferably or exclusively bind to
ligand-receptor complexes. An antibody of the invention can also
act as an agonist of a particular antigen receptor, thereby
potentiating, enhancing or activating either all or partial
activities of the ligand-mediated receptor activation.
[0385] In certain embodiments, an immunoconjugate comprising an
antibody conjugated with a cytotoxic agent is administered to the
patient. In some embodiments, the immunoconjugate and/or antigen to
which it is bound is/are internalized by the cell, resulting in
increased therapeutic efficacy of the immunoconjugate in killing
the target cell to which it binds. In one embodiment, the cytotoxic
agent targets or interferes with nucleic acid in the target cell.
Examples of such cytotoxic agents include any of the
chemotherapeutic agents noted herein (such as a maytansinoid or a
calicheamicin), a radioactive isotope, or a ribonuclease or a DNA
endonuclease.
[0386] Antibodies of the invention can be used either alone or in
combination with other compositions in a therapy. For instance, an
antibody of the invention may be co-administered with another
antibody, chemotherapeutic agent(s) (including cocktails of
chemotherapeutic agents), other cytotoxic agent(s), anti-angiogenic
agent(s), cytokines, and/or growth inhibitory agent(s). Where an
antibody of the invention inhibits tumor growth, it may be
particularly desirable to combine it with one or more other
therapeutic agent(s) which also inhibits tumor growth. For
instance, an antibody of the invention may be combined with an
anti-VEGF antibody (e.g., AVASTIN) and/or anti-ErbB antibodies
(e.g. HERCEPTIN.RTM. anti-HER2 antibody) in a treatment scheme,
e.g. in treating any of the diseases described herein, including
colorectal cancer, metastatic breast cancer and kidney cancer.
Alternatively, or additionally, the patient may receive combined
radiation therapy (e.g. external beam irradiation or therapy with a
radioactive labeled agent, such as an antibody). Such combined
therapies noted above include combined administration (where the
two or more agents are included in the same or separate
formulations), and separate administration, in which case,
administration of the antibody of the invention can occur prior to,
and/or following, administration of the adjunct therapy or
therapies.
[0387] The antibody of the invention (and adjunct therapeutic
agent) is/are administered by any suitable means, including
parenteral, subcutaneous, intraperitoneal, intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In addition, the antibody is suitably administered
by pulse infusion, particularly with declining doses of the
antibody. Dosing can be by any suitable route, for example by
injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic.
[0388] The antibody composition of the invention will be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The antibody need not be, but is optionally formulated with one or
more agents currently used to prevent or treat the disorder in
question. The effective amount of such other agents depends on the
amount of antibodies of the invention present in the formulation,
the type of disorder or treatment, and other factors discussed
above. These are generally used in the same dosages and with
administration routes as used hereinbefore or about from 1 to 99%
of the heretofore employed dosages.
[0389] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with other agents such as chemotherapeutic agents) will
depend on the type of disease to be treated, the type of antibody,
the severity and course of the disease, whether the antibody is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody is an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. One exemplary dosage of the antibody
would be in the range from about 0.05 mg/kg to about 10 mg/kg.
Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or
10 mg/kg (or any combination thereof) may be administered to the
patient. Such doses may be administered intermittently, e.g. every
week or every three weeks (e.g. such that the patient receives from
about two to about twenty, e.g. about six doses of the antibody).
An initial higher loading dose, followed by one or more lower doses
may be administered. An exemplary dosing regimen comprises
administering an initial loading dose of about 4 mg/kg, followed by
a weekly maintenance dose of about 2 mg/kg of the antibody.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and
assays.
Articles of Manufacture
[0390] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is by
itself or when combined with another composition effective for
treating, preventing and/or diagnosing the condition and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is an antibody of the invention. The label or package
insert indicates that the composition is used for treating the
condition of choice, such as cancer. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic agent. The article of manufacture in this embodiment of
the invention may further comprise a package insert indicating that
the first and second antibody compositions can be used to treat a
particular condition, for example cancer. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0391] The following are examples of the methods and compositions
of the invention. It is understood that various other embodiments
may be practiced, given the general description provided above.
EXAMPLES
[0392] The examples herein describe the generation of humanized
anti-beta7 antibodies from a rat anti-mouse antibody that binds to
the beta7 subunit of the alpha4beta7 integrin.
Example 1
Humanization of a Beta7 Antagonist Antibody
[0393] Materials and Methods
[0394] Residue numbers are according to Kabat (Kabat et al.,
Sequences of proteins of immunological interest, 5th Ed., Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Single letter amino acid abbreviations are used. DNA
degeneracies are represented using the IUB code (N=A/C/G/T,
D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T, K=G/T, M=A/C, R=A/G, S=G/C,
W=A/T, Y=C/T).
[0395] Direct Hypervariable Region Grafts onto the Acceptor Human
Consensus Framework
[0396] The phagemid used for this work, pV0350-2b, was a monovalent
Fab-g3 display vector having 2 open reading frames under control of
the phoA promoter, essentially as described in Lee et al., J. Mol.
Biol. (2004), 340(5):1073-93. The first open reading frame consists
of the stII signal sequence fused to the VL and CH1 domains of the
acceptor light chain and the second consists of the stII signal
sequence fused to the VH and CH1 domains of the acceptor heavy
chain followed by a truncated form of the minor phage coat protein
P3 (Lowman, H. et al. (1990) Biochemistry 30:10832).
[0397] The VL and VH domains from rat Fib504 (antibody FIB504.64
produced by hybridoma ATCC HB-293, American Type Culture Collection
(ATCC), P.O. Box 1549, Manassas, Va. 20108, USA) were aligned with
the human consensus kappa I (huKI) and human subgroup III consensus
VH (huIII) domains. To make the hypervariable region (HVR) grafts,
the following frameworks were used: HuKI was used for the light
chain variable domain framework (see FIGS. 1A and 7). For the heavy
chain variable domain framework, the acceptor VH framework, a
modified human subgroup III (humIII) consensus VH domain which
differs from the humIII sequence at 3 positions: R71A, N73T, and
L78A may be used (see Carter et al., Proc. Natl. Acad. Sci. USA
89:4285 (1992)) (see FIG. 1B). In generation of antibodies of the
present invention, the 504K-RF graft was also prepared from the
modified human subgroupIII consensus VH domain by making the
following amino acid substitutions: A71R and A78F.
[0398] Hypervariable regions from rat Fib504 (produced by hybridoma
ATCC HB-293) antibody were engineered into the acceptor human
subgroup III consensus VH framework to generate a direct HVR-graft
(Fib504graft) (see FIG. 1B). In the VL domain the following regions
from rat Fib504 were grafted to the human consensus acceptor, huKI:
positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) (FIG. 1A). In the
VH domain, positions 26-35 (H1), 49-65 (H2) and 94-102 (H3) were
grafted (FIG. 1B). In addition a second HVR graft, Fib504Kgraft,
was constructed that also included within the HVR, VL position 49,
based on an extended definition for L2 (see MacCallum et al. J.
Mol. Biol. 262: 732-745 (1996)). MacCallum et al. have analyzed
antibody and antigen complex crystal structures and found position
49 of the light chain and positions 49 and 94 of the heavy chain
are part of the antigen contact region thus, these positions were
included in the definitions of HVR-L2, HVR-H2 and HVR-H3 for
humanized anti-beta7 antibodies disclosed herein.
[0399] The direct-graft variants were generated by Kunkel
mutagenesis (Kunkel et al. (1987) supra) using a separate
oligonucleotide for each hypervariable region. Correct clones were
assessed by DNA sequencing.
[0400] Soft Randomization of the Hypervariable Regions:--
[0401] The process of "soft randomization" (see U.S. Application
Ser. No. 60/545,840) refers a procedure for biased mutagensis of a
selected protein sequence, such as a hypervariable region of an
antibody. The method maintains a bias towards the murine, rat, or
other starting hypervariable region sequence, while introducing an
approximately 10-50 percent mutation at each selected position.
This technique increases the capacity of the library screening
employed and avoids a change in the antigen epitope recognized by
the antibody. According to this soft randomization technique,
sequence diversity is introduced into each hypervariable region
using a strategy that maintains a bias towards the murine
hypervariable region sequence. This was accomplished using a
poisoned oligonucleotide synthesis strategy first described by
Gallop et al., J. Med. Chem. 37:1233-1251 (1994). However, other
methods for maintaining a bias towards the non-human hypervariable
region residue are available, such as error prone PCR, DNA
shuffling, etc.
