U.S. patent application number 10/719642 was filed with the patent office on 2004-09-23 for modulating immune responses.
This patent application is currently assigned to Celltech R & D Limited. Invention is credited to Andrews, Dawn, Chen, Yuching, Garcia-Martinez, Leon Fernando.
Application Number | 20040185040 10/719642 |
Document ID | / |
Family ID | 32996330 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040185040 |
Kind Code |
A1 |
Garcia-Martinez, Leon Fernando ;
et al. |
September 23, 2004 |
Modulating immune responses
Abstract
The invention provides methods for modulating the immune system
using anti-CD83 antibodies that can influence CD83 function.
Inventors: |
Garcia-Martinez, Leon Fernando;
(Woodinville, WA) ; Chen, Yuching; (Bellevue,
WA) ; Andrews, Dawn; (Lake Forest Park, WA) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE, LLP
2600 CENTURY SQUARE
1501 FOURTH AVENUE
SEATTLE
WA
98101-1688
US
|
Assignee: |
Celltech R & D Limited
Berkshire
GB
|
Family ID: |
32996330 |
Appl. No.: |
10/719642 |
Filed: |
November 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10719642 |
Nov 21, 2003 |
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PCT/US02/37738 |
Nov 21, 2002 |
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60331958 |
Nov 21, 2001 |
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60428130 |
Nov 21, 2002 |
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60473279 |
May 22, 2003 |
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Current U.S.
Class: |
424/132.1 ;
530/387.3 |
Current CPC
Class: |
C07K 2317/73 20130101;
Y02A 50/30 20180101; C07K 16/2803 20130101; C07K 2317/50 20130101;
C07K 2317/565 20130101 |
Class at
Publication: |
424/132.1 ;
530/387.3 |
International
Class: |
A61K 039/395; C07K
016/44 |
Claims
What is claimed:
1. An isolated multimerized antibody that can bind to a CD83
polypeptide comprising amino acid sequence SEQ ID NO:97.
2. The isolated antibody of claim 1, wherein proliferation of a
lymphocyte is decreased when the lymphocyte is contacted with the
multimerized antibody.
3. The isolated antibody claim 1, wherein the multimerized antibody
comprises amino acid sequence SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ
ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71 SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87,
SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ
ID NO:98 or SEQ ID NO:99.
4. An isolated nucleic acid encoding an antibody that can be
multimerized and that can bind to a CD83 polypeptide, wherein the
antibody comprises any one of amino acid sequences SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,
SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,
SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID
NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ
ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67,
SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ
ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90;
SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID
NO:95, SEQ ID NO:96, SEQ ID NO:98 or SEQ ID NO:99.
5. A nucleic acid encoding an anti-cd83 antibody wherein the
nucleic acid comprises any one of amino acid sequences SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65,
SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 or SEQ ID
NO:90.
6. A method of modulating lymphocyte proliferation in a mammal
comprising administering to the mammal a multimerized antibody that
is directed against an extracellular domain of CD83 polypeptide,
wherein the multimerized antibody can modulate lymphocyte
proliferation.
7. The method of claim 6, wherein the multimerized antibody can
bind to an extracellular domain of CD83 polypeptide that comprises
amino acid sequence SEQ ID NO:97.
8. The method of claims 6, wherein the multimerized antibody
comprises amino acid sequence SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ
ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71 SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87,
SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ
ID NO:98 or SEQ ID NO:99.
9. The method of claim 6, wherein the multimerized antibody is
non-covalently multimerized.
10. The method claim 6, wherein the multimerized antibody is
covalently multimerized.
11. The method of claim 6, wherein lymphocyte proliferation is
modulated at a localized site in the mammal.
12. The method of claim 11, wherein the localized site in the
mammal is a joint, a site in a lung, a site in a muscle, a site in
a stomach, a site in an intestine, a site in a thyroid, a site on
the skin, a site in a bladder, a site in a vagina, a site in the
brain, or a site in the prostate.
13. A method for decreasing proliferation of CD4.sup.+ T-cells in a
mammal comprising administering to the mammal a multimerized
antibody that can bind to an extracellular domain of a CD83 gene
product that comprises amino acid sequence SEQ ID NO:97.
14. A method of modulating cytokine production by a lymphocyte by
contacting the lymphocyte with a multimerized antibody that can
modulate cytokine production and wherein the multimerized antibody
can bind to a polypeptide that comprises amino acid sequence SEQ ID
NO:97.
15. A method of modulating granulocyte macrophage colony
stimulating factor production in a mammal by administering to the
mammal a multimerized antibody that can modulate the activity or
expression of CD83 polypeptides, wherein the multimerized antibody
can bind to a polypeptide that comprises amino acid sequence SEQ ID
NO:97.
16. A method of modulating granulocyte macrophage colony
stimulating factor production by a lymphocyte by contacting the
lymphocyte with a multimerized antibody that can modulate the
activity or expression of a CD83 polypeptide, wherein the
multimerized antibody can bind to a polypeptide that comprises
amino acid sequence SEQ ID NO:97.
17. A method of modulating tumor necrosis factor production in a
mammal by administering to the mammal a multimerized antibody that
can modulate the activity or expression of CD83 polypeptides, and
wherein the multimerized antibody can bind to a polypeptide that
comprises amino acid sequence SEQ ID NO:97.
18. A method of inhibiting proliferation of a human peripheral
blood mononuclear cell in a mammal by administering to the mammal a
multimerized antibody that can modulate the activity or expression
of CD83 polypeptides, and wherein the multimerized antibody can
bind to a polypeptide that comprises amino acid sequence SEQ ID
NO:97.
19. A method for placing an immune cell into anergy, comprising
contacting the immune cell that expresses CD83 gene product with a
multimerized antibody that can bind to a polypeptide that comprises
amino acid sequence SEQ ID NO:97.
20. A method for decreasing the activity of a CD83 gene product in
a mammal, comprising administering to the mammal a multimerized
antibody that can bind to a polypeptide that comprises amino acid
sequence SEQ ID NO:97.
21. A method for modulating cytokine levels in a mammal comprising
administering to the mammal a multimerized that can bind to an
extracellular domain of a CD83 gene product that comprises amino
acid sequence SEQ ID NO:97.
22. A method for increasing interleukin-10 levels in a mammal
comprising administering to the mammal a multimerized antibody that
can bind to an extracellular domain of a CD83 gene product that
comprises amino acid sequence SEQ ID NO:97.
23. A method for increasing interleukin-4 levels in a mammal
comprising administering to the mammal a multimerized antibody that
can bind to an extracellular domain of a CD83 gene product that
comprises amino acid sequence SEQ ID NO:97.
24. A method for increasing granulocyte macrophage colony
stimulating factor levels in a mammal comprising administering to
the mammal a multimerized antibody that can bind to an
extracellular domain of a CD83 gene product that comprises amino
acid sequence SEQ ID NO:97.
25. A method for treating an inappropriate immune response in a
mammal comprising administering to the mammal a multimerized
antibody that can bind to an extracellular domain of a CD83 gene
product that comprises amino acid sequence SEQ ID NO:97.
26. The method of claim 25, wherein the inappropriate immune
response is diabetes mellitus, arthritis, rheumatoid arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis,
multiple sclerosis, myasthenia gravis, systemic lupus
erythematosis, autoimmune thyroiditis, dermatitis, atopic
dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome,
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, or interstitial lung fibrosis.
27. The method of claim 25, wherein the inappropriate immune
response is tissue rejection of a transplanted tissue.
28. The method of claim 25, wherein the transplanted tissue is
skin, cardiac or bone marrow.
29. The method of claim 13, wherein the multimerized antibody
comprises amino acid sequence SEQ ID NO:1, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ
ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71 SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87,
SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ
ID NO:98 or SEQ ID NO:99.
30. The method of claim 13, wherein the multimerized antibody is
non-covalently multimerized.
31. The method of claim 13, wherein the multimerized antibody is
covalently multimerized.
32. The method of claim 13, wherein lymphocyte proliferation is
modulated at a localized site in the mammal.
33. The method of claim 32, wherein the localized site in the
mammal is a joint, a site in a lung, a site in a muscle, a site in
a stomach, a site in an intestine, a site in a thyroid, a site on
the skin, a site in a bladder, a site in a vagina, brain or
prostate.
34. The method of claim 22, wherein the interleukin-10 levels are
modulated to treat neoplastic disease.
35. The method of claim 22, wherein the interleukin-10 levels are
modulated to treat a tumor.
36. The method of claim 13, 15, 17, 20, 21, 22, 23, 24 or 25
wherein the mammal is a human.
Description
[0001] This application is a continuation under 35 U.S.C. 111(a) of
International Application No. PCT/US02/37738 filed Nov. 21, 2002
and published in English as WO 03/045318 on Jun. 5, 2003, which
claimed priority under 35 U.S.C. 119(e) from U.S. Provisional
Application Ser. No. 60/331,958 filed Nov. 21, 2001, which
applications and publication are incorporated herein by
reference.
[0002] This application also claims priority to U.S. Provisional
Application Ser. No. 60/428,130 filed Nov. 21, 2002 and U.S.
Provisional Application Ser. No. 60/473,279 filed May 22, 2003
which are incorporate here by reference.
FIELD OF THE INVENTION
[0003] The invention relates to multimerized antibodies directed
against the CD83 gene product, and methods of modulating the immune
response of an animal by using such multimerized antibodies.
BACKGROUND OF THE INVENTION
[0004] CD83 is a 45 kilodalton glycoprotein that is predominantly
expressed on the surface of dendritic cells and other cells of the
immune system. Structural analysis of the predicted amino acid
sequence of CD83 indicates that it is a member of the
immunoglobulin superfamily. See, Zhou et al., J. Immunol. 149:735
(1992)). U.S. Pat. No. 5,316,920 and WO 95/29236 disclose further
information about CD83. While such information suggests that CD83
plays a role in the immune system, that role is undefined, and the
interrelationship of CD83 with cellular factors remains
unclear.
[0005] Moreover, treatment of many diseases could benefit from more
effective methods for increasing or decreasing the immune response.
Hence, new reagents and methods are needed for modulating the
immune system through the CD83 gene and its gene product.
SUMMARY OF THE INVENTION
[0006] The invention provides methods for modulating an immune
response. In one aspect, the invention relates to the surprising
discovery that multimerized antibodies raised against the CD83 gene
product can arrest cellular proliferation of immune cells. Hence,
the invention provides a method of modulating the immune response
by modulating the activity or expression of the CD83 gene products,
for example, by using such multimerized antibodies.
[0007] Also according to the invention, the production of a
cytokine such as interleukin-2, interleukin-4, or interleukin-10
can be modulated by modulating the activity or expression of a CD83
polypeptide. In some embodiments, a multimerized antibody is used
that can modulate the activity or expression of a CD83 polypeptide.
For example, the antibody can be administered to the mammal or the
immune cell can be contacted with the antibody. In some
embodiments, the immune cells are T cells or antigen presenting
cells. In other embodiments, the immune cells are CD4.sup.+ T
cells.
[0008] The invention also provides a method of modulating
granulocyte macrophage colony stimulating factor production in a
mammal or in an immune cell by modulating the activity or
expression of CD83 polypeptides. In some embodiments, an antibody
or a multimerized antibody is used that can modulate the activity
or expression of a CD83 polypeptide. For example, the antibody can
be administered to the mammal or the immune cell can be contacted
with the antibody. In some embodiments, the immune cells are T
cells or antigen presenting cells. In other embodiments, the immune
cells are CD4.sup.+ T cells.
[0009] The invention also provides a method of modulating tumor
necrosis factor production in a mammal or in a mammalian cell by
modulating the activity or expression of CD83 polypeptides. In some
embodiments, an antibody or a multimerized antibody is used that
can modulate the activity or expression of a CD83 polypeptide. For
example, the antibody can be administered to the mammal or the
mammalian cell can be contacted with the antibody. In some
embodiments, the immune cells are T cells or antigen presenting
cells. In other embodiments, the immune cells are CD4.sup.+ T
cells.
[0010] The invention further provides a method of inhibiting
proliferation of a human peripheral blood mononuclear cell by
modulating the activity or expression of CD83 polypeptides. In some
embodiments, an antibody or a multimerized antibody is used that
can modulate the activity or expression of a CD83 polypeptide. For
example, the antibody can be administered to the mammal or the
human peripheral blood mononuclear cell can be contacted with the
antibody.
[0011] The invention also provides an antibody that can bind to a
CD83 polypeptide comprising SEQ ID NO:4, SEQ ID NO:8 or SEQ ID
NO:9, wherein activated CD4.sup.+ T-cells produce lower levels of
interleukin-4 when the T-cells are contacted with the antibody. The
invention further provides an antibody that can bind to a CD83
polypeptide comprising SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:9,
wherein CD4.sup.+ T-cells proliferation is decreased when the
T-cells are contacted with the antibody. The antibody can be a
multimerized antibody. Such multimerized antibodies can be bound to
a solid support, covalently crosslinked or bound together by a
second entity such as a secondary antibody. Examples of antibodies
of the invention include those that have an amino acid sequence
that includes SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ
ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,
SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID
NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ
ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID
NO:71 SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:79, SEQ
ID NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88,
SEQ ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98 or
SEQ ID NO:99. Nucleic acids encoding such an antibody can have, for
example, a sequence that includes SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:59,
SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:74, SEQ ID
NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:83, SEQ
ID NO:84, SEQ ID NO:85 or SEQ ID NO:90.
[0012] The invention also provides a method for decreasing the
activity of a CD83 gene product, comprising contacting the CD83
gene product with an antibody that comprises amino acid sequence
includes SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,
SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ
ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ
ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71
SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:79, SEQ ID
NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ
ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,
SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98 or SEQ ID
NO:99. The antibody can be a multimerized antibody. The activity of
a CD83 gene product can be decreased in a mammal or in a cell that
is involved in an immune response, for example, a T cell.
