U.S. patent application number 12/786095 was filed with the patent office on 2011-01-13 for antibodies that bind il-18 and methods of inhibiting il-18 activity.
This patent application is currently assigned to Abbott Laboratories. Invention is credited to Simon Mark Brocklehurst, Richard W. Dixon, Alexander Robert Duncan, Tariq Ghayer, Boris Labkovsky, Simon Nicholas Lennard, John Mankovich, Michael Roguska, Jochen Salfeld, Celia Patricia Shorrock, Julia Elizabeth Thompson, Michael White.
Application Number | 20110008357 12/786095 |
Document ID | / |
Family ID | 22665007 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110008357 |
Kind Code |
A1 |
Ghayer; Tariq ; et
al. |
January 13, 2011 |
ANTIBODIES THAT BIND IL-18 AND METHODS OF INHIBITING IL-18
ACTIVITY
Abstract
Antibodies that bind human interleukin-18 (hIL-18) are provided,
in particular antibodies that bind epitope(s) of human IL-18. The
antibodies can be, for example, entirely human antibodies,
recombinant antibodies, or monoclonal antibodies. Preferred
antibodies have high affinity for hIL-18 and neutralize hIL-18
activity in vitro and in vivo. An antibody of the invention can be
a full-length antibody or an antigen-binding portion thereof.
Method of making and method of using the antibodies of the
invention are also provided. The antibodies, or antibody portions,
of the invention are useful for detecting hIL-18 and for inhibiting
hIL-18 activity, e.g., in a human subject suffering from a disorder
in which hIL-18 activity is detrimental.
Inventors: |
Ghayer; Tariq; (Holliston,
MA) ; Dixon; Richard W.; (Jefferson, MA) ;
Roguska; Michael; (Ashland, MA) ; White; Michael;
(Framingham, MA) ; Labkovsky; Boris; (Marlborough,
MA) ; Salfeld; Jochen; (North Grafton, MA) ;
Duncan; Alexander Robert; (Little Shelford, GB) ;
Brocklehurst; Simon Mark; (Fulbourn, GB) ; Mankovich;
John; (Andover, MA) ; Shorrock; Celia Patricia;
(Cambridge, GB) ; Thompson; Julia Elizabeth;
(Whittlesford, GB) ; Lennard; Simon Nicholas;
(Linton, GB) |
Correspondence
Address: |
McCarter & English, LLP / Abbott Laboratories Ltd.
265 Franklin Street
Boston
MA
02110
US
|
Assignee: |
Abbott Laboratories
Abbott Park
IL
|
Family ID: |
22665007 |
Appl. No.: |
12/786095 |
Filed: |
May 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09780035 |
Feb 9, 2001 |
7767207 |
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12786095 |
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60181608 |
Feb 10, 2000 |
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Current U.S.
Class: |
424/142.1 ;
435/320.1; 435/325; 435/69.6; 506/9; 530/300; 530/350; 530/387.3;
530/387.9; 530/388.15; 536/23.5 |
Current CPC
Class: |
A61P 3/00 20180101; A61P
9/10 20180101; A61P 25/18 20180101; A61P 5/50 20180101; A61P 15/08
20180101; A61P 37/00 20180101; A61K 2039/505 20130101; A61P 7/06
20180101; A61P 37/08 20180101; A61P 1/00 20180101; A61P 17/00
20180101; A61P 19/00 20180101; A61K 31/519 20130101; A61P 7/04
20180101; A61P 9/04 20180101; A61P 13/00 20180101; A61P 15/00
20180101; A61P 43/00 20180101; A61P 25/08 20180101; A61P 1/04
20180101; A61P 5/18 20180101; A61P 19/02 20180101; C07K 2317/565
20130101; A61P 9/00 20180101; A61P 3/08 20180101; A61P 19/04
20180101; A61P 25/14 20180101; A61P 15/10 20180101; A61P 27/02
20180101; A61P 31/00 20180101; A61P 31/10 20180101; A61P 37/02
20180101; C07K 2317/622 20130101; A61P 39/02 20180101; A61P 19/06
20180101; A61P 31/04 20180101; A61P 11/00 20180101; A61P 31/14
20180101; A61P 5/40 20180101; A61P 21/00 20180101; A61P 25/16
20180101; A61P 5/00 20180101; A61P 7/02 20180101; A61P 17/06
20180101; A61P 25/28 20180101; A61P 35/00 20180101; A61P 39/00
20180101; C07K 2317/92 20130101; C07K 2317/21 20130101; A61P 37/06
20180101; A61K 38/13 20130101; A61P 9/12 20180101; A61P 25/24
20180101; C07K 16/244 20130101; C07K 2317/34 20130101; A61P 3/10
20180101; A61P 31/12 20180101; A61K 31/56 20130101; A61P 5/16
20180101; A61P 29/00 20180101; A61P 31/18 20180101; A61P 25/00
20180101; A61P 33/00 20180101; C07K 2317/76 20130101; A61P 5/14
20180101; A61P 11/06 20180101; A61K 39/3955 20130101; A61P 1/16
20180101; A61P 7/00 20180101; A61P 13/12 20180101; A61P 17/14
20180101; A61K 39/3955 20130101; A61K 39/395 20130101; A61K 39/3955
20130101; A61K 31/00 20130101; A61K 39/3955 20130101; A61K 39/395
20130101; A61K 39/3955 20130101; A61K 31/00 20130101; A61K 39/3955
20130101; A61K 39/395 20130101; A61K 39/3955 20130101; A61K 31/00
20130101; A61K 39/3955 20130101; A61K 39/395 20130101; A61K 39/3955
20130101; A61K 31/00 20130101; A61K 39/3955 20130101; A61K 39/395
20130101; A61K 39/3955 20130101; A61K 31/00 20130101; A61K 39/3955
20130101; A61K 39/395 20130101; A61K 39/3955 20130101; A61K 31/00
20130101; A61K 39/3955 20130101; A61K 39/395 20130101; A61K 39/3955
20130101; A61K 31/00 20130101; A61K 39/3955 20130101; A61K 39/395
20130101; A61K 39/3955 20130101; A61K 31/00 20130101 |
Class at
Publication: |
424/142.1 ;
435/69.6; 435/325; 435/320.1; 506/9; 530/300; 530/350; 530/387.3;
530/387.9; 530/388.15; 536/23.5 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12P 21/00 20060101 C12P021/00; C12N 5/07 20100101
C12N005/07; C12N 15/63 20060101 C12N015/63; C40B 30/04 20060101
C40B030/04; C07K 2/00 20060101 C07K002/00; C07K 14/00 20060101
C07K014/00; C07K 16/24 20060101 C07K016/24; C07H 21/00 20060101
C07H021/00; A61P 1/16 20060101 A61P001/16; A61P 19/02 20060101
A61P019/02; A61P 29/00 20060101 A61P029/00; A61P 3/10 20060101
A61P003/10; A61P 37/06 20060101 A61P037/06; A61P 25/28 20060101
A61P025/28; A61P 25/16 20060101 A61P025/16; A61P 9/10 20060101
A61P009/10; A61P 37/08 20060101 A61P037/08; A61P 17/06 20060101
A61P017/06; A61P 13/12 20060101 A61P013/12; A61P 11/00 20060101
A61P011/00; A61P 9/00 20060101 A61P009/00; A61P 15/08 20060101
A61P015/08; A61P 25/24 20060101 A61P025/24; A61P 11/06 20060101
A61P011/06; A61P 5/16 20060101 A61P005/16; A61P 5/18 20060101
A61P005/18; A61P 5/50 20060101 A61P005/50 |
Claims
1. A compound capable of binding a human IL-18 amino acid sequence,
or portion thereof, wherein said amino acid comprises a sequence
selected from the group consisting of SEQ ID NO: 70 and SEQ ID NO:
71.
2. The compound of claim 1, wherein said compound is selected from
the group consisting of a small molecule, peptide, polypeptide,
antibody, and antibody fragment.
3. The compound of claim 2, wherein said antibody, or antibody
fragment, is fully human.
4. A human monoclonal antibody, or antigen-binding portion thereof,
capable of binding to human IL-18.
5. The antibody of claim 4, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 0.1 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-6M or less.
6. The antibody of claim 4, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 1.times.10-2 s-lor less, as determined by
surface plasmon resonance, or which inhibits human IL-18 activity
with an IC50 of 1.times.10-7M or less.
7. The antibody of claim 4, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 1.times.10-3 s-1 or less, as determined by
surface plasmon resonance, or which inhibits human IL-18 activity
with an IC50 of 1.times.10-8M or less.
8. The antibody of claim 4, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 1.times.10-4 s-1 or less, as determined by
surface plasmon resonance, or which inhibits human IL-18 activity
with an IC50 of 1.times.10-9M or less.
9. The antibody of claim 4, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 1.times.10-5 s-1 or less, as determined by
surface plasmon resonance, or which inhibits human IL-18 activity
with an IC50 of 1.times.10-10M or less.
10. The antibody of claim 4, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 1.times.10-6 s-1 or less, as determined by
surface plasmon resonance, or which inhibits human IL-18 activity
with an IC50 of 1.times.10-11M or less.
11. An isolated antibody, or an antigen-binding portion thereof,
that binds an epitope of human IL-18, or portion thereof,
comprising an amino acid sequence selected from the group
comprising SEQ ID NO: 3 and SEQ ID NO: 33.
12. The antibody, or antigen-binding portion thereof, of claim 11,
wherein the antibody is a neutralizing antibody.
13. The antibody, or antigen-binding portion thereof, of claim 11,
which is a human antibody.
14. The antibody, or antigen-binding portion thereof, of claim 11,
which is a recombinant antibody.
15. The antibody, or antigen-binding portion thereof, of claim 11,
which is a monoclonal antibody.
16. An isolated antibody, or antigen-binding portion thereof, that
binds to an epitope of human IL-18, wherein the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a koff rate constant of 0.1 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-6M or less.
17. The isolated antibody of claim 16, or an antigen-binding
portion thereof, which dissociates from human IL-18 with a koff
rate constant of 1.times.10-2 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-7M or less.
18. The isolated antibody of claim 16, or an antigen-binding
portion thereof, which dissociates from human IL-18 with a koff
rate constant of 1.times.10-3 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-8M or less.
19. The isolated antibody of claim 16, or an antigen-binding
portion thereof, which dissociates from human IL-18 with a koff
rate constant of 1.times.10-4 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-9M or less.
20. The isolated antibody of claim 16, or an antigen-binding
portion thereof, which dissociates from human IL-18 with a koff
rate constant of 1.times.10-5 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-10M or less.
21. The isolated antibody of claim 16, or an antigen-binding
portion thereof, which dissociates from human IL-18 with a koff
rate constant of 1.times.10-6 s-1 or less, as determined by surface
plasmon resonance, or which inhibits human IL-18 activity with an
IC50 of 1.times.10-11M or less.
22. An isolated human antibody, or an antigen-binding portion
thereof, comprising at least one variable region CDR domain capable
of binding an epitope of human IL-18.
23. The isolated antibody, or an antigen-binding portion thereof,
of claim 22, wherein said antibody, or an antigen-binding portion
thereof, contains at least one amino acid substitution or insertion
that improves IL-18 binding as compared to the unmodified antibody
or antigen-binding portion thereof.
24. The isolated antibody, or an antigen-binding portion thereof,
of claim 22, wherein said antibody, or an antigen-binding portion
thereof, contains at least one amino acid substitution or insertion
that improves neutralization of IL-18 as compared to the unmodified
antibody or antigen-binding portion thereof.
25. The isolated antibody, or an antigen-binding portion thereof,
of claim 22, wherein said variable region comprises a CDR domain
selected from the group consisting of: a heavy chain CDR1 domain
having an amino acid sequence of SEQ ID NO: 9, or sequence modified
from SEQ ID NO: 9 by at least one amino acid substitution; a heavy
chain CDR2 domain having an amino acid sequence of SEQ ID NO: 10 ,
or sequence modified from SEQ ID NO: 10 by at least one amino acid
substitution; and a heavy chain CDR3 domain having an amino acid
sequence of SEQ ID NO: 11, or sequence modified from SEQ ID NO: 11
by at least one amino acid substitution.
26. The isolated antibody, or an antigen-binding portion thereof,
of claim 25, wherein said variable region comprises a CDR domain
selected from the group consisting of: a heavy chain CDR1 domain
modified from SEQ ID NO: 9 by at least one amino acid substitution
at position H30, H31, H32, H33, or H35; a heavy chain CDR2 domain
modified from SEQ ID NO: 10 by at least one amino acid substitution
at position H52, H52a, H53, H54, H56, or H58; and a heavy chain
CDR3 domain modified from SEQ ID NO: 11 by at least one amino acid
substitution at position H95, H96, H97, or H98.
27. The isolated antibody, or an antigen-binding portion thereof,
of claim 22, wherein said variable region comprises a CDR domain
selected from the group consisting of: a light chain CDR1 domain
having an amino acid sequence of SEQ ID NO: 12, or sequence
modified from SEQ ID NO: 12 by at least one amino acid
substitution; a light chain CDR2 domain having an amino acid
sequence of SEQ ID NO: 13, or sequence modified from SEQ ID NO: 13
by at least one amino acid substitution; and a light chain CDR3
domain having an amino acid sequence of SEQ ID NO: 14, or sequence
modified from SEQ ID NO: 14 by at least one amino acid
substitution.
28. The isolated antibody, or an antigen-binding portion thereof,
of claim 27, wherein said variable region comprises a CDR domain
selected from the group consisting of: a light chain CDR1 domain
modified from SEQ ID NO: 12 by at least one amino acid substitution
at position L30, L31, L32, or L34; a light chain CDR2 domain
modified from SEQ ID NO: 13 by at least one amino acid substitution
at position L50, L52, L53, or L55; and a light chain CDR3 domain
modified from SEQ ID NO: 14 by at least one amino acid substitution
at position L89, L90, L91, L92, L93, L94, L95, L95a, L95b, L96, or
L97.
29. An isolated antibody, or an antigen-binding portion thereof,
with a variable region comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 15, 16, and 17.
30. An isolated antibody, or an antigen-binding portion thereof,
with a light chain variable region (LCVR) comprising the amino acid
sequence of SEQ ID NO: 15 and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 16.
31. An isolated antibody, or an antigen-binding portion thereof,
with a light chain variable region (LCVR) having the amino acid
sequence of SEQ ID NO: 15 and a heavy chain variable region (HCVR)
having the amino acid sequence of SEQ ID NO: 17.