[0402] According to the method used herein, for a given position
within a hypervariable region to be mutated, the codon encoding the
wild-type amino acid is poisoned with a mixture (e.g. a 70-10-10-10
mixture) of nucleotides resulting in an approximately 50 percent
mutation rate at each selected hypervariable region position. To
achieve this, the codon encoding the wild-type hypervariable region
amino acid to be mutated is synthesized with a low level of
contaminating mixture of the other three nucleotides, such as a
70-10-10-10 mixture of nucleotides. Thus, by way of example, for
soft randomization of PRO (CCG), the first position synthesized is
a mixture of 70% C, and 10% each of G, T and A; the second position
is a mixture of 70% C, and 10% each of A, G, and T; and the third
position is a mixture of 70% G, and 10% each of A, C and T. It is
understood that the bias can be adjusted up or down depending upon
the codon being synthesized at a given position, the number of
codons that code for a particular amino acid, and the degree that
oligonucleotide synthesis is poisoned by the nucleotide composition
of the synthesis mixture.
[0403] Soft randomized oligonucleotides can be patterned after the
murine, rat or other starting hypervariable region sequences and
encompass the same regions defined by the direct hypervariable
region grafts. Optionally, two positions, amino acids at the
beginning of H2 and H3 in the VH domain, may be limited in their
diversity: the codon RGC may be used for position 49 encoding A, G,
S or T and at position 94, the codon AKG may be used encoding M or
R.
[0404] Generation of Phage Libraries
[0405] Randomized oligonucleotide pools designed for each
hypervariable region were phoshorylated separately in six 20 .mu.l
reactions containing 660 ng of oligonucleotide, 50 mM Tris pH 7.5,
10 mM MgCl.sub.2, 1 mM ATP, 20 mM DTT, and 5 U polynucleotide
kinase for 1 h at 37.degree. C. The six phosphorylated
oligonucleotide pools were then combined with 20 .mu.g of Kunkel
template in 50 mM Tris pH 7.5, 10 mM MgCl.sub.2 in a final volume
of 500 .mu.l resulting in an oligonucleotide to template ratio of
3. The mixture was annealed at 90.degree. C. for 4 min, 50.degree.
C. for 5 min and then cooled on ice. Excess, unannealed
oligonucleotide was removed with a QIAQUICK.TM. PCR purification
kit (Qiagen kit 28106, Qiagen, Valencia, Calif.) using a modified
protocol to prevent excessive denaturation of the annealed DNA. To
the 500 .mu.l of annealed mixture, 150 .mu.l of Qiagen buffer PB
was added, and the mixture was split between 2 silica columns.
Following a wash of each column with 750 .mu.l of Qiagen buffer PE
and an extra spin to dry the columns, each column was eluted with
110 .mu.l of 10 mM Tris, 1 mM EDTA, pH 8. The annealed and
cleaned-up template (220 .mu.l) was then filled in by adding 1
.mu.l 100 mM ATP, 10 .mu.l 25 mM dNTPs (25 mM each of dATP, dCTP,
dGTP and dTTP), 15 .mu.l 100 mM DTT, 25 .mu.l 10.times.TM buffer
(0.5 M Tris pH 7.5, 0.1 M MgCl.sub.2), 2400 U T4 ligase, and 30 U
T7 polymerase for 3 h at room temperature.
[0406] The filled-in product was analyzed on
Tris-Acetate-EDTA/agarose gels (Sidhu et al., Methods in Enzymology
328:333-363 (2000)). Three bands are usually visible: the bottom
band is correctly filled and ligated product, the middle band is
filled but unligated and the top band is strand displaced. The top
band is produced by an intrinsic side activity of T7 polymerase and
is difficult to avoid (Lechner et al., J. Biol. Chem.
258:11174-11184 (1983)); however, this band transforms 30-fold less
efficiently than the bottom band and usually contributes little to
the library. The middle band is due to the absence of a 5'
phosphate for the final ligation reaction; this band transforms
efficiently and gives mainly wild type sequence.
[0407] The filled in product was then cleaned-up and electroporated
into SS320 cells and propagated in the presence of M13/KO7 helper
phage as described by Sidhu et al., Methods in Enzymology
328:333-363 (2000). Library sizes ranged from 1-2.times.10.sup.9
independent clones. Random clones from the initial libraries were
sequenced to assess library quality.
[0408] Phage Selection
[0409] Full length human integrin alpha4beta7 was expressed in 293
cells (Graham et al., J. Gen Virol. 36:59 (1977)), purified by
Fib504 affinity chromatography and used as the target for phage
selection. For immobilization on MaxiSorp.TM. microtiter plates
(Nalge Nunc, Rochester, N.Y.), 100 .mu.l of human integrin
alpha4beta7 was coated at 5 .mu.g/ml in 150 mM NaCl, 50 mM Tris pH
7.5, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 2 mM MnCl.sub.2 (TBSM),
overnight at 4 degrees C. Wells were blocked for 1 h using TBSM
containing 1% BSA. For the first round of selection, 8 wells coated
with target were used; a single target coated well was used for
successive rounds of selection. Phage were harvested from the
culture supernatant and suspended in TBSM containing 1% BSA and
0.05% TWEEN.TM. 20 (TBSMBT). After binding to the wells for 2 h,
unbound phage were removed by extensive washing with TBS containing
0.05% TWEEN 20 (TBST). Bound phage were eluted by incubating the
wells with 100 mM HCl for 30 min. Phage were amplified using Top10
cells and M13/KO7 helper phage and grown overnight at 37.degree. C.
in 2YT, 50 .mu.g/ml carbanacillin. The titers of phage eluted from
a target coated well were compared to titers of phage recovered
from a non-target coated well to assess enrichment. After four
rounds of selection were performed, random clones were selected for
sequence analysis.
[0410] Fab Production and Affinity Determination
[0411] To express Fab protein for affinity measurements, a stop
codon was introduced between the heavy chain and g3 in the phage
display vector. Clones were transformed into E. coli 34B8 cells and
grown in AP5 media at 30 C (Presta, L. et al., Cancer Res.
57:4593-4599 (1997)). Cells were harvested by centrifugation,
suspended in 10 mM Tris, 1 mM EDTA pH 8 and broken open using a
microfluidizer. Fab was purified with Protein G affinity
chromatography.
[0412] Affinity determinations were performed by surface plasmon
resonance using a BIAcore.TM.-3000 (Biacore, Piscataway, N.J.).
Humanized Fib504 Fab variants were immobilized in 10 mM acetate pH
4.5 (ranging from 250 to 1500 response units (RU)) on a CM5 sensor
chip and 2-fold dilutions of human integrin alpha4beta7 (1.5 to 770
nM) in TBSM containing 2% n-octylglucoside were injected. Each
sample was analyzed with 5-minute association and 5 to 60-minute
dissociation times. After each injection, the chip was regenerated
using three 1-minute injections of 8 M urea. Binding response was
corrected by subtracting the RU from a blank flow cell. A 1:1
Languir model of simultaneous fitting of k.sub.on and k.sub.off was
used for kinetics analysis.
Results and Discussion
[0413] Humanization of rat Fib504
[0414] The human acceptor framework used for humanization is based
on the framework used for HERCEPTIN.RTM. and consists of the
consensus human kappa I (huKI) VL domain and a variant of the human
subgroup III (humIII) consensus VH domain. This variant VH domain
has 3 changes from the human consensus: R71A, N73T and L78A. The VL
and VH domains of rat Fib504 were each aligned with the human kappa
I and subgroup III domains; each hypervariable region (HVR) was
identified and grafted into the human acceptor framework to
generate a HVR graft (504 graft) that could be displayed as an Fab
on phage (FIGS. 1A and 1B).
[0415] Based on the analysis of available antibody and antigen
complex crystal structures MacCallum et al. (MacCallum et al. J.
Mol. Biol. 262: 732-745 (1996)) proposed HVR definitions based on
variable domain residues that frequently contact antigens. Thus
positions 49G and 94M of the heavy chain were included in the HVR
graft of Fib504 (FIG. 1B). In addition, a second HVR graft,
Fib504Kgraft, was also generated which included position 49K of the
light chain, because this position is also within the contact
definition of HVR-L2 and can serve as an antigen contact (FIG. 1A).
