Alpha-4-beta-7 heterodimer specific antagonist antibody

65 Pages·2013·4.72 MB·English

US 20130302354A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2013/0302354 A1
HSU et al. (43) Pub. Date: NOV. 14, 2013
(54) ALPHA-4-BETA-7 HETERODIMER SPECIFIC (22) Filed: Apr. 22, 2013
ANTAGONIST ANTIBODY Related US. Application Data
(71) APP1iCam53HAILING HSU, MOOrPark, CA (Us); (62) Division of application No. 12/725,031, ?led on Mar.
Ian Foltz, Burnaby (CA); Frederick 16, 2010,110w Pat. No. 8,444,981.
J acobsen, NeWbury Park, CA (US); _ _ _ _
Taruna Arora’ Thousand Oaks’ CA (60) Prov1s1onal applicatlon No. 61/306,829, ?led on Feb.
(Us) 22, 2010, provisional application No. 61/162,154,
?led on Mar. 20, 2009.
(72) Inventors: HAILING HSU, Moorpark, CA (US); Publication Classi?cation
Ian Foltz, Burnaby (CA); Frederick
J acobsen, NeWbury Park, CA (US); (51) Int_ CL
Taruna Arora, Thousand Oaks, CA C07K16/28 (200601)
(Us) (52) us. Cl.
CPC .................................... .. C07K16/28 (2013.01)
USPC ................................... .. 424/1731; 536/2353
(73) Assignee: AMGEN INC., Thousand Oaks, CA
(Us) (57) ABSTRACT
There are disclosed alpha4beta7 heterodimer-speci?c antigen
binding proteins, nucleic acids encoding them, and methods
(21) Appl. No.: 13/867,498 of making and using them.
US 2013/0302354 A1 Nov. 14, 2013
ALPHA-4-BETA-7 HETERODIMER SPECIFIC [0007] In addition to pairing With the alpha4 chain, the
ANTAGONIST ANTIBODY beta7 subunit also partners With alphaE to form alphaEbeta7,
Which is primarily expressed on intraepithelial lymphocytes
CROSS-REFERENCE TO RELATED (IEL) in intestine, lung and genitourinary tract. AlphaEbeta7
APPLICATIONS is also expressed on dendritic cells in the gut. The alphaE
beta7 heterodimer binds to E-cadherin, Which is expressed on
[0001] This application claims the bene?t under 35 U.S.C.
epithelial cells. The IEL cells are thought to provide a mecha
119(e) of US. patent application No. 61/162,154, ?led Mar.
nism for immunosurveillance Within the epithelial compart
20, 2009 and US. patent application No. 61/306,829, ?led
ment.
Feb. 22, 2010, Which are incorporated herein by reference.
[0008] Antibodies that bind alpha4 and inhibit binding of
alpha4beta1 to VCAM-1 and ?bronection mapped to a
REFERENCE TO THE SEQUENCE LISTING
52-amino acid region of alpha4, betWeen residues 152 and
[0002] The present application is being ?led along With a 203 (Schiffer et al., J. Biol. Chem. 270114270; 1995). Tid
Sequence Listing in electronic format. The Sequence Listing sWell et al. (J. Immuno 159: 1497; 1997) identi?ed domains of
is provided as a ?le entitled A-1459-US-NP_Seq_Listing. beta7 that are important in binding to MAdCAM-l, utiliZing
txt., created Mar. 16, 2010, Which is 84.0 KB in siZe. The a panel of antibodies that bind beta7 in a mouse/human chi
information in the electronic format of the Sequence Listing meric beta7 subunit approach. They found that six of seven
is incorporated herein by reference in its entirety. antibodies that inhibited binding to MAdCAM-l and E-cad
herin mapped to a region comprising amino acids 176 through
FIELD OF THE INVENTION 250, Which appears to have homology to the metal-ion depen
dent adhesion site (MIDAS) of other integrin subunits. One of
[0003] This application provides compositions and meth
the antibodies used by TidsWell et al. Was an alpha4beta7
ods relating to alpha4beta7 heterodimer-speci?c antigen heterodimer speci?c antibody referred to as ACT-1.
binding proteins.
[0009] The ACT-1 antibody Was originally described by
LaZarovitZ et al. (J. Immunol. 133:1857; 1984) as an antibody
BACKGROUND developed by immunizing mice With human tetanus toxoid
speci?c T lymphocyte line from PBMC. Later it Was shoWn
[0004] Integrins are heterodimeric Type I transmembrane
that ACT-1 binds to the alpha4beta7 heterodimer speci?cally
proteins formed of tWo subunits (one alpha subunit and one
(SchWeighoffer et al., J. Immunol. 1511717, 1993). While
beta subunit), and mediate many different cell-cell and cell
extracellular matrix interactions. Functionally, integrins have ACT-1 does not bind murine alpha4beta7, it does bind
alpha4beta7 from least some non-human primate species, and
been shoWn to be involved in diverse biological processes,
including leukocyte migration and recirculation and the has been shoWn to attenuate spontaneous colitis in captive
cotton-top tamarins (Hesterberg et al., Gastroenterology 11 1:
immune response. In mammals, there are 18 knoWn alpha
1373; 1996)
subunits and eight knoWn beta subunits, Which combine to
form 24 distinct integrins. Ligand speci?city is determined in [0010] ACT-1 has been humaniZed and evaluated as a
large part by the particular combinations of alpha and beta human therapeutic in ulcerative colitis (Feagan et al., N Engl
subunits expressed, While a?inity for ligand is modulated by J. Med. 352:2499; 2005), and recently in Crohn’s disease
(Feagan et al, Clinical Gastroenterology and Hepatology,
integrin conformational changes and is divalent-cation
dependent. 6:1370, 2008). HumaniZed ACT-1, also knoWn as ved
oliZumab, is described in WO 98/06248 and US. Pat. No.
[0005] The ligands for integrins form a structurally diverse
7,147,85, as Well as WO 07/061,679 and US 2007-0122404.
group that includes extracellular matrix proteins such as col
Another humanized antibody, nataliZumab (Tysabri®), has
lagens, ?bronection, vitronectin and laminins; counter-recep
been used to treat Crohn’s disease. NataliZumab is a human
tors such as the cellular adhesion molecules (for example,
iZed version of an alpha4-speci?c murine antibody. Ved
vascular cellular adhesion molecule or VCAM), and plasma
proteins. Numerous pathogenic microorganisms also utiliZe oliZumab has been shoWn to lead to a neutraliZing anti-hu
maniZed antibody response in a portion of patients, and
integrins to initiate infection or as sites for toxin binding. The
nataliZumab has been associated With progressive multifocal
structurally diverse ligands share an exposed glutamic or
leukoencephalopathy (PML), a neurological disorder that is
aspartic acid residue, usually present in an extended, ?exible
associated With reactivation of prior infection With J C virus in
loop, Which is important for recognition by integrins.
immunocompromised individuals. Accordingly, there is a
[0006] The alpha4 integrins (alpha 4 partnered With either
need for a therapeutic agent that ameliorates these disadvan
the beta1 or beta7 subunit) play an important role in the
tages While disrupting the alpha4beta7/MAdCAM-1 path
immune system. Alpha4beta1 is expressed on lymphocytes
Way.