32. The isolated antibody, or an antigen-binding portion thereof,
of claim 22, wherein said variable region comprises a CDR domain
selected from the group consisting of: a heavy chain CDR1 domain
having an amino acid sequence of SEQ ID NO: 20, or sequence
modified from SEQ ID NO: 20 by at least one amino acid
substitution; a heavy chain CDR2 domain having an amino acid
sequence of SEQ ID NO: 21, or sequence modified from SEQ ID NO: 21
by at least one amino acid substitution; and a heavy chain CDR3
domain having an amino acid sequence of SEQ ID NO: 22, or sequence
modified from SEQ ID NO: 22 by at least one amino acid
substitution.
33. The isolated antibody, or an antigen-binding portion thereof,
of claim 32, wherein said variable region comprises a CDR domain
selected from the group consisting of: a heavy chain CDR1 domain
modified from SEQ ID NO: 20 by at least one amino acid substitution
at position H30, H31, H32, H33, or H35; a heavy chain CDR2 domain
modified from SEQ ID NO: 21 by at least one amino acid substitution
at position HSO, H51, H52, H52a, H53, H54, H56, or H58; and a heavy
chain CDR3 domain modified from SEQ ID NO: 22 by at least one amino
acid substitution at position H96, H96, H97, H98, H99, H100, H100a,
H101, or H102.
34. The isolated antibody, or an antigen-binding portion thereof,
of claim 32, wherein said variable region comprises a CDR domain
selected from the group consisting of: a light chain CDR1 domain
having an amino acid sequence of SEQ ID NO: 23, or sequence
modified from SEQ ID NO: 23 by at least one amino acid substitution
at position; a light chain CDR2 domain having an amino acid
sequence of SEQ ID NO: 24, or sequence modified from SEQ ID NO: 24
by at least one amino acid substitution; and a light chain CDR3
domain having an amino acid sequence of SEQ ID NO: 25, or sequence
modified from SEQ ID NO: 25 by at least one amino acid
substitution.
35. The isolated antibody, or an antigen-binding portion thereof,
of claim 34, wherein said variable region comprises a CDR domain
selected from the group consisting of: a light chain CDR1 domain
modified from SEQ ID NO: 23 by at least one amino acid substitution
at position L30, L31, L32, or L34; a light chain CDR2 domain
modified from SEQ ID NO: 24 by at least one amino acid substitution
at position L50, L52, L53, or L55; and a light chain CDR3 domain
modified from SEQ ID NO: 25 by at least one amino acid substitution
at position L89, L90, L91, L92, L93, L94, L95, L95a, L95b, L96, or
L97.
36. An isolated antibody, or an antigen-binding portion thereof,
with a variable region comprising an amino acid selected from the
group consisting of SEQ ID NO: 26, 27, and 29.
37. An isolated antibody, or an antigen-binding portion thereof,
with a light chain variable region (LCVR) comprising the amino acid
sequence of SEQ ID NO: 29 and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 26.
38. An isolated antibody, or an antigen-binding portion thereof,
with a light chain variable region (LCVR) having the amino acid
sequence of SEQ ID NO: 29 and a heavy chain variable region (HCVR)
having the amino acid sequence of SEQ ID NO: 27.
39. An isolated nucleic acid encoding an antibody CDR amino acid
sequence of claim 4.
40. The isolated nucleic acid of claim 39, which is in a
recombinant expression vector.
41. A host cell into which the recombinant expression vector of
claim 40 has been introduced.
42. A method of synthesizing an antibody that binds human IL-18,
comprising culturing the host cell of claim 41 in a culture medium
until an antibody that binds human IL-18 is synthesized by the
cell.
43. The method of claim 42, wherein said antibody is human.
44. A pharmaceutical composition comprising the antibody, or
antigen-binding portion thereof, of claim 4, and a pharmaceutically
acceptable carrier.
45. The pharmaceutical composition of claim 44 which further
comprises at least one additional therapeutic agent for treating a
disorder in which IL-18 activity is detrimental.
46. The pharmaceutical composition of claim 45, wherein said
additional agent is selected from the group consisting of an
antibody, or fragment thereof, capable of binding human IL-12,
methotrexate anti-TNF, corticosteroids, cyclosporin, rapamycin,
FK506, and non-steroidal anti-inflammatory agents an
47. A method of making an antibody that binds human interleukin-18
(IL-18), comprising: exposing an antibody repertoire to an antigen
comprising an epitope of human IL-18 or portion thereof; and
selecting from the antibody repertoire an antibody that binds the
epitope of human IL-18, or portion thereof.
48. The method of claim 47, wherein the antibody repertoire is an
in vivo repertoire in an animal and the method comprises immunizing
the animal with the antigen comprising an epitope of human IL-18 or
portion thereof.
49. The method of claim 47, wherein said epitope comprises the
amino acid sequence selected from the group consisting of SEQ ID
NO: 3 and 33.
50. The method of claim 47, wherein said in vivo repertoire is a
fully human immunoglobulin repertoire integrated into the genome of
the animal.
51. The method of claim 47, wherein the antibody repertoire is a
recombinant antibody library and the method comprises screening the
library with an antigen comprising the epitope of human IL-18 or
portion thereof.
52. The method of claim 47, wherein the library is a human antibody
library.
53. A method for inhibiting human IL-18activity comprising
contacting human IL-18 with the compound of claim 1 such that human
IL-18 activity is inhibited.
54. A method for inhibiting human IL-18activity comprising
contacting human IL-18 with the antibody, or antigen-binding
portion thereof, of claim 4 such that human IL-18 activity is
inhibited.
55. A method for inhibiting human IL-18 activity in a human subject
suffering from a disorder in which IL-18 activity is detrimental,
comprising administering to the human subject the compound of claim
1 such that human IL-18 activity in the human subject is
inhibited.
56. A method for inhibiting human IL-18 activity in a human subject
suffering from a disorder in which IL-18 activity is detrimental,
comprising administering to the human subject the antibody, or
antigen-binding portion thereof, of claim 4 such that human IL-18
activity in the human subject is inhibited.
57. A method for treating a human subject suffering from a disorder
in which IL-18 activity is detrimental by administering a compound
according to claim 1 such that treatment is achieved.
58. A method for treating a human subject suffering from a disorder
in which IL-18 activity is detrimental by administering an antibody
of claim 4 such that treatment is achieved.
59. The method of claim 58, wherein said disorder is selected from
the group comprising rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive
arthritis, spondyloarthropathy, systemic lupus erythematosus,
Crohn's disease, ulcerative colitis, inflammatory bowel disease,
insulin dependent diabetes mellitus, thyroiditis, asthma, allergic
diseases, psoriasis, dermatitis scleroderma, graft versus host
disease, organ transplant rejection, acute or chronic immune
disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,
microscopic vasculitis of the kidneys, chronic active hepatitis,
uveitis, septic shock, toxic shock syndrome, sepsis syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's
chorea, Parkinson's disease, Alzheimer's disease, stroke, primary
biliary cirrhosis, hemolytic anemia, malignancies, heart failure,
myocardial infarction, Addison's disease, sporadic, polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia,
alopecia areata, seronegative arthopathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis C, common varied immunodeficiency
(common variable hypogammaglobulinaemia), dilated cardiomyopathy,
female infertility, ovarian failure, premature ovarian failure,
fibrotic lung disease, cryptogenic fibrosing alveolitis,
post-inflammatory interstitial lung disease, interstitial
pneumonitis, connective tissue disease associated interstitial lung
disease, mixed connective tissue disease associated lung disease,
systemic sclerosis associated interstitial lung disease, rheumatoid
arthritis associated interstitial lung disease, systemic lupus
erythematosus associated lung disease, dermatomyositis/polymyositis
associated lung disease, Sjogren's disease associated lung disease,
ankylosing spondylitis associated lung disease, vasculitic diffuse
lung disease, haemosiderosis associated lung disease, drug-induced
interstitial lung disease, radiation fibrosis, bronchiolitis
obliterans, chronic eosinophilic pneumonia, lymphocytic
infiltrative lung disease, postinfectious interstitial lung
disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune
hepatitis (classical autoimmune or lupoid hepatitis), type-2
autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune
mediated hypoglycaemia, type B insulin resistance with acanthosis
nigricans, hypoparathyroidism, acute immune disease associated with
organ transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjogren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis,vitiligo, acute
liver disease, chronic liver diseases, allergy and asthma, mental
disorders (e.g., depression and schizophrenia) and Th2 Type and Th1
Type mediated diseases.
60. A method of treating a patient suffering from a disorder in
which IL-18 is detrimental comprising the step of administering an
anti-IL-18 antibody, before, concurrent, or after the
administration of a second agent, wherein the second agent is
selected from the group consisting of an anti-IL-12 antibody or
antigen binding fragment thereof, methotrexate, anti-TNF antibody
or antigen binding fragment thereof, corticosteroids, cyclosporin,
rapamycin, FK506, and non-steroidal anti-inflammatory agents.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of, and claims priority
from, U.S. patent application Ser. No. 09/780,035, filed on Feb. 9,
2001, entitled, "Antibodies that Bind IL-18 and Methods of
Inhibiting IL-18 Activity," which claims the benefit of U.S.
Provisional Application No. 60/181,608, filed Feb. 10, 2000,
entitled, "Antibodies that Bind Human Interleukin-18 and Methods of
Making and Using," the contents of each which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Interleukin-18 (IL-18) was originally described in 1989 as
interferon-gamma inducing factor (IGIF) and is a pro-inflammatory
cytokine with various functions in addition to an ability to induce
interferon gamma. These biological properties include activation of
NF-.kappa.b, Fas ligand expression, the induction of both CC and
CXC chemokines, and increased production of competent human
immunodeficiency virus.
[0003] Due to the ability of IL-18 to induce interferon gamma
production in T cells and macrophages, it plays an important role
in Th1-type immune responses and participates in both innate and
acquired immunity. IL-18 is related to the IL-1 family in terms of
both structure and function. For reviews of IL-18 structure,
function and biological activity, see for example Dinarello, C. et
al. (1998) J. Leukoc. Biol. 63:658-654; Dinarello, C. A. (1999)
Methods 19:121-132; and Dinarello, C. A. (1999) J. Allergy Clin.
Immunol. 103:11-24.
[0004] It would be desirable to use to modulate IL-18 in a variety
of human immune responses. In particular, antibodies that bind to
and neutralize IL-18 are particularly desirable. Moreover, murine
IL-18 antibodies are limited for their use in vivo due to problems
associated with administration of mouse antibodies to humans, such
as short serum half life, an inability to trigger certain human
effector functions and elicitation of an unwanted immune response
against the mouse antibody in a human (the "human anti-mouse
antibody" (HAMA) reaction).
[0005] In general, attempts to overcome the problems associated
with use of fully-murine antibodies in humans, have involved
genetically engineering the antibodies to be more "human-like." For
example, chimeric antibodies, in which the variable regions of the
antibody chains are murine-derived and the constant regions of the
antibody chains are human-derived, have been prepared (Junghans, et
al. (1990) Cancer Res. 50:1495-1502; Brown et al. (1991) Proc.
Natl. Acad. Sci. 88:2663-2667; Kettleborough et al. (1991) Protein
Engineering. 4:773-783). However, because these chimeric and
humanized antibodies still retain some murine sequences, they still
may elicit an unwanted immune reaction, the human anti-chimeric
antibody (HAMA) reaction, especially when administered for
prolonged periods.
[0006] A preferred IL-18 inhibitory agent to murine antibodies or
derivatives thereof (e.g., chimeric or humanized antibodies) would
be an entirely human anti-IL-18 antibody, since such an agent
should not elicit the HAMA reaction, even if used for prolonged
periods. However, such antibodies have not been described in the
art and, therefore are still needed.
SUMMARY OF THE INVENTION
[0007] This invention pertains to compounds, such as antibodies,
that bind human IL-18, as well as methods of making and using such
compounds or antibodies.
[0008] In one aspect, the invention pertains to a compound capable
of binding a human IL-18 amino acid sequence, or portion thereof,
where the amino acid comprises an N- or C-terminal portion of human
IL-18 such as provided in SEQ ID NO: 70 or SEQ ID NO: 71. In one
embodiment, the compound is a small molecule, peptide, polypeptide,
antibody, or antibody fragment, such as a fully human antibody or
fragment.
[0009] In another aspect, the invention pertains to a human
monoclonal antibody, or antigen-binding portion thereof, capable of
binding to human IL-18. In other embodiments, the antibody or
fragment thereof, dissociates from human IL-18, as determined by
plasmon resonance, with a koff rate constant of 0.1 s-1 or less,
1.times.10E-2 s-1 or less, 1.times.10E-3 s-1 or less, 1.times.10E-4
s-1 or less, 1.times.10E-5 s-1 or less, 1.times.10E-6 s-1 or less,
or inhibits human IL-18 activity with an IC50 of 1.times.10E-6 or
less, 1.times.10E-7 or less, 1.times.10E-8 or less, 1.times.10E-9,
1.times.10E-10 or less, or 1.times.10E-11 or less.
[0010] In another aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds an
epitope of human IL-18 comprising amino acids PLFEDMTDSDCRDNA (SEQ
ID NO: 1), VIRNLNDQVLFIDQ (SEQ ID NO: 33), or a portion of either.
Preferably, the antibody is a neutralizing antibody. Preferably,
the antibody is a human antibody. In various embodiments, the
antibody is a recombinant antibody (e.g., a single-chain antibody
(scFv)), or a monoclonal antibody.
[0011] In other embodiments, the isolated antibody, or
antigen-binding portion thereof, binds to an epitope of human
IL-18, or a portion of either, where the antibody, or
antigen-binding portion thereof, dissociates from human IL-18 with
a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by
surface plasmon resonance, or which inhibits human IL-18 activity
with an IC.sub.50 of 1.times.10.sup.-6M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-2 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-7M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-8M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-4 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-9M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-5 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-10M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-6 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-11M or less.
[0012] Another aspect of the invention pertains to an isolated
human antibody, or an antigen-binding portion thereof, containing
at least one variable region CDR domain capable of binding an
epitope of human IL-18. In related embodiments, the isolated
antibody, or an antigen-binding portion thereof, has a variable
region containing a heavy and/or light chain CDR1 domain, CDR2
domain, or CDR3 domain as set forth in Table 6 or 9 which can have,
e.g., one or more amino acid substitutions or insertions at or
adjacent to any of the Kabat positions indicated in Tables 7-8 and
10-11. In a preferred embodiment, the isolated antibody, or an
antigen-binding portion thereof, contains a light chain variable
region (LCVR) containing the amino acid sequence of SEQ ID NO: 29
and a heavy chain variable region (HCVR) containing the amino acid
sequence of SEQ ID NO: 26. In another preferred embodiment, the
isolated antibody, or an antigen-binding portion thereof, contains
a light chain variable region (LCVR) having the amino acid sequence
of SEQ ID NO: 29 and a heavy chain variable region (HCVR) having
the amino acid sequence of SEQ ID NO: 27.