When either the Fib504 or Fib504K grafts were displayed on phage
and tested for binding to immobilized alpha4beta7, no binding was
observed.
[0416] Libraries were generated using both the Fib504 and Fib504-K
HVR grafts in which each of the HVR regions were soft randomized
simultaneously. Each HVR graft library was panned against
immobilized alpha4beta7 for 4 rounds of selection. No enrichment
was observed and clones picked for DNA sequence analysis displayed
only random sequence changes targeted to the 6 HVR regions.
[0417] Two additional VH framework sequences, "RL" and "RF" were
investigated as acceptor frameworks and contained changes at
positions 71 and 78. Position 71 was changed to an Arginine as in
the human subgroup III consensus, and position 78 was changed to a
Leucine as in the human subgroup III consensus (acceptor framework
"RL") or a Phenylalanine as in the human subgroup II consensus and
the rat Fib504 VH framework (acceptor framework "RF") (FIG. 10A).
When either the Fib504 or Fib504K graft in the "RL" (Fib504-RL and
Fib504K-RL) or "RF" (Fib504-RF and Fib504K-RF) acceptor framework
was displayed on phage and tested for binding to immobilized
alpha4beta7. Specific phage binding was only observed for the
Fib504K graft using the "RF" framework (FIG. 10B). The modest
binding of phage displaying the Fib504-RF graft relative to the
other grafts lacking Y49K (light chain) and L78F (heavy chain)
indicates the importance of these positions in selecting a useful
acceptor framework.
[0418] Libraries were generated as before using a soft
randomization strategy simultaneously at each of the 6 HVRs for the
Fib504K-RL and Fib504K-RF grafts and sorted on immobilized
alpha4beta7 for 4 rounds of selection as described above.
Enrichment was only observed for the library based on the
Fib504K-RF graft. Clones from round 4 of the Fib504K-RF library
were selected for sequence analysis and revealed amino acid changes
targeted to HVR-L1. Most clones contained the change Y32L; in
addition position 31 was frequently changed to D, S, P or N (FIG.
1C). In addition to the starting graft, Fib504K-RF, 3 clones were
expressed and purified as Fab protein and further analyzed by
Biacore as described above. Clones hu504-5, hu504-16 and hu504-32
(variants of SEQ ID NO:1 containing substitutions T31S plus Y32L
(variant hu504.5), Y32L (variant hu504.16), or T31D plus Y32L
(variant hu504.32); see FIG. 1C), showed excellent binding to
alpha4beta7 relative to the Fib504K-RF graft and met or exceeded
the affinity of the chimeric Fib504 Fab for binding to alpha4beta7.
The results of the Biacore analysis are shown in Table 3, below,
and indicate that selected variation in the HVRs and/or framework
regions, disclosed herein, generated antagonist antibodies to
alpha4beta7 having improved affinity relative to the starting
antibody. The results in Table 3 indicate that humanized variant
504.32 showed the greatest increase in affinity relative to the
starting rat antibody by binding 3-fold more tightly to
alpha4beta7.
TABLE-US-00007 TABLE 3 Fab Affinity to (BIAcore .TM. analysis)
Alpha4beta7 (nM) Fib504 11 Variant 504.5 9 Variant 504.16 23
Variant 504.32 3
[0419] The results in Table 3 also indicate that the redesign of
HVR-L1 was important to the restoration of high affinity antigen
binding. In particular, the mutation Y32L was most frequent among
the various clones. Other changes at position 31 and numerous other
substitutions throughout HVR-H1 appear to be well tolerated and may
provide additional improvement. From these results it is clear that
there are multiple sequence changes that can improve the affinity
of Fib504 grafted onto a human framework to generate affinities
that meet or exceed that of the initial rat antibody.
[0420] Thus, starting from the graft of the 6 rat Fib504 HVRs into
the human acceptor scaffold, the expansion of HVR-L2 to include
position 49 (Lysine), expansion of HVR-H2 to include position 49
(Glycine), and the expansion of HVR-94 to include position 94
(Methionine) as well as amino acid changes at position 32 of VHR-L1
(where L or I replace Y) and, optionally, at position 31 of the
VHR-L1 (where T is replaced by D or S, for example). Useful
framework amino acid changes were made at positions 71 (A71R) and
78 (L78F) in the VH domain. Such amino acid changes lead to a fully
human antibody, variant hu504.32, for example, with 3-fold improved
binding affinity for alpha4beta7 integrin. Furthermore, selected
humanized antibodies described herein have been determined to have
at least comparable biological activity as the parent rat Fib504
antibody (see Example 3 herein).
Example 2
Additional Humanized Fib504 HVR Variants
[0421] The HVR amino acid sequences of humanized variant Fib504.32
were further modified to generated additional variants capable of
antagonizing the activity of beta7 integrin subunit and/or
integrins containing the beta7 subunit.
[0422] Generating a Broad Amino Acid Scan Library
[0423] A library to scan selected HVR positions for other amino
acid residues capable of generating beta7-binding variants of
variant hu504.32 was generated using 3 oligonucleotides: 504-L1,
designed to soft randomize a portion of HVR-L1 with a bias towards
the hu504.32 HVR-L1 sequence (i.e. the sequence ASESVDDLLH (SEQ ID
NO:47, for relative positions A2-A11) was soft randomized as
described above); and HVR-L3 504-N96 and HVR-H3 504-M94 which
introduce NNS at positions HVR-L3 position 96 in the light chain
and HVR-H3 position 94 in the heavy chain, thus allowing all 20
amino acids at these positions. With these three oligonucleotides,
the broad amino acid scan library was generated as described above
using a template containing three stop codons in the light chain
(positions 31 and 32 in HVR-L1 and position 96 in HVR-L3) and 1
stop codon in the heavy chain (position 94 in HVR-H3).
Broad Amino Acid Scan of Hu504-32
[0424] To more fully explore the preferred sequences allowed in
HVR-L1 and to enhance the stability of 504-32, we designed a phage
library that a) soft randomized HVR-L1 of 504-32 in the region
where changes were observed (i.e. ASESVDDLLH (SEQ ID NO:47, for
relative positions A2-A11) during humanization (FIG. 1C), and b)
allowed all possible amino acids at N96 in HVR-L3 and M94 in
HVR-H3. Following 4 rounds of selection against immobilized
full-length human integrin alpha4beta7 as described above, 96
random clones were selected for sequence analysis. The frequency at
which amino acids were found at each position in the broad amino
acid scan library suggest that the HVR-L1 sequence present in
hu504-32 and the methionine at position 94 in the heavy chain are
optimum for high affinity binding (FIG. 12). The most preferred
amino acids obtained by the selections starting from variant 504.32
(FIG. 12) are shown in yellow. In contrast, although asparagine is
present at position 96 in the light chain of hu504-32, the high
frequency of leucine observed at this position in the broad amino
acid scan suggests the mutation N96L could further improve the
affinity of humanized Fib504 variants for alpha4beta7 and also
eliminate any potential deamidation problems at this position. The
information in FIG. 12 also suggests that a number of replacement
amino acids are likely to be tolerated at most positions without a
substantial loss in affinity. For example, to eliminate oxidation
of M94 in HVR-H3, glutamine or arginine could likely be
substituted.
[0425] Generating the Limited Amino Acid Scan Libraries
[0426] Six libraries for a limited amino acid scan utilized six
different Kunkel templates, each containing one stop codon located
within one of the six HVRs. Each library was generated using a
single oligonucleotide encoding a single HVR and utilizing the
codons listed in FIG. 11A ("codon" column) to alter amino acid
residues for subsequent testing for binding to beta7 or
alpha4beta7. The same procedures are used to alter amino acid
residues of anti-beta7 antibodies and test them for binding to
alphaEbeta7 integrins.
[0427] Limited Amino Acid Scan of Hu504-32
[0428] The limited amino acid scan of hu504-16 was designed to make
hu504-16 even more like the human light and heavy chain consensus
sequence and in the process identify the minimal sequence elements
of rat Fib504 that are required for binding. Six libraries were
generated and targeted at each HVR at positions that differed
between the hu504-16 and human consensus kappa I light or subgroup
III heavy chains (FIGS. 1A and 1B); either the rat or human amino
acid was allowed at these positions in the library (FIG. 11A). In
order to accommodate coding for both amino acids during the
oligonucleotide synthesis and mutagenesis, additional amino acids
were also introduced in some cases (see encoded amino acids, FIG.