and myeloid cells; it appears to be the major binding partner
for vascular cell adhesion molecule (VCAM). VCAM is ubiq
SUMMARY OF THE INVENTION
uitously expressed on vascular endothelium, is up regulated
during in?ammation, and binds alpha4beta7 as Well as [0011] In one aspect, the present invention provides an
alpha4beta1 (albeit Weakly to alpha4beta7). Though also isolated antigen binding protein that speci?cally binds to
detected on d peripheral T cells, B cells, NK cells and eosin human alpha4beta7 (i.e., an alpha4beta7 heterodimer speci?c
phils, alpha4beta7 is most highly expressed on a subpopula antigen binding protein). In another aspect of the invention,
tion of CD4+ CD45RA-memory T cells Which has been the antigen binding protein speci?cally binds to the
shoWn to preferentially home to the gut. The primary ligand alpha4beta7 of a non-human primate, a cynomologous mon
for the alpha4beta7 heterodimer is mucosal addressin cell key, a chimpanZee, a non-primate mammal, a rodent, a
adhesion molecule 1 (MAdCAM-l or MAdCAM), Which is mouse, a rat, a hamster, a guinea pig, a cat, or a dog. In another
expressed in gut endothelium. embodiment, the isolated antigen binding protein comprises a
US 2013/0302354 A1 Nov. 14, 2013
human antibody; a chimeric antibody; a monoclonal anti and CDR3 and the light chain variable region comprises
body; a recombinant antibody; an antigen-binding antibody CDRl, CDR2 and CDR3. In one embodiment, the light chain
fragment; a single chain antibody; a diabody; a triabody; a CDRs are selected from the group consisting of a CDRl,
tetrabody; a Pab fragment; a P(ab')2 fragment; a domain anti CDR2 and CDR3 at least 90% identical to a CDRl, CDR2
body; an IgD antibody; an IgE antibody; an IgM antibody; an and CDR3, respectively, of SEQ ID NO: 3; a CDRl, CDR2
IgGl antibody; an IgG2 antibody; an IgG3 antibody; an IgG4 and CDR3 at least 90% identical to a CDRl, CDR2 and
antibody; or an IgG4 antibody having at least one mutation in CDR3, respectively, of SEQ ID NO: 5; a CDRl, CDR2 and
a hinge region that alleviates a tendency to form intra-H chain CDR3 at least 90% identical to a CDRl, CDR2 and CDR3,
disul?de bond. In another aspect, the isolated antigen binding respectively, of SEQ ID NO: 7; a CDRl, CDR2 and CDR3 at
protein comprises a heavy chain constant region from one of least 90% identical to a CDRl, CDR2 and CDR3, respec
the aforementioned antibodies; in another aspect, the con tively, of SEQ ID NO: 22; and a CDRl, CDR2 and CDR3 at
stant region is a polypeptide comprising SEQ ID NO:72; a least 90% identical to a CDRl, CDR2 and CDR3, respec
polypeptide at least 90% identical to SEQ ID NO:72; a tively, of SEQ ID NO: 24; and the heavy chain variable
polypeptide having an amino acid sequence as set forth in CDRl, CDR2 and CDR3 are from SEQ ID NO:58.
SEQ ID NO:72 from Which one, tWo, three, four or ?ve [0015] In another aspect of the invention, the heavy chain
N-terminal and/or C-terminal amino acids have been variable region further comprises four frameWork regions
removed; or one of the afore-mentioned polypeptides Which (PRs) designated PRl, PR2, PR3 and PR4, and the light chain
incorporates one or more post-translational modi?cations. In variable region further comprises four frameWork regions
one embodiment, the isolated antigen binding protein com (PRs) designated PRl, PR2, PR3 and PR4. In one aspect, the
prises a kappa light chain constant region, in another it com PRs are selected from the same SEQ ID NO as the CDRs; in
prises a lambda light chain region. In one embodiment, the another, the PRs are selected from a different SEQ ID NO. In
light chain constant region is a polypeptide comprising SEQ a further embodiment, the invention provides an alpha4beta7
ID NO:70; a polypeptide at least 90% identical to SEQ ID heterodimer speci?c antigen binding protein Wherein the
NO:70; a polypeptide having an amino acid sequence as set light chain variable region is selected from the group consist
forth in SEQ ID NO:70 from Which one, tWo, three, four or ing of a light chain variable region at least 90% identical to
?ve N-terminal and/ or C-terminal amino acids have been SEQ ID NO:3; a light chain variable region at least 90%
removed; or one of the afore-mentioned polypeptides Which identical to SEQ ID NO:5; a light chain variable region at
incorporates one or more post-translational modi?cations least 90% identical to SEQ ID NO:7; a light chain variable
[0012] One embodiment of the present invention provides region at least 90% identical to SEQ ID NO:22; and a light
an alpha4beta7 heterodimer speci?c antigen binding protein chain variable region at least 90% identical to SEQ ID NO:24;
having a heavy chain and a light chain, each of Which com and the heavy chain variable region comprises SEQ ID
prise one or more complementarity determining regions, or NO:58.
CDRs. In another aspect of the invention, the heavy chain [001 6] Another aspect of the invention provides an isolated,
variable region comprises CDRl, CDR2 and CDR3 and a alpha4beta7 heterodimer speci?c antigen binding protein
light chain variable region comprises CDRl, CDR2 and having a heavy chain variable region comprising CDRl,
CDR3, Wherein each respective CDR is selected from the CDR2 and CDR3 and a light chain variable region compris
group consisting of the light chain CDRl, CDR2 and CDR3 ing CDRl, CDR2 and CDR3, Wherein the light chain CDRl,
from SEQ ID NO:55, and the heavy chain CDRl, CDR2 and CDR2 and CDR3 are selected from the group consisting of a
CDR3 from SEQ ID NO:58; the light chain CDRl, CDR2 and CDRl, CDR2 and CDR3 at least 90% identical to a CDRl,
CDR3 from SEQ ID NO:56, and the heavy chain CDRl, CDR2 and CDR3, respectively, of SEQ ID NO1l2; a CDRl,
CDR2 and CDR3 from SEQ ID NO:59; and the light chain CDR2 and CDR3 at least 90% identical to a CDRl, CDR2
CDRl, CDR2 and CDR3 from SEQ ID NO:57, and the heavy and CDR3, respectively, of SEQ ID NO: 25; and a CDRl,
chain CDRl, CDR2 and CDR3 from SEQ ID NO:60. CDR2 and CDR3 at least 90% identical to a CDRl, CDR2
[0013] In another aspect of the invention, the heavy chain and CDR3, respectively, of SEQ ID NO: 26; and the heavy
variable region further comprises four framework regions chain CDRl, CDR2 and CDR3 are selected from the group
(PRs) designated PRl, PR2, PR3 and PR4, and the light chain consisting of a CDRl, CDR2 and CDR3 at least 90% identi
variable region further comprises four framework regions cal to a CDRl, CDR2 and CDR3, respectively, of SEQ ID
(PRs) designated PRl PR2, PR3 and PR4. In one aspect, the NO141 ; and a CDRl, CDR2 and CDR3 at least 90% identical
PRs are selected from the same SEQ ID NO as the CDRs; in to a CDRl, CDR2 and CDR3, respectively, of SEQ ID
another, the PRs are selected from a different SEQ ID NO. In NO:54. In one embodiment, the light chain variable region is
a further embodiment, the invention provides an alpha4beta7 selected from the group consisting of variable regions that are
heterodimer speci?c antigen binding protein Wherein the at least 90% identical to any one of SEQ ID NOs: 12, 25 and
light chain variable region comprises SEQ ID NO:55, and the 26, and the heavy variable region is selected from the group
heavy chain variable region comprises SEQ ID NO:58; the consisting of variable regions that are at least 90% identical to
light chain variable region comprises SEQ ID NO:56, and the any one of SEQ ID NOsz4l and 54. In another aspect of the
heavy chain variable region comprises SEQ ID NO: 59; or the invention, the heavy chain variable region further comprises
light chain variable region comprises SEQ ID NO:57, and the four frameWork regions (PRs) designated PRl, PR2, PR3 and
heavy chain variable region comprises SEQ ID NO:60. PR4, and the light chain variable region further comprises
[0014] In another aspect of the invention, the present inven four frameWork regions (PRs) designated PRl, PR2, PR3 and
tion provides an isolated alpha4beta7 heterodimer speci?c PR4. In one aspect, the PRs are selected from the same SEQ
antigen binding protein, having a heavy chain and a light ID NO as the CDRs; in another, the PRs are selected from a
chain, each of Which comprise one or more complementarity different SEQ ID NO.
determining regions, or CDRs. In another aspect of the inven [0017] In one embodiment, the invention provides an iso
tion, the heavy chain variable region comprises CDRl, CDR2 lated, alpha4beta7 heterodimer speci?c antigen binding pro
US 2013/0302354 A1 Nov. 14, 2013
tein having a heavy chain variable region comprising CDR1, chain CDRs are identical to the respective CDRs of the
CDR2 and CDR3 and a light chain variable region compris recited SEQ ID NOs. In one embodiment of the invention, the
ing CDR1, CDR2 and CDR3, Wherein each respective CDR heavy chain variable region further comprises four frame
is at least 90% identical to a CDR selected from the group Work regions (PRs) designated PR1, PR2, PR3 and PR4, and
consisting of a light chain CDR1, CDR2 and CDR3 from the light chain variable region further comprises four frame
SEQ ID N0110, and a heavy chain CDR1, CDR2 and CDR3 Work regions (PRs) designated PR1, PR2, PR3 and PR4. In
from SEQ ID N0138; a light chain CDR1, CDR2 and CDR3 one aspect, the PRs are selected from the same SEQ ID NO as
from SEQ ID N012, and a heavy chain CDR1, CDR2 and the CDRs; in another, the PRs are selected from a different
SEQ ID N0.
