U.S. patent application number 13/439413 was filed with the patent office on 2013-04-11 for kim-1 antibodies for treatment of th2-mediated conditions.
This patent application is currently assigned to BIOGEN IDEC MA INC.. The applicant listed for this patent is Veronique Bailly, Alexey Lugovskoy, Patricia McCoon, Paul Rennert. Invention is credited to Veronique Bailly, Alexey Lugovskoy, Patricia McCoon, Paul Rennert.
Application Number | 20130089539 13/439413 |
Document ID | / |
Family ID | 36648475 |
Filed Date | 2013-04-11 |
United States Patent
Application |
20130089539 |
Kind Code |
A1 |
Rennert; Paul ; et
al. |
April 11, 2013 |
KIM-1 ANTIBODIES FOR TREATMENT OF TH2-MEDIATED CONDITIONS
Abstract
Compositions and methods for treating Th2- and Th1-mediated
disease are provided.
Inventors: |
Rennert; Paul; (Holliston,
MA) ; McCoon; Patricia; (Sudbury, MA) ;
Bailly; Veronique; (Boxborough, MA) ; Lugovskoy;
Alexey; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rennert; Paul
McCoon; Patricia
Bailly; Veronique
Lugovskoy; Alexey |
Holliston
Sudbury
Boxborough
Woburn |
MA
MA
MA
MA |
US
US
US
US |
|
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
36648475 |
Appl. No.: |
13/439413 |
Filed: |
April 4, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11817638 |
Dec 22, 2009 |
8206705 |
|
|
PCT/US2006/007441 |
Mar 2, 2006 |
|
|
|
13439413 |
|
|
|
|
60657789 |
Mar 2, 2005 |
|
|
|
Current U.S.
Class: |
424/132.1 ;
424/139.1; 530/387.9 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2803 20130101; C07K 2317/74 20130101; A61K 39/39533
20130101; A61P 11/02 20180101; A61P 37/04 20180101; C07K 2317/34
20130101; C07K 2317/73 20130101; A61P 37/00 20180101; A61P 17/00
20180101; A61P 37/08 20180101; A61P 11/06 20180101; A61P 43/00
20180101 |
Class at
Publication: |
424/132.1 ;
424/139.1; 530/387.9 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating a Th2-mediated disorder in a mammal, the
method comprising administering to the mammal an antibody, or
antigen-binding fragment thereof, that binds the stalk region of
KIM-1.
2. The method of claim 1, wherein the mammal is a human.
3. The method of claim 1, wherein the disorder is atopy.
4. The method of claim 1, wherein the disorder is asthma.
5. The method of claim 1, wherein the antibody binds an epitope at
least partially contained in the peptide
DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID
NO:1).
6. The method of claim 1, wherein the antibody is a humanized or
fully human monospecific antibody.
7. The method of claim 1, wherein the antibody is a humanized or
fully human monospecific antibody and wherein the antibody binds an
epitope at least partially contained in the peptide
DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID
NO:1)
8. The method of claim 1, wherein a full-length antibody is
administered.
9. The method of claim 1, wherein a full length antibody is
administered and wherein the antibody binds an epitope at least
partially contained in the peptide
DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID
NO:1)
10. The method of claim 1, wherein an antigen-binding fragment of
an antibody is administered.
11. The method of claim 1, wherein an antigen-binding fragment of
an antibody is administered and wherein the antibody binds an
epitope at least partially contained in the peptide
DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID
NO:1).
12-13. (canceled)
14. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof is administered in combination with a second
therapeutic agent for the disorder.
15-17. (canceled)
18. An isolated antibody, or antigen binding fragment thereof, that
specifically binds the stalk region of KIM-1, wherein the antibody
does not inhibit shedding of KIM-1 from E293 cells in culture.
19. The method of claim 1, wherein the antibody binds to a peptide
having a sequence selected from the group consisting of: (a) amino
acids 262-270 of SEQ ID NO:1, (b) amino acids 260-269 of SEQ ID
NO:1, (c) amino acids 257-270 of SEQ ID NO:1, (d) amino acids
252-270 of SEQ ID NO:1, (e) amino acids 236-250 of SEQ ID NO:1, and
(f) amino acids 236-258 of SEQ ID NO:1.
20-24. (canceled)
25. A method of treating a Th1-mediated disorder or reducing a
pathogenic Th1 response in a mammal, the method comprising
administering to the mammal an antibody, or antigen-binding
fragment thereof, that binds the sequence
VATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTT of human KIM-1 (amino acids
200-235 of SEQ ID NO:1) or an epitope overlapping said
sequence.
26-31. (canceled)
32. A method of treating a Th2-mediated disorder in a mammal, the
method comprising administering to the mammal an antibody, or
antigen-binding fragment thereof, that binds the sialic acid
binding motif of KIM-1.
33. The method of claim 32, wherein the antibody binds at least
partly to an epitope contained within or overlapping a peptide
having the sequence GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107
of SEQ ID NO:1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID
NO:1).
34. The method of claim 32, wherein the antibody binds a linear
epitope of contiguous amino acid residues of
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1) or
RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO:1).
35. The method of claim 32, wherein the antibody binds a structural
epitope to which one or both of the sequences
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1) and
RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO:1) contribute.
36. The method of claim 32, wherein the antibody protects amino
acids 81-107 of SEQ ID NO:1.
37-46. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Atopic diseases such as allergic asthma and atopic
dermatitis are thought to involve a pathogenic shift to predominant
Th2 immunity (Umetsu et al., 2002, Nat. Immunol. 3:715-20.
[0002] In the asthma setting Th2 cytokine production drives
eosinophil influx into the lung, eosinophil activation, IgE
production and IgE mediated mast cell activation and degranulation,
and mononuclear cell accumulation in lung interstitial space, where
T cells and activated granulocytes continue to secrete Th2
cytokines, chemokines, and effector molecules, thereby fostering
continued lung inflammation. The TAPR locus containing the KIM gene
family has been implicated in the development of atopic
inflammation in mouse, and KIM-1 allelic variation has been
associated with the incidence of atopy in patient population
analyses (McIntire et al., 2001, Nat Immunol 2:1109-16; McIntire et
al., 2003, Nature 425:576).
SUMMARY OF THE INVENTION
[0003] The invention is based, at least in part, on the discovery
that agents, such as antibodies, that bind particular regions of
KIM-1, can differentially modulate Th1 and/or Th2-mediated
immunity. For example, agents that bind the stalk region of KIM-1
or the sialic acid binding regions of KIM-1 can modulate the
expression of Th2 cytokines and can be used to treat a Th2 mediated
disorder, e.g., asthma; and agents that bind particular epitopes
within the mucin region of KIM-1 can reduce a pathogenic Th1
response and can be used to treat a Th1-mediated disorder, e.g.,
inflammatory disorders or autoimmune disorders such as inflammatory
bowel disease (IBD), Crohn's disease, multiple sclerosis, diabetes,
rheumatoid arthritis, psoriasis, acute graft versus host disease
(GVHD), transplant, pancreatitis, delayed type hypersensitivity
(DTH). Compositions and methods useful in the treatment of Th2 and
Th1 mediated disorders are provided.