[0013] Another aspect of the invention pertains to pharmaceutical
compositions comprising an antibody, or antigen-binding portion
thereof, of the invention and a pharmaceutically acceptable
carrier. In one embodiment, the pharmaceutical composition further
comprises at least one additional therapeutic agent for treating a
disorder in which IL-18 activity is detrimental.
[0014] Another aspect of the invention pertains to methods of
making an antibody that binds human interleukin-18 (IL-18). The
invention provides a method comprising exposing an antibody
repertoire to an antigen comprising an epitope of human IL-18
comprising amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1)),
VIRNLNDQVLFIDQ (SEQ ID NO: 33), or a portion of either; and
selecting from the antibody repertoire an antibody that binds the
epitope of human IL-18 comprising amino acids PLFEDMTDSDCRDNA (SEQ
ID NO: 1), VIRNLNDQVLFIDQ (SEQ ID NO: 33), or a portion of
either.
[0015] In one embodiment, the antibody repertoire is an in vivo
repertoire in an animal and the method comprises immunizing the
animal with the antigen comprising the epitope of human IL-18
comprising amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1),
VIRNLNDQVLFIDQ (SEQ ID NO: 33), the N- or C-terminal portion of
human IL-18 (SEQ ID NOS: 70-71), or a portion of any of these
epitopes. In another embodiment, the antibody repertoire is a
recombinant antibody library and the method comprises screening the
library with an antigen containing the epitope of human IL-18
having the amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1),
VIRNLNDQVLFIDQ (SEQ ID NO: 33), peptides represented by SEQ ID NOS:
31-32 and 34-60, or a portion of any of the foregoing. Preferably,
the library is a human antibody library.
[0016] In another aspect, the invention provides an isolated
nucleic acid encoding an antibody of any of the above aspects,
e.g., a heavy and/or light chain variable region, or portion
thereof. In related embodiments, the isolated nucleic acid encoding
the anti-IL-18 antibody, or portion thereof, is in a recombinant
expression vector, e.g., for expression in a host cell.
[0017] Thus, in another aspect, the invention pertains to a method
of using the foregoing host cell into which the recombinant
expression vector has been introduced, for synthesizing an antibody
that binds human IL-18, by culturing the host cell in a culture
medium until an antibody that binds human IL-18 is synthesized by
the cell.
[0018] Another aspect of the invention pertains to a method for
inhibiting human IL-18 activity comprising contacting human IL-18
with the antibody, or antigen-binding portion thereof, of the
invention such that human IL-18 activity is inhibited.
[0019] Yet another aspect of the invention pertains to a method for
inhibiting human IL-18 activity in a human subject suffering from a
disorder in which IL-18 activity is detrimental, comprising
administering to the human subject the antibody, or antigen-binding
portion thereof, of the invention such that human IL-18 activity in
the human subject is inhibited. In one embodiment, the anti-IL-18
antibody may be administered, e.g., before, concurrent, or after,
an additional agent such as an anti-IL-12 antibody or antigen
binding fragment thereof, methotrexate, anti-TNF antibody or
antigen binding fragment thereof, corticosteroids, cyclosporin,
rapamycin, FK506, or a non-steroidal anti-inflammatory agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows the structural model of IL-18 (center) as
compared to IL-1.beta. (left) and IL1RA (right).
[0021] FIG. 2 shows a structural model of IL-18 complexed with the
IL-18 receptor, wherein the peptide epitope comprising amino acids
PLFEDMTDSDCRDNA (SEQ ID NO: 1) of IL-18 is indicated in dark gray.
This peptide epitope is bound by the anti-IL-18 antibody 2E1.
[0022] FIG. 3 shows a structural model of IL-18 complexed with the
IL-18 receptor, wherein the peptide epitope comprising amino acids
YFGKLESKLSVIRN (SEQ ID NO: 33) of IL-18 is indicated in dark gray.
This peptide epitope is bound by the anti-IL-18 antibody LT28.
[0023] FIG. 4 shows a structural model of full length IL-18
complexed with the IL-18 receptor. The spherical light and dark
gray epitopes represent the N and C terminal contact epitopes of
IL-18 (respectively, SEQ ID NOS: 70 and 71).
[0024] FIG. 5 shows the potency of three different anti-IL-18
antibodies in neutralizing the biologic effects of IL-18 as a
function of inhibition of IFN-.gamma. induction in KG1 cells. The
IC50 values for the antibodies 125H (boxes) and the 2E1 antibody as
an IgG antibody (circles) or as a single chain antibody (triangles)
are, respectively, 2.1E-10, 9.0E-10, and 3.3E-9.
DETAILED DESCRIPTION OF THE INVENTION
[0025] This invention pertains to the selection of peptide epitopes
that are capable of generating neutralizing antibodies to IL-18
mediated signal transduction, the preparation of antibodies to
these epitopes and the use of such antibodies, including use to
treat disorders involving IL-18. The strategy of selecting epitopes
entails construction of an homology model of the IL-18 protein and
its corresponding receptor. A combination of visual inspection and
computational evaluation is then used to select representative
peptide segments for synthesis and antibody generation Amino acid
sequences shown herein use the standard one-letter abbreviation
code.
Selection of IL-18 Epitopes
[0026] The program Modeler (Sali, A. et al., Evaluation of
comparative protein modeling by MODELLER. Proteins: Struct.,
Funct., Genet. (1995), 23(3), pp. 318-26. CODEN: PSFGEY; ISSN:
0887-3585) was used to generate homology models for both IL-18 and
the IL-18 receptor. The X-ray crystal structures of IL-1.beta.
(Priestle, J., et al. The three-dimensional structure of human
interleukin-1.beta. refined to 2.0.ANG. resolution. Prog. Clin.
Biol. Res. (1990), 349 (Cytokines Lipocortins Inflammation
Differ.), pp. 297-307) and IL-1RA (Schreuder, H. et al., Refined
crystal structure of the interleukin-1 receptor antagonist:
presence of a disulfide link and a cis-proline. Eur. J. Biochem.
(1995), 227(3), pp. 838-47) are available and were used as
reference coordinates for the model construction of IL-18. The IL-1
receptor structure (Vigers, G., et al. Crystal structure of the
type-I interleukin-1-receptor complexed with interleukin-1.beta.
Nature (London) (1997), 386(6621), pp. 190-194) was used to model
the IL-18 receptor.
[0027] The structural model building for IL-18 and the IL-18
receptor is described further below.
IL-18 Model Building
[0028] The overall sequence homology with these two proteins (i.e.,
IL-1.beta. and IL-18) is low, however, there is compelling evidence
that IL-18 is a member of the IL-1 family (see Dinarello, C. A.
IL-18: a TH1-inducing, proinflammatory cytokine and new member of
the IL-1 family. J. Allergy Clin. Immunol. (1999), 103(1, Pt. 1),
pp. 11-24) and that the overall protein fold is very similar. Like
IL-1.beta., IL-18 is initially secreted in a pro form. Both
pro-IL-1 .beta. and pro-IL-18 are activated by IL-1
.beta.-converting enzyme (ICE) (Fantuzzi, G. and Dinarello, C. A.
Interleukin-18 and interleukin-1.beta.: two cytokine substrates for
ICE (caspase-1). J. Clin. Immunol. (1999), 19(1), pp. 1-11). It is
also known that the IL-1 receptor and the IL-18 receptor are
similar (Dinarello, C. A. et al. Overview of interleukin-18: more
than an interferon-.gamma.inducing factor. J. Leukocyte Biol.
(1998), 63(6), 658-664). IL-1.beta. is capable of binding to the
IL-18 receptor. As a final argument, IL-1 .beta. and IL-1RA display
an identical fold, even though the overall sequence homology
between these two proteins is on a par with the sequence homology
with IL-18. The sequence alignment between the three proteins
(i.e., IL-18, IL-1.beta. and IL1-RA) was constructed manually with
the program InsightII. This alignment can be seen in Table 1:
TABLE-US-00001 TABLE 1 Sequence alignment for IL-18 relative to
IL-1.beta. and IL-1RA 24 IL-18:
YFGK-LESKLS-VIRNLNDQVLFIDQGNRPLFE--DMT-DSDCRD--NAP (SEQ ID NO: 4)
IL-1.beta.: AP-VRS-LNCTLRDSQQKSLVMS-G--P-YELKALHLQGQ--D--MEQ (SEQ
ID NO: 5) IL-1RA: SSKMQA-FR--IWDVNQKTFYLR-N--N--QLVAGYLQGP--NVNLEE
(SEQ ID NO: 6) 80 IL-18:
RTIFIISMY-KDSQPRG-MAVTISVKCEKISTLSC----ENK-IISFKEM IL-1.beta.:
QVVFSMS-FVQGEESNDKIPVALGLK-EKNLYLSCVLK-DDKPTLQLESV IL-1RA:
KI--DV---VP-IEPH---ALFLGIH-GGKMCLSCV-KSGDETRLQLEAV 123 IL-18:
NPPDNI-KDTKSDIIF-FQRSVPGHDNKMQFESSSYEGYFLACE-KERDL IL-1.beta.:
DPKNYP-KK-KMEKRFVFNK-I-EINNKLEFESAQFPNWYISTS-QAENM IL-1RA:
NITDLSENR-KQDKRFAFIR-S-DSGPTTSFESAACPGWFLCTAMEADQ- 170 IL-18:
FKLILKKED-ELGDRSIM-FTVQNED IL-1.beta.: -PVFL--GG-TKGGQDITDFTMQFVSS
IL-1RA: -PVSL--TNMPDEGVMVTKFYFQED
[0029] The sequence homology between these sequences is listed in
Table 2. The upper triangle is percent strict sequence identity and
the lower triangle is percent conservative sequence homology. Only
the portions of the total sequences reported in Table 1 are
considered in Table 2. As was mentioned above, the overall homology
is low but consistent across the family.
TABLE-US-00002 TABLE 2 Sequence homology between IL-1 family
members Molecule IL-18 IL-1.beta. IL-1RA IL-1 Rec IL-18 Rec IL-18
-- 20.0 21.8 -- -- IL-1b 53.5 -- 27.5 -- -- IL-1RA 50.6 54.4 -- --
-- IL-1 Rec -- -- -- -- 26.1 IL-18 Rec -- -- -- 50.5 --
[0030] The resulting IL-18 structure is pictured in FIG. 1 along
with IL-1.beta. and IL-1RA. The overall quality as assessed by the
program What_Check (Hooft, R. W. et al., Errors in Protein
Structures. Nature (1996) 381, pp. 272) is reasonable, but a bit
low (see Table 3 below).
TABLE-US-00003 TABLE 3 Structural Z-scores from What_Check
(Positive is better than average) IL-18 IL-18 Rec IL-1.beta. IL-1RA
IL-1 Rec (model) (model) Packing Quality -1.6 -2.3 -3.6 -5.5 -5.7
Ramachandran -2.0 -1.3 -2.9 -3.3 -2.3 Plot Rotamer -1.6 -0.8 -1.5
-0.6 -0.6 Normality Backbone -1.7 +0.5 -1.6 -5.6 -2.7
Conformation
[0031] However, the assessment of the reference structures by
What_Check is also low, suggesting that this protein fold is poorly
represented in the database of reference structures. Undoubtedly
though, the low sequence homology contributed to a less than
perfect final structure in spite of our confidence in the overall
protein fold. However, for the purpose of choosing epitopes for
antibody generation, this structure is considered to be
sufficient.
IL-18 Receptor Model Building
[0032] The structure of the IL-18 receptor was also generated using
the program Modeler. The reference coordinates were from the IL-1
receptor. As in the case of the cytokines associated with these
receptors, the overall sequence identity is low, but sufficient to
generate an alignment. The sequence homology figures are included
in Table 2 above. The alignment was generated manually using the
program InsightII and is presented in Table 4.
TABLE-US-00004 TABLE 4 Sequence alignment for the IL-18 receptor
relative to the IL-1 Receptor 22 IL-18 Rec:
CTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPR (SEQ ID NO: 7)
IL-1 Rec: CKEREEKIILVSSANEIDVRPCPLNPNEHKGTITWYKDD-SKTPVSTEQA (SEQ
ID NO: 8) 72 IL-18 Rec:
SSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHS--- IL-1 Rec:
S--RIHQHKEKLWFVPAKVEDSGHYYCVVRNSSYCLRIKISAKFVENEPN 119 IL-18 Rec:
-CFTERQVTSKIVEVKKFFQITCENSYYQTLVNST----SLYKNCKKLLL IL-1 Rec:
LCYNAQAIFKQKLPVAGDGGLVCPYMEFFKNENNELPKLQWYKDCKPLLL 163 IL-18 Rec:
EN-----NKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVE IL-1 Rec:
DNIHFSGVKDRLIVMNVAEKHRGNYTCHASYTYLGKQYPITRVIEFITLE 209 IL-18 Rec:
DRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMF-GEE-NG IL-1 Rec:
ENKPTRPVIVSPANETMEVDLGSQIQLICNVTGQLSDIAYWKWNGSVIDE 257 IL-18 Rec:
SDPNIHEE-KEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGG IL-1 Rec:
DDPVLGEDYYSVENPANKRRSTLITVLNISEIESRFYKHPFTCFAKNTHG 306 IL-18 Rec:
TDTKSFILVRKAD IL-1 Rec: IDAAYIQLIYPVT
[0033] The overall quality of the structure of the IL-18 receptor
as determined using the Modeler program is reasonable but again
scores somewhat low according to What_Check (see Table 3 above).
The confidence that can be placed on the overall fold comes
primarily from the fact that IL-1.beta. binds to both the IL-1 and
IL-18 receptor. The low sequence homology certainly contributes to
the quality of the final structure, however, as in the case of the
associated cytokines above, this current structure is considered to
be sufficient. As an additional exercise, the IL-18 peptide epitope
bound by LT28 (SEQ ID NO: 33) was modeled when complexed with the
IL-18 receptor (FIG. 3). As a final exercise, a model of the
IL-18/IL-18 receptor complex was generated based on the
IL-1.beta./IL-1 receptor structure (FIG. 4). This structure was
generated by superimposing the cytokine structures and the receptor
structures. No attempt was made to energy minimize the final
structure.
Peptide Epitope Selection
[0034] The primary purpose of generating structural models was to
be able to select suitable peptides based primarily on a visual
score. Solvent exposed sections of the proteins and portions of the
proteins which were both hydrophilic and buried in the
receptor/cytokine complex were considered. A final element
considered was based on a selectivity criterion. The selected
peptide epitope should be different in sequence from similar
portions of other members of the family. Based on this criteria, a
peptide from IL-18 was selected. In addition, a comprehensive
overlapping panel of peptides (SEQ ID NOS: 31-60) representative of
full length human IL-18 (SEQ ID NO: 61) was also made and the
sequence of all of these IL-18 related peptides is shown in Table
5, below.