[0013] The invention further provides a method for decreasing the
translation of a CD83 gene product in a mammalian cell, comprising
contacting the mammalian cell with a nucleic acid complementary to
a CD83 nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, or SEQ ID NO:10.
[0014] In another embodiment, the invention provides a method for
decreasing the translation of a CD83 gene product in a mammal,
comprising administering to the mammal a nucleic acid complementary
to a CD83 nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, or SEQ ID NO:10.
[0015] The invention further provides a method for decreasing
proliferation of CD4.sup.+ T-cells in a mammal comprising
administering to the mammal an antibody that can bind to a CD83
gene product, wherein the CD83 gene product comprises SEQ ID NO:2
or SEQ ID NO:9. The antibody can have a sequence comprising
includes SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,
SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ
ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ
ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71
SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:79, SEQ ID
NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ
ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,
SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98 or SEQ ID
NO:99. The antibody can be a multimerized antibody.
[0016] The invention also provides a method for decreasing
interleukin-2 levels and increasing interleukin-4 levels in a
mammal comprising administering to the mammal an antibody that can
bind to a CD83 gene product, wherein the CD83 gene product
comprises SEQ ID NO:2 or SEQ ID NO:9. The antibody can have a
sequence comprising includes SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ
ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71 SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:87,
SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90; SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ
ID NO:98 or SEQ ID NO:99. The antibody can be a multimerized
antibody.
[0017] The invention further provides a method for decreasing
interleukin-2 levels and increasing interleukin-4 levels in a
mammal comprising administering to the mammal a nucleic acid
complementary to a CD83 nucleic acid comprising SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, or SEQ ID NO:10. In some embodiments the
interleukin-2 levels are decreased and the interleukin-4 levels are
increased to treat an autoimmune disease. In other embodiments, the
interleukin-2 levels are decreased and the interleukin-4 levels are
increased to stimulate production of Th2-associated cytokines in
transplant recipients, for example, to prolong survival of
transplanted tissues.
[0018] The invention also provides a method for increasing
interleukin-10 levels in a mammal comprising administering to the
mammal an antibody that can bind to a CD83 gene product, wherein
the CD83 gene product comprises SEQ ID NO:2 or SEQ ID NO:9. The
antibody can have a sequence comprising includes SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID
NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ
ID NO:69, SEQ ID NO:70, SEQ ID NO:71 SEQ ID NO:72, SEQ ID NO:73,
SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID
NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90; SEQ
ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,
SEQ ID NO:96, SEQ ID NO:98 or SEQ ID NO:99. The antibody can be a
multimerized antibody.
[0019] The invention further provides a method for increasing
interleukin-10 levels in a mammal comprising administering to the
mammal a nucleic acid complementary to a CD83 nucleic acid
comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:10.
In some embodiments, the interleukin-10 levels are increased to
treat neoplastic disease. In other embodiments, the interleukin-10
levels are increased to treat a tumor.
[0020] The invention also provides a method for increasing
interleukin-2 levels in a mammal comprising administering to the
mammal a functional CD83 polypeptide that comprises SEQ ID
NO:9.
[0021] The invention further provides a method for increasing
interleukin-2 levels in a mammal comprising: (a) transforming a T
cell from the mammal with a nucleic acid encoding a functional CD83
polypeptide operably linked to a promoter functional in a mammalian
cell, to generate a transformed T cell; (b) administering the
transformed T cell to the mammal to provide increased levels of
interleukin-2. In some embodiments, the CD83 polypeptide has a
sequence that comprises SEQ ID NO:9 or the nucleic acid has a
sequence that comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or
SEQ ID NO:10. Such methods for increasing interleukin-2 levels can
be used to treat an allergy or an infectious disease.
[0022] The invention also provides a method for increasing
granulocyte macrophage colony stimulating factor levels in a mammal
comprising administering to the mammal an antibody that can bind to
a CD83 gene product, wherein the CD83 gene product comprises SEQ ID
NO:2 or SEQ ID NO:9.
[0023] Such an antibody can have a sequence comprising includes SEQ
ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,
SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ
ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID
NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ
ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 SEQ ID NO:72,
SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID
NO:81, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ
ID NO:90; SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,
SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98 or SEQ ID NO:99. The
antibody can be a multimerized antibody.
[0024] The invention further provides a method for increasing
granulocyte macrophage colony stimulating factor levels in a mammal
comprising administering to the mammal a nucleic acid complementary
to a CD83 nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, or SEQ ID NO:10.
[0025] The invention also provides a method for increasing tumor
necrosis factor levels at a selected site in a mammal comprising
administering to the site a functional CD83 polypeptide. In another
embodiment, the invention provides a method for increasing tumor
necrosis factor levels in a selected mammalian cell comprising
transforming the cell with a nucleic acid encoding a functional
CD83 polypeptide. The CD83 polypeptide employed can, for example,
have a sequence comprising SEQ ID NO:9.
[0026] Animals such as mammals and birds may be treated by the
methods and compositions described herein. Such mammals and birds
include humans, dogs, cats, and livestock, for example, horses,
cattle, sheep, goats, chickens, turkeys and the like.
[0027] The invention further provides a mutant mouse that can serve
as an animal model of diminished T cell activation or altered
cytokine levels. The mutant mouse has an altered CD83 gene that
produces a larger gene product, having SEQ ID NO:4 or containing
SEQ ID NO:8. Also provided are methods of using the mutant mouse
model to study the effects of cytokines on the immune system,
inflammation, the function and regulation of CD83, T cell and
dendritic cell activity, the immune response and conditions and
treatments related thereto. Hence, the invention further provides a
mutant mouse whose somatic and germ cells comprise a mutant CD83
gene encoding a polypeptide comprising SEQ ID NO:4 or SEQ ID NO:8,
wherein expression of the mutant CD83 gene reduces CD4+ T cell
activation. The mutant CD83 gene can, for example, comprise SEQ ID
NO:3.
[0028] The invention further provides a method of identifying a
compound that can modulate CD4+ T cell activation comprising
administering a test compound to a mouse having a mutant or wild
type transgenic CD83 gene and observing whether CD4+ T cell
activation is decreased or increased. The somatic and/or germ cells
of the mutant mouse can comprise a mutant CD83 gene encoding a
polypeptide comprising SEQ ID NO:4 or SEQ ID NO:8. Alternatively,
the somatic and/or germ cells of the mouse can contain a wild type
CD83 gene, for example, SEQ ID NO:1 or SEQ ID NO:9.
[0029] The invention also provides a mutant CD83 gene encoding a
polypeptide comprising SEQ ID NO:4 or SEQ ID NO:8. The invention
further provides a mutant CD83 gene comprising nucleotide sequence
SEQ ID NO:3.
DESCRIPTION OF THE FIGURES
[0030] FIG. 1 summarizes flow cytometry data for G3 animals. As
shown, reduced numbers of CD4+ T cells are seen in two animals from
Pedigree 9, mouse 9.4.1 and mouse 9.4.9. All other animals analyzed
on that day exhibit normal numbers of CD4+ T cells.
[0031] FIG. 2 provides a graph of flow cytometry data for G3
animals that may have a mutant CD83 gene. Each diamond symbol
represents an individual animal. As shown, multiple animals from
the N2 generation exhibit a reduced percentage of CD4+ T cells.
[0032] FIG. 3 provides the nucleotide sequence of wild type mouse
CD83 (SEQ ID NO:1). The ATG start codon and the TGA stop codon are
underlined.
[0033] FIGS. 4A-B provides the nucleotide sequence of the mutant
CD83 gene (SEQ ID NO:3) of the invention derived from the mutant
LCD4.1 animal. The ATG start codon, the mutation and the TGA stop
codon are underlined.
[0034] FIG. 5 provides the amino acid sequence for wild type (top,
SEQ ID NO:2) and mutant (bottom, SEQ ID NO:4) CD83 coding regions.
The additional C-terminal sequences arising because of the CD83
mutation are underlined.
[0035] FIG. 6A illustrates that dendritic cells from wild type (?,
WT DC) and mutant (.linevert split., mutant DC) mice are capable of
the allogeneic activation of CD4+ T cells. CD4+ T cells were
stimulated with 10,000, 1000 or 100 dendritic cells for 5 days and
proliferation was measured by incorporation of tritiated
thymidine.
[0036] FIG. 6B illustrates that CD4.sup.+ T cells from mutant mice
(.linevert split., mutant CD4) fail to respond to allogeneic
stimulation with BALBc dendritic cells, although wild type animals
(?, WT CD4+) respond normally. CD4+ T cells were stimulated with
10,000, 1000 or 100 dendritic cells for 5 days and proliferation
measured by incorporation of tritiated thymidine.
[0037] FIG. 7 provides a bar graph illustrating IL-2, IL-4, IL-5,
TNFa, and IFN? production from wild type CD4+ T cells (white bar)
or CD83 mutant CD4+ T cells (dark bar) that had been stimulated
with 1 .mu.g/ml of anti-CD3 antibodies and 0.2 .mu.g/ml of
anti-CD28 antibodies for 72 hours. As illustrated, IL-2 levels are
lower, and IL-4 levels are higher in the CD83 mutant T cells.
[0038] FIG. 8 provides a bar graph illustrating IL-10 production
from wild type CD4+ T cells (white bar) or CD83 mutant CD4+ T cells
(dark bar) that had been stimulated with 0.1 .mu.g/ml of anti-CD28
antibodies and 1 to 10 .mu.g/ml of anti-CD3 antibodies for 72
hours. As illustrated, IL-10 levels are higher in the CD83 mutant T
cells.
[0039] FIG. 9 provides a bar graph illustrating GM-CSF production
from wild type CD4+ T cells (white bar) or CD83 mutant CD4+ T cells
(dark bar) that had been stimulated with anti-CD3 and anti-CD28
antibodies. As illustrated, GM-CSF production is higher in the CD83
mutant cells than in wild type cells.
[0040] FIG. 10A provides a bar graph illustrating IL-4 mRNA levels
from wild type CD4+ T cells (white bar) or CD83 mutant CD4+ T cells
(dark bar) that had been stimulated with anti-CD3 and anti-CD28
antibodies. As illustrated, the IL-4 mRNA levels are higher in the
CD83 mutant cells.
[0041] FIG. 10B provides a bar graph illustrating IL-10 mRNA levels
from wild type CD4+ T cells (white bar) or CD83 mutant CD4+ T cells
(dark bar) that had been stimulated with anti-CD3 and anti-CD28
antibodies. As illustrated, the IL-10 mRNA levels are higher in the
CD83 mutant cells.
[0042] FIG. 11 provides a graph illustrating that various
preparations of anti-CD83 antibodies inhibit IL-4 production in
anti-CD3 and anti-CD28 antibody stimulated T cells. The amount of
IL-4 produced by T cells in pg/ml is plotted versus the
concentration of different anti-CD83 antibody preparations,
including the 20B08 (?) anti-CD83 preparation, the 20D04 (.linevert
split.) anti-CD83 preparation, the 14C12 (?) anti-CD83 preparation
and the 11 G05 (X) anti-CD83 antibody preparation.
[0043] FIG. 12 provides a graph illustrating that various
preparations of anti-CD83 antibodies inhibit T cell proliferation.
The graph plots the incorporation of radioactive thymidine in cpms,
which was used as an indicator of the amount of T cell
proliferation, versus the concentration of the different anti-CD83
antibody preparations, including the 20D04 (?) anti-CD83
preparation, the 11G05 (.linevert split.) anti-CD83 antibody
preparation, the 14C12 (?) anti-CD83 preparation and the 6G05
anti-CD83 preparation (X).
[0044] FIG. 13 provides a graph illustrating that transgenic mice
that over-express wild type CD83 have increased T cell
proliferation. The graph plots the incorporation of radioactive
thymidine in cpms, which was used as an indicator of the amount of
T cell proliferation, versus the concentration of OVA peptide. The
transgenic mice utilized had a T-cell receptor specific for chicken
ovalbumin (OVA) 323-339 peptide that can activate T-cells. When
mixed with either transgenic or wild type dendritic cells in the
presence of OVA peptide, transgenic CD4+ T cells had increased
T-cell proliferation. However, transgenic dendritic cells could not
substantially increase wild type CD4+ T cell proliferation.
Transgenic CD83 CD4+T cells mixed with wild type dendritic cells
(?); transgenic CD83 CD4+ T cells mixed with transgenic dendritic
cells (.linevert split.); wild type CD4+ T cells mixed with
transgenic dendritic cells (?); and wild type CD4+ T cells mixed
with wild type dendritic cells (X).
[0045] FIG. 14 provides a schematic diagram of the structural
elements included in the mouse CD83 protein used for generating
antibodies.
[0046] FIG. 15 provides a graph of ELISA data illustrating the
titer obtained for different isolates of polyclonal anti-CD83
anti-sera. The first (?), second (.linevert split.) and third (?)
isolates had similar titers, though the titer of the second isolate
(.linevert split.) was somewhat higher.
[0047] FIG. 16 illustrates that proliferation of PHA-activated
human PBMCs was inhibited by antibodies raised against the external
region of the mouse CD83 protein (?). Pre-immune serum (.linevert
split.) had little effect on the proliferation of human PBMCs.
[0048] FIG. 17A provides a sequence alignment of anti-CD83 heavy
chain variable regions isolated by the invention. Sequences for
isolates 20B08H (SEQ ID NO:52), 6G05H (SEQ ID NO:53), 20D04H (SEQ
ID NO:54), 11 G05 (SEQ ID NO:66) and 14C12 (SEQ ID NO:67) are
provided. The CDR regions are highlighted in bold.