[0004] In one aspect, the invention provides methods for treating
Th2-mediated conditions, e.g., asthma (particularly allergic
asthma), allergic rhinitis, allergy, eczema, and other atopic
conditions. The methods include administering to a mammal,
preferably a human, having a Th2-mediated condition, an agent that
binds the stalk region of KIM-1 or the sialic acid binding motif of
KIM-1. For example, the method can include administering a
pharmaceutical composition containing a monospecific antibody,
e.g., a monoclonal antibody (or antigen-binding fragment thereof)
that binds the stalk region of KIM-1 or the sialic acid binding
motif of KIM-1, in an amount and for a time sufficient to treat the
condition. The stalk region of KIM-1 is identified herein as a
charged domain containing highly conserved N-linked glycosylation
sites, present between the mucin domain and the transmembrane
domain of KIM-1. The stalk region of human KIM-1 and the sialic
acid binding motif are shown in FIG. 1. It is understood that the
N- and C-termini of these regions as defined herein are approximate
and may contain a few (e.g., 1, 2 or 3) more or fewer contiguous
residues from the KIM-1 sequence.
[0005] In one embodiment, the agent is an antibody that binds to
the human KIM-1 stalk region. For example, the antibody binds a
peptide having the sequence DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK
(amino acids 236-269 of SEQ ID NO:1). In one embodiment, the
antibody binds to a peptide having the sequence LLTANTTKG (amino
acids 262-270 of SEQ ID NO:1), HSLLTAN'TTKG (amino acids 260-269 of
SEQ ID NO:1), FLEHSLLTANTTKG (amino acids 257-270 of SEQ ID NO:1)
or NQTQLFLEHSLLTANTTKG (amino acids 252-270 of SEQ ID NO:1). In
other embodiments, the antibody binds to a peptide having the
sequence of amino acid 236-250 or 236-258 of SEQ ID NO:1.
[0006] In one embodiment, the antibody binds to the sialic acid
binding motif of KIM-1. For example, the antibody binds, at least
partly, to an epitope contained within or overlapping a peptide
having the sequence GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107
of SEQ ID NO:1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID
NO:1). In one embodiment, the antibody binds reduced and
non-reduced protein, i.e., it binds a linear epitope of at least 4,
5, 6, 7, 8, 9, or 10 contiguous amino acid residues of
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1) or
RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO:1).
[0007] In another embodiment, the antibody binds a structural
epitope to which one or both of the sequences
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1) and
RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO:1) contribute. In
one embodiment, the epitope may be a structural epitope contained
in the human KIM-1 sequence corresponding to an 8 kDa TPCK trypsin
fragment of recombinant mouse KIM-1 IgV-human IgG1 Fc fusion.
[0008] In one embodiment, the antibody interferes with one or more
of residues R86, W92, and F93 of SEQ ID NO:1, which are required
for sialic acid binding.
[0009] While it is understood that the methods described herein are
not bound by any particular mechanism or theory, the antibody may
have one or more of the following characteristics: (a) it
interferes with an interaction of the sialic acid biding motif on
the IgV domain of KIM-1 with the carbohydrates displayed on one or
more N-glycosylation sites of the stalk region of KIM-1, (b) it
binds or sterically hinders one or more N-linked glycosylation
sites in the stalk region, (c) it inhibits down-regulation of KIM-1
signaling, (d) it is an agonist antibody, e.g., upon binding it
promotes or increases downstream signaling through KIM-1, (e) it
blocks multimerization of KIM-1, (f) it binds or sterically hinders
interaction of the stalk region with a co-receptor or ligand to
disrupt normal function, (g) it interferes with an interaction of
the sialic acid biding motif on the IgV domain of KIM-1 with the
carbohydrates displayed on one or more O-glycosylation sites of the
mucin region adjacent to the stalk region of KIM-1, (h) it alters
structural features of the Ig-domain so as to change protein
conformation, e.g., by altering or interfering with disulfide or
hydrogen bonding, (i) it alters structural or functional features
of the KIM-11 g-domain so as to change binding to other proteins
such as the KIM-1 ligand.
[0010] In one embodiment, the antibody does not inhibit shedding of
KIM-1 from the cell surface, e.g., it does not inhibit shedding of
KIM-1 from 293 Ebna (E293) cells in culture.
[0011] In preferred embodiments the antibody is monospecific, e.g.,
the antibody is a monoclonal antibody, e.g., a humanized or fully
human monoclonal antibody or antigen-binding fragment thereof.
[0012] In one embodiment, the condition is allergic asthma. In this
embodiment, the method optionally also includes identifying a
subject who is at risk for, or has, allergic asthma. Optionally,
the method also includes evaluating a symptom of asthma in the
subject, e.g., IgE levels, airway hyperresponsiveness, coughing,
wheezing, chest tightness, dyspnea, airway smooth muscle
contraction, bronchial mucus secretion, inflammation, vasodilation,
recruitment of inflammatory cells (e.g., neutrophils, monocytes,
macrophages, lymphocytes, eosinophils), goblet cell hyperplasia,
release of inflammatory mediators by mast cells or migrating
inflammatory cells. The evaluation step can be performed before,
during and/or after the administration step. The evaluation can be
performed by a physician, other health care provider or by the
subject. The evaluation can be performed one or more times, e.g.,
one or more times after administration, e.g., at least twice during
a one week, one month, two month, three month, six month period
after the administration, or longer.
[0013] In a preferred embodiment, the method includes determining
whether the administration of the agent (or multiple
administrations) reduced the severity or initiation of one or more
symptoms of airway disease in the subject.
[0014] In some embodiments, the antibody is co-administered with a
second agent effective to treat asthma in the subject, e.g., a
corticosteroid, bronchodilator, leukotriene modifier,
anti-inflammatory agent, anti-IgE agent (e.g., anti-IgE antibody,
e.g., omalizumab (Xolair.RTM.)). "Co-administered" or "administered
in combination" means administration at the same time or within an
interval, e.g., a week, such that the effects of the substances on
the patient overlap.
[0015] In another embodiment, the condition is allergy, e.g., food
allergy or seasonal (e.g., pollen) allergy. A diagnosis of allergy
may be made by one or more of: administration of an allergen skin
test; determination of IgE concentration in serum (e.g., IgE>300
ng/ml); and determination of allergen-specific IgE or IgG
antibodies in serum.
[0016] The antibody can be administered in one or more of the
following periods: prior to an atopic subject's exposure to
allergen; after exposure to allergen but prior to the onset of
symptoms; at the time of onset of symptoms; after onset of
symptoms.
[0017] In one embodiment, the agent is administered as a course of
treatment, e.g., in periodic administrations of predetermined
frequency, e.g., daily, weekly, biweekly or monthly. In some
embodiments, an antibody can be administered for a period of time
and/or in an amount sufficient to reduce (e.g., to substantially
reduce) the frequency or severity of episodes of wheezing,
coughing, shortness of breath, or tightness in the chest, e.g.,
over a period of time, e.g., 3 months, 6 months, a year or
more.
[0018] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof, that specifically
binds the stalk region of KIM-1. The antibody does not inhibit
shedding of KIM-1 from E293 cells in culture. In one embodiment,
the antibody binds to the human KIM-1 stalk region. For example,
the antibody binds a peptide having the sequence
DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID
NO:1). In one embodiment, the antibody binds to a peptide having
the sequence LLTANTTKG (amino acids 262-270 of SEQ ID NO:1),
HSLLTANTTKG (amino acids 260-270 of SEQ ID NO:1), FLEHSLLTANTTKG
(amino acids 257-270 of SEQ ID NO:1) or NQTQLFLEHSLLTANTTKG (amino
acids 252-270 of SEQ ID NO:1). In other embodiments, the antibody
binds to a peptide having the sequence of amino acid 241-254,
242-258, 242-255 of SEQ ID NO:1.