11A). The limited amino acid scan libraries were selected against
immobilized full-length human integrin alpha4beta7 as described
above and approximately 32 random clones were sequenced from each
library after round 3. The frequency of each amino acid found at
each position is shown in FIGS. 11B and 11C.
[0429] Like the broad amino acid scan, the limited amino acid scan
also provides information about what changes are tolerated at many
positions in humanized Fib504. Unlike the broad amino acid scan,
however, the diversity allowed at each position randomized in the
limited amino acid scan was restricted to a couple of amino acids.
Thus the lack of any observed substitution at a given position does
not indicate that a particular residue can not be changed nor does
the high frequency of any particular amino acid at a given position
necessarily indicate that it is the best solution for high
affinity.
[0430] At some positions (positions 27, 29, 30, 53, 54 of the light
chain and 50, 54, 58, 60, 61, and 65 of the heavy chain) the human
consensus amino acid is selected quite frequently suggesting a back
mutation to the human consensus would not dramatically alter
binding to human alpha4beta7. In fact, at position 54 of the light
chain (in HVR-L2), the human consensus amino acid is selected more
frequently than the amino acid from rat Fib504 indicating that this
change made to 504-32 provides a useful beta7 binding antibody.
[0431] Further, as a result of the library design, amino acids that
are not derived from either the human consensus or rat Fib504 are
selected more frequently at some positions and provide potential
substitutions to improve the affinity of humanized Fib504 variants.
These include, without limitation, D30A and I55V in the light chain
and Y50F in the heavy chain The results from these 2 libraries
indicate that many HVR positions tolerate other amino acid
substitutions and still retain comparable biological activity.
[0432] Summaries of observed amino acid changes are shown in FIGS.
13 and 15. FIG. 15 summarizes the various amino acids useful at
each of the positions in the CDRs of the antibody variants of the
invention at positions numbered according to Kabat numbering or a
relative numbering system. Each of the additional antibodies
encompassed by the variants depicted in FIGS. 13 and 15 is an
embodiment of the invention.
Example 3
Cell Adhesion Assays
[0433] The ability of some of the humanized Fib504 variants of the
invention to bind ligands expressed on a cell surface was tested by
cell adhesion assays. Binding to alpha4beta7 and another beta7
integrin, alphaEbeta7 were tested by the ability of the humanized
variants to disrupt binding of the integrin to its natural
receptor. Binding of the humanized Fib504 variants to beta7 subunit
alone expressed on a cell surface was similarly tested. The
procedures and the results are described below.
IgG Production
[0434] Humanized Fib 504 IgG variants were expressed transiently in
293 cells (Graham et al. (1977) supra) using a separate vector for
the light and heavy chains. The vectors were constructed by
subcloning the light or heavy variable domains into suitable
expression vectors for each of the light and heavy chains.
Supernatant from 1.1 L CHO cell culture of a humanized Fib504
variant was filtered through a 0.45 um filter and applied to a new
1 mL HiTrap Protein A HP column (Amersham/Pharmacia) equilibrated
in Buffer A (10 mM tris pH 7.5, 150 mM NaCl). Samples were applied
at 0.8 mL/min, overnight, 4 degrees C. Each column was then washed
and equilibrated with 30 mL Buffer A. Elution of antibody was
accomplished by chromatography at room temperature on an FPLC
(Amersham/Pharmacia) using a linear gradient of 14 min at 1 mL/min
from 0 to 100% Buffer B (100 mM Glycine, pH 3.0). Resulting 1 mL
fractions were immediately neutralized by the addition of 75 uL 1 M
tris, pH 8. Eluted protein was detected by absorption at 280 nm,
and peak fractions were pooled and desalted into PBS on PD10 G-25
sephadex disposable sizing columns (Amersham/Pharmacia). Protein
was detected by OD280 and peak fractions were pooled. The antibody
in PBS was 0.22 um filtered and stored at 4 degrees C. Amino acid
analysis was used to quantify the concentrations of these purified
antibodies, and values were assigned from the average of two
separate determinations.
BCECF Labeling:
[0435] In each of the assays presented in this Example 3, cells
were labeled according to the following procedure. All cells used
in the adhesion assays were labeled with
2',7'-bis-(2-carboxylethyl)-5-(and-6)-carboxyfluorescein,
acetoxymethyl ester (BCECF) at 10 uM in RPMI1640 media containing
10% FBS for RPMI8866 cells and 38C13 cells transfected with beta7
subunit (38C13 beta7 cells) and in F-12:DMEM mix (50:50) containing
10% FBS for alphaEbeta7-transfected 293 cells (alphaEbeta7 293
cells). Cells were labeled for 30 minutes and washed two times with
assay media. Cell density was adjusted to 3.times.10.sup.6 cells
per ml for RPMI8866 and 38C13beta7 cells and 2.2.times.10.sup.6
cells per ml for alphaEbeta7 293 cells.
Humanized Fib504 Variants Disrupt alpha4beta7 Binding to MAdCAM
[0436] RPMI8866/MAdCAM-1-Ig Cell Adhesion:
[0437] RPMI8866 cells express alpha4beta7 on their surface (Roswell
Park Memorial Institute, Buffalo, N.Y.). Humanized Fib504 variants
(hu504 variants) were contacted with a mixture of RMPI8866 cells
and MAdCAM fused to IgG coated on a solid support. Humanized Fib504
variant concentrations resulting in 50% inhibition (IC.sub.50) of
the binding of RPMI8866 cells to MAdCAM-1 were measured by coating
Nunc Maxisorp.TM. 96-well plates with 2 .mu.g/ml in PBS, 100
.mu.l/well MAdCAM-1-Ig (Genentech, Inc., where Ig refers to fusion
of MAdCAM-1 to an Fc region) overnight at 4.degree. C. After
blocking the plates with 200 ul/well of 5 mg/ml BSA for one hour at
room temperature, 50 .mu.l of humanized Fib504 variants in assay
media (RPMI 1640 media, Hyclone.RTM., Logan Utah, USA, supplemented
with 5 mg/mL BSA) were added to each well and 150,000 BCECF-labeled
cells (BCECF, Molecular Probes, Eugene, Oreg.) in 50 .mu.l of assay
media were added to each well and incubated for 15 minutes at
37.degree. C. The wells were washed two times with 150 .mu.l of
assay media to remove unbound cells. The bound cells were
solubilized with 100 .mu.l of 0.1% SDS in 50 mM Tris/HCl pH7.5. The
amount of fluorescence released from lysed cells was measured by
SPECTRAmax GEMINI.TM. (Molecular Devices, Sunnyvale, Calif.) at 485
nm excitation 530 nm emission wavelengths. The fluorescence values
were analyzed as a function of the concentrations of the humanized
Fib504 variants added in each assay, using a four-parameter
nonlinear least squares fit, to obtain the IC.sub.50 values of each
humanized Fib504 variant in the assay. IC.sub.50 and IC.sub.90
values were estimated from the four-parameter fit. FIG. 14 is an
exemplary plot of the results. The IC.sub.90 and IC.sub.50 values
for each of the variants tested are shown below in Table 4.
TABLE-US-00008 TABLE 4 Antibody binding to human MAdCAM-1 Antibody
Tested: IC.sub.50 (nM) IC.sub.90 (nM) Fib504 and hu504 variants Exp
1/Exp 2* Exp 1/Exp 2* Rat Fib504 0.098/0.197 0.483/0.703 Variant
hu504.5 0.067/0.248 0.361/0.880 Variant hu504.16 0.0768/0.206
0.244/0.551 Variant hu504.32 0.036/0.119 0.150/0.396 6B11
(non-blocking control) >100 >100 *Exp 1/Exp 2 refers to the
results of repeated assays.
Humanized Fib504 Variant Disruption of alpha4beta7 Binding to
VCAM
[0438] RPMI8866/7dVCAM-1 Cell Adhesion:
[0439] The RPMI8866/7dVCAM-1 assay is similar format to the
RPMI8866/MAdCAM-1-Ig except that 7dVCAM-1 (ADP5, R&D Systems,
Minneapolis, Minn.) was used at 2 ug/ml to coat plates. The results
were analyzed as described above for the MAdCAM binding
experiments. The IC.sub.50 values for each of the variants tested
are shown below in Table 5.