[0049] FIG. 17B provides a sequence alignment of anti-CD83 light
chain variable regions isolated by the invention. Sequences for
isolates 20B08L (SEQ ID NO:55), 6G05L (SEQ ID NO:56), 20D04L (SEQ
ID NO:57), 11G05L (SEQ ID NO:68) and 14C12L (SEQ ID NO:69) are
provided. The CDR regions are highlighted in bold.
[0050] FIG. 18 graphically illustrates that cells expressing CD83
can be detected and sorted using an anti-CD83 antibody preparation.
In this study, a Hodgkin's lymphoma cell line, KMH2, and a
commercially available anti-CD83 antibody preparation was used for
FACS sorting.
[0051] FIGS. 19A-B shows that two antibody preparations of the
invention can bind to endogenously produced human CD83, as detected
by FACS sorting of KMH2 cells (see also FIG. 18). Note that "Beer"
is another name used for CD83.
[0052] FIG. 20 illustrates that the 95F04 and 96G08 antibody
preparations described herein can inhibit proliferation of human
peripheral blood mononuclear cells as detected by [.sup.3H]
thymidine incorporation. As shown, when 30 .mu.g/ml of the 95F04
(?) antibody preparation was present, incorporation of [.sup.3H]
thymidine dropped to about 2000 cpm. When 30 .mu.g/ml 96G08
antibody preparation (?) was added to human peripheral blood
mononuclear cells, [.sup.3H] thymidine incorporation was reduced to
about 300 cpm. A third antibody preparation (98B 11, .linevert
split.) provided slight inhibition of PBMC proliferation. These
data indicate that the 95F04 and 96G08 antibody preparations can
alter the function of human CD83 in vivo.
[0053] FIG. 21 provides nucleotide and amino acid sequences for the
monoclonal antibody 96G08 light chain.
[0054] FIG. 22 provides nucleotide and amino acid sequences for the
monoclonal antibody 96G08 heavy chain.
[0055] FIG. 23 provides nucleotide and amino acid sequences for the
monoclonal antibody 95F04 light chain.
[0056] FIG. 24 provides nucleotide and amino acid sequences for the
monoclonal antibody 95F04 heavy chain.
[0057] FIGS. 25A-B provides the results of one screen of anti-CD83
antibody preparations that were multimerized by binding them to
microtiter plates. The plate-bound antibodies were screened for an
ability to inhibit lymphocyte proliferation as measured by
tritiated thymidine incorporation. As illustrated in FIG. 25A many
plate-bound anti-CD83 antibody preparations inhibit proliferation
of activated lymphocytes, including the 94c09, 98a02, 94d08, 98d11,
101b08, 6g05, 20d04, 14c12, 11g05, 12g04, 32f12 and 98b11
preparations. FIG. 25B further illustrates that some antibody
preparations are highly effective inhibitors (e.g. 117G12) but
others are not (e.g. 824pb and 98g08).
[0058] FIG. 26 illustrates that the inhibitory activity of the
multimerized (plate-bound) 6g05 antibody preparation is quenched by
soluble mouse CD83 protein (mCD83rFc). Lymphocyte proliferation was
measured by tritiated thymidine incorporation. As shown, the
multimerized 6g05 antibody preparation is strongly inhibitory of
proliferation when no CD83 protein is added. However, when the
mouse CD83 protein is added to assay, little or no inhibition of
lymphocyte proliferation is observed. The 98g08 antibody
preparation was used as a negative control because it exhibited
little or no lymphocyte inhibition in previous tests (see FIG.
25B).
[0059] FIG. 27 is a bar graph showing that both plate-bound and
cross-linked 6g05 antibodies are highly effective inhibitors of
lymphocyte proliferation. Lymphocyte proliferation was measured by
tritiated thymidine incorporation. As shown on the left side of the
graph above "plate-bound" the presence of plate-bound 6g05
antibodies in the lymphocyte proliferation assay cause little
tritiated thymidine incorporation (about 1000 cpm). Similarly, as
shown on the right side of the graph above "1.sup.st Ab (1
.mu.g/ml)" soluble cross-linked 6g05 antibodies also cause little
tritiated thymidine incorporation (about 1800 cpm).
[0060] FIG. 28 is a bar graph showing that several preparations of
soluble cross-linked anti-CD83 antibodies are highly effective
inhibitors of lymphocyte proliferation. Antibody preparations were
cross-linked with the rabbit anti-mouse secondary antibody and
lymphocyte proliferation was measured by tritiated thymidine
incorporation. As shown, soluble cross-linked antibody preparations
including the 6g05, 11g04, 12g04, 14c12, 20d04, 32f12, 94c09,
94d08, 98a02, 98d11(3), 101B08(2.7) and 117g12 preparations caused
little tritiated thymidine incorporation.
[0061] FIG. 29 shows that soluble, multimerized anti-CD83
antibodies exhibit inhibitory activity in mixed lymphocyte reaction
assays. A series of anti-CD83 antibody preparations that were
cross-linked using a rabbit anti-mouse antibody and then screened
for inhibition of CD4.sup.+ T cellular proliferation after
activation of the CD4.sup.+ T cells with CD11 cells in a mixed
lymphocyte reaction assay. As shown, the 98a02, 98d11, 20d04,
14c12, 12g04, and 117g12 inhibit lymphocyte proliferation in this
assay.
[0062] FIG. 30 shows that many anti-CD83 antibody preparations can
inhibit the recall response of lymphocytes. BALBc mice were first
immunized with keyhole limpet hemocyanin (KLH) prior to spleen
removal and CD 11 c and CD4+cell isolation. CD11c and CD4+cells
were mixed and added to microtiter wells coated with anti-CD83
antibodies. Lymphocyte proliferation was measured by tritiated
thymidine incorporation. As shown, the 94c09, 98a02, 6g05, 20d04,
and 117104 antibody preparations inhibited proliferation of
activated lymphocytes exposed to an antigen (KLH) to which they had
been immunized.
[0063] FIGS. 31A-B shows that soluble but cross-linked 6g05 and
14c12 anti-CD83 antibody preparations not only inhibit activated
lymphocyte cell proliferation (FIG. 31B) but also have very low
caspase activity (FIG. 31A). Caspase activity was determined using
a fluorogenic substrate and plotted as relative fluorescent units
(RFU) on the y axis.
[0064] FIG. 32 shows that the percentage of activated lymphocytes
that express annexin V is reduced after treatment with soluble but
cross-linked 6g05 and 14c12 anti-CD83 antibody preparations.
[0065] FIG. 33 shows that the activation marker CD69 is expressed
on splenocytes that were activated with Concavalin A and exposed to
anti-CD83 antibodies. The anti-CD83 antibodies employed were the
6g05, 14c12, 98b11 and 112d08 anti-CD83 antibody preparations that
were shown to inhibit activated splenocyte proliferation.
[0066] FIGS. 34A-E shows that a population of activated splenocytes
mixed with anti-CD83 antibody preparations have lost the blasting
(dividing) cells as detected by FACS sorting. The antibody
preparations employed were the rabbit anti-mouse antibody, called
the 2.sup.nd Ab (FIG. 34A), the 6g05 antibody preparation (FIG.
34B), the 98b11 antibody preparation (FIG. 34C), the 14c12 antibody
preparation (FIG. 34D), and the 112d08 antibody preparation (FIG.
34E). Almost all cells exposed to the 6g05 or 98b11 antibody
preparations sort as small cells with a 2N content of DNA as
illustrated by the high proportion of cells towards the left
(smaller) side of the population distribution compared to cells
exposed to the control 2.sup.nd Ab, 14c12 and 112d08 preparations
in FIGS. 34A, C and E.
[0067] FIG. 35A shows that the proportion of cells in the G1/G0
phase of the cell cycle is increased when a population of activated
splenocytes is treated with anti-CD83 antibody preparations. The
antibody preparations employed were the control rabbit anti-mouse
antibody, called the 2.sup.nd Ab, the 6g05 antibody preparation,
the 14c12 antibody preparation, and the negative control 112d08
antibody preparation. Both of the 6g05 and 14c12 antibody
preparations arrest the activated splenocytes in the G1/G0 phase of
the cell cycle.
[0068] FIG. 35B shows the proportion of cells in the G2/M phase of
the cell cycle after a population of activated splenocytes is
treated with anti-CD83 antibody preparations. The antibody
preparations employed were the control rabbit anti-mouse antibody,
called the 2.sup.nd Ab, the 6g05 antibody preparation, the 14c12
antibody preparation, and the negative control 112d08 antibody
preparation.
[0069] FIG. 35C shows that the proportion of cells in the S phase
of the cell cycle is decreased when a population of activated
splenocytes is treated with anti-CD83 antibody preparations. The
antibody preparations employed were the control rabbit anti-mouse
antibody, called the 2.sup.nd Ab, the 6g05 antibody preparation,
the 14c12 antibody preparation, and the negative control 112d08
antibody preparation. Activated splenocytes treated with either of
the 6g05 or 14c12 antibody preparations have lesser numbers of
cells in the S phase of the cell cycle.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The invention provides methods for modulating the immune
system. For example, according to the invention, loss or reduction
of CD83 activity in vivo results in decreased numbers of immune
cells, for example, decreased numbers of T cells. In some
embodiments, binding entities such as monoclonal antibodies that
are capable of inhibiting the function of CD83 are provided for use
in the invention. In other embodiments the binding entities or
antibodies are multimerized. The compositions and methods of the
invention can be used for treating conditions involving an
inappropriate immune response, for example, autoimmune diseases,
inflammation, tissue rejection, arthritis, atherosclerosis and the
like.
[0071] CD83
[0072] CD83 is a lymphocyte and dendritic cell activation antigen
that is expressed by activated lymphocytes and dendritic cells.
CD83 is also a single-chain cell-surface glycoprotein with a
molecular weight of about 45,000 that is believed to be a member of
the Ig superfamily. The structure predicted from the CD83 amino
acid sequence indicates that CD83 is a membrane glycoprotein with a
single extracellular Ig-like domain, a transmembrane domain and
cytoplasmic domain of approximately forty amino acids. The mature
CD83 protein has about 186 amino acids and is composed of a single
extracellular V type immunoglobulin (Ig)-like domain, a
transmembrane domain and a thirty nine amino acid cytoplasmic
domain. Northern blot analysis has revealed that CD83 is translated
from three mRNA transcripts of about 1.7, 2.0 and 2.5 kb that are
expressed by lymphoblastoid cell lines. It is likely that CD83
undergoes extensive post-translational processing because CD83 is
expressed as a single chain molecule, but the determined molecular
weight is twice the predicted size of the core protein. See U.S.
Pat. No. 5,766,570.
[0073] An example of a human CD83 gene product that can be used in
the invention is provided below (SEQ ID NO:9):
1 1 MSRGLQLLLL SCAYSLAPAT PEVKVACSED VDLPCTAPWD 41 PQVPYTVSWV
KLLEGGEERM ETPQEDHLRG QHYHQKGQNG 81 SFDAPNERPY SLKIRNTTSC
NSGTYRCTLQ DPDGQRNLSG 121 KVILRVTGCP AQRKEETFKK YRAEIVLLLA
LVIFYLTLII 161 FTCKFARLQS IFPDFSKAGM ERAFLPVTSP NKHLGLVTPH 201
KTELV
[0074] Such a CD83 gene product can be encoded by a number of
different nucleic acids. One example of a human CD83 nucleic acid
is provided below (SEQ ID NO:10).