[0019] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof that specifically
binds to a sialic acid binding motif of KIM-1. For example, the
antibody binds, at least partly, to an epitope contained within or
overlapping the peptide having the sequence
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1) or
RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO:1). In one
embodiment, the antibody binds reduced and non-reduced protein,
i.e., it binds a linear epitope of at least 4, 5, 6, 7, 8, 9, or 10
contiguous amino acid residues of GVYCCRVEHRGWFNDMKITVSLEIVPP
(amino acids 81-107 of SEQ ID NO:1) or RGSCSLFTCQNGIV (amino acids
29-42 of SEQ ID NO:1). In another embodiment, the antibody binds a
structural epitope to which one or both of the sequences
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1) and
RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO:1) contribute. In
some embodiments, the epitope is a structural epitope contained in
a human KIM-1 region corresponding to an 8 kDa TPCK trypsin
fragment of recombinant mouse KIM-1 IgV-human IgG1 Fc fusion. In
one embodiment, the antibody interferes with one or more of
residues R86, W92, and F93 of SEQ ID NO:1, which are required for
sialic acid binding.
[0020] The isolated antibody may have one or more of the following
characteristics: (a) it interferes with an interaction of the
sialic acid biding motif on the IgV domain of KIM-1 with the
carbohydrates displayed on one or more N-glycosylation sites of the
stalk region of KIM-1, (b) it binds or sterically hinders one or
both N-linked glycosylation sites in the stalk region, (c) it
inhibits down-regulation of KIM-1 signaling, (d) it is an agonist
antibody, e.g., upon binding it promotes or increases downstream
signaling through KIM-1, (e) it blocks multimerization of KIM-1,
(f) it binds or sterically hinders interaction of the stalk region
with a co-receptor or ligand to disrupt normal function, (g) it
interferes with an interaction of the sialic acid biding motif on
the IgV domain of KIM-1 with the carbohydrates displayed on one or
more O-glycosylation sites of the mucin region adjacent to the
stalk region of KIM-1, (h) it alters structural features of the
Ig-domain so as to change protein conformation, e.g., by altering
or interfering with disulfide or hydrogen bonding, (i) it alters
structural or functional features of the KIM-11 g-domain so as to
change binding to other proteins such as the KIM-1 ligand.
[0021] In another aspect, the invention features a method of
treating a Th1-mediated condition, e.g., a condition characterized
by a pathogenic or increased Th1 response. Such conditions include
inflammatory and/or autoimmune disorders such as inflammatory bowel
disease (IBD), Crohn's disease, multiple sclerosis, diabetes,
rheumatoid arthritis, psoriasis, acute graft versus host disease
(GVHD), transplant, pancreatitis, delayed type hypersensitivity
(DTH). The method includes administering to a mammal, preferably a
human, having a Th1-mediated condition, an agent, e.g., an
antibody, that binds an epitope contained in the sequence
VATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTT (residues 200-235 of SEQ ID
NO:1). For example, the method can include administering a
pharmaceutical composition containing a monospecific antibody,
e.g., a monoclonal antibody (or antigen-binding fragment thereof)
that binds the specified region of KIM-1, in an amount and for a
time sufficient to treat the condition. The specified region of
KIM-1 is found in an alternatively spliced variant of KIM-1 in the
mouse. It is understood that the N- and C-termini of this region as
defined herein are approximate and may contain a few (e.g., 1 or 2)
more or fewer contiguous residues from the KIM-1 sequence.
[0022] In one embodiment, the antibody binds reduced and
non-reduced protein.
[0023] In preferred embodiments the antibody is monospecific, e.g.,
the antibody is a monoclonal antibody, e.g., a humanized or fully
human monoclonal antibody or antigen-binding fragment thereof.
[0024] In one embodiment, the condition is inflammatory bowel
disease (IBD), Chron's disease, rheumatoid arthritis, psoriasis,
acute graft versus host disease (GVHD), transplant, pancreatitis,
or delayed type hypersensitivity (DTH). The method optionally also
includes identifying a subject who is at risk for, or has, any of
the listed conditions.
[0025] In a preferred embodiment, the method includes determining
whether the administration of the agent (or multiple
administrations) reduced the severity or initiation of one or more
symptoms of the condition the subject.
[0026] In some embodiments, the antibody is co-administered with a
second agent effective to treat the condition in the subject, e.g.,
a corticosteroid or other anti-inflammatory agent, DMARD, anti-TNF
therapy or anti-CD20 therapy. "Co-administered" or "administered in
combination" means administration at the same time or within an
interval, e.g., a week, such that the effects of the substances on
the patient overlap.
[0027] In one embodiment, the agent is administered as a course of
treatment, e.g., in periodic administrations of predetermined
frequency, e.g., daily, weekly, biweekly or monthly. In some
embodiments, an antibody can be administered for a period of time
and/or in an amount sufficient to reduce (e.g., to substantially
reduce) the frequency or severity of symptoms, e.g., over a period
of time, e.g., 3 months, 6 months, a year or more.
[0028] As used herein, the terms "to treat," "treating," and
"treatment" refer to administering a therapy in an amount, manner,
and/or mode effective to improve or ameliorate a symptom or
parameter that characterizes a pathological condition; to reduce
the severity of a symptom or parameter that characterizes a
pathological condition; to prevent, slow or reverse progression of
the pathological condition; or to prevent one or more symptom or
parameter of the pathological condition.
[0029] As used herein, an "agent that binds" a particular domain of
KIM-1 refers to any compound that binds to the specified domain
with a K.sub.d of less than 10.sup.-6 M. An example of a KIM-1
binding agent is a KIM-1 binding protein, e.g., a KIM-1 binding
antibody, preferably a monospecific antibody.
[0030] As used herein the terms "sialic-acid binding region",
"sialic-acid binding motif", and "required for sialic-acid
binding", and variants of those terms, refer to amino acid
residues, amino acid sequences, and amino acid secondary or
tertiary structures that are similar or homologous to those amino
acid residues, amino acid sequences, and amino acid secondary and
tertiary structures identified in the family of sialic-acid-binding
Ig-like lectins (Siglecs) that are required for carbohydrate
binding.
[0031] The term "antibody or antigen binding fragment thereof"
encompasses proteins that include at least one immunoglobulin
variable region, e.g., an amino acid sequence that provides an
immunoglobulin variable domain or immunoglobulin variable domain
sequence, sufficient to specifically bind an antigen. For example,
the term includes an antigen binding protein that has a heavy (H)
chain variable region (abbreviated herein as VH), and a light (L)
chain variable region (abbreviated herein as VL). In another
example, the term includes an antigen binding protein that includes
two heavy (H) chain variable regions and two light (L) chain
variable regions. The term encompasses antigen-binding fragments of
antibodies (e.g., single chain antibodies, Fab fragments, F(ab')2
fragments, Fd fragments, Fv fragments, and dAb fragments) as well
as complete antibodies, e.g., intact immunoglobulins of types IgA,
IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chains
of the immunoglobulin may be of types kappa or lambda. In one
embodiment, the antibody is glycosylated. An antibody can be
functional for antibody-dependent cytotoxicity and/or
complement-mediated cytotoxicity, or may be non-functional for one
or both of these activities. The VH and VL regions can be further
subdivided into regions of hypervariability, termed
"complementarity determining regions" ("CDR"), interspersed with
regions that are more conserved, termed "framework regions" (FR).
The extent of the FR's and CDR's has been precisely defined (see,
Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, US Department of Health and Human
Services, NIH Publication No. 91-3242; and Chothia, C. et al.
(1987) J. Mol. Biol. 196:901-917). Kabat definitions are used
herein. Each VH and VL is typically composed of three CDR's and
four FR's, arranged from amino-terminus to carboxyl-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0032] The foregoing summary and the following description are not
restrictive of the invention as claimed.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 is an annotated polypeptide sequence (SEQ ID NO:1) of
human KIM-1 (without the signal sequence and without the
insertional polymorphism MTTVP) showing the various domains
described herein.