TABLE-US-00005 TABLE 5 Selected Peptides Representative of IL-18
Peptide Sequence SEQ ID NO: PLFEDMTDSDCRDNA (SEQ ID NO: 1)
CPLFEDMTDSDCRDNA (SEQ ID NO: 2) PLFEDMTDSDCR (SEQ ID NO: 3)
YFGKLESKLSVIRN (SEQ ID NO: 31) ESKLSVIRNLNDQV (SEQ ID NO: 32)
VIRNLNDQVLFIDQ (SEQ ID NO: 33) (LT28 binding epitope)
NDQVLFIDQGNRPL (SEQ ID NO: 34) FIDQGNRPLFEDMT (SEQ ID NO: 35)
NRPLFEDMTDSDCR (SEQ ID NO: 36) (2E1 binding epitope) EDMTDSDCRDNAPR
(SEQ ID NO: 37) SDCRDNAPRTIFII (SEQ ID NO: 38) NAPRTIFIISMYKD (SEQ
ID NO: 39) IFIISMYKDSQPRG (SEQ ID NO: 40) MYKDSQPRGMAVTI (SEQ ID
NO: 41) QPRGMAVTISVKCE (SEQ ID NO: 42) AVTISVKCEKISTL (SEQ ID NO:
43) VKCEKISTLSCENK (SEQ ID NO: 44) ISTLSCENKIISFK (SEQ ID NO: 45)
CENKIISFKEMNPP (SEQ ID NO: 46) ISFKEMNPPDNIKD (SEQ ID NO: 47)
MNPPDNIKDTKSDI (SEQ ID NO: 48) NIKDTKSDIIFFQR (SEQ ID NO: 49)
KSDIIFFQRSVPGH (SEQ ID NO: 50) FFQRSVPGHDNKMQ (SEQ ID NO: 51)
VPGHDNKMQFESSS (SEQ ID NO: 52) NKMQFESSSYEGYF (SEQ ID NO: 53)
ESSSYEGYFLACEK (SEQ ID NO: 54) EGYFLACEKERDLF (SEQ ID NO: 55)
ACEKERDLFKLILK (SEQ ID NO: 56) RDLFKLILKKEDEL (SEQ ID NO: 57)
LILKKEDELGDRSI (SEQ ID NO: 58) EDELGDRSIMFTVQ (SEQ ID NO: 59)
DRSIMFTVQNED (SEQ ID NO: 60) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFED (SEQ
ID NO: 61) MTDSDCRDNAPRTIFIISMYKDSQPRGMAVTI LT28 and 2E1
SVKCEKISTLSCENKIISFKEMNPPDNIKDTK epitopes
SDIIFFQRSVPGHDNKMQFESSSYEGYFLACE indicated
KERDLFKLILKKEDELGDRSIMFTVQNED in bold
[0035] The N-terminal cysteine of the IL-18 peptide represented by
SEQ ID NO: 2 is not part of the native IL-18 sequence, but was
added as a conjugation site. Accordingly, within the native IL-18
amino acid sequence, the region corresponding to the selected
epitope comprises amino acid residues having the amino acid
sequence PLFEDMTDSDCRDNA (SEQ ID NO: 1).
[0036] A schematic model of the IL-18 peptide (SEQ ID NO: 1)
complexed with the IL-18 receptor is shown in FIG. 2, with this
peptide epitope indicated in dark gray.
[0037] Subsequent antigenicity calculations were performed on IL-18
peptide sequences, with the result that this peptide scored
particularly highly. This peptide was synthesized and used as an
epitope to generate antibodies in a rabbit host. Molecular modeling
data obtained using the IL-18 peptide PLFEDMTDSDCRDNA (SEQ ID NO:
1) or YFGKLESKLSVIRN (SEQ ID NO: 31) as compared to a cognate
receptor, i.e., the IL-18 receptor, as depicted in FIGS. 2 and 3,
provides an indication as to what residues a neutralizing antibody
or compound may interact with.
[0038] An alternative method for peptide epitope selection can be
accomplished in the absence of any molecular modeling by screening
a panel of representative peptides using immunoselection. In one
approach, overlapping peptides representative of the entire protein
sequence can be used. In a more limited approach, only certain
epitopes are represented in the panel of peptides. In a combined
approach, molecular modeling can be used to identify epitopes
likely to be important. The identified epitope(s) sequence can then
be used to construct a panel of peptides (e.g., overlapping
peptides) that are representative of the identified epitope(s).
Methods for manufacturing desired peptide sequences can be carried
out using standard techniques known in the art.
[0039] Once the binding peptide or peptides (e.g., panel of
overlapping peptides) has been selected, an immunoscreen for a
cognate receptor can be performed. Alternatively, an immunoscreen
can be performed with a selected cognate receptor such that a
peptide having a certain binding affinity can be identified. Any
number of immunoscreens can be employed such that, either a desired
receptor or desired peptide can be identified as a candidate
binding molecule for further study. Such "bait" and "prey"
techniques for analyzing protein-protein interactions, for
identifying candidate biding molecules, and/or for scoring binding
affinities are described in the art. One preferred technique
utilizes phage display as described herein.
Anti-IL-18 Antibodies
[0040] The invention provides antibodies, as well as antibody
portions thereof, that bind IL-18. Preferably, the antibodies, or
portions thereof, are isolated antibodies. Preferably, the
antibodies, or portions thereof, are neutralizing antibodies.
[0041] The term "antibody", as used herein, is intended to refer to
immunoglobulin molecules comprised of four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (abbreviated herein as LCVR or VL)
and a light chain constant region. The light chain constant region
is comprised of one domain, CL. 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).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0042] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., hIL-18). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising 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). 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) .
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. Other
forms of single chain antibodies, such as diabodies are also
encompassed. Diabodies are bivalent, bispecific antibodies in which
VH and VL domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)
Structure 2:1121-1123).
[0043] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecules, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described
herein.
[0044] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds hIL-18 is substantially free of
antibodies that specifically bind antigens other than hIL-18). An
isolated antibody that specifically binds hIL-18 may, however, have
cross-reactivity to other antigens, such as IL-18 molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals. Further, an
isolated antibody, e.g., an isolated human antibody, can be a
chimeric antibody wherein, e.g., variable regions, CDR domains, or
isotypes derived from a different human source are grafted to the
parent human antibody.
[0045] A "compound" as used herein, refers to binding molecules
such as antibodies, e.g., polyclonal antibodies, monoclonal
antibodies, binding fragments thereof (e.g., Fab fragments), single
chain antibodies (e.g., scFv), peptides or peptide mimetics, as
well as non-peptide based molecules, such as small molecules having
ligand binding activity.
[0046] A "neutralizing antibody", as used herein (or an "antibody
that neutralized hIL-18 activity"), is intended to refer to an
antibody whose binding to hIL-18 results in inhibition of the
biological activity of hIL-18. This inhibition of the biological
activity of hIL-18 can be assessed by measuring one or more
indicators of hIL-18 biological activity. These indicators of
hIL-18 biological activity can be assessed by one or more of
several standard in vitro or in vivo assays known in the art.
[0047] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0048] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0049] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction.
[0050] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds an
epitope of human IL-18 comprising amino acids PLFEDMTDSDCRDNA (SEQ
ID NO: 1) or VIRNLNDQVLFIDQ (SEQ ID NO: 33), or a portion of any of
these epitopes. Preferably, the antibody is a neutralizing
antibody. Preferably, the antibody is a human antibody. In various
embodiments, the antibody is a recombinant antibody or a monoclonal
antibody.
[0051] In other embodiments, the isolated antibody, or
antigen-binding portion thereof, binds to an epitope of human IL-18
comprising amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1), wherein the
antibody, or antigen-binding portion thereof, dissociates from
human IL-18 with a k.sub.off rate constant of 0.1 s.sup.-1 or less,
as determined by surface plasmon resonance, or which inhibits human
IL-18 activity with an IC.sub.50 of 1.times.10.sup.-6M or less.
Alternatively, the antibody, or an antigen-binding portion thereof,
may dissociate from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-2 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-7M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1.times.10.sup.-8M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human IL-18 with a k.sub.off rate constant of
1.times.10.sup.-4 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-18 activity with an
IC.sub.50 of 1 x 10.sup.-9M or less. Alternatively, the antibody,
or an antigen-binding portion thereof, may dissociate from human
IL-18 with a k.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1
or less, as determined by surface plasmon resonance, or may inhibit
human IL-18 activity with an IC.sub.50 of 1.times.10.sup.-10M or
less. Alternatively, the antibody, or an antigen-binding portion
thereof, may dissociate from human IL-18 with a k.sub.off rate
constant of 1.times.10.sup.-6 s.sup.-1 or less, as determined by
surface plasmon resonance, or may inhibit human IL-18 activity with
an IC.sub.50 of 1.times.10.sup.-11M or less.
Affinity Maturation of Identified Anti-IL-18 Antibodies
[0052] The invention also provides for the further modification of
an antibody identified as binding to an IL-18 epitope. The
modification of the identified anti-IL-18 antibody is to improve
binding and/or neutralization activity.
Therapeutic Compositions and Methods for Administering
[0053] The invention also provides pharmaceutical compositions
comprising an antibody, or antigen-binding portion thereof, of the
invention and a pharmaceutically acceptable carrier. In one
embodiment, the pharmaceutical composition further comprises at
least one additional therapeutic agent for treating a disorder in
which IL-18 activity is detrimental.
[0054] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0055] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. Preferably, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0056] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0057] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0058] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, the preferred
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0059] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0060] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which IL-18 activity is detrimental. For
example, an anti-hIL-18 antibody or antibody portion of the
invention may be coformulated and/or coadministered with one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies.
Therapeutic Uses
[0061] Interleukin 18 plays a critical role in the pathology
associated with a variety of diseases involving immune and
inflammatory elements. These diseases include, but are not limited
to, rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,
spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitis scleroderma, graft versus host disease, organ
transplant rejection, acute or chronic immune disease associated
with organ transplantation, sarcoidosis, atherosclerosis,
disseminated intravascular coagulation, Kawasaki's disease, Grave's
disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis
of the kidneys, chronic active hepatitis, uveitis, septic shock,
toxic shock syndrome, sepsis syndrome, cachexia, infectious
diseases, parasitic diseases, acquired immunodeficiency syndrome,
acute transverse myelitis, Huntington's chorea, Parkinson's
disease, Alzheimer's disease, stroke, primary biliary cirrhosis,
hemolytic anemia, malignancies, heart failure, myocardial
infarction, Addison's disease, sporadic, polyglandular deficiency
type I and polyglandular deficiency type II, Schmidt's syndrome,
adult (acute) respiratory distress syndrome, alopecia, alopecia
areata, seronegative arthopathy, arthropathy, Reiter's disease,
psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic
synovitis, chlamydia, yersinia and salmonella associated
arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis C, common varied immunodeficiency
(common variable hypogammaglobulinaemia), dilated cardiomyopathy,
female infertility, ovarian failure, premature ovarian failure,
fibrotic lung disease, cryptogenic fibrosing alveolitis,
post-inflammatory interstitial lung disease, interstitial
pneumonitis, connective tissue disease associated interstitial lung
disease, mixed connective tissue disease associated lung disease,
systemic sclerosis associated interstitial lung disease, rheumatoid
arthritis associated interstitial lung disease, systemic lupus
erythematosus associated lung disease, dermatomyositis/polymyositis
associated lung disease, Sjogren's disease associated lung disease,
ankylosing spondylitis associated lung disease, vasculitic diffuse
lung disease, haemosiderosis associated lung disease, drug-induced
interstitial lung disease, radiation fibrosis, bronchiolitis
obliterans, chronic eosinophilic pneumonia, lymphocytic
infiltrative lung disease, postinfectious interstitial lung
disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune
hepatitis (classical autoimmune or lupoid hepatitis), type-2
autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune
mediated hypoglycaemia, type B insulin resistance with acanthosis
nigricans, hypoparathyroidism, acute immune disease associated with
organ transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjogren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis and vitiligo. The
human antibodies, and antibody portions of the invention can be
used to treat humans suffering from autoimmune diseases, in
particular those associated with inflammation, including,
rheumatoid spondylitis, allergy, autoimmune diabetes, autoimmune
uveitis, acute liver disease, chronic liver diseases, allergy and
asthma, mental disorders (e.g., depression and schizophrenia), and
Th2 Type and Th1 Type mediated diseases.
[0062] Preferably, the antibodies of the invention or
antigen-binding portions thereof, are used to treat rheumatoid
arthritis, Crohn's disease, multiple sclerosis, insulin dependent
diabetes, mellitus, and psoriasis.
[0063] An antibody, or antibody portion, of the invention also can
be administered with one or more additional therapeutic agents
useful in the treatment of autoimmune and inflammatory
diseases.
[0064] Antibodies of the invention, or antigen binding portions
thereof can be used alone or in combination to treat such diseases.
It should be understood that the antibodies of the invention or
antigen binding portion thereof can be used alone or in combination
with an additional agent, e.g., a therapeutic agent, said
additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent which imparts
a beneficial attribute to the therapeutic composition e.g., an
agent which effects the viscosity of the composition.
[0065] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited . The
combinations which are part of this invention can be the antibodies
of the present invention and at least one additional agent selected
from the lists below. The combination can also include more than
one additional agent, e.g., two or three additional agents if the
combination is such that the formed composition can perform its
intended function.
[0066] Preferred combinations are non-steroidal anti-inflammatory
drug(s) also referred to as NSAIDS which include drugs like
ibuprofen. Other preferred combinations are corticosteroids
including prednisolone; the well known side-effects of steroid use
can be reduced or even eliminated by tapering the steroid dose
required when treating patients in combination with the anti-IL-18
antibodies of this invention. Non-limiting examples of therapeutic
agents for rheumatoid arthritis with which an antibody, or antibody
portion, of the invention can be combined include the following:
cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies
to or antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15,
IL-16, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention,
or antigen binding portions thereof, can be combined with
antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,
CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90,
or their ligands including CD154 (gp39 or CD40L).