CDR3 from SEQ ID N0130; a light chain CDR1, CDR2 and
CDR3 from SEQ ID N0120, and a heavy chain CDR1, CDR2 [0018] In another embodiment, an alpha4beta7 het
and CDR3 from SEQ ID N0151; a light chain CDR1, CDR2 erodimer speci?c antigen binding protein comprises a light
and CDR3 from SEQ ID N0111, and a heavy chain CDR1, chain variable region and a heavy chain variable region,
CDR2 and CDR3 from SEQ ID N0139; a light chain CDR1, Wherein the light chain variable region is at least 90% iden
CDR2 and CDR3 from SEQ ID N0113, and a heavy chain tical to SEQ ID N0110, and the heavy chain variable region is
CDR1, CDR2 and CDR3 from SEQ ID N0142; a light chain at least 90% identical to SEQ ID N0138; the light chain
CDR1, CDR2 and CDR3 from SEQ ID N0117, and a heavy variable region is at least 90% identical to SEQ ID N012, and
chain CDR1, CDR2 and CDR3 from SEQ ID N0146; a light the heavy chain variable region is at least 90% identical to
chain CDR1, CDR2 and CDR3 from SEQ ID N018, and a SEQ ID N0130; the light chain variable region is at least 90%
heavy chain CDR1, CDR2 and CDR3 from SEQ ID N0136; identical to SEQ ID N0120, and the heavy chain variable
a light chain CDR1, CDR2 and CDR3 from SEQ ID N0119, region is at least 90% identical to SEQ ID N0151; the light
and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID chain variable region is at least 90% identical to SEQ ID
N0149; a light chain CDR1, CDR2 and CDR3 from SEQ ID N0111, and the heavy chain variable region is at least 90%
N0118, and a heavy chain CDR1, CDR2 and CDR3 from identical to SEQ ID N0139; the light chain variable region is
SEQ ID N0147; a light chain CDR1, CDR2 and CDR3 from at least 90% identical to SEQ ID N0113, and the heavy chain
SEQ ID N0121, and a heavy chain CDR1, CDR2 and CDR3 variable region is at least 90% identical to SEQ ID N0142; the
from SEQ ID N0152; a light chain CDR1, CDR2 and CDR3 light chain variable region is at least 90% identical to SEQ ID
from SEQ ID N013, and a heavy chain CDR1, CDR2 and N0117, and the heavy chain variable region is at least 90%
CDR3 from SEQ ID N0131; a light chain CDR1, CDR2 and identical to SEQ ID N0146; the light chain variable region is
CDR3 from SEQ ID N017, and a heavy chain CDR1, CDR2 at least 90% identical to SEQ ID N018, and the heavy chain
and CDR3 from SEQ ID N0135; a light chain CDR1, CDR2 variable region is at least 90% identical to SEQ ID N0136; the
and CDR3 from SEQ ID N016, and a heavy chain CDR1, light chain variable region is at least 90% identical to SEQ ID
CDR2 and CDR3 from SEQ ID N0134; a light chain CDR1, N0119, and the heavy chain variable region is at least 90%
CDR2 and CDR3 from SEQ ID N011, and a heavy chain identical to SEQ ID N0149; the light chain variable region is
CDR1, CDR2 and CDR3 from SEQ ID N0129; a light chain at least 90% identical to SEQ ID N0118, and the heavy chain
CDR1, CDR2 and CDR3 from SEQ ID N0122, and a heavy variable region is at least 90% identical to SEQ ID N0147; the
chain CDR1, CDR2 and CDR3 from SEQ ID N0150; a light light chain variable region is at least 90% identical to SEQ ID
chain CDR1, CDR2 and CDR3 from SEQ ID N0124, and a N0121, and the heavy chain variable region is at least 90%
heavy chain CDR1, CDR2 and CDR3 from SEQ ID N0140; identical to SEQ ID N0152; the light chain variable region is
a light chain CDR1, CDR2 and CDR3 from SEQ ID N019, at least 90% identical to SEQ ID N013, and the heavy chain
and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID variable region is at least 90% identical to SEQ ID N0131 ; the
N0137; a light chain CDR1, CDR2 and CDR3 from SEQ ID light chain variable region is at least 90% identical to SEQ ID
N014, and a heavy chain CDR1, CDR2 and CDR3 from SEQ N017, and the heavy chain variable region is at least 90%
ID N0132; a light chain CDR1, CDR2 and CDR3 from SEQ identical to SEQ ID N0135; the light chain variable region is
ID N0128, and a heavy chain CDR1, CDR2 and CDR3 from at least 90% identical to SEQ ID N016, and the heavy chain
SEQ ID N0153; a light chain CDR1, CDR2 and CDR3 from variable region is at least 90% identical to SEQ ID N0134; the
SEQ ID N0116, and a heavy chain CDR1, CDR2 and CDR3 light chain variable region is at least 90% identical to SEQ ID
from SEQ ID N0145; a light chain CDR1, CDR2 and CDR3 N011, and the heavy chain variable region is at least 90%
from SEQ ID N0115, and a heavy chain CDR1, CDR2 and identical to SEQ ID N0129; the light chain variable region is
CDR3 from SEQ ID N0144; a light chain CDR1, CDR2 and at least 90% identical to SEQ ID N0122, and the heavy chain
CDR3 from SEQ ID N0114, and a heavy chain CDR1, CDR2 variable region is at least 90% identical to SEQ ID N0150; the
and CDR3 from SEQ ID N0143; a light chain CDR1, CDR2 light chain variable region is at least 90% identical to SEQ ID
and CDR3 from SEQ ID N0127, and a heavy chain CDR1, N0124, and the heavy chain variable region is at least 90%
CDR2 and CDR3 from SEQ ID N0143; a light chain CDR1, identical to SEQ ID N0140; the light chain variable region is
CDR2 and CDR3 from SEQ ID N015, and a heavy chain at least 90% identical to SEQ ID N019, and the heavy chain
CDR1, CDR2 and CDR3 from SEQ ID N0133; a light chain variable region is at least 90% identical to SEQ ID N0137; the
CDR1, CDR2 and CDR3 from SEQ ID N0112, and a heavy light chain variable region is at least 90% identical to SEQ ID
chain CDR1, CDR2 and CDR3 from SEQ ID N0141; a light N014, and the heavy chain variable region is at least 90%
chain CDR1, CDR2 and CDR3 from SEQ ID N0123, and a identical to SEQ ID N0132; the light chain variable region is
heavy chain CDR1, CDR2 and CDR3 from SEQ ID N0148; at least 90% identical to SEQ ID N0128, and the heavy chain
a light chain CDR1, CDR2 and CDR3 from SEQ ID N0125, variable region is at least 90% identical to SEQ ID N0153; the
and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID light chain variable region is at least 90% identical to SEQ ID
N0154; and a light chain CDR1, CDR2 and CDR3 from SEQ N0116, and the heavy chain variable region is at least 90%
ID N0126, and a heavy chain CDR1, CDR2 and CDR3 from identical to SEQ ID N0145; the light chain variable region is
SEQ ID N0154. In another aspect, the heavy chain and light at least 90% identical to SEQ ID N0115, and the heavy chain
US 2013/0302354 A1 Nov. 14, 2013
variable region is at least 90% identical to SEQ ID NO:44; the invention, the isolated antigen binding protein inhibits adhe
light chain variable region is at least 90% identical to SEQ ID sion of cells expressing alpha4beta7 to cells expressing MAd
NO: 14, and the heavy chain variable region is at least 90% CAM-l. In yet another aspect of the invention, the isolated
identical to SEQ ID NO:43; the light chain variable region is antigen binding protein inhibits tra?icking of cells expressing
at least 90% identical to SEQ ID NO:27, and the heavy chain alpha4beta7 to areas or tissues populated by cells expressing
variable region is at least 90% identical to SEQ ID NO:43; the MAdCAM-l; in one example of such an embodiment, the
light chain variable region is at least 90% identical to SEQ ID isolated antigen binding proteins inhibit traf?cking of lym
N015, and the heavy chain variable region is at least 90% phocytes to the gut.