[0034] FIG. 2 is a graph showing the percent of eosinophils (y
axis) present in bronchial lavage fluid (BAL) after aerosol
challenge with OVA and treatment with 3A2.
[0035] FIG. 3 is a graph showing the proliferation of draining
(bronchial) lymph node cells after stimulation ex vivo with the OVA
antigen and treatment with 3A2. Y-axis is tritiated thymidine
incorporation in cpm.
[0036] FIG. 4 is a graph showing the response of draining
(bronchial) lymph node cells to stimulation ex vivo with the OVA
antigen. LN were harvested after OVA aerosol challenge, and then
cultured with OVA ex vivo and treatment with 3A2. Supernatants were
taken from these cultures and analyzed for TH2 cytokine (IL-4, IL-5
and IL-10) production. Y-axis is picagrams/ml.
[0037] FIG. 5 shows binding curves generated for the interaction of
3A2 with the immobilized proteins. MAb 3A2 bound equivalently to
mKIM-1-ECD-Fc (circles), mKIM-1-137-216-Fc (triangles), and
mKIM-1-196-216-Fc (diamonds), but failed to bind to mKIM-1-IgV-Fc
(squares).
[0038] FIG. 6 is a graph showing the percent of eosinophils and
lymphocytes (y axes) present in bronchial lavage fluid (BAL) after
aerosol challenge with OVA and treatment with 4A2.
[0039] FIG. 7 is a graph showing the response of draining
(bronchial) lymph node cells to stimulation ex vivo with the OVA
antigen and treatment with 4A2. LN were harvested after OVA aerosol
challenge, and then cultured with OVA ex vivo. Supernatants were
taken from these cultures and analyzed for Th2 cytokines (IL-4,
IL-5, IL-10 and IL-13) production. Y-axis is picagrams/ml.
[0040] FIG. 8 is a graph showing the percent of eosinophils (y
axis) present in bronchial lavage fluid (BAL) after aerosol
challenge with OVA and therapeutic treatment with 4A2.
[0041] FIG. 9 shows the binding curve of mAb 4A2 to purified KIM-1
proteins.
DETAILED DESCRIPTION OF THE INVENTION
[0042] As described herein, targeting specific regions of KIM-1
with antibody therapy exerts critical control over the expression
of Th2 and Th1 cytokines and provides therapeutic strategies for
treating Th2 mediated diseases and other atopic disorders, and for
Th1 mediated diseases.
[0043] Antibody Generation
[0044] Antibodies described herein (e.g., antibodies that bind to
the stalk region of KIM-1 or to the sialic acid binding motif of
KIM-1) can be generated by immunization, e.g., using an animal, or
by in vitro methods such as phage display. A polypeptide that
includes the target epitope of KIM-1 (e.g., the stalk region of
KIM-1 or to the sialic acid binding motif of KIM-1) can be used as
an immunogen. In other embodiments, a larger portion of the KIM-1
polypeptide, such as the extracellular domain, can be used as an
immunogen and resulting antibodies can be screened for reactivity
to the desired KIM-1 region or domain.
[0045] In one embodiment, the immunized animal contains
immunoglobulin producing cells with natural, human, or partially
human immunoglobulin loci. In one embodiment, the non-human animal
includes at least a part of a human immunoglobulin gene. For
example, it is possible to engineer mouse strains deficient in
mouse antibody production with large fragments of the human Ig
loci. Using the hybridoma technology, antigen-specific monoclonal
antibodies derived from the genes with the desired specificity may
be produced and selected. See, e.g., XenoMouse.TM., Green et al.
Nature Genetics 7:13-21 (1994), US 2003-0070185, U.S. Pat. No.
5,789,650, and WO 96/34096.
[0046] Non-human antibodies to KIM-1 can also be produced, e.g., in
a rodent. The non-human antibody can be humanized, e.g., as
described in U.S. Pat. No. 6,602,503, EP 239 400, U.S. Pat. No.
5,693,761, and U.S. Pat. No. 6,407,213.
[0047] EP 239 400 (Winter et al.) describes altering antibodies by
substitution (within a given variable region) of their
complementarity determining regions (CDRs) for one species with
those from another. CDR-substituted antibodies can be less likely
to elicit an immune response in humans compared to true chimeric
antibodies because the CDR-substituted antibodies contain
considerably less non-human components. (Riechmann et al., 1988,
Nature 332, 323-327; Verhoeyen et al., 1988, Science 239,
1534-1536). Typically, CDRs of a murine antibody substituted into
the corresponding regions in a human antibody by using recombinant
nucleic acid technology to produce sequences encoding the desired
substituted antibody. Human constant region gene segments of the
desired isotype (usually gamma I for CH and kappa for CL) can be
added and the humanized heavy and light chain genes can be
co-expressed in mammalian cells to produce soluble humanized
antibody.
[0048] Queen et al., 1989 and WO 90/07861 have described a process
that includes choosing human V framework regions by computer
analysis for optimal protein sequence homology to the V region
framework of the original murine antibody, and modeling the
tertiary structure of the murine V region to visualize framework
amino acid residues that are likely to interact with the murine
CDRs. These murine amino acid residues are then superimposed on the
homologous human framework. See also U.S. Pat. Nos. 5,693,762;
5,693,761; 5,585,089; and 5,530,101. Tempest et al., 1991,
Biotechnology 9, 266-271, utilize, as standard, the V region
frameworks derived from NEWM and REI heavy and light chains,
respectively, for CDR-grafting without radical introduction of
mouse residues. An advantage of using the Tempest et al. approach
to construct NEWM and REI based humanized antibodies is that the
three dimensional structures of NEWM and REI variable regions are
known from x-ray crystallography and thus specific interactions
between CDRs and V region framework residues can be modeled.
[0049] Non-human antibodies can be modified to include
substitutions that insert human immunoglobulin sequences, e.g.,
consensus human amino acid residues at particular positions, e.g.,
at one or more (preferably at least five, ten, twelve, or all) of
the following positions: (in the FR of the variable domain of the
light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L,
65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and/or (in
the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H,
37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H,
75H, 78H, 91H, 92H, 93H, and/or 103H (according to the Kabat
numbering). See, e.g., U.S. Pat. No. 6,407,213.
[0050] Fully human monoclonal antibodies that bind to desired
regions of KIM-1 can be produced, e.g., using in vitro-primed human
splenocytes, as described by Boerner et al., 1991, J. Immunol.,
147, 86-95. They may be prepared by repertoire cloning as described
by Persson et al., 1991, Proc. Nat. Acad. Sci. USA, 88: 2432-2436
or by Huang and Stollar, 1991, J. Immunol. Methods 141, 227-236;
also U.S. Pat. No. 5,798,230. Large nonimmunized human phage
display libraries may also be used to isolate high affinity
antibodies that can be developed as human therapeutics using
standard phage technology (see, e.g., Vaughan et al, 1996;
Hoogenboom et al. (1998) Immunotechnology 4:1-20; and Hoogenboom et
al. (2000) Immunol Today 2:371-8; US 2003-0232333).
[0051] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence that can fowl the structure of an
immunoglobulin variable domain. For example, the sequence may
include all or part of the amino acid sequence of a
naturally-occurring variable domain. For example, the sequence may
omit one, two or more N- or C-terminal amino acids, internal amino
acids, may include one or more insertions or additional terminal
amino acids, or may include other alterations. In one embodiment, a
polypeptide that includes an immunoglobulin variable domain
sequence can associate with another immunoglobulin variable domain
sequence to form a target binding structure (or "antigen binding
site"), e.g., a structure that interacts with a specified region of
KIM-1.