2 1 CCTGGCGCAG CCGCAGCAGC GACGCGAGCG AACTCGGCCG 41 GGCCCGGGCG
CGCGGGGGCG GGACGCGCAC GCGGCGAGGG 81 CGGCGGGTGA GCCGGGGGCG
GGGACGGGGG CGGGACGGGG 121 GCGAAGGGGG CGGGGACGGG GGCGCCCGCC
GGCCTAACGG 161 GATTAGGAGG GCGCGCCACC CGCTTCCGCT GCCCGCCGGG 201
GAATCCCCCG GGTGGCGCCC AGGGAAGTTC CCGAACGGGC 241 GGGCATAAAA
GGGCAGCCGC GCCGGCGCCC CACAGCTCTG 281 CAGCTCGTGG CAGCGGCGCA
GCGCTCCAGC CATGTCGCGC 321 GGCCTCCAGC TTCTGCTCCT GAGCTGCGCC
TACAGCCTGG 361 CTCCCGCGAC GCCGGAGGTG AAGGTGGCTT GCTCCGAAGA 401
TGTGGACTTG CCCTGCACCG CCCCCTGGGA TCCGCAGGTT 441 CCCTACACGG
TCTCCTGGGT CAAGTTATTG GAGGGTGGTG 481 AAGAGAGGAT GGAGACACCC
CAGGAAGACC ACCTCAGGGG 521 ACAGCACTAT CATCAGAAGG GGCAAAATGG
TTCTTTCGAC 561 GCCCCCAATG AAAGGCCCTA TTCCCTGAAG ATCCGAAACA 601
CTACCAGCTG CAACTCGGGG ACATACAGGT GCACTCTGCA 641 GGACCCGGAT
GGGCAGAGAA ACCTAAGTGG CAAGGTGATC 681 TTGAGAGTGA CAGGATGCCC
TGCACAGCGT AAAGAAGAGA 721 CTTTTAAGAA ATACAGAGCG GAGATTGTCC
TGCTGCTGGC 761 TCTGGTTATT TTCTACTTAA CACTCATCAT TTTCACTTGT 801
AAGTTTGCAC GGCTACAGAG TATCTTCCCA GATTTTTCTA 841 AAGCTGGCAT
GGAACGAGCT TTTCTCCCAG TTACCTCCCC 881 AAATAAGCAT TTAGGGCTAG
TGACTCCTCA CAAGACAGAA 921 CTGGTATGAG CAGGATTTCT GCAGGTTCTT
CTTCCTGAAG 961 CTGAGGCTCA GGGGTGTGCC TGTCTGTTAC ACTGGAGGAG 1001
AGAAGAATGA GCCTACGCTG AAGATGGCAT CCTGTGAAGT 1041 CCTTCACCTC
ACTGAAAACA TCTGGAAGGG GATCCCACCC 1081 CATTTTCTGT GGGCAGGCCT
CGAAAACCAT CACATGACCA 1121 CATAGCATGA GGCCACTGCT GCTTCTCCAT
GGCCACCTTT 1161 TCAGCGATGT ATGCAGCTAT CTGGTCAACC TCCTGGACAT 1201
TTTTTCAGTC ATATAAAAGC TATGGTGAGA TGCAGCTGGA 1241 AAACGGTCTT
GGGAAATATG AATGCCCCCA GCTGGCCCGT 1281 GACAGACTCC TGAGGACAGC
TGTCCTCTTC TGCATCTTGG 1321 GGACATCTCT TTGAATTTTC TGTGTTTTGC
TGTACCAGCC 1361 CAGATGTTTT ACGTCTGGGA GAAATTGACA GATCAAGCTG 1401
TGAGACAGTG GGAAATATTT AGCAAATAAT TTCCTGGTGT 1441 GAAGGTCCTG
CTATTACTAA GGAGTAATCT GTGTACAAAG 1481 AAATAACAAG TCGATGAACT
ATTCCCCAGC AGGGTCTTTT 1521 CATCTGGGAA AGACATCCAT AAAGAAGCAA
TAAAGAAGAG 1561 TGCCACATTT ATTTTTATAT CTATATGTAC TTGTCAAAGA 1601
AGGTTTGTGT TTTTCTGCTT TTGAAATCTG TATCTGTAGT 1641 GAGATAGCAT
TGTGAACTGA CAGGCAGCCT GGACATAGAG 1681 AGGGAGAAGA AGTCAGAGAG
GGTGACAAGA TAGAGAGCTA 1721 TTTAATGGCC GGCTGGAAAT GCTGGGCTGA
CGGTGCAGTC 1761 TGGGTGCTCG CCCACTTGTC CCACTATCTG GGTGCATGAT 1801
CTTGAGCAAG TTCCTTCTGG TGTCTGCTTT CTCCATTGTA 1841 AACCACAAGG
CTGTTGCATG GGCTAATGAA GATCATATAC 1881 GTGAAAATTA TTTGAAAACA
TATAAAGCAC TATACAGATT 1921 CGAAACTCCA TTGAGTCATT ATCCTTGCTA
TGATGATGGT 1961 GTTTTGGGGA TGAGAGGGTG CTATCCATTT CTCATGTTTT 2001
CCATTGTTTG AAACAAAGAA GGTTACCAAG AAGCCTTTCC 2041 TGTAGCCTTC
TGTAGGAATT CTTTTGGGGA AGTGAGGAAG 2081 CCAGGTCCAC GGTCTGTTCT
TGAAGCAGTA GCCTAACACA 2121 CTCCAAGATA TGGACACACG GGAGCCGCTG
GCAGAAGGGA 2161 CTTCACGAAG TGTTGCATGG ATGTTTTAGC CATTGTTGGC 2201
TTTCCCTTAT CAAACTTGGG CCCTTCCCTT CTTGGTTTCC 2241 AAAGGCATTT
ATTGCTGAGT TATATGTTCA CTGTCCCCCT 2281 AATATTAGGG AGTAAAACGG
ATACCAAGTT GATTTAGTGT 2321 TTTTACCTCT GTCTTGGCTT TCATGTTATT
AAACGTATGC 2361 ATGTGAAGAA GGGTGTTTTT CTGTTTTATA TTCAACTCAT 2401
AAGACTTTGG GATAGGAAAA ATGAGTAATG GTTACTAGGC 2441 TTAATACCTG
GGTGATTACA TAATCTGTAC AACGAACCCC 2481 CATGATGTAA GTTTACCTAT
GTAACAAACC TGCACTTATA 2521 CCCATGAACT TAAAATGAAA GTTAAAAATA
AAAAACATAT 2561 ACAAATAAAA AAAA
[0075] A sequence of a wild type mouse CD83 gene that can be used
in the invention is provided herein as SEQ ID NO:1. SEQ ID NO:1 is
provided below with the ATG start codon and the TGA stop codon
identified by underlining.
3 1 GCGCTCCAGC CGCATGTCGC AAGGCCTCCA GCTCCTGTTT 41 CTAGGCTGCG
CCTGCAGCCT GGCACCCGCG ATGGCGATGC 81 GGGAGGTGAC GGTGGCTTGC
TCCGAGACCG CCGACTTGCC 121 TTGCACAGCG CCCTGGGACC CGCAGCTCTC
CTATGCAGTG 161 TCCTGGGCCA AGGTCTCCGA GAGTGGCACT GAGAGTGTGG 201
AGCTCCCGGA GAGCAAGCAA AACAGCTCCT TCGAGGCCCC 241 CAGGAGAAGG
GCCTATTCCC TGACGATCCA AAACACTACC 281 ATCTGCAGCT CGGGCACCTA
CAGGTGTGCC CTGCAGGAGC 321 TCGGAGGGCA GCGCAACTTG AGCGGCACCG
TGGTTCTGAA 361 GGTGACAGGA TGCCCCAAGG AAGCTACAGA GTCAACTTTC 401
AGGAAGTACA GGGCAGAAGC TGTGTTGCTC TTCTCTCTGG 441 TTGTTTTCTA
CCTGACACTC ATCATTTTCA CCTGCAAATT 481 TGCACGACTA CAAAGCATTT
TCCCAGATAT TTCTAAACCT 521 GGTACGGAAC AAGCTTTTCT TCCAGTCACC
TCCCCAAGCA 561 AACATTTGGG GCCAGTGACC CTTCCTAAGA CAGAAACGGT 601
ATGAGTAGGA TCTCCACTGG TTTTTACAAA GCCAAGGGCA 641 CATCAGATCA
GTGTGCCTGA ATGCCACCCG GACAAGAGAA 681 GAATGAGCTC CATCCTCAGA
TGGCAACCTT TCTTTGAAGT 721 CCTTCACCTG ACAGTGGGCT CCACACTACT
CCCTGACACA 761 GGGTCTTGAG CACCATCATA TGATCACGAA GCATGGAGTA 801
TCACCGCTTC TCTGTGGCTG TCAGCTTAAT GTTTCATGTG 841 GCTATCTGGT
CAACCTCGTG AGTGCTTTTC AGTCATCTAC 881 AAGCTATGGT GAGATGCAGG
TGAAGCAGGG TCATGGGAAA 921 TTTGAACACT CTGAGCTGGC CCTGTGACAG
ACTCCTGAGG 961 ACAGCTGTCC TCTCCTACAT CTGGGATACA TCTCTTTGAA 1001
TTTGTCCTGT TTCGTTGCAC CAGCCCAGAT GTCTCACATC 1041 TGGCGGAAAT
TGACAGGCCA AGCTGTGAGC CAGTGGGAAA 1081 TATTTAGCAA ATAATTTCCC
AGTGCGAAGG TCCTGCTATT 1121 AGTAAGGAGT ATTATGTGTA CATAGAAATG
AGAGGTCAGT 1161 GAACTATTCC CCAGCAGGGC CTTTTCATCT GGAAAAGACA 1201
TCCACAAAAG CAGCAATACA GAGGGATGCC ACATTTATTT 1241 TTTTAATCTT
CATGTACTTG TCAAAGAAGA ATTTTTCATG 1281 TTTTTTCAAA GAAGTGTGTT
TCTTTCCTTT TTTAAAATAT 1321 GAAGGTCTAG TTACATAGCA TTGCTAGCTG
ACAAGCAGCC 1361 TGAGAGAAGA TGGAGAATGT TCCTCAAAAT AGGGACAGCA 1401
AGCTAGAAGC ACTGTACAGT GCCCTGCTGG GAAGGGCAGA 1441 CAATGGACTG
AGAAACCAGA AGTCTGGCCA CAAGATTGTC 1481 TGTATGATTC TGGACGAGTC
ACTTGTGGTT TTCACTCTCT 1521 GGTTAGTAAA CCAGATAGTT TAGTCTGGGT
TGAATACAAT 1561 GGATGTGAAG TTGCTTGGGG AAAGCTGAAT GTAGTGAATA 1601
CATTGGCAAC TCTACTGGGC TGTTACCTTG TTGATATCCT 1641 AGAGTTCTGG
AGCTGAGCGA ATGCCTGTCA TATCTCAGCT 1681 TGCCCATCAA TCCAAACACA
GGAGGCTACA AAAAGGACAT 1721 GAGCATGGTC TTCTGTGTGA ACTCCTCCTG
AGAAACGTGG 1761 AGACTGGCTC AGCGCTTTGC GCTTGAAGGA CTAATCACAA 1801
GTTCTTGAAG ATATGGACCT AGGGGAGCTA TTGCGCCACG 1841 ACAGGAGGAA
GTTCTCAGAT GTTGCATTGA TGTAACATTG 1881 TTGCATTTCT TTAATGAGCT
GGGCTCCTTC CTCATTTGCT 1921 TCCCAAAGAG ATTTTGTCCC ACTAATGGTG
TGCCCATCAC 1961 CCACACTATG AAAGTAAAAG GGATGCTGAG CAGATACAGC 2001
GTGCTTACCT CTCAGCCATG ACTTTCATGC TATTAAAAGA 2041 ATGCATGTGA A
[0076] Nucleic acids having SEQ ID NO:1 encode a mouse polypeptide
having SEQ ID NO:2, provided below.
4 1 MSQGLQLLFL GCACSLAPAM AMREVTVACS ETADLPCTAP 41 WDPQLSYAVS
WAKVSESGTE SVELPESKQN SSFEAPRRRA 81 YSLTIQNTTI CSSGTYRCAL
QELGGQRNLS GTVVLKVTGC 121 PKEATESTFR KYRAEAVLLF SLVVFYLTLI
IFTCKFARLQ 161 SIFPDISKPG TEQAFLPVTS PSKHLGPVTL PKTETV
[0077] According to the invention, loss or reduction of CD83
activity in vivo results in a decreased immune response, for
example, decreased numbers of T cells. The effect of CD83 on the
immune response was initially ascertained through use of a mutant
mouse that encodes a mutant CD83. Such a mutant mouse has a CD83
gene encoding SEQ ID NO:4, with added C-terminal sequences provided
by SEQ ID NO:8. In contrast to these wild type CD83 nucleic acids
and polypeptides, the mutant CD83 gene of the invention has SEQ ID
NO:3. SEQ ID NO:3 is provided below with the ATG start codon, the
mutation, and the TGA stop codon are identified by underlining.
5 1 GCGCTCCAGC CGCATGTCGC AAGGCCTCCA GCTCCTGTTT 41 CTAGGCTGCG
CCTGCAGCCT GGCACCCGCG ATGGCGATGC 81 GGGAGGTGAC GGTGGCTTGC
TCCGAGACCG CCGACTTGCC 121 TTGCACAGCG CCCTGGGACC CGCAGCTCTC
CTATGCAGTG 161 TCCTGGGCCA AGGTCTCCGA GAGTGGCACT GAGAGTGTGG 201
AGCTCCCGGA GAGCAAGCAA AACAGCTCCT TCGAGGCCCC 241 CAGGAGAAGG
GCCTATTCCC TGACGATCCA AAACACTACC 281 ATCTGCAGCT CGGGCACCTA
CAGGTGTGCC CTGCAGGAGC 321 TCGGAGGGCA GCGCAACTTG AGCGGCACCG
TGGTTCTGAA 361 GGTGACAGGA TGCCCCAAGG AAGCTACAGA GTCAACTTTC 401
AGGAAGTACA GGGCAGAAGC TGTGTTGCTC TTCTCTCTGG 441 TTGTTTTCTA
CCTGACACTC ATCATTTTCA CCTGCAAATT 481 TGCACGACTA CAAAGCATTT
TCCCAGATAT TTCTAAACCT 521 GGTACGGAAC AAGCTTTTCT TCCAGTCACC
TCCCCAAGCA 561 AACATTTGGG GCCAGTGACC CTTCCTAAGA CAGAAACGGT 601
AAGAGTAGGA TCTCCACTGG TTTTTACAAA GCCAAGGGCA 641 CATCAGATCA
GTGTGCCTGA ATGCCACCCG GACAAGAGAA 681 GAATGAGCTC CATCCTCAGA
TGGCAACCTT TCTTTGAAGT 721 CCTTCACCTG ACAGTGGGCT CCACACTACT
CCCTGACACA 761 GGGTCTTGAG CACCATCATA TGATCACGAA GCATGGAGTA 801
TCACCGCTTC TCTGTGGCTG TCAGCTTAAT GTTTCATGTG 841 GCTATCTGGT
CAACCTCGTG AGTGCTTTTC AGTCATCTAC 881 AAGCTATGGT GAGATGCAGG
TGAAGCAGGG TCATGGGAAA 921 TTTGAACACT CTGAGCTGGC CCTGTGACAG
ACTCCTGAGG 961 ACAGCTGTCC TCTCCTACAT CTGGGATACA TCTCTTTGAA 1001
TTTGTCCTGT TTCGTTGCAC CAGCCCAGAT GTCTCACATC 1041 TGGCGGAAAT
TGACAGGCCA AGCTGTGAGC CAGTGGGAAA 1081 TATTTAGCAA ATAATTTCCC
AGTGCGAAGG TCCTGCTATT 1121 AGTAAGGAGT ATTATGTGTA CATAGAAATG
AGAGGTCAGT 1161 GAACTATTCC CCAGCAGGGC CTTTTCATCT GGAAAAGACA 1201
TCCACAAAAG CAGCAATACA GAGGGATGCC ACATTTATTT 1241 TTTTAATCTT
CATGTACTTG TCAAAGAAGA ATTTTTCATG 1281 TTTTTTCAAA GAAGTGTGTT
TCTTTCCTTT TTTAAAATAT 1321 GAAGGTCTAG TTACATAGCA TTGCTAGCTG
ACAAGCAGCC 1361 TGAGAGAAGA TGGAGAATGT TCCTCAAAAT AGGGACAGCA 1401
AGCTAGAAGC ACTGTACAGT GCCCTGCTGG GAAGGGCAGA 1441 CAATGGACTG
AGAAACCAGA AGTCTGGCCA CAAGATTGTC 1481 TGTATGATTC TGGACGAGTC
ACTTGTGGTT TTCACTCTCT 1521 GGTTAGTAAA CCAGATAGTT TAGTCTGGGT
TGAATACAAT 1561 GGATGTGAAG TTGCTTGGGG AAAGCTGAAT GTAGTGAATA 1601
CATTGGCAAC TCTACTGGGC TGTTACCTTG TTGATATCCT 1641 AGAGTTCTGG
AGCTGAGCGA ATGCCTGTCA TATCTCAGCT 1681 TGCCCATCAA TCCAAACACA
GGAGGCTACA AAAAGGACAT 1721 GAGCATGGTC TTCTGTGTGA ACTCCTCCTG
AGAAACGTGG 1761 AGACTGGCTC AGCGCTTTGC GCTTGAAGGA CTAATCACAA 1801
GTTCTTGAAG ATATGGACCT AGGGGAGCTA TTGCGCCACG 1841 ACAGGAGGAA
GTTCTCAGAT GTTGCATTGA TGTAACATTG 1881 TTGCATTTCT TTAATGAGCT
GGGCTCCTTC CTCATTTGCT 1921 TCCCAAAGAG ATTTTGTCCC ACTAATGGTG
TGCCCATCAC 1961 CCACACTATG AAAGTAAAAG GGATGCTGAG CAGATACAGC 2001
GTGCTTACCT CTCAGCCATG ACTTTCATGC TATTAAAAGA 2041 ATGCATGTGA A
[0078] The change from a thymidine in SEQ ID NO:1 to an adenine in
SEQ ID NO:3 at the indicated position (602) leads to read-through
translation because the stop codon at positions 602-604 in SEQ ID
NO:1 is changed to a codon that encodes an arginine. Accordingly,
mutant CD83 nucleic acids having SEQ ID NO:3 encode an elongated
polypeptide having SEQ ID NO:4, provided below, where the extra
amino acids are underlined.