identical to SEQ ID NOz33; the light chain variable region is [0023] In another aspect, the present invention provides a
at least 90% identical to SEQ ID NO:l2, and the heavy chain pharmaceutical composition comprising the antigen binding
variable region is at least 90% identical to SEQ ID NO:4l ; the protein. In one embodiment, the present invention provides a
light chain variable region is at least 90% identical to SEQ ID method of treating a condition in a subject comprising admin
NO:23, and the heavy chain variable region is at least 90% istering the pharmaceutical composition to the subject,
identical to SEQ ID NO:48; the light chain variable region is Wherein the condition is treatable by reducing the activity
at least 90% identical to SEQ ID NO:25, and the heavy chain (partially or fully) of alpha4beta7 in the subject. In another
variable region is at least 90% identical to SEQ ID NO:54; or embodiment, the subject is a human being. In another
the light chain variable region is at least 90% identical to SEQ embodiment, the condition is an in?ammatory condition of
ID NO:26, and the heavy chain variable region is at least 90% the gastrointestinal system. Thus, there is provided a method
identical to SEQ ID NO: 54. In another aspect, the heavy chain of treating an individual afflicted with a condition character
and light chain variable regions are identical to the respective iZed by inappropriate traf?cking of cells expressing
variable regions of the recited SEQ ID NOs. alpha4beta7 to tissues comprising cells expressing MAd
[0019] One aspect of the invention provides an isolated, CAM, comprising administering to the individual an
alpha4beta7 heterodimer speci?c antigen binding protein alpha4beta7 heterodimer speci?c antigen binding protein in
having an EC50 of less than 35 ng/ml in a CD4+ memory T am amount suf?cient to inhibit (partially or fully) the traf?ck
cell binding assay; another provides an isolated, alpha4beta7 ing of cells expressing alpha4beta7 to tissues comprising
heterodimer speci?c antigen binding Which has an EC50 of cells expressing MAdCAM. In one embodiment, the condi
less than 10 ng/ml in a CD4+ memory T cell binding assay. In tion is in?ammatory boWel disease, for example, ulcerative
another embodiment, the invention provides an isolated, colitis, Crohn’s disease, Celiac disease (nontropical Sprue),
alpha4beta7 heterodimer speci?c antigen binding protein enteropathy associated With seronegative arthropathies,
having an IC50 in a MAdCAM competition assay of less than microscopic or collagenous colitis, eosinophilic gastroenteri
30 ng/m; in another is provided an isolated, alpha4beta7 tis, or pouchitis resulting after proctocolectomy and ileoanal
heterodimer speci?c antigen binding Which has an IC50 of anastomosis. In another embodiment, the condition is s pan
less than 10 ng/ml in a MAdCAM competition assay. One creatitis, insulin-dependent diabetes mellitus, mastitis, chole
aspect of the invention provides an isolated, alpha4beta7 het cystitis, cholangitis, pericholangitis, chronic bronchitis,
erodimer speci?c antigen binding protein that binds an chronic sinusitis, asthma or graft versus host disease.
S250N mutant of alha4beta7. [0024] In another embodiment, the method further com
prises administering to the subject a second treatment. In
[0020] In one aspect of the invention, the present invention
provides nucleic acids encoding the aforementionedpolypep another embodiment, the second treatment is administered to
tides. In another aspect of the invention the nucleic acid is a the subject before and/or simultaneously With and/ or after the
pharmaceutical composition is administered to the subject. In
vector. In another embodiment of the invention, the invention
provides host cells transformed or transfected With the inven another embodiment, the second treatment comprises an anti
tive nucleic acids. In another aspect of the invention, there is in?ammatory agent. In another embodiment, the second
provided a method of preparing a polypeptide comprising pharmaceutical composition comprises an agent selected
incubating the host cells under conditions promoting expres from the group consisting of non-steroidal anti-in?ammatory
sion of the polypeptides and harvesting the polypeptides. drugs, steroids, and immunomodulating agents. In another
embodiment, the method comprises administering to the sub
[0021] In another aspect, the present invention provides an
ject a third treatment.
isolated cell that secretes an antigen binding protein that
[0025] In another aspect, the present invention provides a
binds alpha4beta7. In another embodiment, the cell is a hybri
method of increasing the longevity of a subject comprising
doma. In another embodiment, the present invention provides
administering to the subject the pharmaceutical composition.
a method of making an antigen binding protein that speci?
cally binds alpha4beta7 (i.e., human alpha4beta7), compris In another aspect, the present invention provides a method of
decreasing alpha4beta7 activity in a subject in need thereof
ing incubating said isolated cell under conditions that alloW it
comprising administering to the subject the pharmaceutical
to express said antigen binding protein.
composition. In another aspect, the present invention pro
[0022] In one aspect, the present invention provides an
vides a method of decreasing alpha4beta7-mediated traf?ck
isolated antigen binding protein that speci?cally binds to an
ing (for example, alpha4beta7mediated gut homing) in a sub
alpha4beta7 heterodimer. In another embodiment, the iso
ject in need thereof comprising administering to the subject
lated antigen binding protein, When bound to a human
the pharmaceutical composition.
alpha4beta7, inhibits binding of alpha4beta7 to MAdCAM-l .
Accordingly, one embodiment of the invention provides a
DETAILED DESCRIPTION OF THE INVENTION
method of inhibiting at least one activity of alpha4beta7,
comprising contacting a cell expressing alpha4beta7 With an [0026] The present invention provides compositions, kits,
alpha4beta7 heterodimer-speci?c antigen binding protein and methods relating to molecules that bind to the integrin
such that the activity is partially or fully inhibited. In one alpha4beta7 (“alpha4beta7”), including molecules that ago
aspect, such method is carried out in vivo. In one aspect of the niZe or antagoniZe alpha4beta7, such as anti-alpha4beta7
US 2013/0302354 A1 Nov. 14, 2013
antibodies, antibody fragments, and antibody derivatives, [0029] The folloWing terms, unless otherWise indicated,
e.g., antagonistic anti-alpha4beta7 antibodies, antibody frag shall be understood to have the folloWing meanings:
ments, or antibody derivatives. Also provided are nucleic [0030] The term “isolated molecule” (Where the molecule
acids, and derivatives and fragments thereof, comprising a is, for example, a polypeptide, a polynucleotide, or an anti
sequence of nucleotides that encodes all or a portion of a body) is a molecule that by virtue of its origin or source of
polypeptide that binds to alpha4beta7, e.g., a nucleic acid derivation (1 ) is not associated With naturally associated com
encoding all orpart of an anti-alpha4beta7 antibody, antibody ponents that accompany it in its native state, (2) is substan
fragment, or antibody derivative, plasmids and vectors com tially free of other molecules from the same species (3) is
prising such nucleic acids, and cells or cell lines comprising expressed by a cell from a different species, or (4) does not
such nucleic acids and/ or vectors and plasmids. The provided occur in nature Without human intervention. Thus, a molecule
methods include, for example, methods of making, identify that is chemically synthesiZed, or synthesiZed in a cellular
system different from the cell from Which it naturally origi
ing, or isolating molecules that bind to alpha4beta7, such as
nates, Will be “isolated” from its naturally associated compo
anti-alpha4beta7 antibodies, methods of determining
nents. A molecule also may be rendered substantially free of
Whether a molecule binds to alpha4beta7, methods of deter
naturally associated components by isolation, using puri?ca
mining Whether a molecule agoniZes or antagoniZes
tion techniques Well knoWn in the art. Molecule purity or
alpha4beta7, methods of making compositions, such as phar
homogeneity may be assayed by a number of means Well
maceutical compositions, comprising a molecule that binds
knoWn in the art. For example, the purity of a polypeptide
to alpha4beta7, and methods for administering a molecule
sample may be assayed using polyacrylamide gel electro
that binds alpha4beta7 to a subject, for example, methods for
phoresis and staining of the gel to visualiZe the polypeptide
treating a condition mediated by alpha4beta7, and for ago
using techniques Well knoWn in the art. For certain purposes,
niZing or antagonizing a biological activity of alpha4beta7, in
higher resolution may be provided by using HPLC or other
vivo or in vitro.