[0052] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region, to thereby form
a heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains. The heavy and light
immunoglobulin chains can be connected by disulfide bonds. The
heavy chain constant region typically includes three constant
domains, CH1, CH2 and CH3. The light chain constant region
typically includes a CL domain. The variable region of the heavy
and light chains contains a binding domain that interacts with an
antigen. The constant regions of the antibodies typically mediate
the binding of the antibody to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system.
[0053] One or more regions of an antibody can be human, effectively
human, or humanized. For example, one or more of the variable
regions can be human or effectively human. For example, one or more
of the CDRs, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and
LC CDR3, can be human. Each of the light chain CDRs can be human.
HC CDR3 can be human. One or more of the framework regions can be
human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one
embodiment, all the framework regions are human, e.g., derived from
a human somatic cell, e.g., a hematopoietic cell that produces
immunoglobulins or a non-hematopoietic cell. In one embodiment, the
human sequences are germline sequences, e.g., encoded by a germline
nucleic acid. One or more of the constant regions can be human,
effectively human, or humanized. In another embodiment, at least
70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g.,
FR1, FR2, and FR3, collectively, or FR1, FR2, FR3, and FR4,
collectively) or the entire antibody can be human, effectively
human, or humanized. For example, FR1, FR2, and FR3 collectively
can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical to
a human sequence encoded by a human germline segment.
[0054] An "effectively human" immunoglobulin variable region is an
immunoglobulin variable region that includes a sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. An "effectively human" antibody is an antibody that includes
a sufficient number of human amino acid positions such that the
antibody does not elicit an immunogenic response in a normal
human.
[0055] A "humanized" immunoglobulin variable region is an
immunoglobulin variable region that is modified such that the
modified form elicits less of an immune response in a human than
does the non-modified form, e.g., is modified to include a
sufficient number of human framework amino acid positions such that
the immunoglobulin variable region does not elicit an immunogenic
response in a normal human. Descriptions of "humanized"
immunoglobulins include, for example, U.S. Pat. No. 6,407,213 and
U.S. Pat. No. 5,693,762. In some cases, humanized immunoglobulins
can include a non-human amino acid at one or more framework amino
acid positions.
[0056] All or part of an antibody can be encoded by an
immunoglobulin gene or a segment thereof. Exemplary human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 Kd or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes, e.g.,
gamma (encoding about 330 amino acids).
[0057] The term "antigen-binding fragment" of a full length
antibody refers to one or more fragments of a full-length antibody
that retain the ability to specifically bind to a target of
interest. Examples of binding fragments encompassed within the term
"antigen-binding fragment" of a full length antibody include (i) a
Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
including two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR) that retains
functionality. Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules known as single
chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883.
[0058] Antibody Production
[0059] Antibodies can be produced in prokaryotic and eukaryotic
cells. In one embodiment, the antibodies (e.g., scFv's) are
expressed in a yeast cell such as Pichia (see, e.g., Powers et al.
(2001) J Immunol Methods. 251:123-35), Hanseula, or
Saccharomyces.
[0060] In one embodiment, antibodies, particularly full length
antibodies, e.g., IgG's, are produced in mammalian cells. Exemplary
mammalian host cells for recombinant expression include Chinese
Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in
Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,
used with a DHFR selectable marker, e.g., as described in Kaufman
and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines,
e.g., NS0 myeloma cells and SP2 cells, COS cells, K562, and a cell
from a transgenic animal, e.g., a transgenic mammal. For example,
the cell is a mammary epithelial cell.
[0061] In addition to the nucleic acid sequence encoding the
immunoglobulin domain, the recombinant expression vectors may carry
additional nucleic acid sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). Exemplary selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0062] In an exemplary system for recombinant expression of an
antibody (e.g., a full length antibody or an antigen-binding
portion thereof), a recombinant expression vector encoding both the
antibody heavy chain and the antibody light chain is introduced
into dhfr-CHO cells by calcium phosphate-mediated transfection.
Within the recombinant expression vector, the antibody heavy and
light chain genes are each operatively linked to enhancer/promoter
regulatory elements (e.g., derived from SV40, CMV, adenovirus and
the like, such as a CMV enhancer/AdMLP promoter regulatory element
or an SV40 enhancer/AdMLP promoter regulatory element) to drive
high levels of transcription of the genes. The recombinant
expression vector also carries a DHFR gene, which allows for
selection of CHO cells that have been transfected with the vector
using methotrexate selection/amplification. The selected
transformant host cells are cultured to allow for expression of the
antibody heavy and light chains and intact antibody is recovered
from the culture medium. Standard molecular biology techniques are
used to prepare the recombinant expression vector, to transfect the
host cells, to select for transformants, to culture the host cells,
and to recover the antibody from the culture medium. For example,
some antibodies can be isolated by affinity chromatography with a
Protein A or Protein G.
[0063] Antibodies may also include modifications, e.g.,
modifications that alter Fc function, e.g., to decrease or remove
interaction with an Fc receptor or with Clq, or both. For example,
the human IgG1 constant region can be mutated at one or more
residues, e.g., one or more of residues 234 and 237, e.g.,
according to the numbering in U.S. Pat. No. 5,648,260. Other
exemplary modifications include those described in U.S. Pat. No.
5,648,260.
[0064] For some antibodies that include an Fc domain, the antibody
production system may be designed to synthesize antibodies in which
the Fc region is glycosylated. For example, the Fc domain of IgG
molecules is glycosylated at asparagine 297 in the CH2 domain. This
asparagine is the site for modification with biantennary-type
oligosaccharides. This glycosylation participates in effector
functions mediated by Fc.quadrature. receptors and complement Clq
(Burton and Woof (1992) Adv. Immunol. 51:1-84; Jefferis et al.
(1998) Immunol. Rev. 163:59-76). The Fc domain can be produced in a
mammalian expression system that appropriately glycosylates the
residue corresponding to asparagine 297. The Fc domain can also
include other eukaryotic post-translational modifications.
[0065] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method for expressing
an antibody in the mammary gland of a transgenic mammal. A
transgene is constructed that includes a milk-specific promoter and
nucleic acid sequences encoding the antibody of interest, e.g., an
antibody described herein, and a signal sequence for secretion. The
milk produced by females of such transgenic mammals includes,
secreted-therein, the antibody of interest, e.g., an antibody
described herein. The antibody can be purified from the milk, or
for some applications, used directly.
[0066] Antibodies can be modified, e.g., with a moiety that
improves its stabilization and/or retention in circulation, e.g.,
in blood, serum, lymph, bronchoalveolar lavage, or other tissues,
e.g., by at least 1.5, 2, 5, 10, or 50 fold.
[0067] In one example, a KIM-1 binding antibody can be associated
with a polymer, e.g., a substantially non-antigenic polymer, such
as a polyalkylene oxide or a polyethylene oxide. Suitable polymers
will vary substantially by weight. Polymers having molecular number
average weights ranging from about 200 to about 35,000 daltons (or
about 1,000 to about 15,000, and 2,000 to about 12,500) can be
used.
[0068] In another example, a KIM-1 binding antibody can be
conjugated to a water soluble polymer, e.g., a hydrophilic
polyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone. A
non-limiting list of such polymers include polyalkylene oxide
homopolymers such as polyethylene glycol (PEG) or polypropylene
glycols, polyoxyethylenated polyols, copolymers thereof and block
copolymers thereof, provided that the water solubility of the block
copolymers is maintained. Additional useful polymers include
polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and
block copolymers of polyoxyethylene and polyoxypropylene
(Pluronics); polymethacrylates; carbomers; branched or unbranched
polysaccharides that comprise the saccharide monomers D-mannose, D-
and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin or
heparon.