6 1 MSQGLQLLFL GCACSLAPAM AMREVTVACS ETADLPCTAP 41 WDPQLSYAVS
WAKVSESGTE SVELPESKQN SSFEAPRRRA 81 YSLTIQNTTI CSSGTYRCAL
QELGGQRNLS GTVVLKVTGC 121 PKEATESTFR KYRAEAVLLF SLVVFYLTLI
IFTCKFARLQ 161 SIFPDISKPG TEQAFLPVTS PSKHLGPVTL PKTETVRVGS 201
PLVFTKPRAH QISVPECHPD KRRMSSILRW QPFFEVLHLT 241 VGSTLLPDTG S
[0079] In another embodiment, the invention provides mutant CD83
nucleic acids that include SEQ ID NO:5.
7 1 ATGTCGCAAG GCCTCCAGCT CCTGTTTCTA GGCTGCGCCT 41 GCAGCCTGGC
ACCCGCGATG GCGATGCGGG AGGTGACGGT 81 GGCTTGCTCC GAGACCGCCG
ACTTGCCTTG CACAGCGCCC 121 TGGGACCCGC AGCTCTCCTA TGCAGTGTCC
TGGGCCAAGG 161 TCTCCGAGAG TGGCACTGAG AGTGTGGAGC TCCCGGAGAG 201
CAAGCAAAAC AGCTCCTTCG AGGCCCCCAG GAGAAGGGCC 241 TATTCCCTGA
CGATCCAAAA CACTACCATC TGCAGCTCGG 281 GCACCTACAG GTGTGCCCTG
CAGGAGCTCG GAGGGCAGCG 321 CAACTTGAGC GGCACCGTGG TTCTGAAGGT
GACAGGATGC 361 CCCAAGGAAG CTACAGAGTC AACTTTCAGG AAGTACAGGG 401
CAGAAGCTGT GTTGCTCTTC TCTCTGGTTG TTTTCTACCT 441 GACACTCATC
ATTTTCACCT GCAAATTTGC ACGACTACAA 481 AGCATTTTCC CAGATATTTC
TAAACCTGGT ACGGAACAAG 521 CTTTTCTTCC AGTCACCTCC CCAAGCAAAC
ATTTGGGGCC 561 AGTGACCCTT CCTAAGACAG AAACGGTAAG AGTAGGATCT 601
CCACTGGTTT TTACAAAGCC AAGGGCACAT CAGATCAGTG 641 TGCCTGAATG
CCACCCGGAC AAGAGAAGAA TGAGCTCCAT 681 CCTCAGATGG CAACCTTTCT
TTGAAGTCCT TCACCTGACA 721 GTGGGCTCCA CACTACTCCC TGACACAGGG
TCTTGA
[0080] Nucleic acids having SEQ ID NO:5 also encode a polypeptide
having SEQ ID NO:4.
[0081] In another embodiment, the invention provides mutant CD83
nucleic acids that include SEQ ID NO:7.
8 1 AGAGTAGGAT CTCCACTGGT TTTTACAAAG CCAAGGGCAC 41 ATCAGATCAG
TGTGCCTGAA TGCCACCCGG ACAAGAGAAG 81 AATGAGCTCC ATCCTCAGAT
GGCAACCTTT CTTTGAAGTC 121 CTTCACCTGA CAGTGGGCTC CACACTACTC
CCTGACACAG 161 GGTCTTGA
[0082] The invention also provides a mutant CD83 containing SEQ ID
NO:8, provided below.
9 1 RVGSPLVFTK PRAHQISVPE CHPDKRRMSS ILRWQPPFEV 41 LHLTVGSTLL
PDTGS
[0083] SEQ ID NO:8 contains read through sequences that are not
present in the wild type CD83 polypeptide but are present in the
mutant CD83 gene product provided by the invention.
[0084] In some embodiments, the CD83 gene product is used for
generating antibodies. While any of the CD83 gene products
described herein can be employed for immunization of animal, in
some embodiments the extracellular Ig-like domain of the CD83 gene
product is used for immunization, or antibodies are screened for
reactivity with the extracellular Ig-like domain. The extracellular
Ig-like domain of the human CD83 gene product spans amino acids
21-127, and is provided below (SEQ ID NO:97):
10 21 PEVKVACSED VDLPCTAPWD 41 PQVPYTVSWV KLLEGGEERM ETPQEDHLRG
QHYHQKGQNG 81 SFDAPNERPY SLKIRNTTSC NSGTYRCTLQ DPDGQRNLSG 121
KVILRVT
[0085] CD83 Antibodies
[0086] The invention provides antibody preparations directed
against the mutant and wild type CD83 polypeptides of the
invention, for example, against a polypeptide having SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. Other
antibodies of interest can bind to the cytoplasmic tail of
CD83.
[0087] In some embodiments, the anti-CD83 antibodies are
multimerized antibodies. According to the invention, multimerized
anti-CD83 antibodies are surprisingly effective inhibitors of
lymphocyte cell proliferation. As used herein, an "multimerized"
anti-CD83 antibody is a collection of anti-CD83 antibodies that are
in close proximity. While such multimerized antibodies can be
covalently linked, no such covalent linkage is necessary. Instead,
multimerization of anti-CD83 antibodies can simply involve bringing
the antibodies into close proximity, for example, by attachment to
a solid support such as a plate or a bead. Alternatively, the
antibodies can be non-covalently linked together through another
entity, for example, any convenient non-covalent binding entity or
secondary antibody. Hence, any available means for bringing the
anti-CD83 antibodies into proximity can be used to generate the
multimerized antibodies of the invention.
[0088] In some embodiments, the anti-CD83 binding proteins or
antibodies can be chemically cross-linked or genetically fused with
any available crosslinking reagent. Crosslinking can be achieved
using one or a combination of a wide variety of multifunctional
reagents. Such crosslinking agents include glutaraldehyde,
succinaldehyde, octanedialdehyde and glyoxal. Additional
multifunctional crosslinking agents include halo-triazines, e.g.,
cyanuric chloride; halo-pyrimidines, e.g.,
2,4,6-trichloro/bromo-pyrimidine; anhydrides or halides of
aliphatic or aromatic mono- or di-carboxylic acids, e.g., maleic
anhydride, (meth)acryloyl chloride, chloroacetyl chloride;
N-methylol compounds, e.g., N-methylol-chloro acetamide;
di-isocyanates or di-isothiocyanates, e.g.,
phenylene-1,4-di-isocyanate and aziridines. Other crosslinking
agents include epoxides, such as, for example, di-epoxides,
tri-epoxides and tetra-epoxides. Other crosslinking agents include,
for example, dimethyl 3,3'-dithiobispropionimidate-HCl (DTBP);
dithiobis (succinimidylpropionate) (DSP); bismaleimidohexane (BMH);
bis[Sulfosuccinimidyl]suberate (BS);
1,5-difluoro-2,4-dinitrobenzene (DFDNB); dimethylsuberimidate-2HCl
(DMS); disuccinimidyl glutarate (DSG); disulfosuccinimidyl
tartarate (Sulfo-DST); 1-ethyl-3-[3-dimethylaminoprop-
yl]carbodiimide hydrochloride (EDC); ethylene glycolbis
[sulfo-succinimidylsuccinate] (Sulfo-EGS);
N-[?-maleimido-butyryloxy]succ- inimide ester (GMBS);
N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB);
sulfosuccinimidyl-6-[a-methyl-a-(2-pyridyldithio)
toluamido]hexanoate (Sulfo-LC-SMPT);
bis-[.beta.-(4-azidosalicylamido) ethyl]disulfide (BASED); and
NHS-PEG-Vinylsulfone (NHS-PEG-VS).
[0089] In some embodiments, crosslinkers useful with various
preparations of anti-CD83 antibodies of this invention include (1)
those which create covalent links from one cysteine side chain of a
protein to another cysteine side chain, (2) those which create
covalent links from one lysine side chain of a protein to another,
or (3) those which create covalent links from one cysteine side
chain of a protein to a lysine side chain.
[0090] In other embodiments, the anti-CD83 antibodies are
reversibly crosslinked. Such reversibly crosslinked antibodies are
useful for short term use, for example, for short term control of
the immune response either in vivo or in vitro, or for controlled
dissipation of the anti-CD83 antibodies at a localized site after
administration for short term therapeutic purposes. Examples of
reversible crosslinkers are described in T. W. Green, Protective
Groups in Organic Synthesis, John Wiley & Sons (Eds.) (1981).
Other types of reversible crosslinkers are disulfide
bond-containing crosslinkers. The crosslinks formed by such
crosslinkers can be broken by the addition of reducing agent, such
as cysteine, to the environment of the crosslinked anti-CD83
antibodies. Disulfide crosslinkers are described in the Pierce
Catalog and Handbook (1994-1995).
[0091] Examples of crosslinkers that may be used also include:
Homobifunctional (Symmetric);
DSP--Dithiobis(succinimidylpropionate), also know as Lomant,'s
Reagent; DTSSP--3-3'-Dithiobis (sulfosuccinimidyl-propionate),
water soluble version of DSP; DTBP--Dimethyl
3,3'-dithiobispropionimidate-HCl; BASED--Bis-(13-[4-azidos-
alicylamido] ethyl)disulfide;
DPDPB--1,4-Di-(3'-[2'-pyridyldithio]-propion- amido)butane;
Heterobifunctional (Asymmetric); SPDP--N-Succinimidyl-3-(2-p-
yridyldithio)propionate;
LC-SPDP--Succinimidyl-6-(3-[2-pyridyldithio] propionate)hexanoate;
Sulfo-LC-SPDP--Sulfosuccinimidyl-6-(3-[2-pyridyldlt- hio]
propionate)hexanoate, water soluble version of LC-SPDP;
APDP--N-(4-[p-azidosalicylamido]butyl)-3'-(2'-pyridyldithio)
propionamide;
SADP--N-Succinimidyl(4-azidophenyl)1,3'-dithiopropionate;
Sulfo-SADP--Sulfosuccinimidyl(4-azidophenyl) 1,3'-dithiopropionate,
water soluble version of SADP;
SAED--Sulfosuccinimidyl-2-(7-azido-4-methycoumar-
in-3-acetamide)ethyl-1,3'dithiopropionate;
SAND--Sulfosuccinimidyl-2-(m-az-
ido-o-nitrobenzamido)ethyl-1,3'-dithiopropionate;
SASD--Sulfosuccinimidyl--
2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate;
SMPB--Succinimidyl-4-(p-maleimidophenyl)butyrate;
Sulfo-SMPB--Sulfosuccin- imidyl-4-(p-maleimidophenyl)butyrate;
SMPT--4-Succinimidyloxycarbonyl-meth- yl-a-(2-pyridylthio) toluene;
Sulfo-LC-SMPT--Sulfosuccinimidyl-6-(a-methyl-
-a-(2-pyridylthio)toluamido)hexanoate.