[0067] Preferred combinations of therapeutic agents may interfere
at different points in the autoimmune and subsequent inflammatory
cascade; preferred examples include TNF antagonists like chimeric,
humanized or human TNF antibodies, D2E7, (PCT Publication No. WO
97/29131), CA2 (Remicade.TM.), CDP 571, and soluble p55 or p75 TNF
receptors, derivatives, thereof, (p75TNFR1gG (Enbrel.TM.) or
p55TNFR1gG (Lenercept), and also TNF.alpha. converting enzyme
(TACE) inhibitors; similarly IL-1 inhibitors
(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be
effective for the same reason. Other preferred combinations include
Interleukin 11. Yet another preferred combination are other key
players of the autoimmune response which may act parallel to,
dependent on or in concert with IL-18 function; especially
preferred are IL-12 antagonists including IL-12 antibodies or
soluble IL-12 receptors, or IL-12 binding proteins. It has been
shown that IL-12 and IL-18 have overlapping but distinct functions
and a combination of antagonists to both may be most effective. Yet
another preferred combination are non-depleting anti-CD4
inhibitors. Yet other preferred combinations include antagonists of
the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including
antibodies, soluble receptors or antagonistic ligands.
[0068] The antibodies of the invention, or antigen binding portions
thereof, may also be combined with agents, such as methotrexate,
6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, cochicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen, cox-2
inhibitors, cox-2 selective inhibitors (e.g., rofecoxib (VIOXX.TM.;
Merck & Co., Inc.)) corticosteroids such as prednisolone,
phosphodiesterase inhibitors, adensosine agonists, antithrombotic
agents, complement inhibitors, adrenergic agents, agents which
interfere with signalling by proinflammatory cytokines such as
TNF.alpha. or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TNF.alpha.
converting enzyme (TACE) inhibitors, T-cell signalling inhibitors
such as kinase inhibitors, metalloproteinase inhibitors,
sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin
converting enzyme inhibitors, soluble cytokine receptors and
derivatives thereof (e.g. soluble p55 or p75 TNF receptors and the
derivatives p75TNFRIgG (Enbrel.TM. and p55TNFRIgG (Lenercept)),
sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines (e.g.
IL-4, IL-10, IL-11, IL-13 and TGF.beta.. Preferred combinations
include methotrexate or leflunomide and in moderate or severe
rheumatoid arthritis cases, cyclosporine.
[0069] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which an antibody, or antibody portion, of the
invention can be combined include the following: budenoside;
epidermal growth factor; corticosteroids; cyclosporin,
sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;
balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor
antagonists; anti-IL-1.beta. monoclonal antibodies; anti-IL-6
monoclonal antibodies; growth factors; elastase inhibitors;
pyridinyl-imidazole compounds; antibodies to or antagonists of
other human cytokines or growth factors, for example, TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, EMAP-II, GM-CSF,
FGF, and PDGF. Antibodies of the invention, or antigen binding
portions thereof, can be combined with antibodies to cell surface
molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,
CD69, CD90 or their ligands. The antibodies of the invention, or
antigen binding portions thereof, may also be combined with agents,
such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme inhibitors, T-cell
signalling inhibitors such as kinase inhibitors, metalloproteinase
inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g. soluble p55 or p75 TNF
receptors, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory
cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGF.beta.).
[0070] Preferred examples of therapeutic agents for Crohn's disease
in which an antibody or an antigen binding portion can be combined
include the following: TNF antagonists, for example, anti-TNF
antibodies, D2E7 (PCT Publication No. WO 97/29131), CA2
(Remicade.TM.), CDP 571, TNFR-Ig constructs, (p75TNFRIgG
(Enbrel.TM.) and p55TNFRIgG (Lenercept)) inhibitors and PDE4
inhibitors. Antibodies of the invention or antigen binding portions
thereof, can be combined with corticosteroids, for example,
budenoside and dexamethasone. Antibodies of the invention or
antigen binding portions thereof, may also be combined with agents
such as sulfasalazine, 5-aminosalicylic acid and olsalazine, and
agents which interfere with synthesis or action of proinflammatory
cytokines such as IL-1, for example, IL-1.beta. converting enzyme
inhibitors and IL-lra. Antibodies of the invention or antigen
binding portion thereof may also be used with T cell signaling
inhibitors, for example, tyrosine kinase inhibitors
6-mercaptopurines. Antibodies of the invention or antigen binding
portions thereof, can be combined with IL-11.
[0071] Non-limiting examples of therapeutic agents for multiple
sclerosis with which an antibody, or antibody portion, of the
invention can be combined include the following: corticosteroids;
prednisolone; methylprednisolone; azathioprine; cyclophosphamide;
cyclosporine; methotrexate; 4-aminopyridine; tizanidine;
interferon-.beta.1a (Avonex; Biogen); interferon-.beta.1b
(Betaseron; Chiron/Berlex); Copolymer 1 (Cop-1; Copaxone; Teva
Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous
immunoglobulin; clabribine; antibodies to or antagonists of other
human cytokines or growth factors, for example, TNF, LT, IL-1,
IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, EMAP-II, GM-CSF, FGF,
and PDGF. Antibodies of the invention, or antigen binding portions
thereof, can be combined with antibodies to cell surface molecules
such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. The antibodies of the invention,
or antigen binding portions thereof, may also be combined with
agents, such as methotrexate, cyclosporine, FK506, rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TACE inhibitors, T-cell signalling inhibitors
such as kinase inhibitors, metalloproteinase inhibitors,
sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin
converting enzyme inhibitors, soluble cytokine receptors and
derivatives thereof (e.g. soluble p55 or p75 TNF receptors,
sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines (e.g.
IL-4, IL-10, IL-13 and TGF.beta.).
[0072] Preferred examples of therapeutic agents for multiple
sclerosis in which the antibody or antigen binding portion thereof
can be combined to include interferon-.beta., for example,
IFN.beta.1a and IFN.beta.1b; copaxone, corticosteroids, IL-1
inhibitors, TNF inhibitors, and antibodies to CD40 ligand and
CD80.
[0073] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the antibody or antibody portion to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
antibody or antibody portion are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0074] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0075] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 0.1-20 mg/kg, more preferably 1-10
mg/kg. It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
Methods of Making Anti-IL-18 Antibodies
[0076] The anti-IL-18 antibodies of the invention are made using
any one of a variety of techniques known in the art for preparing
antibodies and using antigens comprising the IL-18 peptide epitope
described in subsection I, i.e., an epitope of human IL-18
comprising amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1).
[0077] In general, the methods of the invention for making an
antibody that binds human interleukin-18 (IL-18) involve:
[0078] exposing an antibody repertoire to an antigen comprising an
epitope of human IL-18 comprising amino acids PLFEDMTDSDCRDNA (SEQ
ID NO: 1), or portion thereof (e.g., SEQ ID NO: 3 or 33); and
[0079] selecting from the antibody repertoire an antibody that
binds the epitope of human IL-18 comprising amino acids
PLFEDMTDSDCRDNA (SEQ ID NO: 1), or portion thereof (e.g., SEQ ID
NO: 3 or 33).
[0080] In one embodiment, the antibody repertoire is an in vivo
repertoire in an animal and the method comprises immunizing the
animal with the antigen comprising the epitope of human IL-18
comprising amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1). In another
embodiment, the antibody repertoire is a recombinant antibody
library and the method comprising screening the library with the
antigen comprising the epitope of human IL-18 comprising amino
acids PLFEDMTDSDCRDNA (SEQ ID NO: 1). Preferably, the library is a
human antibody library.
[0081] Methods for immunizing an animal with an antigen to thereby
raise specific antibodies to the antigen are well known in the art.
An IL-18 antigen comprising an epitope of human IL-18 comprising
amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1) can be administered to
an animal to elicit polyclonal antibodies and specific antibodies
that bind the epitope can be isolated by selecting from the
polyclonal antibodies those antibodies that bind to the epitope
(e.g., by passing the polyclonal antisera over a column that
comprises a peptide comprising amino acids PLFEDMTDSDCRDNA (SEQ ID
NO: 1) of hIL-18). The antigen used to elicit the polyclonal
antibodies can be intact (i.e., full-length) hIL-18 or can be a
portion of hIL-18 that includes the epitope of interest, e.g., a
synthetic peptide comprising amino acids PLFEDMTDSDCRDNA (SEQ ID
NO: 1) of hIL-18. Furthermore, monoclonal antibodies to the epitope
can be made from the aforementioned animals using standard
hybridoma technology and selection for those hybridomas secreting
an antibody that specifically binds the epitope of interest, e.g.,
by screening the hybridomas with a peptide comprising amino acids
PLFEDMTDSDCRDNA (SEQ ID NO: 1) of hIL-18 and selecting for
antibodies that bind specifically to the peptide.
[0082] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, Ladner et
al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO
92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et
al. PCT Publication No. WO 92/20791; Markland et al. PCT
Publication No. WO 92/15679; Breitling et al. PCT Publication No.
WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047;
Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
McCafferty et al., Nature (1990) 348:552-554; Griffiths et al.
(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol
226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.
(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology
9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137;
and Barbas et al. (1991) PNAS 88:7978-7982, and PCT Publication No.
WO 97/29131, the contents of each of which are incorporated herein
by reference.
[0083] The recombinant antibody library may be from a subject
immunized with IL-18, or a portion of IL-18 comprising the epitope
of amino acids PLFEDMTDSDCRDNA (SEQ ID NO: 1). Alternatively, the
recombinant antibody library may be from a naive subject, i.e., one
who has not been immunized with IL-18, such as a human antibody
library from a human subject who has not been immunized with human
IL-18. Antibodies of the invention are selected by screening the
recombinant antibody library with the epitope of amino acids
PLFEDMTDSDCRDNA (SEQ ID NO: 1) of human IL-18 to thereby select
those antibodies that recognize this epitope. Methods for
conducting such screening and selection are well known in the art,
such as described in the references in the preceding paragraph.
[0084] To select antibodies of the invention having a particular
binding affinity for hIL-18, the art-known method of surface
plasmon resonance can be used. To select antibodies having a
particular neutralizing activity for hIL-18, standard methods known
in the art for assessing the inhibition of hIL-18 activity may be
used. In addition, methods for immunizing mice that have been
transgenically altered to encode a human immunoglobulin repertoire
thereby enabling the organism to express fully human antibodies in
response to an immunogen, are known in the art (see, e.g., U.S.
Pat. Nos. 5,877,397 and 6,150,584).
Uses of Anti-IL-18 Antibodies
[0085] Given their ability to bind to hIL-18, the anti-hIL-18
antibodies, or portions thereof, of the invention can be used to
detect hIL-18 (e.g., in a biological sample, such as serum or
plasma), using a conventional immunoassay, such as an enzyme linked
immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue
immunohistochemistry. The invention provides a method for detecting
hIL-18 in a biological sample comprising contacting a biological
sample with an antibody, or antibody portion, of the invention and
detecting either the antibody (or antibody portion) bound to hIL-18
or unbound antibody (or antibody portion), to thereby detect hIL-18
in the biological sample. The antibody is directly or indirectly
labeled with a detectable substance to facilitate detection of the
bound or unbound antibody. Suitable detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; and examples of suitable
radioactive material include .sup.125I, .sup.131I, .sup.35S,
.sup.32P, .sup.33P, or .sup.3H.
[0086] Alternative to labeling the antibody, hIL-18 can be assayed
in biological fluids by a competition immunoassay utilizing rhIL-18
standards labeled with a detectable substance and an unlabeled
anti-hIL-18 antibody. In this assay, the biological sample, the
labeled rhIL-18 standards and the anti-hIL-18 antibody are combined
and the amount of labeled rhIL-18 standard bound to the unlabeled
antibody is determined The amount of hIL-18 in the biological
sample is inversely proportional to the amount of labeled rhIL-18
standard bound to the anti-hIL-18 antibody.
[0087] The antibodies and antibody portions of the invention
preferably are capable of neutralizing hIL-18 activity both in
vitro and in vivo. Accordingly, such antibodies and antibody
portions of the invention can be used to inhibit hIL-18 activity,
e.g., in a cell culture containing hIL-18, in human subjects or in
other mammalian subjects having IL-18 with which an antibody of the
invention cross-reacts. In one embodiment, the invention provides a
method for inhibiting IL-18 activity comprising contacting IL-18
with an antibody or antibody portion of the invention such that
IL-18 activity is inhibited. Preferably, the IL-18 is human IL-18.
For example, in a cell culture containing, or suspected of
containing hIL-18, an antibody or antibody portion of the invention
can be added to the culture medium to inhibit hIL-18 activity in
the culture.
[0088] In another embodiment, the invention provides a method for
inhibiting IL-18 activity in a subject suffering from a disorder in
which IL-18 activity is detrimental. The invention provides methods
for inhibiting IL-18 activity in a subject suffering from such a
disorder, which method comprises administering to the subject an
antibody or antibody portion of the invention such that IL-18
activity in the subject is inhibited. Preferably, the IL-18 is
human IL-18 and the subject is a human subject. Alternatively, the
subject can be a mammal expressing an IL-18 with which an antibody
of the invention cross-reacts. Still further the subject can be a
mammal into which has been introduced hIL-18 (e.g., by
administration of hIL-18 or by expression of an hIL-18 transgene).
An antibody of the invention can be administered to a human subject
for therapeutic purposes. Moreover, an antibody of the invention
can be administered to a non-human mammal expressing an IL-18 with
which the antibody cross-reacts for veterinary purposes or as an
animal model of human disease. Regarding the latter, such animal
models may be useful for evaluating the therapeutic efficacy of
antibodies of the invention (e.g., testing of dosages and time
courses of administration).
[0089] In particular, one animal model for modulating IL-18
activity in an animal uses NOD-SCID mice which are transplanted
with human peripheral blood mononuclear cells. Then, two to four
weeks after engraftment (as measured by human IgG titers in serum)
the mice are injected with LPS (lipopolysaccharide). Four to six
hours later LPS-induced human interferon-gamma serum titers are
determined. The efficacy (potency) of anti-IL-18 antibodies (e.g.,
IL-18 neutralizing antibodies) is determined by injecting the
antibodies (ip) one day prior to LPS challenge followed by
monitoring the test animals for a reduction in interferon-gamma
serum titers (a function of IL-18 in vivo activity) as compared to
controls (see, e.g., Holmes et al., Hybridoma, 19:363367
(2000)).
[0090] As used herein, the term "a disorder in which IL-18 activity
is detrimental" is intended to include diseases and other disorders
in which the presence of IL-18 in a subject suffering from the
disorder has been shown to be, or is suspected of being, either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which IL-18 activity is detrimental is a disorder in
which inhibition of IL-18 activity is expected to alleviate the
symptoms and/or progression of the disorder. Such disorders may be
evidenced, for example, by an increase in the concentration of
IL-18 in a biological fluid of a subject suffering from the
disorder (e.g., an increase in the concentration of IL-18 in serum,
plasma, synovial fluid, etc. of the subject), which can be
detected, for example, using an anti-IL-18 antibody as described
above.
[0091] Non-limiting examples of disorders that can be treated with
the antibodies of the invention include those disorders discussed
in the section above pertaining to pharmaceutical compositions of
the antibodies of the invention.