[0069] Uses and Methods of Administration
[0070] In the methods described herein, an agent, such as an
antibody that binds a particular region of KIM-1, is administered
to a subject to treat a Th2-mediated condition or Th1 mediated
condition. The subject treated is a mammal, e.g., human.
[0071] "Administration" is not limited to any particular
formulation, delivery system, or route and may include, for
example, intrabronchial, parenteral (including subcutaneous,
intravenous, intramedullary, intraarticular, intramuscular, or
intraperitoneal injection) rectal, topical, transdermal, or oral
(for example, in capsules, suspensions, or tablets). Administration
may be provided in a single dose or repeatedly, and in any of a
variety of pharmaceutical compositions containing physiologically
acceptable salt forms, and/or with an acceptable pharmaceutical
excipients. Physiologically acceptable salt forms and
pharmaceutical formulations and excipients are known (see, e.g.,
2004 Physicians' Desk Reference.RTM. (PDR) (2003) Thomson
Healthcare, 58th ed; Gennado et al., (2000), 20th ed, Lippincott,
Williams & Wilkins) Remington; The Science and Practice of
Pharmacy.
[0072] A number of therapeutic agents are useful in the management
and treatment of asthma. These include, but are not limited to,
bronchodilators, e.g., anticholinergic bronchodilators to relax the
airway (e.g., ipratropium bromide, albuterol/ipratropium bromide);
beta agonists to relax airway muscles (e.g., epinephrine,
metaproterenol, terbutaline, isoetharinemesylate, isoetharine,
isuprel, pirbuterol, albuterol, salmeterol, bitolterol); oral or
inhaled corticosteroids to reduce inflammation (e.g.,
hydrocortisone, cortisone, dexamethasone, prednisolone, prednisone,
methylprednisolone, flunisolide, triamcinolone, beclomethosone,
dexamethasone, fluticasone, budesonide); leukotriene modifiers to
prevent the airways from swelling and blocking airflow and decrease
mucus production (e.g., zafirlukast, montelukast sodium, zileuton);
and theophylline, which helps, inter alia, to open the airways and
reduce release of phlegm. Anti-asthma agents also include
therapeutic antibodies (or functional fragments thereof),
including, but not limited to, anti-IgE, anti-IL-9, anti-IL-3,
anti-IL-4, anti-IL-5, anti-IL-13, anti-VLA proteins, and
anti-migration inhibitory factor (MIF). An antibody described
herein can be administered in combination with one or more of the
aforementioned agents to treat allergic asthma.
[0073] Therapeutic agents are useful in the management and
treatment of Th1-mediated inflammatory conditions include
anti-inflammatory compounds, e.g., steroids and NSAIDs.
[0074] Therapeutically effective dosages achieved in one animal
model may be converted for use in another animal, including humans,
using known conversion factors (see, e.g., Freireich et al. (1966)
Cancer Chemother. Reports, 50(4):219-244.
[0075] The following Examples provide illustrative embodiments. The
Examples do not in any way limit the invention. One of ordinary
skill in the art will recognize numerous modifications and
variations that may be performed within the scope of the present
invention. Such modifications and variations are therefore
encompassed by the invention.
EXAMPLES
Example 1
Characterization of Rat Monoclonal Antibodies to Mouse KIM-1
[0076] Using standard PCR and cloning techniques, full-length
extracellular domain and IgV domain-only murine 1 expression
constructs were generated and stably transfected into CHO cells,
These fusion proteins were purified from CHO cell line supernatants
by protein-A and SEC chromatography. Full length KIM-1 fused to a
human IgG1-Fc domain appeared as a doublet, consistent with
differential glycosylation. Rats were immunized with the
full-length mouse KIM-1-Ig fusion protein consisting of the entire
extracellular domain. A panel of rat monoclonal antibodies to
mKIM-1 was identified by ELISA assay and FACS screening, and a set
of these were further characterized by Biacore and by domain
specific ELISA and Western blot analysis, which demonstrated that
multiple antibodies binding distinct epitopes were represented in
the panel. Thus, 7 antibodies bound full-length protein in Biacore
and ELISA analyses, while 4 of these 7 failed to bind to a protein
encoding the IgV-domain only (Table 1). Of the 4 antibodies which
appeared to require the presence of the mucin-stalk domains to bind
in the Biacore format, 3 were further defined by ELISA and Western
blot analysis to bind within the mucin domain, while 1 bound in the
stalk domain (Table 1). Within the mucin domain several antibodies
recognized a distinct region encoded by exon 4 (Table 1). Thus,
antibodies recognizing the IgV, mucin, and stalk domains were
identified. Table 1 shows results of gross epitope mapping of the
rat-anti-mKIM-1 mAbs. Table 1 data was compiled from multiple
assays (Biacore, ELISA, Western blot, and FACS) using the full ECD
of KIM-1-Ig, KIM-1-IgV-Ig, and proteolytic fragments of KIM-1-Ig
protein.
TABLE-US-00001 TABLE 1 ECD minus region encoded by ECD domain IgV
domain alternatively spliced exon stalk only 1H9 + + + - 1D9 + - -
- 1C11 + - - - 3A2 + - + + 1H8 + - - - 4A2 + + + -* 2A7 + + + -*
*predicted
Example 2
Induction of KIM-1 Expression in Hyperactive Lung
[0077] Balb/c mice were primed with OVA/alum twice, then rested for
3 weeks, at which time the mice received 3 days of exposure to OVA
aerosol using a nebulizer. Lung tissue, draining (bronchial) lymph
node and spleen were harvested and examined for induction of KIM-1
expression by RT-PCR. KIM-1 message was induced in both bronchial
LN and lung tissue by 24 hours post nebulization. In contrast to
KIM-1 mRNA levels, KIM-3 mRNA levels were not modulated after
challenge with OVA aerosol. KIM-2 levels were upregulated in a
manner similar to KIM-1.
Example 3
Effect of Anti-KIM-1 Antibodies on Ova-Induced
Hyper-Responsiveness
[0078] Anti-KIM-1 antibodies with different epitope specificities
were tested for the ability to influence the development of lung
inflammation using the OVA aerosol model, and using both
prophylactic and therapeutic dosing regimens.
[0079] For prophylactic studies, OVA-induced lung inflammation and
recall assays were performed as follows: Balb/c mice were given ip
injections of 100 .mu.l 0.5 mg/ml OVA (Grade V, Sigma) mixed with
100 .mu.l ImjectAlum (Pierce, Rockford Ill. USA) on days 1 and 7.
Three weeks after the second injection, mice were exposed for 20
minutes daily for 3 days to an aerosal of 1% OVA in PBS using an
ultrasonic nebulizer (Devilbiss, Carlsbad Calif. USA). Dosing with
mAbs was as follows: 200 .mu.g was given ip on days 1, 3, 6, and 9,
and then 500 .mu.g was given ip the day that nebulizations
began.
[0080] For therapeutic studies, mice were immunized with OVA in
alum as described above, but no mAb was administered until just
prior to the nebulization series: thus 250 .mu.g of mAb 4A2 was
given the day prior to the first nebulization, and 250 .mu.g of mAb
4A2 was given the morning of the second nebulization.