means Well knoWn in the art for puri?cation.
[0027] Polynucleotide and polypeptide sequences are indi [0031] The terms “alpha4beta7 inhibitor” and
cated using standard one- or three-letter abbreviations. “alpha4beta7 antagonist” are used interchangeably. Each is a
Unless otherWise indicated, each polypeptide sequence has molecule that detectably inhibits at least one function of
amino terminus at the left and a carboxy terminus at the right; alpha4beta7. Conversely, an “alpha4beta7 agonist” is a mol
each single-stranded nucleic acid sequence, and the top strand ecule that detectably increases at least one function of
of each double-stranded nucleic acid sequence has a 5' termi alpha4beta7. The inhibition caused by an alpha4beta7 inhibi
nus at the left and a 3' terminus at the right. A particular tor need not be complete so long as it is detectable, for
polypeptide orpolynucleotide sequence also can be described example by using an assay. Any assay of a function of
by explaining hoW it differs from a reference sequence. alpha4beta7 can be used, examples of Which are provided
herein. Examples of functions of alpha4beta7 that can be
[0028] Unless otherWise de?ned herein, scienti?c and tech
inhibited by an alpha4beta7 inhibitor (or increased by an
nical terms used in connection With the present invention
alpha4beta7 agonist) include ligand binding (i.e., binding to
shall have the meanings that are commonly understood by
MAdCAM-1), adhesion to ligand-expressing cells, traf?ck
those of ordinary skill in the art. Further, unless otherWise
required by context, singular terms shall include pluralities ing to a particular compartment such as the gut, release of
cytokines, chemokines and other mediators, enhancing or
and plural terms shall include the singular. Generally, nomen
exacerbating in?ammatory response and tissue damage, and
clatures used in connection With, and techniques of, cell and
tissue culture, molecular biology, immunology, microbiol so on. Examples of types of alpha4beta7 inhibitors and
ogy, genetics and protein and nucleic acid chemistry and alpha4beta7 agonists include, but are not limited to,
alpha4beta7 binding polypeptides such as antigen binding
hybridiZation described herein are those Well knoWn and
proteins (e.g., alpha4beta7 antigen binding proteins), anti
commonly used in the art. The methods and techniques of the
bodies, antibody fragments, and antibody derivatives.
present invention are generally performed according to con
ventional methods Well knoWn in the art and as described in [0032] The terms “peptide, polypeptide” and “protein”
various general and more speci?c references that are cited and each refers to a molecule comprising tWo or more amino acid
discussed throughout the present speci?cation unless other residues joined to each other by peptide bonds. These terms
Wise indicated. See, e.g., Sambrook et al. Molecular Cloning: encompass, e.g., native and arti?cial proteins, protein frag
A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory ments and polypeptide analogs (such as muteins, variants,
Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., and fusion proteins) of a protein sequence as Well as post
Current Protocols in Molecular Biology, Greene Publishing translationally, or otherWise covalently or non-covalently,
Associates (1992), and HarloW and LaneAntibodies: A Labo modi?ed proteins. A peptide, polypeptide, or protein may be
ratory Manual Cold Spring Harbor Laboratory Press, Cold monomeric or polymeric.
Spring Harbor, N.Y. (1990), Which are incorporated herein by [0033] The term “polypeptide fragment” as used herein
reference. Enzymatic reactions and puri?cation techniques refers to a polypeptide that has an amino-terminal and/or
are performed according to manufacturer’s speci?cations, as carboxy-terminal deletion as compared to a corresponding
commonly accomplished in the art or as described herein. The full-length protein. Fragments can be, for example, at least 5,
terminology used in connection With, and the laboratory pro 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150
cedures and techniques of, analytical chemistry, synthetic or 200 amino acids in length. Fragments can also be, for
organic chemistry, and medicinal and pharmaceutical chem example, at most 1,000, 750, 500, 250, 200, 175, 150, 125,
istry described herein are those Well knoWn and commonly 100, 90, 80, 70, 60, 50, 40, 30, 20, 15,14,13, 12, 11, or 10
used in the art. Standard techniques can be used for chemical amino acids in length. Fragments can also result from pro
syntheses, chemical analyses, pharmaceutical preparation, teolytic (or other) processing, Which, for example, results in
formulation, and delivery, and treatment of patients. variation in the amino and/ or carboxy terminus of from one to
US 2013/0302354 A1 Nov. 14, 2013
?ve amino acids from that predicted. A fragment can further [0037] A “variant” of a polypeptide (e.g., an antibody)
comprise, at either or both of its ends, one or more additional comprises an amino acid sequence Wherein one or more
amino acids, for example, a sequence of amino acids from a amino acid residues are inserted into, deleted from and/or
different naturally-occurring protein (e.g., an Fc or leucine substituted into the amino acid sequence relative to another
Zipper domain) or an arti?cial amino acid sequence (e.g., an polypeptide sequence. Variants of the invention include
arti?cial linker sequence or a tag protein). fusion proteins.
[0034] Polypeptides of the invention include polypeptides [0038] A “derivative” of a polypeptide is a polypeptide
that have been modi?ed in any Way and for any reason, for (e. g., an antibody) that has been chemically modi?ed, e. g., via
example, to: (1) reduce susceptibility to proteolysis, (2) conjugation to another chemical moiety (such as, for
reduce susceptibility to oxidation, (3) alter binding a?inity example, polyethylene glycol or albumin, e.g., human serum
for forming protein complexes, (4) alter binding a?inities, albumin), phosphorylation, and/or glycosylation. Unless oth
and (4) confer or modify other physicochemical or functional erWise indicated, the term “antibody” includes, in addition to
properties. Analogs include muteins of a polypeptide. For antibodies comprising tWo full-length heavy chains and tWo
example, single or multiple amino acid substitutions (e.g., full-length light chains, derivatives, variants, fragments, and
conservative amino acid substitutions) may be made in the muteins thereof, examples of Which are described beloW.
naturally occurring sequence (e.g., in the portion of the [0039] An “antigen binding protein” is a protein compris
polypeptide outside the domain(s) forming intermolecular ing a portion that binds to an antigen and, optionally, a scaf
contacts). Consensus sequences can be used to select amino fold or frameWork portion that alloWs the antigen binding
acid residues for substitution; those of skill in the art recog portion to adopt a conformation that promotes binding of the
niZe that additional amino acid residues may also be substi antigen binding protein to the antigen. Examples of antigen
tuted. binding proteins include antibodies, antibody fragments
[0035] A “conservative amino acid substitution” is one that (e.g., an antigen binding portion of an antibody), antibody
does not substantially change the structural characteristics of derivatives, and antibody analogs. The antigen binding pro
the parent sequence (e.g., a replacement amino acid should tein can comprise, for example, an alternative protein scaffold
not tend to break a helix that occurs in the parent sequence, or or arti?cial scaffold With grafted CDRs or CDR derivatives.