TABLE-US-00009 TABLE 5 Antibody binding to human VCAM Antibody
Tested: IC.sub.50 (nM) IC.sub.90 (nM) Fib504 and hu504 variants Exp
1/Exp 2* Exp 1/Exp 2* Rat Fib504 0.107/0.193 0.396/0.580 Variant
hu504.5 0.088/0.270 0.396/0.726 Variant hu504.16 0.098/0.223
0.261/0.774 Variant hu504.32 0.059/0.110 0.183/0.337 6B11
(non-blocking control) >100 >100 *Exp 1/Exp 2 refers to the
results of repeated assays.
Humanized Fib504 Variant Disruption of alphaEbeta7 Binding to Human
E-Cadherin
[0440] AlphaEbeta7 293/huE-Cadherin Cell Adhesion:
[0441] 293 cells (Graham et al. (1977) supra) were transfected with
alphaE and beta7 (Genentech, Inc.). The assay format is similar to
the RPMI8866/MAdCAM-1-Ig assay except that the huE-Cadherin
(648-EC, R&D Systems, Minneapolis, Minn.) was used at 2
.mu.g/ml to coat the plates. Plates were then blocked with 5 mg/ml
BSA as mentioned above and 50 .mu.l of FIB504 variants in assay
media (F-12:DMEM (50:50) supplemented with 5 mg/ml BSA) were add to
each well and 110,000 BCECF labeled cells in 50 ul of assay media
were added to each well and incubated for 15 minutes at 37.degree.
C. The wells were washed two times with 150 .mu.l of assay media
and the amount of fluorescence released by lysed cells was measured
and analyzed as described above. Assay results from three
experiments are shown in Table 6.
TABLE-US-00010 TABLE 6 Antibody binding to human E-Cadherin
Antibody Tested: Fib504 and hu504 variants IC.sub.50 (nM) IC.sub.90
(nM) Rat Fib504 2.047/7.89/4.19 8.80/24.5/9.95 Variant hu504.5
2.132/10.18/4.77 7.99/28.7/10.19 Variant hu504.16 1.957/10.05/4.58
7.03/33.7/13.51 Variant hu504.32 1.814/6.99/3.47 8.8/24.5/11.73
HP2/1 (anti-alpha4, control) >100/>100/>100
>100/>100/>100
Humanized Fib504 Variant Disruption of Beta7 Binding to MAdCAM
[0442] 38C13beta7/muMAdCAM-1-Ig Cell Adhesion Assay:
[0443] The 38C13beta7/muMAdCAM-1-Ig assay was similar format to the
RPMI8866/MAdCAM-1-Ig except that muMAdCAM-1-Ig (Genentech, Inc.)
was used at 2 .mu.g/ml to coat plates. 38C13 alpha4+ murine
lymphoma cells (Crowe, D. T. et al., J. Biol. Chem. 269:14411-14418
(1994)) were transfected with DNA encoding integrin beta7 such that
alpha4beta7 was expressed on the cell surface. The ability of the
antibodies variants to disrupt interaction between the cell
membrane associated alpha4beta7 and MAdCAM was performed as above.
Assay results are shown in Table 7. Assay results are shown in
Table 7 (IC50 and IC90 values for 2 experiments are shown).
TABLE-US-00011 TABLE 7 Activity of hu504 variant antibodies in
38C13-beta7 expressing cells Binding to murine MAdCAM Antibody
Tested: Fib504 and hu504 variants IC.sub.50 (nM) IC.sub.90 (nM) Rat
Fib504 0.682/0.306 2.869/1.51 Variant hu504.5 0.8587/0.466
2.322/2.61 Variant hu504.16 0.998/0.610 3.717/4.08 Variant hu504.32
0.718/0.458 4.08/1.51
Humanized Fib504 Variant Disruption of Beta7 Binding to Murine
VCAM
[0444] 38C13beta7/muVCAM-1-Ig Cell Adhesion Assay:
[0445] The 38C13beta7/muVCAM-1-Ig assay was performed according to
the murine MAdCAM-1-Ig/RPMI8866 cell binding assay above, except
that muVCAM-1-Ig (Genentech, Inc.) was used at 2 .mu.g/ml to coat
plates. Results of the assay are shown in Table 8. (IC50 and IC90
values for 2 experiments are shown).
TABLE-US-00012 TABLE 8 Activity of hu504 variant antibodies in
38C13-beta7 expressing cells Binding to murine VCAM-1-Ig Antibody
Tested: Fib504 and hu504 variants IC.sub.50 (nM) IC.sub.90 (nM) Rat
Fib504 0.845/0.447 2.903/2.30 Variant hu504.5 0.763/0.407
3.074/2.30 Variant hu504.16 0.835/0.584 2.857/1.84 Variant hu504.32
0.562/0.330 2.004/1.84
[0446] The results of the humanized Fib504 variant binding studies
demonstrate that the humanized antibody of the invention binds its
target beta7 integrin subunit as well as the alpha4beta7 and
alphaEbeta7 integrin with about the affinity of the starting rat
antibody and, in some embodiments, with greater affinity. Thus, a
humanized anti-beta7 antibody according to the invention has uses
in anti-beta7 integrin therapies, particularly human therapies.
[0447] Relative Activity of Hu504.32 Variants of the Invention
[0448] Different amino acid variants of the hu504.32 antibody were
tested in human and mouse cell adhesion assay for their ability to
inhibit beta7-containing receptor binding to its ligand according
to the cell adhesion assay methods disclosed herein. The
RPMI8866/MAdCAM-1-Fc assay was performed as described herein above.
The alphaEbeta7-293/hu E-cadherein assay was modified by the use of
human E-cadherin-Fc as the ligand (human E-cadherin-Fc, 648-EC,
R&D Systems, Minneapolis, Minn.). The relative ability of
hu504.32 variants to inhibit interaction of human fibronectin
(huFN40) with human alpha4beta7 receptor on PRMI8866 cells was also
examined. The RPMI8866/hu Fibronectin (huFN40) assay used for these
studies was similar in format to the RPMI8866/MAdCAM-1-Ig assay
disclosed herein except that human fibronectin alpha-chymotryptic
fragment 40 kDa (F1903, Chemicon International, Temecula, Calif.)
was used at 2 .mu.g/ml to coat plates.
[0449] The ability of the hu504.32 variants to inhibit interaction
of murine beta7-containing receptors with murine MAdCAM-1 or murine
VCAM-1 was examined. Murine MAdCAM-1-Fc and murine VCAM-1-Fc were
inhibited from interacting with murine lymphoma alpha4+ cells
expressing murine beta7 (38C13beta7 cells) by the hu504.32
variants. The murine MAdCAM-1-Fc and VCAM-1-Fc cell adhesion assays
were performed similarly to those described herein above for human
MAdCAM and VCAM. Where ligands were fused to Fc regions, the Fc
receptors on the cells were blocked with 0.5 .mu.g anti-CD16/32
antibody (anti-Fcgamma III/II receptor antibody, catalog No.
553142, BD Biosciences, San Jose, Calif.) per1 million cells for 5
minutes at room temperature. 150,000 labeled cells in 50 .mu.l of
assay medium were added to each well and incubated for 13 minutes
at 37.degree. C. The wells were washed and the amount of
fluorescence released from lysed cells was measured as disclosed
herein above. The control antibody for the human cell adhesion
assays was the mouse monoclonal antibody to human serum albumin,
6B11 (Catalog No. ab10244, Novus Biologicals, Littleton, Colo.,
USA). The control antibody for the murine cell adhesion assays was
the rat anti-mouse integrin beta7 antibody, M293 (BD Biosciences,
San Jose, Calif.), which does not compete with ligand or with
Fib504 for binding to integrin beta7.
[0450] The results of triplicate assays for the human and murine
cell adhesion assays are provided in Tables 9 and 10,
respectively.