[0092] In another embodiment, a fusion protein can be made with a
selected anti-CD83 antibody to allow a domain to be attached to one
or both of the polypeptides comprising the anti-CD83 antibody to be
bound to a solid substrate. For example,
glutathione-S-transferase/anti-CD83 fusion proteins can be linked
to another anti-CD83 preparation having glutathione attached
thereto or the glutathione-S-transferase/anti-CD83 fusion proteins
can be adsorbed onto a solid support having glutathione attached
thereto, such as glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtiter plate. In another
embodiment, DSP-crosslinked antibodies can be linked to protein G
agarose beads. Other techniques for immobilizing polypeptides on
solid support materials can also be used. For example, an anti-CD83
antibody can be immobilized utilizing conjugation of biotin and
streptavidin. Biotinylated anti-CD83 polypeptides can be prepared
from biotin-NHS(N-hydroxy-succinimide) using techniques known in
the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized with a streptavidin-linked antiCD83 antibody
preparation, streptavidin-coated beads or another solid support
material.
[0093] Therefore, in one embodiment, the invention provides
antibodies capable of reducing CD83 activity and decreasing an
immune response in a mammal. Such antibodies can be multimerized
antibodies. These antibodies may be used as CD83 inhibitory agents
in the methods of the invention as described herein. In another
embodiment, the antibodies of the invention can activate CD83
activity. Such activating antibodies may be used as CD83
stimulatory agents.
[0094] All antibody molecules belong to a family of plasma proteins
called immunoglobulins, whose basic building block, the
immunoglobulin fold or domain, is used in various forms in many
molecules of the immune system and other biological recognition
systems. A typical immunoglobulin has four polypeptide chains,
containing an antigen binding region known as a variable region and
a non-varying region known as the constant region.
[0095] Native antibodies and immunoglobulins are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (VH) followed by a number of constant domains. Each light
chain has a variable domain at one end (VL) and a constant domain
at its other end. The constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light and heavy chain variable domains
(Clothia et al., J. Mol. Biol. 186, 651-66, 1985); Novotny and
Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596(1985).
[0096] Depending on the amino acid sequences of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are at least five (5) major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG-1,
IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chains constant
domains that correspond to the different classes of immunoglobulins
are called alpha (a), delta (d), epsilon (e), gamma (?) and mu
(.mu.), respectively. The light chains of antibodies can be
assigned to one of two clearly distinct types, called kappa (?) and
lambda (?), based on the amino sequences of their constant domain.
The subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0097] The term "variable" in the context of variable domain of
antibodies, refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies.
The variable domains are for binding and determine the specificity
of each particular antibody for its particular antigen. However,
the variability is not evenly distributed through the variable
domains of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) also known as
hypervariable regions both in the light chain and the heavy chain
variable domains.
[0098] The more highly conserved portions of variable domains are
called the framework (FR). The variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies. The constant domains are not involved directly in
binding an antibody to an antigen, but exhibit various effector
function, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0099] An antibody that is contemplated for use in the present
invention thus can be in any of a variety of forms, including a
whole immunoglobulin, an antibody fragment such as Fv, Fab, and
similar fragments, a single chain antibody that includes the
variable domain complementarity determining regions (CDR), and the
like forms, all of which fall under the broad term "antibody," as
used herein. Moreover, the multimerized antibodies of the invention
can be an aggregation or multimerization of whole immunoglobulins.
Alternatively, the multimerized antibodies of the invention can be
an aggregation or multimerization of antibody fragments such as Fv,
Fab, single chain antibodies that include the variable domain
complementarity determining regions (CDR), CDRs and the like. Such
intact antibodies or antibody fragments can be multimerized by any
convenient means, including covalent linkage or non-covalent
association.
[0100] The present invention contemplates the use of any
specificity of an antibody, polyclonal or monoclonal, and is not
limited to antibodies that recognize and immunoreact with a
specific epitope. In preferred embodiments, in the context of both
the therapeutic and screening methods described below, an antibody
or fragment thereof is used that is immunospecific for an
extracellular portion of the CD83 protein.
[0101] The term "antibody fragment" refers to a portion of a
full-length antibody, generally the antigen binding or variable
region. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2 and Fv fragments. Papain digestion of antibodies
produces two identical antigen binding fragments, called the Fab
fragment, each with a single antigen binding site, and a residual
"Fc" fragment, so-called for its ability to crystallize readily.
Pepsin treatment yields an F(ab').sub.2 fragment that has two
antigen binding fragments, which are capable of cross-linking
antigen, and a residual other fragment (which is termed pFc').
Additional fragments can include diabodies, linear antibodies,
single-chain antibody molecules, and multispecific antibodies
formed from antibody fragments. As used herein, "functional
fragment" with respect to antibodies, refers to Fv, F(ab) and
F(ab').sub.2 fragments.
[0102] Antibody fragments retain some ability to selectively bind
with its antigen or receptor and are defined as follows:
[0103] (1) Fab is the fragment that contains a monovalent
antigen-binding fragment of an antibody molecule. A Fab fragment
can be produced by digestion of whole antibody with the enzyme
papain to yield an intact light chain and a portion of one heavy
chain.
[0104] (2) Fab' is the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain. Two Fab' fragments are obtained per antibody molecule.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH 1 domain
including one or more cysteines from the antibody hinge region.
[0105] (3) (Fab').sub.2 is the fragment of an antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction. F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds.
[0106] (4) Fv is the minimum antibody fragment that contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in a
tight, non-covalent association (VH-V L dimer). It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen binding site on the surface of the VH-V L
dimer. Collectively, the six CDRs confer antigen binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0107] (5) Single chain antibody ("SCA"), defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain molecule.
Such single chain antibodies are also referred to as "single-chain
Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide
further comprises a polypeptide linker between the VH and VL
domains that enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).
[0108] The term "diabodies" refers to a small antibody fragments
with two antigen-binding sites, which fragments comprise a heavy
chain variable domain (VH) connected to a light chain variable
domain (VL) in the same polypeptide chain (VH-VL). By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90: 6444-6448 (1993).
[0109] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, for example, Green, et al.,
Production of Polyclonal Antisera, in: Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press); Coligan, et al.,
Production of Polyclonal Antisera in Rabbits, Rats Mice and
Hamsters, in: Current Protocols in Immunology, section 2.4.1
(1992), which are hereby incorporated by reference.
[0110] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature,
256:495 (1975); Coligan, et al., sections 2.5.1-2.6.7; and Harlow,
et al., in: Antibodies: A Laboratory Manual, page 726 (Cold Spring
Harbor Pub. (1988)), which are hereby incorporated by reference.
Methods of in vitro and in vivo manipulation of monoclonal
antibodies are also available to those skilled in the art. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler and Milstein, Nature 256, 495 (1975), or they
may be made by recombinant methods, for example, as described in
U.S. Pat. No. 4,816,567. The monoclonal antibodies for use with the
present invention may also be isolated from antibody libraries
using the techniques described in Clackson et al. Nature 352:
624-628 (1991), as well as in Marks et al., J. Mol. Biol. 222:
581-597 (1991).
[0111] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography. See, e.g., Coligan, et al., sections
2.7.1-2.7.12 and sections 2.9.1-2.9.3; Bames, et al., Purification
of Immunoglobulin G (IgG), in: Methods in Molecular Biology, Vol.
10, pages 79-104 (Humana Press (1992).
[0112] Another method for generating antibodies involves a Selected
Lymphocyte Antibody Method (SLAM). The SLAM technology permits the
generation, isolation and manipulation of monoclonal antibodies
without the process of hybridoma generation. The methodology
principally involves the growth of antibody forming cells, the
physical selection of specifically selected antibody forming cells,
the isolation of the genes encoding the antibody and the subsequent
cloning and expression of those genes.
[0113] More specifically, an animal is immunized with a source of
specific antigen. The animal can be a rabbit, mouse, rat, or any
other convenient animal. This immunization may consist of purified
protein, in either native or recombinant form, peptides, DNA
encoding the protein of interest or cells expressing the protein of
interest. After a suitable period, during which antibodies can be
detected in the serum of the animal (usually weeks to months),
blood, spleen or other tissues are harvested from the animal.
Lymphocytes are isolated from the blood and cultured under specific
conditions to generate antibody-forming cells, with antibody being
secreted into the culture medium. These cells are detected by any
of several means (complement mediated lysis of antigen-bearing
cells, fluorescence detection or other) and then isolated using
micromanipulation technology. The individual antibody forming cells
are then processed for eventual single cell PCR to obtain the
expressed Heavy and Light chain genes that encode the specific
antibody. Once obtained and sequenced, these genes are cloned into
an appropriate expression vector and recombinant, monoclonal
antibody produced in a heterologous cell system. These antibodies
are then purified via standard methodologies such as the use of
protein A affinity columns. These types of methods are further
described in Babcook, et al., Proc. Natl. Acad. Sci. (USA) 93:
7843-7848 (1996); U.S. Pat. No. 5,627,052; and PCT WO 92/02551 by
Schrader.
[0114] Another method involves humanizing a monoclonal antibody by
recombinant means to generate antibodies containing human specific
and recognizable sequences. See, for review, Holmes, et al., J.
Immunol., 158:2192-2201 (1997) and Vaswani, et al., Annals Allergy,
Asthma & Immunol., 81:105-115 (1998). The term "monoclonal
antibody" as used herein refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
additional to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the antibody is obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method.
[0115] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567); Morrison et
al. Proc. Natl. Acad. Sci. 81, 6851-6855 (1984).
[0116] Methods of making antibody fragments are also known in the
art (see for example, Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York, (1988),
incorporated herein by reference). Antibody fragments of the
present invention can be prepared by proteolytic hydrolysis of the
antibody or by expression in E. Coli of DNA encoding the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies conventional methods. For example, antibody
fragments can be produced by enzymatic cleavage of antibodies with
pepsin to provide a 5S fragment denoted F(ab').sub.2. This fragment
can be further cleaved using a thiol reducing agent, and optionally
a blocking group for the sulfhydryl groups resulting from cleavage
of disulfide linkages, to produce 3.5S Fab=monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, in U.S. Pat. No. 4,036,945 and
No. 4,331,647, and references contained therein. These patents are
hereby incorporated in their entireties by reference.
[0117] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody. For
example, Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent or the variable chains can be
linked by an intermolecular disulfide bond or cross-linked by
chemicals such as glutaraldehyde. Preferably, the Fv fragments
comprise VH and VL chains connected by a peptide linker. These
single-chain antigen binding proteins (sFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow, et al., Methods: a
Companion to Methods in Enzmmology, Vol. 2, page 97 (1991); Bird,
et al., Science 242:423-426 (1988); Ladner, et al, U.S. Pat. No.
4,946,778; and Pack, et al., Bio/Technology 11:1271-77 (1993).
[0118] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick, et al., Methods: a Companion to
Methods in Enzymology, Vol. 2, page 106 (1991).
[0119] The invention further contemplates human and humanized forms
of non-human (e.g. murine) antibodies. Such humanized antibodies
can be chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a nonhuman
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity.
[0120] In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and optimize antibody performance. In general, humanized antibodies
can comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the Fv regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see: Jones et al., Nature 321, 522-525 (1986); Reichmann
et al., Nature 332, 323-329 (1988); Presta, Curr. Op. Struct. Biol.
2, 593-596 (1992); Holmes, et al., J. Immunol., 158:2192-2201
(1997) and Vaswani, et al., Annals Allergy, Asthma & Immunol.,
81:105-115 (1998); U.S. Pat. Nos. 4,816,567 and 6,331,415;
PCT/GB84/00094; PCT/US86/02269; PCT/US89/00077; PCT/US88/02514; and
WO91/09967, each of which is incorporated herein by reference in
its entirety.
[0121] The invention also provides methods of mutating antibodies
to optimize their affinity, selectivity, binding strength or other
desirable property. A mutant antibody refers to an amino acid
sequence variant of an antibody. In general, one or more of the
amino acid residues in the mutant antibody is different from what
is present in the reference antibody. Such mutant antibodies
necessarily have less than 100% sequence identity or similarity
with the reference amino acid sequence. In general, mutant
antibodies have at least 75% amino acid sequence identity or
similarity with the amino acid sequence of either the heavy or
light chain variable domain of the reference antibody. Preferably,
mutant antibodies have at least 80%, more preferably at least 85%,
even more preferably at least 90%, and most preferably at least 95%
amino acid sequence identity or similarity with the amino acid
sequence of either the heavy or light chain variable domain of the
reference antibody.
[0122] The antibodies of the invention are isolated antibodies. An
isolated antibody is one that has been identified and separated
and/or recovered from a component of the environment in which it
was produced. Contaminant components of its production environment
are materials that would interfere with diagnostic or therapeutic
uses for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. The term "isolated
antibody" also includes antibodies within recombinant cells because
at least one component of the antibody's natural environment will
not be present. Ordinarily, however, isolated antibody will be
prepared by at least one purification step.
[0123] If desired, the antibodies of the invention can be purified
by any available procedure. For example, the antibodies can be
affinity purified by binding an antibody preparation to a solid
support to which the antigen used to raise the antibodies is bound.
After washing off contaminants, the antibody can be eluted by known
procedures. Those of skill in the art will know of various
techniques common in the immunology arts for purification and/or
concentration of polyclonal antibodies, as well as monoclonal
antibodies (see for example, Coligan, et al., Unit 9, Current
Protocols in Immunology, Wiley Interscience, 1991, incorporated by
reference).
[0124] In preferred embodiments, the antibody will be purified as
measurable by at least three different methods: 1) to greater than
95% by weight of antibody as determined by the Lowry method, and
most preferably more than 99% by weight; 2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator; or 3) to homogeneity
by SDS-PAGE under reducing or non-reducing conditions using
Coomasie blue or, preferably, silver stain.