disrupt other types of secondary structure that characteriZe Such scaffolds include, but are not limited to, antibody-de
the parent sequence or are necessary for its functionality). rived scaffolds comprising mutations introduced to, for
Examples of art-recognized polypeptide secondary and ter example, stabiliZe the three-dimensional structure of the anti
tiary structures are described in Proteins, Structures and gen binding protein as Well as Wholly synthetic scaffolds
Molecular Principles (Creighton, Ed., W. H. Freeman and comprising, for example, a biocompatible polymer. See, for
Company, NeW York (1984)); Introduction to Protein Struc example, Korndorfer et al., 2003, Proteins: Structure, Func
ture (C. Branden and J. TooZe, eds., Garland Publishing, NeW tion, and Bioinformatics, Volume 53, Issue 1:121-129; Roque
York, N.Y. (1991)); and Thornton et at. Nature 354:105 et al., 2004, Biotechnol. Prog. 20:639-654. In addition, pep
(1991), Which are each incorporated herein by reference. tide antibody mimetics (“PAMs”) can be used, as Well as
[0036] The present invention also provides non-peptide scaffolds based on antibody mimetics utiliZing ?bronection
analogs of alpha4beta7 binding polypeptides. Non-peptide components as a scaffold.
analogs are commonly used in the pharmaceutical industry as [0040] An antigen binding protein can have, for example,
drugs With properties analogous to those of the template the structure of a naturally occurring immunoglobulin. An
peptide. These types of non-peptide compound are termed “immunoglobulin” is a tetrameric molecule. In a naturally
“peptide mimetics” or “peptidomimetics,” see, for example, occurring immuno globulin, each tetramer is composed of tWo
Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Fre identical pairs of polypeptide chains, each pair having one
idinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. “light” (about 25 kDa) and one “heavy” chain (about 50-70
30:1229 (1987), Which are incorporated herein by reference. kDa). The amino-terminal portion of each chain includes a
Peptide mimetics that are structurally similar to therapeuti variable region of about 100 to 110 or more amino acids
cally useful peptides may be used to produce an equivalent primarily responsible for antigen recognition. The carboxy
therapeutic or prophylactic effect. Generally, peptidomimet terminal portion of each chain de?nes a constant region pri
ics are structurally similar to a paradigm polypeptide (i.e., a marily responsible for effector function. Human light chains
polypeptide that has a desired biochemical property or phar are classi?ed as kappa or lambda light chains. Heavy chains
macological activity), such as a human antibody, but have one are classi?ed as mu, delta, gamma, alpha, or epsilon, and
or more peptide linkages optionally replaced by a linkage de?ne the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE,
selected from the group consisting of: 4CH2NHi, respectively. Within light and heavy chains, the variable and
iCHZSi, 4CH:CH-(cis and trans), 4COCH2i, ‘CH constant regions are joined by a “J” region of about 12 or more
(OH)CH2i, and ‘CH2 SOi, by methods Well knoWn in the amino acids, With the heavy chain also including a “D” region
art. Systematic substitution of one or more amino acids of a of about 10 more amino acids. See generally, Fundamental
consensus sequence With a D-amino acid of the same type Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.
(e.g., D-lysine in place of L-lysine) may also be used to (1989)) (incorporated by reference in its entirety for all pur
generate more stable peptides. In addition, constrained pep poses). The variable regions of each light/heavy chain pair
tides comprising a consensus sequence or a substantially form the antibody binding site such that an intact immuno
identical consensus sequence variation may be generated by globulin has tWo binding sites.
methods knoWn in the art (RiZo and Gierasch Ann. Rev. [0041] The variable regions of naturally occurring immu
Biochem. 61 :387 (1992), incorporated herein by reference), noglobulin chains exhibit the same general structure of rela
for example, by adding internal cysteine residues capable of tively conserved frameWork regions (FR) joined by three
forming intramolecular disul?de bridges Which cycliZe the hypervariable regions, also called complementarity deter
peptide. mining regions or CDRs. From N-terminus to C-terminus,
US 2013/0302354 A1 Nov. 14, 2013
both light and heavy chains comprise the domains PR1, Poljak et al., 1994, Structure 2: 1 121-23). Ifthe tWo polypep
CDR1, PR2, CDR2, PR3, CDR3 and PR4. The assignment of tide chains of a diabody are identical, then a diabody resulting
amino acids to each domain is in accordance With the de?ni from their pairing Will have tWo identical antigen binding
tions of Kabat et al. in Sequences ofProZeins oflmmunologi sites. Polypeptide chains having different sequences can be
cal Interest, 5”’ Ed., US Dept. of Health and Human Services, used to make a diabody With tWo different antigen binding
PHS, NIH, NIH. Publication no. 91-3242, 1991. Other num sites. Similarly, triabodies and tetrabodies are antibodies
bering systems for the amino acids in immunoglobulin chains comprising three and four polypeptide chains, respectively,
include IMGTO (the international ImMunoGeneTics infor and forming three and four antigenbinding sites, respectively,
mation system; Lefranc et al, Dev. Comp. Immunol. 29:185 Which can be the same or different.
203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. [0046] Complementarity determining regions (CDRs) and
309(3):657-670; 2001). frameWork regions (PR) of a given antibody may be identi?ed
[0042] Antibodies can be obtained from sources such as using the system described by Kabat et al. supra; Lefranc et
serum or plasma that contain immunoglobulins having varied al., supra and/ or Honegger and Pluckthun, supra. One or more
antigenic speci?city. If such antibodies are subjected to a?in CDRs may be incorporated into a molecule either covalently
ity puri?cation, they can be enriched for a particular antigenic or noncovalently to make it an antigen binding protein. An
speci?city. Such enriched preparations of antibodies usually antigen binding protein may incorporate the CDR(s) as part
are made of less than about 10% antibody having speci?c of a larger polypeptide chain, may covalently link the CDR(s)
binding activity for the particular antigen. Subjecting these to another polypeptide chain, or may incorporate the CDR(s)
preparations to several rounds of a?inity puri?cation can noncovalently. The CDRs permit the antigen binding protein
increase the proportion of antibody having speci?c binding to speci?cally bind to a particular antigen of interest.
activity for the antigen. Antibodies prepared in this manner [0047] An antigen binding protein may have one or more
are often referred to as “monospeci?c.” Monospec?c anti binding sites. If there is more than one binding site, the
body preparations can be made up of about 10%, 20%, 30%, binding sites may be identical to one another or may be
40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, different. For example, a naturally occurring human immu
99%, or 99.9% antibody having speci?c binding activity for noglobulin typically has tWo identical binding sites, While a
the particular antigen. “bispeci?c” or “bifunctional” antibody has tWo different
[0043] An “antibody” refers to an intact immunoglobulin binding sites.
or to an antigen binding portion thereof that competes With [0048] The term “human antibody” includes all antibodies
the intact antibody for speci?c binding, unless otherwise that have one or more variable and constant regions derived
speci?ed. Antigen binding portions may be produced by from human immunoglobulin sequences. In one embodi
recombinant DNA techniques or by enZymatic or chemical ment, all of the variable and constant domains are derived
cleavage of intact antibodies. Antigen binding portions from human immunoglobulin sequences (a fully human anti
include, inter alia, Pab, Pab', P(ab')2, Pv, domain antibodies body). These antibodies may be prepared in a variety of Ways,
(dAbs), and complementarity determining region (CDR) examples of Which are described beloW, including through
fragments, variable region fragments, single-chain antibodies the immunization With an antigen of interest of a mouse that
(scPv), chimeric antibodies, diabodies, triabodies, tetrabod is genetically modi?ed to express antibodies derived from
ies, and polypeptides that contain at least a portion of an human heavy and/ or light chain-encoding genes.