TABLE-US-00013 TABLE 9 Activity of hu504.32 Variant Antibodies in
Human Cell Adhesion Assays IC50 Ave .+-. SD Antibody RPMI8866/
RPMI8866/ .alpha.E.beta.7-293/ Variant huMAdCAM-1-Fc hu7dVCAM-1
huE-Cadherin-Fc RPMI8866/huFN40 hu504.32 0.088 .+-. 0.035 0.101
.+-. 0.021 3.970 .+-. 1.664 0.100 .+-. 0.046 hu504.32M94Q 0.090
.+-. 0.045 0.111 .+-. 0.035 4.130 .+-. 1.212 0.124 .+-. 0.056
hu504.32M94R 0.075 .+-. 0.034 0.089 .+-. 0.009 3.963 .+-. 1.776
0.119 .+-. 0.056 Control (6B11) >100 >100 >100 >100
TABLE-US-00014 TABLE 10 Activity of hu504.32 Variant Antibodies in
Murine Cell Adhesion Assays IC50 Ave SD .+-. 38C13beta7/
38C13beta7/ Antibody Variant muMAdCAM-1-Fc mu7dVCAM-1-Fc hu504.32
0.270 .+-. 0.041 0.228 .+-. 0.065 hu504.32M94Q 0.370 .+-. 0.102
0.264 .+-. 0.083 hu504.32M94R 0.391 .+-. 0.112 0.228 .+-. 0.081
Control (M293) >100 >100
[0451] The hu504.32 antibody has a methionine at position 94 of the
heavy chain CDR3. The variants M94Q (or hu504.32Q) and M94R (or
hu504.32R) have glutamine or arginine, respectively, at position 94
of the hu504.32 antibody variant. The hu504.32M, Q, and R
antibodies substantially reduced integrin beta7 receptor-ligand
interaction in each of the assays and are, thus, potent inhibitors
of beta7-mediated cell adhesion.
[0452] Antibody hu504.32R Activity In Vivo
[0453] The hu504.32R antibody variant was tested in vivo for its
ability to reduce integrin beta7 receptor-ligand interaction and
reduce lymphocyte recruitment to inflamed colon in an in vivo
murine inflammatory bowel disease model. BALB/c mice and CB17 SCID
mice were obtained from Charles River Laboratories International,
Inc. (Wilmington, Mass., USA). CD4.sup.+CD45Rb high T cell
reconstituted SCID colitic mice were prepared by isolating
CD4.sup.+CD45Rb high T cells from donor BALB/c mice and
transferring 3.times.10.sup.5 cells in 100 .mu.l PBS intravenously.
Control SCID mice did not receive CD4.sup.+CD45Rb high T cells.
Reconstituted CD4+ mice meeting the treatment group enrollment
criteria of 10% weight loss from baseline or 15% from peak weight
at week 4 were considered to have induced inflammatory bowel
disease and were selected for treatment.
[0454] On the day of treatment with test antibodies, donor BALB/c
mice mesenteric lymph node (MLN) cells were harvested and
radiolabelled with Cr.sup.51. Treatment involved prior
administration of anti-GP120 antibody, hu504.32 anti-beta7
antibody, hu504.32R anti-beta7 antibody, or no antibody (control)
intravenously, 200 .mu.g/100 .mu.l PBS. Thirty minutes after
antibody administration, Cr.sup.51-labelled MLN cells were
injected, 4.times.10.sup.6 cells/100 .mu.l. One hour post-injection
of labelled cells, mice were euthanized and spleen, colon, and
peyers patch were collected, weighed, and total Cr.sup.51
radioactivity per organ was determined. FIG. 16 is a bar graph of
the results of these tests showing the relative ability of the
antibodies to block homing of radiolabelled T cells to the colon of
mice experiencing inflammatory bowel disease. Homing of T cells to
inflamed colon was inhibited by hu504.32 and hu504.32R anti-beta7
antibodies relative to negative control, anti-GP120 antibody.
Distribution to spleen was similar for all of the antibodies. Thus,
the hu504.32 and hu504.32R anti-beta7 antibodies effectively
inhibit homing of T cells to inflamed colon in vivo.
[0455] Antibody glycation does not affect the ability of hu504.32R
variant to block MAdCAM-1 binding to alpha4beta7 receptor.
[0456] Glycation, the non-enzymatic glycosylation of proteins, can
affect antibody-ligand interactions (see, for example, Kennedy, D.
M. et al., Clin Exp Immunol. 98(2):245-51 (1994). Glycation of
lysine at position 49 of the 504.32R Glycation of the lysine at
light chain position 49 of the hu504.32R variant (HVR-L2 relative
position B1) was observed but had no significant affect on the
ability of the antibody variant to block the binding of MAdCAM-1 to
alpha4beta7 receptor-expressing RPMI8866 cells. Determination of
glycation and glycation levels was performed using standard
electrospray ionization-mass spectroscopy (ESI-MS) and by boronate
affinity chromatography. Boronate affinity HPLC methods useful to
test for glycation are found at, for example, Quan C. P. et al.,
Analytical Chemistry 71(20):4445-4454 (1999) and Li Y. C. et al.,
J. Chromatography A, 909:137-145 (2001). The cell adhesion assay
was performed according to the RPMI8866/MAdCAM-1-Fc cell adhesion
assay disclosed herein.
[0457] In alternative embodiments of the invention, glycation at
position 49 is reduced or eliminated where position 49 comprises an
amino acid other than lysine. The polypeptide or antibody of the
invention encompasses as an amino acid at position 49 (HVR-L2
relative position B1) any of amino acids A, C, D, E, F, G, H, I, L,
M, N, P, Q, R, S, T, V, W, or Y, where each letter refers to a
amino acid according to the standard single-letter amino acid
designation. Alternatively, the amino acid at position 49 of the
light chain of a 504.32R variant (or other 504 variant) is selected
from the group consisting of R, N, V, A, F, Q, H, P, I, or L. An
amino acid useful at position 49 is selected, for example, by
displaying (preparing a phage library of) the hu504.32R Fab on
phage (variant) and substituting separately, at the codon for
position 49, a codon for each of the 20 naturally occurring amino
acids. Phage expressing the hu504.32R variants altered at position
49 are tested for binding to integrin beta7 and/or to a receptor
comprising integrin beta7, such as alpha4beta7 or alphaEbeta7
receptors. Those variants which bind to beta7 integrin or the
alpha4beta7 or alphaEbeta7 receptors are further screened for the
ability to inhibit integrin beta7 receptor-ligand binding and in
vivo efficacy as described herein. Alternative, naturally or
non-naturally occurring amino acids may be substituted at position
49 by standard mutagenesis techniques and tested in the cell
adhesion and in vivo assays described herein. Alternatively, the
amino acid at position 49 of the light chain is an amino acid other
than lysine (K), and amino acids at any other HVR or framework
position or positions in the light chain and/or heavy chain is
altered to select for a variant anti-beta7 binding polypeptide or
antibody that exhibits binding affinity, in vitro and in vivo
biological activity, pharmacokinetics, drug clearance and
immunogenicity useful for reduction of inflammation by reducing the
biological activity of integrin beta7. Mutagenesis and selection of
such a polypeptide or antibody variant is performed as disclosed
herein and according to other standard techniques. Such a variant
anti-beta7 binding polypeptide or antibody exhibits integrin beta7
binding affinity within 10,000-fold, 1000-fold, alternatively
within 100-fold, alternatively within 10-fold, alternatively within
5-fold, alternatively within 2-fold of the binding affinity
exhibited by the any of the humanized Fib504 variants disclosed
herein.