[0125] The invention also provides antibodies that can bind to CD83
polypeptides. Sequences of complementarity determining regions
(CDRs) or hypervariable regions from light and heavy chains of
these anti-CD83 antibodies are provided. For example, a heavy chain
variable region having a CDR1 sequence of SYDMT (SEQ ID NO:23),
SYDMS (SEQ ID NO:24), DYDLS (SEQ ID NO:25) or SYDMS (SEQ ID NO:26)
can be used in an antibody, multimerized antibody or other single-
or multi-valent binding moiety to bind to CD83 gene products and/or
modulate the immune response. In other embodiments, a heavy chain
variable region having a CDR2 sequence of YASGSTYY (SEQ ID NO:27),
SSSGTTYY (SEQ ID NO:28), YASGSTYY (SEQ ID NO:29), AIDGNPYY (SEQ ID
NO:30) or STAYNSHY (SEQ ID NO:31) can be used in an antibody,
multimerized antibody or other single- or multi-valent binding
moiety to bind to CD83 gene products or modulate the immune system.
In further embodiments of the invention, a heavy chain variable
region having a CDR3 sequence of EHAGYSGDTGH (SEQ ID NO:32),
EGAGVSMT (SEQ ID NO:33), EDAGFSNA (SEQ ID NO:34), GAGD (SEQ ID
NO:35) or GGSWLD (SEQ ID NO:36) can be used in an antibody,
multimerized antibody or other single- or multi-valent binding
moiety to bind to CD83 gene products or modulate the immune
system.
[0126] Moreover, a light chain variable region having a CDR1
sequence of RCAYD (SEQ ID NO:37), RCADVV (SEQ ID NO:38), or RCALV
(SEQ ID NO:39) can be used in an antibody, multimerized antibody or
other single- or multi-valent binding moiety to bind to CD83 gene
products or modulate the immune system. In other embodiments, a
light chain variable region having a CDR2 sequence of QSISTY (SEQ
ID NO:40), QSVSSY (SEQ ID NO:41), ESISNY (SEQ ID NO:42), KNVYNNNW
(SEQ ID NO:43), or QSVYDNDE (SEQ ID NO:98) can be used in an
antibody, multimerized antibody or other single- or multi-valent
binding moiety to bind to CD83 gene products or modulate the immune
system. In further embodiments, a light chain variable region
having a CDR3 sequence of QQGYTHSNVDNV (SEQ ID NO:44), QQGYSISDIDNA
(SEQ ID NO:45), QCTSGGKFISDGAA (SEQ ID NO:46), AGDYSSSSDNG (SEQ ID
NO:47), or QATHYSSDWLTY (SEQ ID NO:48) can be used in an antibody,
multimerized antibody or other single- or multi-valent binding
moiety to bind to CD83 gene products.
[0127] Light and heavy chains that can bind CD83 polypeptides are
also provided by the invention. For example, in one embodiment, the
invention provides a 20D04 light chain that can bind to CD83
polypeptides. The amino acid sequence for this 20D04 light chain is
provided below (SEQ ID NO:11).
11 1 MDMRAPTQLL GLLLLWLPGA RCADVVMTQT PASVSAAVGG 41 TVTINCQASE
SISNYLSWYQ QKPGQPPKLL IYRTSTLASG 81 VSSRFKGSGS GTEYTLTISG
VQCDDVATYY CQCTSGGKFI 121 SDGAAFGGGT EVVVKGDPVA PTVLLFPPSS
DEVATGTVTI 161 VCVANKYFPD VTVTWEVDGT TQTTGIENSK TPQNSADCTY 201
NLSSTLTLTS TQYNSHKEYT CKVTQGTTSV VQSFSRKNC
[0128] A nucleic acid sequence for this 20D04 anti-CD83 light chain
is provided below (SEQ ID NO:12).
12 1 ATGGACATGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC AGATGTGCCG ATGTCGTGAT 81 GACCCAGACT CCAGCCTCCG
TGTCTGCAGC TGTGGGAGGC 121 ACAGTCACCA TCAATTGCCA GGCCAGTGAA
AGCATTAGCA 161 ACTACTTATC CTGGTATCAG CAGAAACCAG GGCAGCCTCC 201
CAAGCTCCTG ATCTACAGGA CATCCACTCT GGCATCTGGG 241 GTCTCATCGC
GGTTCAAAGG CAGTGGATCT GGGACAGAGT 281 ACACTCTCAC CATCAGCGGC
GTGCAGTGTG ACGATGTTGC 321 CACTTACTAC TGTCAATGCA CTTCTGGTGG
GAAGTTCATT 361 AGTGATGGTG CTGCTTTCGG CGGAGGGACC GAGGTGGTGG 401
TCAAAGGTGA TCCAGTTGCA CCTACTGTCC TCCTCTTCCC 441 ACCATCTAGC
GATGAGGTGG CAACTGGAAC AGTCACCATC 481 GTGTGTGTGG CGAATAAATA
CTTTCCCGAT GTCACCGTCA 521 CCTGGGAGGT GGATGGCACC ACCCAAACAA
CTGGCATCGA 561 GAACAGTAAA ACACCGCAGA ATTCTGCAGA TTGTACCTAC 601
AACCTCAGCA GCACTCTGAC ACTGACCAGC ACACAGTACA 641 ACAGCCACAA
AGAGTACACC TGCAAGGTGA CCCAGGGCAC 681 GACCTCAGTC GTCCAGAGCT
TCAGTAGGAA GAACTGTTAA
[0129] In another embodiment, the invention provides a 20D04 heavy
chain that can bind to CD83 polypeptides. The amino acid sequence
for this 20D04 heavy chain is provided below (SEQ ID NO:13).
13 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGFSLSNN
AINWVRQAPG KGLEWIGYIW SGGLTYYANW 81 AEGRFTISKT STTVDLKMTS
PTIEDTATYF CARGINNSAL 121 WGPGTLVTVS SGQPKAPSVF PLAPCCGDTP
SSTVTLGCLV 161 KGYLPEPVTV TWNSGTLTNG VRTFPSVRQS SGLYSLSSVV 201
SVTSSSQPVT CNVAHPATNT KVDKTVAPST CSKPTCPPPE 241 LLGGPSVFIF
PPKPKDTLMI SRTPEVTCVV VDVSQDDPEV 281 QFTWYINNEQ VRTARPPLRE
QQFNSTIRVV STLPIAHQDW 321 LRGKEFKCKV HNKALPAPIE KTISKARGQP
LEPKVYTMGP 361 PREELSSRSV SLTCMINGFY PSDISVEWEK NGKAEDNYKT 401
TPAVLDSDGS YFLYNKLSVP TSEWQRGDVF TCSVMHEALH 441 NHYTQKSISR SPGK
[0130] A nucleic acid sequence for this 20D04 anti-CD83 heavy chain
is provided below (SEQ ID NO:14).
14 1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACCGTCTCTG GATTCTCCCT CAGTAACAAT
GCAATAAACT 161 GGGTCCGCCA GGCTCCAGGG AAGGGGCTAG AGTGGATCGG 201
ATACATTTGG AGTGGTGGGC TTACATACTA CGCGAACTGG 241 GCGGAAGGCC
GATTCACCAT CTCCAAAACC TCGACTACGG 281 TGGATCTGAA GATGACCAGT
CCGACAATCG AGGACACGGC 321 CACCTATTTC TGTGCCAGAG GGATTAATAA
CTCCGCTTTG 361 TGGGGCCCAG GCACCCTGGT CACCGTCTCC TCAGGGCAAC 401
CTAAGGCTCC ATCAGTCTTC CCACTGGCCC CCTGCTGCGG 441 GGACACACCC
TCTAGCACGG TGACCTTGGG CTGCCTGGTC 481 AAAGGCTACC TCCCGGAGCC
AGTGACCGTG ACCTGGAACT 521 CGGGCACCCT CACCAATGGG GTACGCACCT
TCCCGTCCGT 561 CCGGCAGTCC TCAGGCCTCT ACTCGCTGAG CAGCGTGGTG 601
AGCGTGACCT CAAGCAGCCA GCCCGTCACC TGCAACGTGG 641 CCCACCCAGC
CACCAACACC AAAGTGGACA AGACCGTTGC 681 GCCCTCGACA TGCAGCAAGC
CCACGTGCCC ACCCCCTGAA 721 CTCCTGGGGG GACCGTCTGT CTTCATCTTC
CCCCCAAAAC 761 CCAAGGACAC CCTCATGATC TCACGCACCC CCGAGGTCAC 801
ATGCGTGGTG GTGGACGTGA GCCAGGATGA CCCCGAGGTG 841 CAGTTCACAT
GGTACATAAA CAACGAGCAG GTGCGCACCG 881 CCCGGCCGCC GCTACGGGAG
CAGCAGTTCA ACAGCACGAT 921 CCGCGTGGTC AGCACCCTCC CCATCGCGCA
CCAGGACTGG 961 CTGAGGGGCA AGGAGTTCAA GTGCAAAGTC CACAACAAGG 1001
CACTCCCGGC CCCCATCGAG AAAACCATCT CCAAAGCCAG 1041 AGGGCAGCCC
CTGGAGCCGA AGGTCTACAC CATGGGCCCT 1081 CCCCGGGAGG AGCTGAGCAG
CAGGTCGGTC AGCCTGACCT 1121 GCATGATCAA CGGCTTCTAC CCTTCCGACA
TCTCGGTGGA 1161 GTGGGAGAAG AACGGGAAGG CAGAGGACAA CTACAAGACC 1201
ACGCCGGCCG TGCTGGACAG CGACGGCTCC TACTTCCTCT 1241 ACAACAAGCT
CTCAGTGCCC ACGAGTGAGT GGCAGCGGGG 1281 CGACGTCTTC ACCTGCTCCG
TGATGCACGA GGCCTTGCAC 1321 AACCACTACA CGCAGAAGTC CATCTCCCGC
TCTCCGGGTA 1361 AA
[0131] In another embodiment, the invention provides a 111 G05
light chain that can bind to CD83 polypeptides. The amino acid
sequence for this 11 G05 light chain is provided below (SEQ ID
NO:15).
15 1 MDTRAPTQLL GLLLLWLPGA RCADVVMTQT PASVSAAVGG 41 TVTINCQSSK
NVYNNNWLSW FQQKPGQPPK LLIYYASTLA 81 SGVPSRFRGS GSGTQFTLTI
SDVQCDDAAT YYCAGDYSSS 121 SDNGFGGGTE VVVKGDPVAP TVLLFPPSSD
EVATGTVTIV 161 CVANKYFPDV TVTWEVDGTT QTTGIENSKT PQNSADCTYN 201
LSSTLTLTST QYNSHKEYTC KVTQGTTSVV QSFSRKNC
[0132] A nucleic acid sequence for this 11G05 anti-CD83 light chain
is provided below (SEQ ID NO:16).
16 1 ATGGACACCA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC AGATGTGCCG ACGTCGTGAT 81 GACCCAGACT CCAGCCTCCG
TGTCTGCAGC TGTGGGAGGC 121 ACAGTCACCA TCAATTGCCA GTCCAGTAAG
AATGTTTATA 161 ATAACAACTG GTTATCCTGG TTTCAGCAGA AACCAGGGCA 201
GCCTCCCAAG CTCCTGATCT ATTATGCATC CACTCTGGCA 241 TCTGGGGTCC
CATCGCGGTT CAGAGGCAGT GGATCTGGGA 281 CACAGTTCAC TCTCACCATT
AGCGACGTGC AGTGTGACGA 321 TGCTGCCACT TACTACTGTG CAGGCGATTA
TAGTAGTAGT 361 AGTGATAATG GTTTCGGCGG AGGGACCGAG GTGGTGGTCA 401
AAGGTGATCC AGTTGCACCT ACTGTCCTCC TCTTCCCACC 441 ATCTAGCGAT
GAGGTGGCAA CTGGAACAGT CACCATCGTG 481 TGTGTGGCGA ATAAATACTT
TCCCGATGTC ACCGTCACCT 521 GGGAGGTGGA TGGCACCACC CAAACAACTG
GCATCGAGAA 561 CAGTAAAACA CCGCAGAATT CTGCAGATTG TACCTACAAC 601
CTCAGCAGCA CTCTGACACT GACCAGCACA CAGTACAACA 641 GCCACAAAGA
GTACACCTGC AAGGTGACCC AGGGCACGAC 681 CTCAGTCGTC CAGAGCTTCA
GTAGGAAGAA CTGTTAA
[0133] In another embodiment, the invention provides a 111 G05
heavy chain that can bind to CD83 polypeptides. The amino acid
sequence for this 11G05 heavy chain is provided below (SEQ ID
NO:17).
17 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGFTISDY
DLSWVRQAPG EGLKYIGFIA IDGNPYYATW 81 AKGRFTISKT STTVDLKITA
PTTEDTATYF CARGAGDLWG 121 PGTLVTVSSG QPKAPSVFPL APCCGDTPSS
TVTLGCLVKG 161 YLPEPVTVTW NSGTLTNGVR TFPSVRQSSG LYSLSSVVSV 201
TSSSQPVTCN VAHPATNTKV DKTVAPSTCS KPTCPPPELL 241 GGPSVFIFPP
KPKDTLMISR TPEVTCVVVD VSQDDPEVQF 281 TWYINNEQVR TARPPLREQQ
FNSTIRVVST LPIAHQDWLR 321 GKEFKCKVHN KALPAPIEKT ISKARGQPLE
PKVYTMGPPR 361 EELSSRSVSL TCMINGFYPS DISVEWEKNG KAEDNYKTTP 401
AVLDSDGSYF LYNKLSVPTS EWQRGDVFTC SVMHEALHNH 441 YTQKSISRSP GK
[0134] A nucleic acid sequence for this 11 G05 anti-CD83 heavy
chain is provided below (SEQ ID NO:18).