immunoglobulin that is su?icient to confer speci?c antigen [0049] A humaniZed antibody has a sequence that differs
binding to the polypeptide. from the sequence of an antibody derived from a non-human
[0044] A Pab fragment is a monovalent fragment having species by one or more amino acid substitutions, deletions,
the VL, VH, CL and CH1 domains; a P(ab')2 fragment is a and/or additions, such that the humaniZed antibody is less
bivalent fragment having tWo Fab fragments linked by a dis likely to induce an immune response, and/or induces a less
ul?de bridge at the hinge region; a Pd fragment has theVH and severe immune response, as compared to the non-human
C H1 domains; an Pv fragment has the VL and VH domains of species antibody, When it is administered to a human subject.
a single arm of an antibody; and a dAb fragment has a VH In one embodiment, certain amino acids in the frameWork and
domain, aVL domain, or an antigen-binding fragment of aVH constant domains of the heavy and/or light chains of the
orVL domain (US. Pat. Nos. 6,846,634, 6,696,245, US appli non-human species antibody are mutated to produce the
cation Pub. Ser. Nos. 05/0202512, 04/0202995, 04/0038291, humaniZed antibody. In another embodiment, the constant
04/0009507, 03/0039958, Ward et al., Nature 341:544-546, domain(s) from a human antibody are fused to the variable
1 989). domain(s) of a non-human species. In another embodiment,
[0045] A single-chain antibody (scPv) is an antibody in one or more amino acid residues in one or more CDR
Which a VL and a VH region are joined via a linker (e.g., a sequences of a non-human antibody are changed to reduce the
synthetic sequence of amino acid residues) to form a continu likely immunogenicity of the non-human antibody When it is
ous protein chain Wherein the linker is long enough to alloW administered to a human subject, Wherein the changed amino
the protein chain to fold back on itself and form a monovalent acid residues either are not critical for immunospeci?c bind
antigen binding site (see, e.g., Bird et al., 1988, Science ing of the antibody to its antigen, or the changes to the amino
242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. acid sequence that are made are conservative changes, such
USA 85:5879-83). Diabodies are bivalent antibodies com that the binding of the humaniZed antibody to the antigen is
prising tWo polypeptide chains, Wherein each polypeptide not signi?cantly Worse than the binding of the non-human
chain comprises VH and VL domains joined by a linker that is antibody to the antigen. Examples of hoW to make humaniZed
too short to alloW for pairing betWeen tWo domains on the antibodies may be found in US. Pat. Nos. 6,054,297, 5,886,
same chain, thus alloWing each domain to pair With a comple 152 and 5,877,293.
mentary domain on another polypeptide chain (see, e. g., Hol [0050] The term “chimeric antibody” refers to an antibody
liger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and that contains one or more regions from one antibody and one
US 2013/0302354 A1 Nov. 14, 2013
or more regions from one or more other antibodies. In one example, an antibody that is alpha4beta7 heterodimer speci?c
embodiment, one or more of the CDRs are derived from a Will bind to alpha4beta7 but not to alpha4beta1 or alphaE
human anti-alpha4beta7 antibody. In another embodiment, beta7.
all of the CDRs are derived from a human anti-alpha4beta7 [0057] Integrins are knoWn to adapt different conforma
antibody. In another embodiment, the CDRs from more than tions, depending on the activation state of the cell(s) express
one human anti-alpha4beta7 antibodies are mixed and ing them and on the presence or absence of certain metal ions.
matched in a chimeric antibody. For instance, a chimeric An integrin in “active” conformation binds to its cognate
antibody may comprise a CDRl from the light chain of a ?rst ligand With higher a?inity than the same integrin in “inactive”
human anti-alpha4beta7 antibody, a CDR2 and a CDR3 from conformation. An antigen binding protein may bind to an
the light chain of a second human anti-alpha4beta7 antibody, integrin in only its active conformation, in only its inactive
and the CDRs from the heavy chain from a third anti conformation, or in both or either conformations. For
alpha4beta7 antibody. Other combinations are possible and example, an alpha4beta7 heterodimer speci?c antigen bind
are included Within the embodiments of the invention. ing protein may bind alpha4beta7 in the presence or absence
[0051] Further, the framework regions may be derived from of the divalent cation manganese2+ (Mn2+), indicating that the
one of the same anti-alpha4beta7 antibodies, from one or antigen binding protein binds both active and inactive
alpah4beta7.
more different antibodies, such as a human antibody, or from
a humaniZed antibody. In one example of a chimeric antibody, [0058] An “antigen binding domain,” “antigen binding
a portion of the heavy and/or light chain is identical With, region,” or “antigen binding site” is a portion of an antigen
homologous to, or derived from an antibody from a particular binding protein that contains amino acid residues (or other
species or belonging to a particular antibody class or subclass, moieties) that interact With an antigen and contribute to the
While the remainder of the chain(s) is/are identical With, antigen binding protein’s speci?city and af?nity for the anti
homologous to, or derived from an antibody (-ies) from gen. For an antibody that speci?cally binds to its antigen, this
another species or belonging to another antibody class or Will include at least part of at least one of its CDR domains.
subclass. Also included are fragments of such antibodies that [0059] An “epitope” is the portion of a molecule that is
exhibit the desired biological activity (i.e., the ability to spe bound by an antigenbinding protein (e.g., by an antibody). An
ci?cally bind alpha4beta7). See, e.g., U.S. Pat. No. 4,816,567 epitope can comprise non-contiguous portions of the mol
and Morrison, 1985, Science 229:1202-07. ecule (e. g., in a polypeptide, amino acid residues that are not
[0052] A “neutralizing antibody” or an “inhibitory anti contiguous in the polypeptide’s primary sequence but that, in
body” is an antibody that inhibits the interaction of the context of the polypeptide’ s tertiary and quaternary struc
alpha4beta7 With MAdCAM-l When an excess of the anti ture, are near enough to each other to be bound by an antigen
alpha4beta7 antibody reduces the amount of interaction by at binding protein).
least about 20% using an assay such as those described herein [0060] The “percent identity” of tWo polynucleotide or tWo
in the Examples. In various embodiments, the antigen bind polypeptide sequences is determined by comparing the
ing protein reduces the interaction of alpha4beta7 With MAd sequences using the GAP computer program (a part of the
CAM-l alpha4beta7 by at least 30%, 40%, 50%, 60%, 70%, GCG Wisconsin Package, version 10.3 (Accelrys, San Diego,
75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%. Calif.)) using its default parameters.
[0053] Fragments or analogs of antibodies can be readily [0061] The terms “polynucleotide, oligonucleotide” and
prepared by those of ordinary skill in the art folloWing the “nucleic acid” are used interchangeably throughout and
teachings of this speci?cation and using techniques Well include DNA molecules (e.g., cDNA or genomic DNA), RNA
knoWn in the art Amino- and carboxy-termini of fragments or molecules (e.g., mRNA), analogs of the DNA or RNA gen
analogs occur near boundaries of functional domains. Struc erated using nucleotide analogs (e.g., peptide nucleic acids
tural and functional domains can be identi?ed by comparison and non-naturally occurring nucleotide analogs), and hybrids
of the nucleotide and/ or amino acid sequence data to public or thereof. The nucleic acid molecule can be single-stranded or
proprietary sequence databases. Computerized comparison double-stranded. In one embodiment, the nucleic acid mol
methods can be used to identify sequence motifs or predicted ecules of the invention comprise a contiguous open reading
protein conformation domains that occur in other proteins of frame encoding an antibody, or a fragment, derivative,
knoWn structure and/ or function. Methods to identify protein mutein, or variant thereof, of the invention.
sequences that fold into a knoWn three-dimensional structure [0062] TWo single-stranded polynucleotides are “the
are knoWn. See, e.g., BoWie et al., 1991, Science 253: 164. complement” of each other if their sequences can be aligned
[0054] A “CDR grafted antibody” is an antibody compris in an anti-parallel orientation such that every nucleotide in
ing one or more CDRs derived from an antibody of a particu one polynucleotide is opposite its complementary nucleotide
lar species or isotype and the frameWork of another antibody in the other polynucleotide, Without the introduction of gaps,
of the same or different species or isotype. and Without unpaired nucleotides at the 5' or the 3' end of
[0055] A “multi-speci?c antibody” is an antibody that rec either sequence. A polynucleotide is “complementary” to
ogniZes more than one epitope on one or more antigens. A anotherpolynucleotide if the tWo polynucleotides can hybrid
subclass of this type of antibody is a “bi-speci?c antibody” iZe to one another under moderately stringent conditions.