[0092] Other features of the invention will be apparent from the
following examples which should not be construed as limiting.
[0093] The contents of all cited references, including literature
references, issued patents, and published patent applications, as
cited throughout this application are hereby expressly incorporated
by reference.
Exemplification
[0094] In general, the practice of the present invention employs,
unless otherwise indicated, conventional techniques of chemistry,
molecular biology, recombinant DNA technology, PCR technology,
immunology (especially, e.g., antibody technology), and any
necessary cell culture or animal husbandry techniques, which are
within the skill of the art and are explained fully in the
literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular
Cloning: Cold Spring Harbor Laboratory Press (1989); DNA Cloning,
Vols. 1 and 2, (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis
(M. J. Gait, Ed. 1984); PCR Handbook Current Protocols in Nucleic
Acid Chemistry, Beaucage, Ed. John Wiley & Sons (1999)
(Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed.,
Oxford Univ Press (1999); PCR Protocols: A Guide to Methods and
Applications, Innis et al., Academic Press (1990); PCR Essential
Techniques: Essential Techniques, Burke, Ed., John Wiley & Son
Ltd (1996); The PCR Technique: RT-PCR, Siebert, Ed., Eaton Pub. Co.
(1998); Quantitative PCR Protocols, Kochanowski et al., Eds.,
Humana Press (1999); Clinical Applications of PCR, Lo, Ed., Humana
Press (1998); Antibody Engineering Protocols (Methods in Molecular
Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A
Practical Approach (Practical Approach Series, 169), McCafferty,
Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al.,
C. S. H. L. Press, Pub. (1999); Current Protocols in Molecular
Biology, eds. Ausubel et al., John Wiley & Sons (1992);
Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed.,
Marcel Dekker, Pub., (1990); and Manipulating the Mouse Embryo,
Hogan et al., C. S. H. L. Press, Pub (1994).
[0095] Throughout the examples, unless otherwise indicated, the
above materials and methods were used.
Example 1
Isolation of Anti-IL-18 Antibodies
[0096] Antibodies to hIL-18 were isolated by screening separate
scFv phage display libraries prepared using human VL and VH cDNAs
from mRNA derived from human B cells (e.g., tonsils and spleen).
Construction of the library and methods for selection are described
in Vaughan et al. (1996) Nature Biotech. 14: 309-314.
[0097] The libraries were screened using either full length human
IL-18 (SEQ ID NO: 61), a peptide epitope of IL-18 (SEQ ID NOS:
1-3), or a panel of overlapping 15 amino acid peptides representing
IL-18 (the epitope sequence of which is presented in Table 5; SEQ
ID NOS: 31-60). IL-18 specific antibodies were selected by coating
the antigen onto immunotubes using standard procedures (Marks et
al., (1991) J. Mol. Biol. 222: 581-597). The scFv libraries were
screened using either IL-18, a peptide epitope of IL-18, or an
IL-18 peptide panel to generate a significant number of IL-18
specific binders. Several different clonotypes were selected,
determined by restriction enzyme digestion patterns, and confirmed
by DNA sequencing.
[0098] In order to identify IL-18 antibodies which preferentially
bind either full length IL-18 or a representative peptide thereof,
the supernatant containing scFv was titrated on biotin-captured
IL-18 in an ELISA and binding characteristics were determined.
[0099] Two anti-IL-18 single chain antibodies were obtained, one
termed 2E1, independently isolated using a peptide epitope and the
peptide panel, and a second anti-IL-18 antibody termed LT28,
isolated using full length IL-18. These parent anti-IL-18
antibodies were selected for further study and modification.
Example 2
Affinity Maturation of an Anti-18 Antibodies
[0100] A single chain Fv version of antibody 2E1 having an
identified IL-18 binding activity and the heavy chain and light
chain sequence shown in Table 6 was further modified for improved
neutralization of IL-18 activity.
TABLE-US-00006 TABLE 6 Sequence of Single-Chain Anti-IL-18 Antibody
2E1 2E1 Heavy Chain (SEQ ID NO: 18) CDR1 (SEQ ID NO: 9)
QVQLVQSGAEVKKPGASMKVSCKTSGYTFTGYYIHWVRQAHGQGFEWI CDR2 (SEQ ID NO:
10) CDR3 (SEQ ID NO: 11)
GRLNPTTGDANFAEKFQGRVALTRDTSISTAYLQLDSLKSDDTAVYYCAGKEGAWGQGTLVTVSS
2E1 Light Chain (SEQ ID NO: 19) CDR1 (SEQ ID NO: 12) CDR2 (SEQ ID
NO: 13) SSELTQDPAVSVALGQTVRITCQGDSLRHFYPNWYQQKPGQAPVLVIYGKNNRPS
CDR3 (SEQ ID NO: 14)
GIPDRFSGSGSGNTGSLTITGAQAEDEADYYCGSRDSSGIHVVFGGGTKVTVLG
[0101] The anti-IL-18 antibody 2E1 was independently selected using
an IL-18 peptide and sequential, overlapping, peptide panel
representative of IL-18 (see Table 6).
[0102] The specific amino acid residues of the heavy chain variable
region selected for mutagenesis are summarized in Table 7. In
particular, with respect to the heavy chain region, individual
amino acid substitutions were tested at positions H30, H31, H32,
H33, and H35 of CDR1, positions H52, H52a, H53, H54, H56, and H58
of CDR2, and H95, H96, H97, and H98 of CDR3.
[0103] With regards to light chain amino acid residues selected for
mutagenesis, individual amino acid substitutions were tested at
positions L30, L31, L32, and L34 of CDR1, positions L50, L52, L53,
and L55 of CDR2 and positions L89, L90, L91, L92, L93, L94, L95,
L95a, L95b, L96, and L97 of CDR 3.
TABLE-US-00007 TABLE 7 Heavy Chain Amino Acid Substitutions
Introduced Into 2E1 Heavy Chain Mutations CDR/Kabat Position
substituted residue CDR1 H30 A, R, N, D, C, G, H, I, F, P, S, or V
H31 A, C, H, S, T, or Y H32 R, N, C, H, P, S, or T H33 N, D, C, Q,
H, L, M, F, S, or V H35 N, D, L, or F CDR2 H52 T H52a R, Q, L, S,
T, or W H53 A, R, N, L, P, S, or Y H54 A, R, N, D, Q, L, K, M, P,
S, or Y H56 A, R, N, C, G, H, I, L, or F H58 A, R, Q, E, H, I, L,
K, M, F, S, T, Y, P, S, T, W, Y, or V CDR3 H95 A, R, E, Q, S, Y, V,
H, P, W, or C H96 A, R, Q, S, Y, V, H, P, W, or C H97 A, R, E, Q,
S, Y, V, H, P, W, or C H98 R, E, Q, S, Y, V, H, P, W, or C
TABLE-US-00008 TABLE 8 Light Chain Amino Acid Substitutions
Introduced Into 2E1 Light Chain Mutations CDR/Kabat Position
substituted residue CDR1 L30 N, D, C, G, I, L, S, W, or Y L31 R, N,
D, C, G, H, I, L, P, S, T, or Y L32 R, N, D, E, G, I, L, P, S, T,
or V L34 A, R, N, D, E, H, I, L, K, M, F, P, S, T, Y, or V CDR2 L50
A, N, I, L, F, P, S, W, Y, or V L52 A, R, D, E, H, I, L, M, F, P,
S, T, or V L53 A, R, C, I, L, K, M, P, S, or T L55 A, R, N, D, C,
G, H, I, L, S, T, or Y CDR3 L89 A, R, E, Q, S, Y, V, H, P, W, or C
L90 A, R, E, Q, Y, V, H, P, W, or C L91 R, E, Q, S, Y, V, H, P, W,
or C L92 A, R, E, Q, S, Y, V, H, P, W, or C L93 A, R, E, Q, Y, V,
H, P, W, or C L94 A, R, E, Q, Y, V, H, P, W, or C L95 A, R, E, Q,
S, Y, V, H, P, W, or C L95a A, R, E, Q, S, Y, V, H, P, W, or C L95b
A, R, E, Q, S, Y, V, P, W, or C L96 A, R, E, Q, S, Y, H, P, W, or C
L97 A, R, E, Q, S, Y, H, P, W, or C
[0104] Substitutions were introduced using standard techniques
(e.g., as described in Taylor et al., Nucleic Acids Res. 13:
8764-8758 (1985); Nakamaye et al., Nucleic Acids Res. 14: 9679-9698
(1986); and Olsen et al., Methods in Enzymology, 217: 189 (1993)).
In brief, oligonucleotides degenerate for a given codon were
synthesized for each of the positions to be mutagenized. A
single-stranded DNA template was prepared from the original plasmid
containing a single-chain Fv version of the antibody 2E1 gene. The
nucleic acid sequence of the parent 2E1 antibody heavy and light
chain is provided in SEQ ID NOS: 62 and 64. The mutant
oligonucleotides were then used to create a complementary DNA
strand and eventually a double-stranded plasmid, thus incorporating
the degeneracy or the different mutations in a given codon of the
antibody. In particular, the CDR3 region of the heavy and light
chain of 2E1 was altered using the QuikChange Kit (Stratagene)
according to the manufactures instruction.
[0105] A representative number of clones were then sequenced from
each mutagenesis reaction (i.e., 7 to 36 clones) and those
representing a change from the parent 2E1 single chain antibody
sequence were expressed in bacteria and purified for further in
vitro and in vivo testing as described infra.
[0106] In another screen using a full length IL-18 ligand, a second
anti-IL-18 antibody was identified and selected for further
improvement using affinity maturation. In particular, using the
techniques described above, the LT28 antibody having the heavy
chain and light chain sequence shown in Table 9 (and nucleic
sequence provided in SEQ ID NOS: 66 and 68) was further
modified.
TABLE-US-00009 TABLE 9 Sequence of Single-Chain Anti-IL-18 Antibody
LT28 LT28 Heavy Chain (SEQ ID NO: 28) CDR1 (SEQ ID NO: 20) CDR2
(SEQ ID NO: 21)
LVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG CDR3 (SEQ
ID NO: 22) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDDDYDFDYWGRGTMVTVSS
LT28 Light Chain (SEQ ID NO: 29) CDR1 (SEQ ID NO: 23) CDR2 (SEQ ID
NO: 24) QSVLTQPPSASGTPGQRVTISCSGSSSNIGINAVNWYQQLPGTAPKLLIYGNDQRPS
CDR3 (SEQ ID NO: 25)
GVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLSGPVFGGGTKLTVLG
[0107] With respect to the heavy chain region, amino acid
substitutions were introduced at positions H31, H32, H33, and H35
of CDR1, positions HSO, H51, H52, H52a, H53, H54, H56, and H58 of
CDR2, and H95, H96, H97, H98, H99, H100, H100a, H101, and H102 of
CDR3.
[0108] With regards to light chain residues selected for mutation,
amino acid substitutions were introduced at positions L30, L31,
L32, L34 of CDR1, positions L50, L52, L53, L55 of CDR2 and
positions L89, L90, L91, L92, L93, L94, L95, L95a, L95b, L96,
L97.
TABLE-US-00010 TABLE 10 Heavy Chain Amino Acid Substitutions
Introduced into LT28 Heavy Chain Mutations CDR/Kabat Position
substituted residue CDR1 H31 A, R, E, Q, S, Y, V, H, P, W, or C H32
A, R, E, Q, S, Y, V, H, P, W, or C H33 A, R, E, Q, S, Y, V, H, P,
W, or C H35 A, R, E, Q, S, Y, V, H, P, W, or C CDR2 H50 A, R, E, Q,
S, Y, V, H, P, W, or C H51 A, R, E, Q, S, Y, V, H, P, W, or C H52
A, R, E, Q, S, Y, V, H, P, W, or C H52a A, R, E, Q, S, Y, V, H, P,
W, or C H53 A, R, E, Q, S, Y, V, H, P, W, or C H54 A, R, E, Q, S,
Y, V, H, P, W, or C H56 A, R, E, Q, S, Y, V, H, P, W, or C H58 A,
R, E, Q, S, Y, V, H, P, W, C CDR3 H95 A, R, E, Q, S, Y, V, H, P, W,
or C H96 A, R, E, Q, S, Y, V, H, P, W, or C H97 A, R, E, Q, S, Y,
V, H, P, W, or C H98 A, R, E, Q, S, Y, V, H, P, W, or C H99 A, R,
E, Q, S, Y, V, H, P, W, or C H100 A, R, E, Q, S, Y, V, H, P, W, or
C H100a A, R, E, Q, S, Y, V, H, P, W, or C H101 A, R, E, Q, S, Y,
V, H, P, W, or C H102 A, R, E, Q, S, Y, V, H, P, W, or C
TABLE-US-00011 TABLE 11 Light Chain Amino Acid Substitutions
Introduced into LT28 Light Chain Mutations CDR/Position substituted
residue CDR1 L30 A, R, E, Q, S, Y, V, H, P, W, C L31 A, R, E, Q, S,
Y, V, H, P, W, C L32 R, E, Q, S, Y, V, H, P, W, C, G L34 A, R, E,
Q, S, Y, V, H, P, W, C CDR2 L50 A, R, E, Q, S, Y, V, H, P, C L52 A,
R, E, S, Y, V, H, P, W, C L53 A, R, E, S, Y, V, H, P, W, C, N L55
A, E, Q, S, Y, V, H, P, W, C CDR3 L89 A, R, E, Q, S, Y, V, H, P, W,
C L90 A, R, E, Q, S, Y, V, H, P, W, C L91 A, R, E, Q, S, Y, V, H,
P, W, C L92 A, R, E, Q, S, Y, V, H, P, W, C L93 A, R, E, Q, S, Y,
V, H, P, W, C L94 A, R, E, Q, S, Y, V, H, P, W, C L95 A, R, E, Q,
S, Y, V, H, P, W, C L95a A, R, E, Q, S, Y, V, H, P, W, C L95b A, R,
E, Q, S, Y, V, H, P, W, C L96 A, R, E, Q, S, Y, V, H, P, W, C L97
A, R, E, Q, S, Y, V, H, P, W, C
[0109] Substitutions were introduced as described above. A
representative number of clones were then sequenced from each
mutagenesis reaction and those representing a change from the
parent LT28 single chain antibody sequence were expressed in
bacteria and purified for further testing as described below.