[0458] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
68111PRTArtificial sequencesequence is synthesized 1Arg Ala Ser Glu
Ser Val Asp Thr Tyr Leu His 5 1028PRTArtificial sequencesequence is
synthesized 2Lys Tyr Ala Ser Gln Ser Ile Ser 539PRTArtificial
sequencesequence is synthesized 3Gln Gln Gly Asn Ser Leu Pro Asn
Thr 5410PRTArtificial sequencesequence is synthesized 4Gly Phe Phe
Ile Thr Asn Asn Tyr Trp Gly 5 10517PRTArtificial sequencesequence
is synthesized 5Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu1 5 10 15Lys Ser610PRTArtificial sequencesequence is
synthesized 6Met Thr Gly Ser Ser Gly Tyr Phe Asp Phe 5
10711PRTArtificial sequencesequence is synthesized 7Arg Ala Ser Glu
Ser Val Asp Ser Leu Leu His 5 10811PRTArtificial sequencesequence
is synthesized 8Arg Ala Ser Glu Ser Val Asp Thr Leu Leu His 5
10911PRTArtificial sequencesequence is synthesized 9Arg Ala Ser Glu
Ser Val Asp Asp Leu Leu His 5 1010108PRTArtificial sequencesequence
is synthesized 10Asp Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser
Val Thr Pro1 5 10 15Gly Glu Arg Ile Ser Leu Ser Cys Arg Ala Ser Glu
Ser Val Asp 20 25 30Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Asn Glu
Ser Pro Arg 35 40 45Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly
Ile Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Ser Ile 65 70 75Asn Gly Val Glu Leu Glu Asp Leu Ser Ile Tyr Tyr
Cys Gln Gln 80 85 90Gly Asn Ser Leu Pro Asn Thr Phe Gly Ala Gly Thr
Lys Leu Glu 95 100 105Leu Lys Arg11117PRTArtificial
sequencesequence is synthesized 11Glu Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser1 5 10 15Gln Ser Leu Ser Leu Thr Cys Ser
Val Thr Gly Phe Phe Ile Thr 20 25 30Asn Asn Tyr Trp Gly Trp Ile Arg
Lys Phe Pro Gly Asn Lys Met 35 40 45Glu Trp Met Gly Tyr Ile Ser Tyr
Ser Gly Ser Thr Ser Tyr Asn 50 55 60Pro Ser Leu Lys Ser Arg Ile Ser
Ile Thr Arg Asp Thr Ser Lys 65 70 75Asn Gln Phe Phe Leu Gln Leu Asn
Ser Val Thr Thr Glu Asp Thr 80 85 90Ala Thr Tyr Tyr Cys Ala Met Thr
Gly Ser Ser Gly Tyr Phe Asp 95 100 105Phe Trp Gly Pro Gly Thr Met
Val Thr Val Ser Ser 110 11512174PRTArtificial sequencesequence is
synthesized 12Asp Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val
Thr Pro1 5 10 15Gly Glu Arg Ile Ser Leu Ser Cys Arg Ala Ser Glu Ser
Val Asp 20 25 30Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Asn Glu Ser
Pro Arg 35 40 45Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile
Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Ser Ile 65 70 75Asn Gly Val Glu Leu Glu Asp Leu Ser Ile Tyr Tyr Cys
Gln Gln 80 85 90Gly Asn Ser Leu Pro Asn Thr Phe Gly Ala Gly Thr Lys
Leu Glu 95 100 105Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro 110 115 120Ser Met Glu Gln Leu Thr Ser Gly Gly Ala Thr
Val Val Cys Phe 125 130 135Val Asn Asn Phe Tyr Pro Arg Asp Ile Ser
Val Lys Trp Lys Ile 140 145 150Asp Gly Ser Glu Gln Arg Asp Gly Val
Leu Asp Ser Val Thr Asp 155 160 165Gln Asp Ser Lys Asp Ser Thr Tyr
Ser 17013146PRTArtificial sequencesequence is synthesized 13Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser1 5 10 15Gln Ser
Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Phe Ile Thr 20 25 30Asn Asn
Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met 35 40 45Glu Trp
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn 50 55 60Pro Ser
Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys 65 70 75Asn Gln
Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr 80 85 90Ala Thr
Tyr Tyr Cys Ala Met Thr Gly Ser Ser Gly Tyr Phe Asp 95 100 105Phe
Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Ala Glu Thr 110 115
120Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Gly Thr Ala Leu Lys 125
130 135Ser Asn Ser Met Val Thr Leu Gly Cys Leu Val 140
1451480PRTArtificial sequencesequence is synthesized 14Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly 20 25 30Lys Ala Pro
Lys Leu Leu Ile Tyr Gly Val Pro Ser Arg Phe Ser 35 40 45Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 50 55 60Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Phe Gly Gln Gly Thr 65 70 75Lys Val Glu
Ile Lys 801579PRTArtificial sequencesequence is synthesized 15Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly
Asp Arg Val Thr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly 20 25 30Lys
Ala Pro Lys Leu Leu Ile Gly Val Pro Ser Arg Phe Ser Gly 35 40 45Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 50 55 60Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gly Gln Gly Thr Lys 65 70 75Val
Glu Ile Lys1680PRTArtificial sequencesequence is synthesized 16Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro1 5 10 15Gly
Glu Pro Ala Ser Ile Ser Cys Trp Tyr Leu Gln Lys Pro Gly 20 25 30Gln
Ser Pro Gln Leu Leu Ile Tyr Gly Val Pro Asp Arg Phe Ser 35 40 45Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val 50 55 60Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gly Gln Gly Thr 65 70 75Lys
Val Glu Ile Lys 801780PRTArtificial sequencesequence is synthesized
17Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro1 5 10
15Gly Glu Arg Ala Thr Leu Ser Cys Trp Tyr Gln Gln Lys Pro Gly 20 25
30Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ile Pro Asp Arg Phe Ser 35 40
45Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 50 55
60Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gly Gln Gly Thr 65 70
75Lys Val Glu Ile Lys 801880PRTArtificial sequencesequence is
synthesized 18Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val
Ser Leu1 5 10 15Gly Glu Arg Ala Thr Ile Asn Cys Trp Tyr Gln Gln Lys
Pro Gly 20 25 30Gln Pro Pro Lys Leu Leu Ile Tyr Gly Val Pro Asp Arg
Phe Ser 35 40 45Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu 50 55 60Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gly Gln
Gly Thr 65 70 75Lys Val Glu Ile Lys 801987PRTArtificial
sequencesequence is synthesized 19Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly1 5 10 15Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr 20 25 30Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met Gly Arg 35 40 45Val Thr Ile Thr Ala Asp Thr Ser
Thr Ser Thr Ala Tyr Met Glu 50 55 60Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 65 70 75Arg Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 80 852081PRTArtificial sequencesequence is
synthesized 20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly1 5 10 15Ala Ser Val Lys Val Ser Cys Lys Ala Ser Trp Val Arg
Gln Ala 20 25 30Pro Gly Gln Gly Leu Glu Trp Met Arg Val Thr Ile Thr
Ala Asp 35 40 45Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gln
Gly Thr 65 70 75Leu Val Thr Val Ser Ser 802180PRTArtificial
sequencesequence is synthesized 21Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly1 5 10 15Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Gln Gly Leu Glu Trp Met
Arg Val Thr Ile Thr Ala Asp 35 40 45Thr Ser Thr Ser Thr Ala Tyr Met
Glu Leu Ser Ser Leu Arg Ser 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Trp Gly Gln Gly Thr Leu 65 70 75Val Thr Val Ser Ser
802279PRTArtificial sequencesequence is synthesized 22Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly1 5 10 15Ala Ser Val
Lys Val Ser Cys Lys Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Gln
Gly Leu Glu Trp Met Arg Val Thr Ile Thr Ala Asp 35 40 45Thr Ser Thr
Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser 50 55 60Glu Asp Thr
Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val 65 70 75Thr Val Ser
Ser23108PRTArtificial sequencesequence is synthesized 23Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30Asn Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Asn Ser
Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg24113PRTArtificial sequencesequence is synthesized 24Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Ser Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50 55 60Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Gly Phe Asp Tyr Trp Gly Gln 95 100 105Gly Thr
Leu Val Thr Val Ser Ser 11025108PRTArtificial sequencesequence is
synthesized 25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser
Val Asp 20 25 30Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys 35 40 45Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val
Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln 80 85 90Gly Asn Ser Leu Pro Asn Thr Phe Gly Gln Gly Thr Lys
Val Glu 95 100 105Ile Lys Arg26117PRTArtificial sequencesequence is
synthesized 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Phe
Ile Thr 20 25 30Asn Asn Tyr Trp Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 35 40 45Glu Trp Val Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser
Tyr Asn 50 55 60Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys 65 70 75Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr 80 85 90Ala Val Tyr Tyr Cys Ala Met Thr Gly Ser Ser Gly Tyr
Phe Asp 95 100 105Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
110 11527214PRTArtificial sequencesequence is synthesized 27Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Asn
Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115
120Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 125
130 135Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
140 145 150Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu 155 160 165Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 170 175 180Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu 185 190 195Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn 200 205 210Arg Gly Glu Cys28448PRTArtificial
sequencesequence is synthesized 28Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Ser Val Ile Ser Gly
Asp Gly Gly Ser Thr Tyr Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser 65 70 75Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Met
Thr Gly Ser Ser Gly Tyr Phe 95 100 105Asp Phe Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser 110 115 120Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser 125
130 135Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
140 145 150Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr 155 160 165Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu 170 175 180Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly 185 190 195Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 200 205 210Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His 215 220 225Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 230 235 240Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 320 325 330Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 335 340 345Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 350 355
360Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 365
370 375Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
380 385 390Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe 395 400 405Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln 410 415 420Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn 425 430 435His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 440 44529214PRTArtificial sequencesequence is synthesized
29Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp 20 25
30Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45Leu Leu Ile Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Gly Asn Ser Leu Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
110 115 120Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu 125 130 135Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val 140 145 150Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu 155 160 165Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 170 175 180Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu 185 190 195Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn 200 205 210Arg Gly Glu
Cys30447PRTArtificial sequencesequence is synthesized 30Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Phe Ile Thr 20 25 30Asn Asn Tyr
Trp Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn 50 55 60Pro Ser Leu
Lys Ser Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 65 70 75Asn Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 80 85 90Ala Val Tyr