Which recogniZes tWo distinct epitopes on the same or differ Thus, a polynucleotide can be complementary to another
ent antigens. polynucleotide Without being its complement.
[0056] An antigen binding protein “speci?cally binds” to [0063] A “vector” is a nucleic acid that can be used to
an antigen (e.g., human alpha4beta7) if it binds to the antigen introduce another nucleic acid linked to it into a cell. One type
With a dissociation constant of 1 nanomolar or less. As used of vector is a “plasmid,” Which refers to a linear or circular
herein, an antigen binding protein is “heterodimer speci?c” if double stranded DNA molecule into Which additional nucleic
it binds to a ?rst heterodimeric integrin but not to other acid segments can be ligated. Another type of vector is a viral
integrins that share one chain With the ?rst integrin. For vector (e.g., replication defective retroviruses, adenoviruses
US 2013/0302354 A1 Nov. 14, 2013
and adeno-associated viruses), wherein additional DNA seg environmental in?uence, such progeny may not, in fact, be
ments can be introduced into the viral genome. Certain vec identical to the parent cell, but are still included Within the
tors are capable of autonomous replication in a host cell into scope of the term as used herein.
Which they are introduced (e.g., bacterial vectors comprising
a bacterial origin of replication and episomal mammalian Antigen Binding Proteins
vectors). Other vectors (e. g., non-episomal mammalian vec
[0066] In one aspect, the present invention provides antigen
tors) are integrated into the genome of a host cell upon intro binding proteins (e.g., antibodies, antibody fragments, anti
duction into the host cell, and thereby are replicated along
body derivatives, antibody muteins, and antibody variants)
With the host genome. An “expression vector” is a type of
that bind to alpha4beta7, e.g., human alpha4beta7.
vector that can direct the expression of a chosen polynucle
[0067] Antigen binding proteins in accordance With the
otide.
present invention include antigen binding proteins that inhibit
[0064] A nucleotide sequence is “operably linked” to a a biological activity of alpha4beta7. Examples of such bio
regulatory sequence if the regulatory sequence affects the logical activities include binding of alpha4beta7 to MAd
expression (e.g., the level, timing, or location of expression) CAM-1, and adhesion betWeen cells expressing alpha4beta7
of the nucleotide sequence. A “regulatory sequence” is a and those expressing MAdCAM-l. Other biological activi
nucleic acid that affects the expression (e.g., the level, timing, ties include those mediated by alpha4beta7 in vivo, such as
or location of expression) of a nucleic acid to Which it is traf?cking or homing; in particular, alpha4beta7 is involved
operably linked. The regulatory sequence can, for example, in the tra?icking of lymphocytes to the gut, Increased MAd
exert its effects directly on the regulated nucleic acid, or CAM-1 expression in the in?amed gut enhances recruitment
through the action of one or more other molecules (e.g., of alpha4beta7 expressing lymphocytes to the gut, Where
polypeptides that bind to the regulatory sequence and/ or the aberrant lymphocyte activation augments in?ammatory
nucleic acid). Examples of regulatory sequences include pro response and tissue damage.
moters, enhancers and other expression control elements [0068] Different antigen binding proteins may bind to dif
(e.g., polyadenylation signals). Further examples of regula
ferent domains or epitopes of alpha4beta7 or act by different
tory sequences are described in, for example, Goeddel, 1990, mechanisms of action. Examples include but are not limited
Gene Expression Technology: Methods in EnZymology 185, to antigen binding proteins that interfere With the ability of
Academic Press, San Diego, Calif. and Baron et al., 1995, alpha4beta7 to bind MAdCAM-l or that inhibit cellular inter
Nucleic Acids Res. 23:3605-06. actions such as adhesion betWeen cells expressing
[0065] A “host cell” is a cell that can be used to express a alpha4beta7 and cells expressing MAdCAM-l. The site of
nucleic acid, e.g., a nucleic acid of the invention. A host cell action may be, for example, intracellular (e. g., by interfering
can be a prokaryote, for example, E. coli, or it can be a With an intracellular signaling cascade) or extracellular. An
eukaryote, for example, a single-celled eukaryote (e.g., a antigen binding protein need not completely inhibit
yeast or other fungus), a plant cell (e.g., a tobacco or tomato alpha4beta7 induced activity to ?nd use in the present inven
plant cell), an animal cell (e.g., a human cell, a monkey cell, tion; rather, antigen binding proteins that reduce a particular
a hamster cell, a rat cell, a mouse cell, or an insect cell) or a activity of alpha4beta7 are contemplated for use as Well.
hybridoma. Examples of host cells include the COS-7 line of (Discussions herein of particular mechanisms of action for
monkey kidney cells (ATCC CRL 1651) (see GluZman et al., alpha4beta7 -binding antigen binding proteins in treating par
1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL ticular diseases are illustrative only, and the methods pre
163), Chinese hamster ovary (CHO) cells or their derivatives sented herein are not bound thereby.)
such as Veggie CHO and related cell lines Which groW in [0069] Other derivatives of anti-alpha4beta7 antibodies
serum-free media (see Rasmussen et al., 1998, Cytotechnol Within the scope of this invention include covalent or aggre
ogy 28:31) or CHO strain DX-Bll, Which is de?cient in gative conjugates of anti-alpha4beta7 antibodies, or frag
DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA ments thereof, With other proteins or polypeptides, such as by
77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the expression of recombinant fusion proteins comprising heter
CV1/EBNA cell line derived from the African green monkey ologous polypeptides fused to the N-terminus or C-terminus
kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., of an anti-alpha4beta7 antibody polypeptide. For example,
1991, EMBO J. 10:2821), human embryonic kidney cells the conjugated peptide may be a heterologous signal (or
such as 293, 293 EBNA or MSR 293, human epidermal A431 leader) polypeptide, e.g., the yeast alpha-factor leader, or a
cells, human Colo205 cells, other transformed primate cell peptide such as an epitope tag. Antigen binding protein-con
lines, normal diploid cells, cell strains derived from in vitro taining fusion proteins can comprise peptides added to facili
culture of primary tissue, primary explants, HL-60, U937, tate puri?cation or identi?cation of antigen binding protein
HaK or Jurkat cells. Typically, a host cell is a cultured cell that (e.g., poly-His). An antigen binding protein also can be linked
can be transformed or transfected With a polypeptide-encod to the FLAG® peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
ing nucleic acid, Which can then be expressed in the host cell. (DYKDDDDK) (SEQ ID NO:62) as described in Hopp et al.,
The phrase “recombinant host cell” can be used to denote a Bio/Technology 6:1204, 1988, and US. Pat. No. 5,011,912.
host cell that has been transformed or transfected With a The FLAG® peptide is highly antigenic and provides an
nucleic acid to be expressed. A host cell also can be a cell that epitope reversibly bound by a speci?c monoclonal antibody
comprises the nucleic acid but does not express it at a desired (mAb), enabling rapid assay and facile puri?cation of
level unless a regulatory sequence is introduced into the host expressed recombinant protein. Reagents useful for prepar
cell such that it becomes operably linked With the nucleic ing fusion proteins in Which the FLAG® peptide is fused to a
acid. It is understood that the term host cell refers not only to given polypeptide are commercially available (Sigma-Ald
the particular subject cell but also to the progeny or potential rich, St. Louis Mo.).
progeny of such a cell. Because certain modi?cations may [0070] Oligomers that contain one or more antigen binding
occur in succeeding generations due to, e. g., mutation or proteins may be employed as alpha4beta7 antagonists. Oli