Example 3
Binding Activity of Human Antibodies to IL-18
[0110] Real-time binding interactions between ligand (biotinylated
recombinant human IL-18 (rhIL-18) immobilized on a biosensor
matrix) and analyte (antibodies in solution) were measured by
surface plasmon resonance (SPR) using the BIAcore system (Pharmacia
Biosensor, Piscataway, N.J.). The system utilizes the optical
properties of SPR to detect alterations in protein concentrations
within a dextran biosensor matrix. Proteins are covalently bound to
the dextran matrix at known concentrations. Antibodies are injected
through the dextran matrix and specific binding between injected
antibodies and immobilized ligand results in an increased matrix
protein concentration and resultant change in the SPR signal. These
changes in SPR signal are recorded as resonance units (RU) and are
displayed with respect to time along the y-axis of a
sensorgram.
[0111] To facilitate immobilization of biotinylated rhIL-18 on the
biosensor matrix, streptavidin is covalently linked via free amine
groups to the dextran matrix by first activating carboxyl groups on
the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400 mM
N-ethyl-N'-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC).
Next, streptavidin is injected across the activated matrix.
Thirty-five microliters of streptavidin (25 .mu.g/ml), diluted in
sodium acetate, pH 4.5, is injected across the activated biosensor
and free amines on the protein are bound directly to the activated
carboxyl groups. Unreacted matrix EDC-esters are deactivated by an
injection of 1 M ethanolamine Streptavidin-coupled biosensor chips
also are commercially available (Pharmacia BR-1000-16, Pharmacia
Biosensor, Piscataway, N.J.).
[0112] Biotinylated rhIL-18 was prepared by first dissolving 5.0 mg
of biotin (D-biotinyl-.epsilon.-aminocaproic acid
N-hydroxysuccinimide ester; Boehringer Mannheim Cat. No. 1008 960)
in 500 .mu.l dimethylsulfoxide to make a 10 mg/ml solution. Ten
microliters of biotin was added per ml of rhIL-18 (at 2.65 mg/ml)
for a 2:1 molar ratio of biotin to rhIL-18. The reaction was mixed
gently and incubated for two hours at room temperature in the dark.
A PD-10 column, Sephadex G-25M (Pharmacia Catalog No. 17-0851-01)
was equilibrated with 25 ml of cold PBS and loaded with 2 ml of
rhIL-18-biotin per column. The column was eluted with 10.times.1 ml
cold PBS. Fractions were collected and read at OD280 (1.0 OD=1.25
mg/ml). The appropriate fractions were pooled and stored at
-80.degree. C. until use.
[0113] Biotinylated rhIL-18 to be immobilized on the matrix via
streptavidin was diluted in PBS running buffer (Gibco Cat. No.
14190-144, Gibco BRL, Grand Island, N.Y.) supplemented with 0.05%
(BIAcore) surfactant P20 (Pharmacia BR-1000-54, Pharmacia
Biosensor, Piscataway, N.J.). To determine the capacity of
rhIL-18-specific antibodies to bind immobilized rhIL-18, a binding
assay was conducted as follows. Aliquots of biotinylated rhIL-18
(25 nM; 10 .mu.l aliquots) were injected through the
streptavidin-coupled dextran matrix at a flow rate of 5 .mu.l/min.
Before injection of the protein and immediately afterward, PBS
buffer alone flowed through each flow cell. The net difference in
signal between baseline and approximately 30 sec. after completion
of biotinylated rhIL-18 injection was taken to represent the
binding value. Direct rhIL-18-specific antibody binding to
immobilized biotinylated rhIL-18 was measured. Antibodies (20
.mu.g/ml) were diluted in PBS running buffer and 25 .mu.l aliquots
were injected through the immobilized protein matrices at a flow
rate of 5 .mu.l/min Prior to injection of antibody, and immediately
afterwards, PBS buffer alone flowed through each flow cell. The net
difference in baseline signal and signal after completion of
antibody injection was taken to represent the binding value of the
particular sample. Biosensor matrices were regenerated using 100 mM
HCl before injection of the next sample. To determine the off rate
(K.sub.off), on rate (K.sub.on), association rate (K.sub.a) and
dissociation rate (K.sub.d) constants, BIAcore kinetic evaluation
software (version 2.1) was used.
[0114] Representative results of improved candidate anti-IL-18
antibodies binding to biotinylated rhIL-18, as compared to the
parent antibodies 2E1 and LT28 (and murine controls), are shown
below in Table 12. For comparison, IC50 values from the cell-based
neutralization assay are also included and these are described in
Example 4. All clones were prepared as single-chain Fv antibodies
for testing using Biacore analysis and the cell-based assay
described below. Parental clones listed comprise an unmutated
parental heavy and light chain, whereas single chain mutants
contain one parental chain and one mutated chain where the mutated
chan is indicated as being either heavy (H) or light (L) followed
by the Kabat position and nature of the amino acid
substitution.
TABLE-US-00012 TABLE 12 Binding of Anti-IL-18 Antibodies Derived
From 2E1 and LT28 Antibody On-rates Off-rates Clone
(M.sup.-1s.sup.-1) (s.sup.-1) Kd (M) IC50 Value* 2E1 parent and
mutants 2E1 (parent) ScFv 2.6E+3 6.42E-03 1.5E-07 3.3E-8M 2E1
(parent) IgG 9.0E-10M L34S 1.69E-04 1.5E-8M H53R 2.34E-03 2.5E-8M
H53Y -- 1.5E-8M H58Q -- 1.6E-8M L34S + H53R 2.7E+03 6.82E-05
2.3E-08 3.0E-09M (2E1RS) L34S + H58Q -- 1.5E-8M L34S + H53Y
5.28E-05 6.7E-9M H53R + H58Q -- 1.2E-8M H53Y + H58Q -- 1.2E-8M L34S
+ H53R + H58Q 6.18E-05 2.8E-9M L34S + H53Y + H58Q -- 8.0E-9M L90C
4x L93C 2-4x L94P, Q, or R 2-4x L95R, Y 3-8x L95bE, W 2-4x LT28
parent and mutants LT28 (parent) 1.3E+04 4.8E-04 3.9E-08 9.0E-8M
H540 2-3x H58W 2-3x 1 125-2H 1.7E+05 1.1E-04 6.2E-10 2.E-10M 318-M
1.2E+04 1.1E-04 9.6E-09 4.0E-9M *Some values presented as fold
improvement compared to the parent.
Example 4
Neutalizing Activity of Anti-IL-18 Antibodies
[0115] To examine the neutralizing activity of the anti-human IL-18
antibodies of the invention, an art recognized assay for monitoring
IL-18 activity was used. Briefly, the assay employs KG1 cells (ATCC
#CCL-246, myelogenous leukemia bone marrow cells) which were
cultured according to standard techniques (e.g., using RPMI 1640
Culture Medium Gibco #21870-076; (supplemented with 10% Fetal
Bovine Serum (BioWhittaker #14-501F); 2 mM L-glutamine (Gibco
#25030-081); 50 units/ml Penicillin, 50 ug/ml Streptomycin (Gibco
#15070-063); and 0.075% Sodium Bicarbonate).
[0116] To test for IL-18 neutralization, 3.times.10E5 KG-1 cells
stimulated with 20 ng/ml hTNF-alpha (Lot #19130132) was incubated
with 50 ul of anti-IL-18 antibody (4.times.Conc.) and 50 ul of
IL-18 (4.times.Conc.=8 ng/ml) and incubated for 37.degree. C. for 1
hr or 16-20 hrs. To determine the amount of IL-18 neutralization
that occurred as a function of induced hIFN-gamma production, an
ELISA was performed using commercially available Elisa Kits (R
& D #DIF00/Endogen #EH-IFNG), according to the manufacturers
instructions, and hIFN-gamma production was calculated (pg/ml) off
a standard curve.
[0117] In all, four mutant antibodies, i.e., the 2E1 derived L34S,
H53R, H53Y, and H58Q, were shown to have greater IL-18
neutralization potency than the parent 2E1 antibody (see Table 12).
The improvements in IC50 values using the KG-1 assay were in the
range of 2 to 5 fold, and similar improved binding results were
determined using BIAcore analysis.
[0118] Various mutation combination clones were also prepared and
tested, and this data is summarized in the Table 12. The best
combination clone L34S-H53R showed a greater than ten fold
improvement over the parent antibody 2E1 in both the KG-1
cell-based assay and using the BIAcore analysis. The resulting
antibody was designated the name of 2E1RS.
[0119] Several other mutant clones of 2E1 showed an improvement in
potency, i.e., IL-18 neutralization, as determined using the KG-1
assay. The mutant L95Y offered 5 to 8 fold better IC50 values than
the parent 2E1 antibody. Several other mutants offered a 2 to 3
fold improvement and they are 2E1 mutants H96A, H96Q, H96S, H98S,
L90C, L90W, L93C, L94P, L94P, L94Q, L94R, L94W, L95R, L95aA, L95aH,
L95aP, L95aR, L95aW, L95bE, L95bW, L95bY, L97C, and L97E.
[0120] The binding of 2E1 in the form of an ScFv antibody or IgG
antibody was also compared (see FIG. 5).
[0121] Still further, two mutants derived from the LT28 parent
should improved 1L-18 neutralization activity compared to the
parent antibody.
[0122] These results demonstrate that fully human IL-18
neutralizing antibodies can be obtained using the methods and
compositions of the invention.
Equivalents
[0123] 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
71115PRTHomo sapiens 1Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys
Arg Asp Asn Ala1 5 10 15216PRTHomo sapiens 2Cys Pro Leu Phe Glu Asp
Met Thr Asp Ser Asp Cys Arg Asp Asn Ala1 5 10 15312PRTHomo sapiens
3Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg1 5 104157PRTHomo
sapiens 4Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn
Leu Asn1 5 10 15Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu
Phe Glu Asp 20 25 30Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg
Thr Ile Phe Ile 35 40 45Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly
Met Ala Val Thr Ile 50 55 60Ser Val Lys Cys Glu Lys Ile Ser Thr Leu
Ser Cys Glu Asn Lys Ile65 70 75 80Ile Ser Phe Lys Glu Met Asn Pro
Pro Asp Asn Ile Lys Asp Thr Lys 85 90 95Ser Asp Ile Ile Phe Phe Gln
Arg Ser Val Pro Gly His Asp Asn Lys 100 105 110Met Gln Phe Glu Ser
Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu 115 120 125Lys Glu Arg
Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu 130 135 140Gly
Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp145 150
1555153PRTHomo sapiens 5Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg
Asp Ser Gln Gln Lys1 5 10 15Ser Leu Val Met Ser Gly Pro Tyr Glu Leu
Lys Ala Leu His Leu Gln 20 25 30Gly Gln Asp Met Glu Gln Gln Val Val
Phe Ser Met Ser Phe Val Gln 35 40 45Gly Glu Glu Ser Asn Asp Lys Ile
Pro Val Ala Leu Gly Leu Lys Glu 50 55 60Lys Asn Leu Tyr Leu Ser Cys
Val Leu Lys Asp Asp Lys Pro Thr Leu65 70 75 80Gln Leu Glu Ser Val
Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu 85 90 95Lys Arg Phe Val
Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe 100 105 110Glu Ser
Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu 115 120
125Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr
130 135 140Asp Phe Thr Met Gln Phe Val Ser Ser145 1506145PRTHomo
sapiens 6Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln
Lys Thr1 5 10 15Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu
Gln Gly Pro 20 25 30Asn Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro
Ile Glu Pro His 35 40 45Ala Leu Phe Leu Gly Ile His Gly Gly Lys Met
Cys Leu Ser Cys Val 50 55 60Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu
Glu Ala Val Asn Ile Thr65 70 75 80Asp Leu Ser Glu Asn Arg Lys Gln
Asp Lys Arg Phe Ala Phe Ile Arg 85 90 95Ser Asp Ser Gly Pro Thr Thr
Ser Phe Glu Ser Ala Ala Cys Pro Gly 100 105 110Trp Phe Leu Cys Thr
Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr 115 120 125Asn Met Pro
Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu 130 135
140Asp1457297PRTHomo sapiens 7Cys Thr Ser Arg Pro His Ile Thr Val
Val Glu Gly Glu Pro Phe Tyr1 5 10 15Leu Lys His Cys Ser Cys Ser Leu
Ala His Glu Ile Glu Thr Thr Thr 20 25 30Lys Ser Trp Tyr Lys Ser Ser
Gly Ser Gln Glu His Val Glu Leu Asn 35 40 45Pro Arg Ser Ser Ser Arg
Ile Ala Leu His Asp Cys Val Leu Glu Phe 50 55 60Trp Pro Val Glu Leu
Asn Asp Thr Gly Ser Tyr Phe Phe Gln Met Lys65 70 75 80Asn Tyr Thr
Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn Lys His 85 90 95Ser Cys
Phe Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu Val Lys 100 105
110Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr Leu Val
115 120 125Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu
Glu Asn 130 135 140Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe
Glu Asp Gln Gly145 150 155 160Tyr Tyr Ser Cys Val His Phe Leu His
His Asn Gly Lys Leu Phe Asn 165 170 175Ile Thr Lys Thr Phe Asn Ile
Thr Ile Val Glu Asp Arg Ser Asn Ile 180 185 190Val Pro Val Leu Leu
Gly Pro Lys Leu Asn His Val Ala Val Glu Leu 195 200 205Gly Lys Asn
Val Arg Leu Asn Cys Ser Ala Leu Leu Asn Glu Glu Asp 210 215 220Val
Ile Tyr Trp Met Phe Gly Glu Glu Asn Gly Ser Asp Pro Asn Ile225 230
235 240His Glu Glu Lys Glu Met Arg Ile Met Thr Pro Glu Gly Lys Trp
His 245 250 255Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly Glu Ser
Asn Leu Asn 260 265 270Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly
Gly Thr Asp Thr Lys 275 280 285Ser Phe Ile Leu Val Arg Lys Ala Asp
290 2958310PRTHomo sapiens 8Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu
Val Ser Ser Ala Asn Glu1 5 10 15Ile Asp Val Arg Pro Cys Pro Leu Asn
Pro Asn Glu His Lys Gly Thr 20 25 30Ile Thr Trp Tyr Lys Asp Asp Ser
Lys Thr Pro Val Ser Thr Glu Gln 35 40 45Ala Ser Arg Ile His Gln His
Lys Glu Lys Leu Trp Phe Val Pro Ala 50 55 60Lys Val Glu Asp Ser Gly
His Tyr Tyr Cys Val Val Arg Asn Ser Ser65 70 75 80Tyr Cys Leu Arg
Ile Lys Ile Ser Ala Lys Phe Val Glu Asn Glu Pro 85 90 95Asn Leu Cys
Tyr Asn Ala Gln Ala Ile Phe Lys Gln Lys Leu Pro Val 100 105 110Ala
Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe Phe Lys Asn 115 120
125Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys Asp Cys Lys Pro
130 135 140Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp Arg
Leu Ile145 150 155 160Val Met Asn Val Ala Glu Lys His Arg Gly Asn
Tyr Thr Cys His Ala 165 170 175Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr
Pro Ile Thr Arg Val Ile Glu 180 185 190Phe Ile Thr Leu Glu Glu Asn
Lys Pro Thr Arg Pro Val Ile Val Ser 195 200 205Pro Ala Asn Glu Thr
Met Glu Val Asp Leu Gly Ser Gln Ile Gln Leu 210 215 220Ile Cys Asn