[0081] In both prophylactic and therapeutic studies, two days after
the final nebulization session, the mice were sacrificed for
analysis. Bronchial lavage fluid (BAL) was collected via
tracheotomy using 3 washes with PBS containing 0.1% BSA and 0.02 mM
EDTA. BAL cells were pelleted using a cytospin and coated slides
(Shandon, Pittsburgh, Pa. USA) then air dried and stained with
Hema3 stain (Fisher Scientific, Pittsburgh Pa. USA) for
identification of different cell populations. The lung tissue was
harvested into neutral buffered formalin for routine histology, or
was snap-frozen in trizol for subsequent RNA isolation. Draining
(bronchial) lymph nodes and the spleen were harvested for isolation
of mononuclear cells, which were placed into culture in RPMI/10%
FBS with varying concentrations of OVA. 72 hours later the
supernatants were harvested and cells were pulsed for 8 hours with
1 uCi tritiated thymidine (Amersham Biosciences, Piscataway, N.J.
USA) and the plates were counted using the Microbetajet system
(Wallac, Gaithersburg, Md. USA). Supernatants were analyzed using
CBA Th1/Th2 and Inflammation kits (BD Biosciences) and IL-13 ELISA
assays (R&D Systems, Minneapolis Minn. USA).
[0082] Results:
[0083] Mice were dosed with antibodies during the OVA priming and
challenge phases. Post challenge, bronchial lavage fluid (BAL),
bronchial lymph node, spleen, and lung tissue were harvested. The
percent eosinophils, neutrophils, and lymphocytes present in BAL
were calculated. mAb 1H8 induced robust eosinophil counts in the
BAL of treated mice, such that the percent eosinophils present more
than doubled as compared to control. Modest increases in the
percent of neutrophils and lymphocytes, which constitute a small
fraction of BAL cellularity, were also noted. Consistent with this
result, bronchial LN cells isolated from 1H8 treated mice and
challenged with OVA ex vivo proliferated more, and expressed higher
levels of Th2 associated cytokines than did control cultures. In
particular, very high levels of IL-5 and IL-13 were produced, as
compared to controls, although levels of IL-4, IL-6, and IL-10 were
also elevated. Of interest, IFN-gamma levels were also increased,
although overall, levels of this cytokine were low. Preferential
induction of Th2 cytokines would be efficacious in settings of Th1
cytokine dependent pathology, such as MS, RA, Crohn's.
[0084] In contrast with 1H8, mAb 3A2 reduced the percentage of
eosinophils in the BAL, and reduced production of Th2-associated
cytokines in the bronchial lymph node recall assay (FIG. 2). Thus,
the antibodies 1H8 and 3A2 have opposing effects in this assay.
Several other antibodies, including 1H9, which recognizes an
epitope within the IgV domain, had no effect on lung inflammation
or cytokine production in this model. Analysis of the response of
draining (bronchial) lymph node cells to stimulation ex vivo with
the OVA antigen with 3A2 showed a reduction in the proliferation of
cells to antigen stimulation (FIG. 3). Furthermore, the analysis of
the supernatants from these cultures showed a marked reduction in
the expression of Th2 cytokines including IL-4, IL-5, and IL-10
(FIG. 4). Levels of IL-13 were also reduced in this assay.
[0085] Treatment with the anti-Ig domain mAb 4A2 resulted in a
pronounced reduction in eosinophil and lymphocyte influx into the
BAL after sensitization with OVA and nebulization treatment (FIG.
6). The average decrease in eosinophil percentage in BAL was 84%
compared to control (p<0.0001, test of mean equivalence) and the
average decrease in lymphocyte percentage was 90% compared to
control (p<0.001, test of mean equivalence). When the bronchial
lymph node cells were restimulated with OVA ex vivo there was a
dramatic decrease in the production of Th2 cytokines including
IL-4, IL-5, IL-10, and 11-13 (FIG. 7). Therefore treatment with mAb
4A2 reduced lung inflammation and the production of cytokines
associated with asthma responses.
[0086] To further characterize the clinical efficacy of mAb 4A2, a
therapeutic dosing experiment was performed. In this model, the
mice were immunized to develop sensitivity to the OVA antigen,
without any mAb treatment being given. The mice were then rested
for 3 weeks, again without any treatment, and then dosed with 4A2
mAb the day prior to the first of 3 sessions of nebulization with
1% OVA. The dosing with mAb was repeated prior to the second
session. This treatment protocol resulted in the reduction of lung
inflammation as measured by the influx of eosinophils into the BAL
(FIG. 8). The percent of eosinophils was reduced an average of 70%
(p<0.001, test of mean equivalence). Therefore mAb 4A2 was
efficacious in both a prophylactic and therapeutic dosing regimen
in the OVA-induced lung inflammation model. This suggests that the
epitope recognized by 4A2 is a therapeutically relevant target for
the treatment of Th2 mediated disorders.
[0087] Other anti-KIM-1 mAbs were also demonstrated to have
therapeutic activity in the OVA-induced lung inflammation model,
including, for example, mAb 2A7 and mAb 2B3. mAb 2A7 was shown to
compete with 4A2 for binding to immobilized KIM-1 in a Biocore.RTM.
assay, suggesting they have shared or overlapping epitopes.
Example 4
Effect of KIM-1 Antibodies on the CD4 T Cell Response to
Antigen
[0088] The activity of anti-KIM-1 mAbs using the KLH antigen recall
assay was evaluated. Mice were treated with anti-KIM-1 mAbs,
control mAb, or PBS, then immunized with KLH and 6 days later the
draining LN were excised. LN CD4+ T cells were isolated and
restimulated ex vivo with purified OVA in the presence of
irradiated whole splenocytes isolated from untreated mice. 48 hours
after ex vivo stimulation cellular proliferation and cytokine
production was assayed. In this assay several of the anti-KIM-1
mAbs had a marked effect. mAb 1118 dramatically increased T cell
proliferation in response to KLH challenge ex vivo. In contrast mAb
3A2 reduced T cell proliferation in the assay. Cytokines produced
in the cultures from cells treated with mAb 1H8 were measured.
Treated cultures were found to contain more IFN-gamma and
TH2-associated cytokines that controls. In contrast levels of TNF
and IL-2 were similar to controls.
[0089] This data indicates that 1H8 can reduce the pathogenic Th1
response. This data also indicates that 1H8 and other antibodies
that bind to the KIM-1 region as defined herein for 1H8 can act as
an adjuvant by increasing the immune response. The invention also
covers methods of increasing the immune response, e.g., to increase
the effectiveness of a vaccine. Such adjuvancy can also have use in
vaccination, immunodeficiency, and anti-tumor immunity.
[0090] 1H8 binds KIM-1 ECD of the Balb/C sequence but not the Dba/2
sequence in Western blots. This indicates that the antibody binds
to the alternatively spliced mouse allelic variant containing the
sequence EPTTFCPHETTAEVTGIPSHTPT (SEQ ID NO:2). This sequence
corresponds to the sequence VATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTT of
human KIM-1 (amino acids 200-235 of SEQ ID NO:1).
Example 5
Characterization of mAb 3A2
[0091] Because 3A2 mAb had a therapeutic effect in the OVA model,
its binding to KIM-1 was characterized in more detail.
[0092] Various purified proteins were used in an ELISA assay to
determine the epitope of mAb 3A2 (Table 2). Full binding curves
were generated for the interaction of 3A2 with the immobilized
proteins (FIG. 5). MAb 3A2 bound equivalently to the following
mouse proteins: murine KIM-1 extracellular domain
(KIM-1-ECD-1-216), mKIM-1-137-216 and mKIM-1-196-216-Fc. In
contrast, mAb 3A2 failed to bind to the mKIM-1-IgV domain alone,
which is lacking the entire mucin and stalk domain. This data shows
that the epitope for 3A2 resides within the 21 amino acid residues
196 to 216 from mKIM-1, which maps to a portion of the stalk region
of KIM-1. This epitope equates to residues 247-272 of human KIM-1
as shown in FIG. 1.