Tyr Cys Ala Met Thr Gly Ser Ser Gly Tyr Phe Asp 95 100 105Phe Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 110 115 120Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 125 130
135Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 140
145 150Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
155 160 165Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr 170 175 180Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr 185 190 195Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys 200 205 210Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 215 220 225Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 320 325 330Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 335 340 345Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 350 355 360Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 365 370
375Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 380
385 390Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
395 400 405Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 410 415 420Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 425 430 435Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
440 44531214PRTArtificial sequencesequence is synthesized 31Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp 20 25 30Thr Tyr
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Gly Asn
Ser Leu Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115
120Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 125
130 135Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
140 145 150Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu 155 160 165Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 170 175 180Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu 185 190 195Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn 200 205 210Arg Gly Glu Cys32447PRTArtificial
sequencesequence is synthesized 32Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Phe Ile Thr 20 25 30Asn Asn Tyr Trp Gly Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Tyr Ile Ser Tyr
Ser Gly Ser Thr Ser Tyr Asn 50 55 60Pro Ser Leu Lys Ser Arg Phe Thr
Ile Ser Arg Asp Thr Ser Lys 65 70 75Asn Thr Phe Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr 80 85 90Ala Val Tyr Tyr Cys Ala Met Thr
Gly Ser Ser Gly Tyr Phe Asp 95 100 105Phe Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 110 115 120Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr 125 130 135Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 140 145 150Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 155 160 165Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 170 175 180Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 185 190 195Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 200 205
210Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 215
220 225Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 305 310 315Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 320 325 330Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu 335 340 345Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys 350 355 360Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser 365 370 375Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 380 385 390Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 395 400 405Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 410 415 420Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 425 430 435Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 440
44533214PRTArtificial sequencesequence is synthesized 33Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp 20 25 30Asp Leu Leu
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Gly Asn Ser
Leu Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115 120Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 125 130
135Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 140
145 150Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
155 160 165Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr 170 175 180Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys Glu 185 190 195Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn 200 205 210Arg Gly Glu Cys3423PRTArtificial
sequencesequence is synthesized 34Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys
203514PRTArtificial sequencesequence is synthesized 35Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 5 103632PRTArtificial
sequencesequence is synthesized 36Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe1 5 10 15Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr 20 25 30Tyr Cys3711PRTArtificial
sequencesequence is synthesized 37Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 5 103825PRTArtificial sequencesequence is synthesized
38Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10
15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 253913PRTArtificial
sequencesequence is synthesized 39Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 5 104031PRTArtificial sequencesequence is
synthesized 40Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu1 5 10 15Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 20 25 30Ala4111PRTArtificial sequencesequence is
synthesized 41Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 5
104231PRTArtificial sequencesequence is synthesized 42Arg Phe Thr
Ile Ser Xaa Asp Xaa Ser Lys Asn Thr Xaa Tyr Leu1 5 10 15Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25
30Ala4331PRTArtificial sequencesequence is synthesized 43Arg Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu1 5 10 15Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25
30Ala4431PRTArtificial sequencesequence is synthesized 44Arg Phe
Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Ala Tyr Leu1 5 10 15Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25
30Ala4531PRTArtificial sequencesequence is synthesized 45Arg Phe
Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr Leu1 5 10 15Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25
30Ala4631PRTArtificial sequencesequence is synthesized 46Arg Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Phe Tyr Leu1 5 10 15Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25
30Ala4710PRTArtificial sequencesequence is synthesized 47Ala Ser
Glu Ser Val Asp Asp Leu Leu His 5 104887PRTArtificial
sequencesequence is synthesized 48Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser1 5 10 15Gln Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Val Ser 20 25 30Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Arg 35 40 45Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 50 55 60Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 65 70 75Arg
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 80 854981PRTArtificial
sequencesequence is synthesized 49Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser1 5 10 15Gln Thr Leu Ser Leu Thr Cys Thr
Val Ser Trp Ile Arg Gln Pro 20 25 30Pro Gly Lys Gly Leu Glu Trp Ile
Arg Val Thr Ile Ser Val Asp 35 40 45Thr Ser Lys Asn Gln Phe Ser Leu
Lys Leu Ser Ser Val Thr Ala 50 55 60Ala Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Trp Gly Gln Gly Thr 65 70 75Leu Val Thr Val Ser Ser
805080PRTArtificial sequencesequence is synthesized 50Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser1 5 10 15Gln Thr Leu
Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro 20 25 30Pro Gly Lys
Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp 35 40 45Thr Ser Lys
Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala 50 55 60Ala Asp Thr
Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu 65 70 75Val Thr Val
Ser Ser 805179PRTArtificial sequencesequence is synthesized 51Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser1 5 10 15Gln
Thr Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro 20 25 30Pro
Gly Lys Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp 35 40 45Thr
Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala 50 55 60Ala
Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val 65 70 75Thr
Val Ser Ser5287PRTArtificial sequencesequence is synthesized 52Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg 35 40 45Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 50 55 60Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 65 70 75Arg
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 80 855381PRTArtificial
sequencesequence is synthesized 53Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys Gly Leu Glu Trp Val
Arg Phe Thr Ile Ser Arg Asp 35 40 45Asn Ser Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Trp Gly Gln Gly Thr 65 70 75Leu Val Thr Val Ser Ser
805480PRTArtificial sequencesequence is synthesized 54Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys
Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Arg Asp 35 40 45Asn Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu 65 70 75Val Thr Val
Ser Ser 805579PRTArtificial sequencesequence is synthesized 55Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Ala 20 25 30Pro
Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Arg Asp 35 40 45Asn
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 50 55 60Glu
Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val 65 70 75Thr
Val Ser Ser5687PRTArtificial sequencesequence is synthesized 56Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg 35 40 45Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 50 55 60Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 65 70 75Arg
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 80 855781PRTArtificial
sequencesequence is synthesized 57Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys Gly Leu Glu Trp Val
Arg Phe Thr Ile Ser Ala Asp 35 40 45Thr Ser Lys Asn Thr Ala Tyr Leu
Gln Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys
Ser Arg Trp Gly Gln Gly Thr 65 70 75Leu Val Thr Val Ser Ser
805880PRTArtificial sequencesequence is synthesized 58Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys
Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp 35 40 45Thr Ser Lys
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr
Ala Val Tyr Tyr Cys Ser Trp Gly Gln Gly Thr Leu 65 70 75Val Thr Val
Ser Ser 805987PRTArtificial sequencesequence is synthesized 59Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg 35 40 45Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 50 55 60Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 65 70 75Arg
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 80 856081PRTArtificial
sequencesequence is synthesized 60Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys Gly Leu Glu Trp Val
Arg Phe Thr Ile Ser Ala Asp 35 40 45Thr Ser Lys Asn Thr Ala Tyr Leu
Gln Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Trp Gly Gln Gly Thr 65 70 75Leu Val Thr Val Ser Ser
806180PRTArtificial sequencesequence is synthesized 61Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys
Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp 35 40 45Thr Ser Lys
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu 65 70 75Val Thr Val
Ser Ser 806279PRTArtificial sequencesequence is synthesized 62Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Ala 20 25 30Pro
Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp 35 40 45Thr
Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 50 55 60Glu
Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val 65 70 75Thr
Val Ser Ser6311PRTArtificial sequencesequence is synthesized 63Ala
Met Thr Gly Ser Ser Gly Tyr Phe Asp Phe 5 106411PRTArtificial
sequencesequence is synthesized 64Ala Arg Thr Gly Ser Ser Gly Tyr
Phe Asp Phe 5 106511PRTArtificial sequencesequence is synthesized
65Ala Gln Thr Gly Ser Ser Gly Tyr Phe Asp Phe 5 106610PRTArtificial
sequencesequence is synthesized 66Arg Thr Gly Ser Ser Gly Tyr Phe
Asp Phe 5 10678PRTArtificial sequencesequence is synthesized 67Arg
Tyr Ala Ser Gln Ser Ile Ser 5688PRTArtificial sequencesequence is
synthesized 68Xaa Tyr Ala Ser Gln Ser Ile Ser 5
* * * * *