Val Thr Gly Gln Leu Ser Asp Ile Ala Tyr Trp Lys Trp225 230 235
240Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val Leu Gly Glu Asp Tyr
245 250 255Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser Thr Leu
Ile Thr 260 265 270Val Leu Asn Ile Ser Glu Ile Glu Ser Arg Phe Tyr
Lys His Pro Phe 275 280 285Thr Cys Phe Ala Lys Asn Thr His Gly Ile
Asp Ala Ala Tyr Ile Gln 290 295 300Leu Ile Tyr Pro Val Thr305
31096PRTHomo sapiens 9Thr Gly Tyr Tyr Ile His1 51017PRTHomo sapiens
10Gly Arg Leu Asn Pro Thr Thr Gly Asp Ala Asn Phe Ala Glu Lys Phe1
5 10 15Gln114PRTHomo sapiens 11Lys Glu Gly Ala11211PRTHomo sapiens
12Gln Gly Asp Ser Leu Arg His Phe Tyr Pro Asn1 5 10137PRTHomo
sapiens 13Gly Lys Asn Asn Arg Pro Ser1 51411PRTHomo sapiens 14Gly
Ser Arg Asp Ser Ser Gly Ile His Val Val1 5 101511PRTHomo sapiens
15Gln Gly Asp Ser Leu Arg His Phe Tyr Ser Asn1 5 101617PRTHomo
sapiens 16Gly Arg Leu Asn Pro Arg Thr Gly Asp Ala Asn Phe Ala Glu
Lys Phe1 5 10 15Gln1717PRTHomo sapiens 17Gly Arg Leu Asn Pro Arg
Thr Gly Asp Ala Gln Phe Ala Glu Lys Phe1 5 10 15Gln18113PRTHomo
sapiens 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Met Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe
Thr Gly Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Ala His Gly Gln Gly
Phe Glu Trp Ile 35 40 45Gly Arg Leu Asn Pro Thr Thr Gly Asp Ala Asn
Phe Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Ala Leu Thr Arg Asp Thr
Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Leu Asp Ser Leu Lys Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly Lys Glu Gly Ala Trp
Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ser19109PRTHomo
sapiens 19Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu
Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg His
Phe Tyr Pro 20 25 30Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val
Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Asn Thr Gly Ser Leu Thr Ile
Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gly
Ser Arg Asp Ser Ser Gly Ile His 85 90 95Val Val Phe Gly Gly Gly Thr
Lys Val Thr Val Leu Gly 100 105204PRTHomo sapiens 20Ser Tyr Ala
Met12117PRTHomo sapiens 21Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val Lys1 5 10 15Gly229PRTHomo sapiens 22Asp Asp Asp
Asp Tyr Asp Phe Asp Tyr1 52313PRTHomo sapiens 23Ser Gly Ser Ser Ser
Asn Ile Gly Ile Asn Ala Val Asn1 5 10246PRTHomo sapiens 24Gly Asn
Asp Gln Arg Pro1 52511PRTHomo sapiens 25Ala Ala Trp Asp Asp Ser Leu
Ser Gly Pro Val1 5 102617PRTHomo sapiens 26Ala Ile Ser Gly Ser Gln
Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly2717PRTHomo
sapiens 27Ala Ile Ser Gly Ser Gly Gly Ser Thr Trp Tyr Ala Asp Ser
Val Lys1 5 10 15Gly28108PRTHomo sapiens 28Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly1 5 10 15Phe Thr Phe Ser Ser
Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 20 25 30Lys Gly Leu Glu
Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 35 40 45Tyr Tyr Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 50 55 60Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp65 70 75
80Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Asp Asp Tyr Asp Phe Asp
85 90 95Tyr Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 100
10529111PRTHomo sapiens 29Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ile Asn 20 25 30Ala Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
11030235PRTHomo sapiens 30Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly1 5 10 15Phe Thr Phe Ser Ser Tyr Ala Met Ser
Trp Val Arg Gln Ala Pro Gly 20 25 30Lys Gly Leu Glu Trp Val Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr 35 40 45Tyr Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn 50 55 60Ser Lys Asn Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp65 70 75 80Thr Ala Val Tyr
Tyr Cys Ala Arg Asp Asp Asp Asp Tyr Asp Phe Asp 85 90 95Tyr Trp Gly
Arg Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly 100 105 110Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Ser Val Leu 115 120
125Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile
130 135 140Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ile Asn Ala Val
Asn Trp145 150 155 160Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu Ile Tyr Gly Asn 165 170 175Asp Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser Gly Ser Lys Ser 180 185 190Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Gln Ser Glu Asp Glu 195 200 205Ala Asp Tyr Tyr Cys
Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val 210 215 220Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly225 230 2353114PRTHomo sapiens 31Tyr
Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn1 5
103214PRTHomo sapiens 32Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn
Asp Gln Val1 5 103314PRTHomo sapiens 33Val Ile Arg Asn Leu Asn Asp
Gln Val Leu Phe Ile Asp Gln1 5 103414PRTHomo sapiens 34Asn Asp Gln
Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu1 5 103514PRTHomo
sapiens 35Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr1
5 103614PRTHomo sapiens 36Asn Arg Pro Leu Phe Glu Asp Met Thr Asp
Ser Asp Cys Arg1 5 103714PRTHomo sapiens 37Glu Asp Met Thr Asp Ser
Asp Cys Arg Asp Asn Ala Pro Arg1 5 103814PRTHomo sapiens 38Ser Asp
Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile1 5 103914PRTHomo
sapiens 39Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp1
5 104014PRTHomo sapiens 40Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser
Gln Pro Arg Gly1 5 104114PRTHomo sapiens 41Met Tyr Lys Asp Ser Gln
Pro Arg Gly Met Ala Val Thr Ile1 5 104214PRTHomo sapiens 42Gln Pro
Arg Gly Met Ala Val Thr Ile Ser Val Lys Cys Glu1 5 104314PRTHomo
sapiens 43Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu1
5 104414PRTHomo sapiens 44Val Lys Cys Glu Lys Ile Ser Thr Leu Ser
Cys Glu Asn Lys1 5 104514PRTHomo sapiens 45Ile Ser Thr Leu Ser Cys
Glu Asn Lys Ile Ile Ser Phe Lys1 5 104614PRTHomo sapiens 46Cys Glu
Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro1 5 104714PRTHomo
sapiens 47Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp1
5 104814PRTHomo sapiens 48Met Asn Pro Pro Asp Asn Ile Lys Asp Thr
Lys Ser Asp Ile1 5 104914PRTHomo sapiens 49Asn Ile Lys Asp Thr Lys
Ser Asp Ile Ile Phe Phe Gln Arg1 5 105014PRTHomo sapiens 50Lys Ser
Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His1 5 105114PRTHomo
sapiens 51Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln1
5 105214PRTHomo sapiens 52Val Pro Gly His Asp Asn Lys Met Gln Phe
Glu Ser Ser Ser1 5 105314PRTHomo sapiens 53Asn Lys Met Gln Phe Glu
Ser Ser Ser Tyr Glu Gly Tyr Phe1 5 105414PRTHomo sapiens 54Glu Ser
Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys1 5 105514PRTHomo
sapiens 55Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe1
5 105614PRTHomo sapiens 56Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys
Leu Ile Leu Lys1 5 105714PRTHomo sapiens 57Arg Asp Leu Phe Lys Leu
Ile Leu Lys Lys Glu Asp Glu Leu1 5 105814PRTHomo sapiens 58Leu Ile
Leu Lys Lys Glu Asp Glu Leu
Gly Asp Arg Ser Ile1 5 105914PRTHomo sapiens 59Glu Asp Glu Leu Gly
Asp Arg Ser Ile Met Phe Thr Val Gln1 5 106012PRTHomo sapiens 60Asp
Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp1 5 1061157PRTHomo
sapiens 61Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn
Leu Asn1 5 10 15Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu
Phe Glu Asp 20 25 30Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg
Thr Ile Phe Ile 35 40 45Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly
Met Ala Val Thr Ile 50 55 60Ser Val Lys Cys Glu Lys Ile Ser Thr Leu
Ser Cys Glu Asn Lys Ile65 70 75 80Ile Ser Phe Lys Glu Met Asn Pro
Pro Asp Asn Ile Lys Asp Thr Lys 85 90 95Ser Asp Ile Ile Phe Phe Gln
Arg Ser Val Pro Gly His Asp Asn Lys 100 105 110Met Gln Phe Glu Ser
Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu 115 120 125Lys Glu Arg
Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu 130 135 140Gly
Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp145 150
15562341DNAHomo sapiensCDS(1)..(339) 62cag gtc cag ctg gtg cag tct
ggg gct gag gtg aag aag cct ggg gcc 48Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15tcg atg aaa gtc tcc tgt
aag act tct gga tac acc ttc acc ggc tat 96Ser Met Lys Val Ser Cys
Lys Thr Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30tat atc cac tgg gtg
cga cag gcc cct gga cag gga ttc gag tgg ata 144Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Gln Gly Phe Glu Trp Ile 35 40 45gga cgg ctc aac
ccc acc act ggt gac gca aat ttt gca gaa aag ttt 192Gly Arg Leu Asn
Pro Thr Thr Gly Asp Ala Asn Phe Ala Glu Lys Phe 50 55 60cag ggc agg
gtc gcc ctg acc aga gac acg tcc atc agc aca gcc tat 240Gln Gly Arg
Val Ala Leu Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80tta
caa cta gac agc ctc aaa tct gac gac acg gcc gta tat tat tgt 288Leu
Gln Leu Asp Ser Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95gcg gga aaa gag ggt gcc tgg ggc cag ggc acc ctg gtc acc gtc tcg
336Ala Gly Lys Glu Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110agt gg 341Ser63113PRTHomo sapiens 63Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Met Lys Val
Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Gly Phe Glu Trp Ile 35 40 45Gly Arg
Leu Asn Pro Thr Thr Gly Asp Ala Asn Phe Ala Glu Lys Phe 50 55 60Gln
Gly Arg Val Ala Leu Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Leu Asp Ser Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Gly Lys Glu Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110Ser64327DNAHomo sapiensCDS(1)..(327) 64tct tct gag
ctg act cag gac cct gct gtg tct gtg gcc ttg gga cag 48Ser Ser Glu
Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15aca gtc
agg atc aca tgc caa gga gac agc ctc aga cac ttt tat cca 96Thr Val
Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg His Phe Tyr Pro 20 25 30aac
tgg tac cag cag aag cca gga cag gcc cct gta ctt gtc atc tat 144Asn
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40
45ggt aaa aac aat cgg ccc tca ggg atc cca gac cga ttc tct ggc tcc
192Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser
50 55 60ggc tca gga aac aca ggt tcc ttg acc atc act ggg gcc cag gcg
gaa 240Gly Ser Gly Asn Thr Gly Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80gat gag gct gac tat tac tgt ggc tcc cgg gac agc agt
ggt atc cat 288Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Arg Asp Ser Ser
Gly Ile His 85 90 95gtg gta ttc ggc gga ggg acc aag gtc acc gtc cta
ggt 327Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100
10565109PRTHomo sapiens 65Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp
Ser Leu Arg His Phe Tyr Pro 20 25 30Asn Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser
Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Asn Thr Gly
Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp
Tyr Tyr Cys Gly Ser Arg Asp Ser Ser Gly Ile His 85 90 95Val Val Phe
Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 10566354DNAHomo
sapiensCDS(1)..(354) 66gag gtg cag ctg ttg gag tct ggg gga ggc ttg
gta cag cct ggg ggg 48Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15tcc ctg aga ctc tcc tgt gca gcc tct gga
ttc acc ttt agc agc tat 96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30gcc atg agc tgg gtc cgc cag gct cca
ggg aag ggg ctg gag tgg gtc 144Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45tca gct att agt ggt agt ggt ggt
agc aca tac tac gca gac tcc gtg 192Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60aag ggc cgg ttc acc atc tcc
aga gac aat tcc aag aac acg ctg tat 240Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80ctg caa atg aac agc
ctg aga gcc gag gac acg gcc gtg tat tac tgt 288Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95gcg aga gat gac
gat gac tac gac ttt gac tac tgg ggc cgg ggg aca 336Ala Arg Asp Asp
Asp Asp Tyr Asp Phe Asp Tyr Trp Gly Arg Gly Thr 100 105 110atg gtc
acc gtc tcg agt 354Met Val Thr Val Ser Ser 11567118PRTHomo sapiens
67Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Asp Asp Asp Tyr Asp Phe
Asp Tyr Trp Gly Arg Gly Thr 100 105 110Met Val Thr Val Ser Ser
11568334DNAHomo sapiensCDS(1)..(333) 68cag tct gtg ttg acg cag ccg
ccc tca gcg tct ggg gcc ccc ggt cag 48Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ala Pro Gly Gln1 5 10 15agg gtc acc atc tct tgt
tct gga agc agc tcc aac atc gga att aat 96Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ile Asn 20 25 30gct gta aac tgg tac
cag cag ctc cca gga acg gcc ccc aaa ctc ctc 144Ala Val Asn Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45atc tat ggt aat
gat cag cgg ccc tca ggg gtc cct gac cga ttc tct 192Ile Tyr Gly Asn
Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60ggc tcc aag
tct ggc acc tca gcc tcc ctg gcc atc agt ggg ctc cag 240Gly Ser Lys
Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80tct
gag gat gag gct gat tat aac tgt gca gca tgg gat gac agc ctg 288Ser
Glu Asp Glu Ala Asp Tyr Asn Cys Ala Ala Trp Asp Asp Ser Leu 85 90
95agt ggt ccg gtg ttc ggc gga ggg acc aag ctg acc gtc cta ggt g
334Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 11069111PRTHomo sapiens 69Gln Ser Val Leu Thr Gln Pro Pro Ser
Ala Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ile Asn 20 25 30Ala Val Asn Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp
Glu Ala Asp Tyr Asn Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly
Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
1107066PRTHomo sapiens 70Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser
Val Ile Arg Asn Leu Asn1 5 10 15Asp Gln Val Leu Phe Ile Asp Gln Gly
Asn Arg Pro Leu Phe Glu Asp 20 25 30Met Thr Asp Ser Asp Cys Arg Asp
Asn Ala Pro Arg Thr Ile Phe Ile 35 40 45Ile Ser Met Tyr Lys Asp Ser
Gln Pro Arg Gly Met Ala Val Thr Ile 50 55 60Ser Val657134PRTHomo
sapiens 71Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile
Leu Lys1 5 10 15Lys Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr
Val Gln Asn 20 25 30Glu Asp
* * * * *