TABLE-US-00002 TABLE 2 3A2 DOMAIN SEQUENCE BINDING mKIM-1-ECD
SYVEVKGVVGHPVTLPCTYSTYRGITTTCWG yes (1-216)
RGQCPSSACQNTLIWTNGHRVTYQKSSRYN LKGHISEGDVSLTIENSVESDSGLYCCRVEI
PGWFNDQKVTFSLQVKPEIPTRPPTRPTTTR PTATGRPTTISTRSTHVPTSIRVSTSTPPTS
THTWTHKPEPTTFCPHETTAEVTGIPSHTPT DWNGTATSSGDTWSNHTEAIPPGKPQKNPTK
(underlined region is encoded by alternatively spliced exon)
mKIM-1-IgV SYVEVKGVVGHPVTLPCTYSTYRGITTTCWG no (1-109)
RGQCPSSACQNTLIWTNGHRVTYQKSSRYN LKGHISEGDVSLTIENSVESDSGLYCCRVEI
PGWFNDQKVTFSLQVKP mKIM-1-137- STHVPTSIRVSTSTPPTSTHTWTHKPEPTTF yes
216 CPHETTAEVTGIPSHTPTDWNGTATSSGDT (137-216) WSNHTEAIPPGKPQKNPTK
mKIM-1-196- DTWSNHTEAIPPGKPQKNPTK yes 216 (196-216)
[0093] Because the stalk region that includes amino acids 196 to
216 of mouse KIM-1 (corresponding to amino acids 247-272 of human
KIM-1) contains N-glycosylation sites, it was of interest to
determine if a sugar moiety bound to an N-glycosylation site was
required for binding of 3A2. Western blot analysis of glycosylated
and deglycosylated KIM-1-196-216-Fc was performed (FIG. 2). This
analysis showed that deglycosylation did not impact the ability of
3A2 to bind to KIM-1-196-216-Fc. Therefore a sugar moiety is not
required for 3A2 to recognize its epitope.
[0094] To determine if 3A2 inhibits shedding of KIM-1 from the cell
surface, E293 cells transfected with KIM-1 were treated with 5
ug/mL 3A2, 25 ug/mL 3A2 or no 3A2 (control). Supernatants from both
sets of 3A2-treated cells showed no difference in KIM-1 staining
from control when run on Western blot probed with biotinylated 1H8
antibody. This suggests that 3A2 does not preventing shedding of
KIM-1.
Example 6
Characterization of the 4A2 mAb
[0095] Using ELISA and Biacore analyses it was determined that mAb
4A2 recognized the Ig-domain of murine KIM-1 (FIG. 9, Table 1). To
further characterize the epitope recognized by mAb 4A2, a
recombinant murine KIM-1 IgV-human IgG1 Fc fusion, alone and in
complex with 4A2, was digested with TPCK trypsin. A band of 8 kDa
was generated from KIM-1 alone and is not generated when 4A2 is
bound. This indicates that binding of 4A2 to KIM-1 blocks access of
trypsin to the cleavage site required to generate this band.
Digestion of the 8 Kda band under non-reducing conditions revealed
that 4A2 protects a fragment corresponding to human KIM-1 sequence
GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO:1).
Thus, the 4A2 antibody protects a TPCK trypsin site at least partly
within, or overlapping amino acids 81-107 of SEQ ID NO:1.
[0096] The same TPCK tryptic digest experiment was performed with
2A7 and a band of approximately the same size was obtained. The
band was not obtained when the same experiment was performed with
1H9 (a non-efficacious mAb in the asthma model that also binds to
the Ig domain), indicating that the epitope tracks with efficacy in
asthma.
[0097] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification. The
embodiments within the specification provide an illustration of
embodiments of the invention and should not be construed to limit
the scope of the invention. The skilled artisan readily recognizes
that many other embodiments are encompassed by the invention. All
publications, patents, and biological sequences cited in this
disclosure are incorporated by reference in their entirety. To the
extent the material incorporated by reference contradicts or is
inconsistent with the present specification, the present
specification will supersede any such material. The citation of any
references herein is not an admission that such references are
prior art to the present invention.
[0098] Unless otherwise indicated, all numbers expressing
quantities of ingredients, cell culture, treatment conditions, and
so forth used in the specification, including claims, are to be
understood as being modified in all instances by the term "about."
Accordingly, unless otherwise indicated to the contrary, the
numerical parameters are approximations and may vary depending upon
the desired properties sought to be obtained by the present
invention. Unless otherwise indicated, the term "at least"
preceding a series of elements is to be understood to refer to
every element in the series. Those skilled in the art will
recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described herein. Such equivalents are intended to be
encompassed by the following claims.
Sequence CWU 1
1
21339PRThuman 1Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val Thr
Leu Pro Cys1 5 10 15His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn
Arg Gly Ser Cys 20 25 30Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp
Thr Asn Gly Thr His 35 40 45Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys
Leu Leu Gly Asp Leu Ser 50 55 60Arg Arg Asp Val Ser Leu Thr Ile Glu
Asn Thr Ala Val Ser Asp Ser65 70 75 80Gly Val Tyr Cys Cys Arg Val
Glu His Arg Gly Trp Phe Asn Asp Met 85 90 95Lys Ile Thr Val Ser Leu
Glu Ile Val Pro Pro Lys Val Thr Thr Thr 100 105 110Pro Ile Val Thr
Thr Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr 115 120 125Thr Val
Pro Thr Thr Thr Thr Val Pro Thr Thr Thr Val Pro Thr Thr 130 135
140Met Ser Ile Pro Thr Thr Thr Thr Val Pro Thr Thr Met Thr Val
Ser145 150 155 160Thr Thr Thr Ser Val Pro Thr Thr Thr Ser Ile Pro
Thr Thr Thr Ser 165 170 175Val Pro Val Thr Thr Thr Val Ser Thr Phe
Val Pro Pro Met Pro Leu 180 185 190Pro Arg Gln Asn His Glu Pro Val
Ala Thr Ser Pro Ser Ser Pro Gln 195 200 205Pro Ala Glu Thr His Pro
Thr Thr Leu Gln Gly Ala Ile Arg Arg Glu 210 215 220Pro Thr Ser Ser
Pro Leu Tyr Ser Tyr Thr Thr Asp Gly Asn Asp Thr225 230 235 240Val
Thr Glu Ser Ser Asp Gly Leu Trp Asn Asn Asn Gln Thr Gln Leu 245 250
255Phe Leu Glu His Ser Leu Leu Thr Ala Asn Thr Thr Lys Gly Ile Tyr
260 265 270Ala Gly Val Cys Ile Ser Val Leu Val Leu Leu Ala Leu Leu
Gly Val 275 280 285Ile Ile Ala Lys Lys Tyr Phe Phe Lys Lys Glu Val
Gln Gln Leu Ser 290 295 300Val Ser Phe Ser Ser Leu Gln Ile Lys Ala
Leu Gln Asn Ala Val Glu305 310 315 320Lys Glu Val Gln Ala Glu Asp
Asn Ile Tyr Ile Glu Asn Ser Leu Tyr 325 330 335Ala Thr Asp223PRTMus
musculus 2Glu Pro Thr Thr Phe Cys Pro His Glu Thr Thr Ala Glu Val
Thr Gly1 5 10 15Ile Pro Ser His Thr Pro Thr 20
* * * * *