18 1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGTCTCTG GATTCACCAT CAGTGACTAC
GACTTGAGCT 161 GGGTCCGCCA GGCTCCAGGG GAGGGGCTGA AATACATCGG 201
ATTCATTGCT ATTGATGGTA ACCCATACTA CGCGACCTGG 241 GCAAAAGGCC
GATTCACCAT CTCCAAAACC TCGACCACGG 281 TGGATCTGAA AATCACCGCT
CCGACAACCG AAGACACGGC 321 CACGTATTTC TGTGCCAGAG GGGCAGGGGA
CCTCTGGGGC 361 CCAGGGACCC TCGTCACCGT CTCTTCAGGG CAACCTAAGG 401
CTCCATCAGT CTTCCCACTG GCCCCCTGCT GCGGGGACAC 441 ACCCTCTAGC
ACGGTGACCT TGGGCTGCCT GGTCAAAGGC 481 TACCTCCCGG AGCCAGTGAC
CGTGACCTGG AACTCGGGCA 521 CCCTCACCAA TGGGGTACGC ACCTTCCCGT
CCGTCCGGCA 561 GTCCTCAGGC CTCTACTCGC TGAGCAGCGT GGTGAGCGTG 601
ACCTCAAGCA GCCAGCCCGT CACCTGCAAC GTGGCCCACC 641 CAGCCACCAA
CACCAAAGTG GACAAGACCG TTGCGCCCTC 681 GACATGCAGC AAGCCCACGT
GCCCACCCCC TGAACTCCTG 721 GGGGGACCGT CTGTCTTCAT CTTCCCCCCA
AAACCCAAGG 761 ACACCCTCAT GATCTCACGC ACCCCCGAGG TCACATGCGT 801
GGTGGTGGAC GTGAGCCAGG ATGACCCCGA GGTGCAGTTC 841 ACATGGTACA
TAAACAACGA GCAGGTGCGC ACCGCCCGGC 881 CGCCGCTACG GGAGCAGCAG
TTCAACAGCA CGATCCGCGT 921 GGTCAGCACC CTCCCCATCG CGCACCAGGA
CTGGCTGAGG 961 GGCAAGGAGT TCAAGTGCAA AGTCCACAAC AAGGCACTCC 1001
CGGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAGAGGGCA 1041 GCCCCTGGAG
CCGAAGGTCT ACACCATGGG CCCTCCCCGG 1081 GAGGAGCTGA GCAGCAGGTC
GGTCAGCCTG ACCTGCATGA 1120 TCAACGGCTT CTACCCTTCC GACATCTCGG
TGGAGTGGGA 1161 GAAGAACGGG AAGGCAGAGG ACAACTACAA GACCACGCCG 1201
GCCGTGCTGG ACAGCGACGG CTCCTACTTC CTCTACAACA 1241 AGCTCTCAGT
GCCCACGAGT GAGTGGCAGC GGGGCGACGT 1281 CTTCACCTGC TCCGTGATGC
ACGAGGCCTT GCACAACCAC 1321 TACACGCAGA AGTCCATCTC CCGCTCTCCG
GGTAAA
[0135] In another embodiment, the invention provides a 14C 12 light
chain that can bind to CD83 polypeptides. The amino acid sequence
for this 14C12 light chain is provided below (SEQ ID NO:19).
19 1 MDXRAPTQLL GLLLLWLPGA RCALVMTQTP ASVSAAVGGT 41 VTINCQSSQS
VYDNDELSWY QQKPGQPPKL LIYLASKLAS 81 GVPSRFKGSG SGTQFALTIS
GVQCDDAATY YCQATHYSSD 121 WYLTFGGGTE VVVKGDPVAP TVLLFPPSSD
EVATGTVTIV 161 CVANKYFPDV TVTWEVDGTT QTTGIENSKT PQNSADCTYN 201
LSSTLTLTST QYNSHKEYTC KVTQGTTSVV QSFSRKNC
[0136] A nucleic acid sequence for this 14C12 anti-CD83 light chain
is provided below (SEQ ID NO:20).
20 1 ATGGACATRA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC AGATGTGCCC TTGTGATGAC 81 CCAGACTCCA GCCTCCGTGT
CTGCAGCTGT GGGAGGCACA 121 GTCACCATCA ATTGCCAGTC CAGTCAGAGT
GTTTATGATA 161 ACGACGAATT ATCCTGGTAT CAGCAGAAAC CAGGGCAGCC 201
TCCCAAGCTC CTGATCTATC TGGCATCCAA GTTGGCATCT 241 GGGGTCCCAT
CCCGATTCAA AGGCAGTGGA TCTGGGACAC 281 AGTTCGCTCT CACCATCAGC
GGCGTGCAGT GTGACGATGC 321 TGCCACTTAC TACTGTCAAG CCACTCATTA
TAGTAGTGAT 361 TGGTATCTTA CTTTCGGCGG AGGGACCGAG GTGGTGGTCA 401
AAGGTGATCC AGTTGCACCT ACTGTCCTCC TCTTCCCACC 441 ATCTAGCGAT
GAGGTGGCAA CTGGAACAGT CACCATCGTG 481 TGTGTGGCGA ATAAATACTT
TCCCGATGTC ACCGTCACCT 521 GGGAGGTGGA TGGCACCACC CAAACAACTG
GCATCGAGAA 561 CAGTAAAACA CCGCAGAATT CTGCAGATTG TACCTACAAC 601
CTCAGCAGCA CTCTGACACT GACCAGCACA CAGTACAACA 641 GCCACAAAGA
GTACACCTGC AAGGTGACCC AGGGCACGAC 681 CTCAGTCGTC CAGAGCTTCA
GTAGGAAGAA CTGTTAA
[0137] In another embodiment, the invention provides a 14C 12 heavy
chain that can bind to CD83 polypeptides. The amino acid sequence
for this 14C 12 heavy chain is provided below (SEQ ID NO:21).
21 1 METGLRWLLL VAVLKGVHCQ SVEESGGRLV TPGTPLTLTC 41 TASGFSRSSY
DMSWVRQAPG KGLEWVGVIS TAYNSHYASW 81 AKGRFTISRT STTVDLKMTS
LTTEDTATYF CARGGSWLDL 121 WGQGTLVTVS SGQPKAPSVF PLAPCCGDTP
SSTVTLGCLV 161 KGYLPEPVTV TWNSGTLTNG VRTFPSVRQS SGLYSLSSVV 201
SVTSSSQPVT CNVAHPATNT KVDKTVAPST CSKPTCPPPE 241 LLGGPSVFIF
PPKPKDTLMI SRTPEVTCVV VDVSQDDPEV 281 QFTWYINNEQ VRTARPPLRE
QQFNSTIRVV STLPIAHQDW 321 LRGKEFKCKV HNKALPAPIE KTISKARGQP
LEPKVYTMGP 361 PREELSSRSV SLTCMINGFY PSDISVEWEK NGKAEDNYKT 401
TPAVLDSDGS YFLYNKLSVP TSEWQRGDVF TCSVMHEALH 441 NHYTQKSISR SPGK
[0138] A nucleic acid sequence for this 14C12 anti-CD83 heavy chain
is provided below (SEQ ID NO:22).
22 1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCACTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGCCTCTG GATTCTCCCG CAGCAGCTAC
GACATGAGCT 161 GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATGGGTCGG 201
AGTCATTAGT ACTGCTTATA ACTCACACTA CGCGAGCTGG 241 GCAAAAGGCC
GATTCACCAT CTCCAGAACC TCGACCACGG 281 TGGATCTGAA AATGACCAGT
CTGACAACCG AAGACACGGC 321 CACCTATTTC TGTGCCAGAG GGGGTAGTTG
GTTGGATCTC 361 TGGGGCCAGG GCACCCTGGT CACCGTCTCC TCAGGGCAAC 401
CTAAGGCTCC ATCAGTCTTC CCACTGGCCC CCTGCTGCGG 441 GGACACACCC
TCTAGCACGG TGACCTTGGG CTGCCTGGTC 481 AAAGGCTACC TCCCGGAGCC
AGTGACCGTG ACCTGGAACT 521 CGGGCACCCT CACCAATGGG GTACGCACCT
TCCCGTCCGT 561 CCGGCAGTCC TCAGGCCTCT ACTCGCTGAG CAGCGTGGTG 601
AGCGTGACCT CAAGCAGCCA GCCCGTCACC TGCAACGTGG 641 CCCACCCAGC
CACCAACACC AAAGTGGACA AGACCGTTGC 681 GCCCTCGACA TGCAGCAAGC
CCACGTGCCC ACCCCCTGAA 721 CTCCTGGGGG GACCGTCTGT CTTCATCTTC
CCCCCAAAAC 761 CCAAGGACAC CCTCATGATC TCACGCACCC CCGAGGTCAC 801
ATGCGTGGTG GTGGACGTGA GCCAGGATGA CCCCGAGGTG 841 CAGTTCACAT
GGTACATAAA CAACGAGCAG GTGCGCACCG 881 CCCGGCCGCC GCTACGGGAG
CAGCAGTTCA ACAGCACGAT 921 CCGCGTGGTC AGCACCCTCC CCATCGCGCA
CCAGGACTGG 961 CTGAGGGGCA AGGAGTTCAA GTGCAAAGTC CACAACAAGG 1001
CACTCCCGGC CCCCATCGAG AAAACCATCT CCAAAGCCAG 1041 AGGGCAGCCC
CTGGAGCCGA AGGTCTACAC CATGGGCCCT 1081 CCCCGGGAGG AGCTGAGCAG
CAGGTCGGTC AGCCTGACCT 1121 GCATGATCAA CGGCTTCTAC CCTTCCGACA
TCTCGGTGGA 1161 GTGGGAGAAG AACGGGAAGG CAGAGGACAA CTACAAGACC 1200
ACGCCGGCCG TGCTGGACAG CGACGGCTCC TACTTCCTCT 1241 ACAACAAGCT
CTCAGTGCCC ACGAGTGAGT GGCAGCGGGG 1281 CGACGTCTTC ACCTGCTCCG
TGATGCACGA GGCCTTGCAC 1321 AACCACTACA CGCAGAAGTC CATCTCCCGC
TCTCCGGGTA 1361 AA
[0139] In another embodiment, the invention provides a M83 020B08L
light chain that can bind to CD83 polypeptides. The amino acid
sequence for this M83 020B08L light chain is provided below (SEQ ID
NO:58).
23 1 MDMRAPTQLL GLLLLWLPGA RCAYDMTQTP ASVEVAVGGT 41 VTIKCQASQS
ISTYLDWYQQ KPGQPPKLLI YDASDLASGV 81 PSRFKGSGSG TQFTLTTSDL
ECADAATYYC QQGYTHSNVD 121 NVFGGGTEVV VKGDPVAPTV LLFPPSSDEV
ATGTVTIVCV 161 ANKYFPDVTV TWEVDGTTQT TGIENSKTPQ NSADCTYNLS 201
STLTLTSTQY NSHKEYTCKV TQGTTSVVQS FSRKNC
[0140] A nucleic acid sequence for this M83 020B08L anti-CD83 light
chain is provided below (SEQ ID NO:59).
24 1 ATGGACATGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC AGATGTGCCT ATGATATGAC 81 CCAGACTCCA GCCTCTGTGG
AGGTAGCTGT GGGAGGCACA 121 GTCACCATCA AGTGCCAGGC CAGTCAGAGC
ATTAGTACCT 161 ACTTAGACTG GTATCAGCAG AAACCAGGGC AGCCTCCCAA 201
GCTCCTGATC TATGATGCAT CCGATCTGGC ATCTGGGGTC 241 CCATCGCGGT
TCAAAGGCAG TGGATCTGGG ACACAGTTCA 281 CTCTCACCAT CAGCGACCTG
GAGTGTGCCG ATGCTGCCAC 321 TTACTACTGT CAACAGGGTT ATACACATAG
TAATGTTGAT 361 AATGTTTTCG GCGGAGGGAC CGAGGTGGTG GTCAAAGGTG 401
ATCCAGTTGC ACCTACTGTC CTCCTCTTCC CACCATCTAG 441 CGATGAGGTG
GCAACTGGAA CAGTCACCAT CGTGTGTGTG 481 GCGAATAAAT ACTTTCCCGA
TGTCACCGTC ACCTGGGAGG 521 TGGATGGCAC CACCCAAACA ACTGGCATCG
AGAACAGTAA 561 AACACCGCAG AATTCTGCAG ATTGTACCTA CAACCTCAGC 601
AGCACTCTGA CACTGACCAG CACACAGTAC AACAGCCACA 641 AAGAGTACAC
CTGCAAGGTG ACCCAGGGCA CGACCTCAGT 681 CGTCCAGAGC TTCAGTAGGA
AGAACTGTTA A
[0141] In another embodiment, the invention provides a M83 020B08H
heavy chain that can bind to CD83 polypeptides. The amino acid
sequence for this M83 020B08H heavy chain is provided below (SEQ ID
NO:60).
25 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGFSLSSY
DMTWVRQAPG KGLEWIGIIY ASGTTYYANW 81 AKGRFTISKT STTVDLKVTS
PTIGDTATYF CAREGAGVSM 121 TLWGPGTLVT VSSGQPKAPS VFPLAPCCGD
TPSSTVTLGC 161 LVKGYLPEPV TVTWNSGTLT NGVRTFPSVR QSSGLYSLSS 201
VVSVTSSSQP VTCNVAHPAT NTKVDKTVAP STCSKPTCPP 241 PELLGGPSVF
IFPPKPKDTL MISRTPEVTC VVVDVSQDDP 281 EVQFTWYINN EQVRTARPPL
REQQFNSTIR VVSTLPIAHQ 321 DWLRGKEFKC KVHNKALPAP IEKTISKARG
QPLEPKVYTM 361 GPPREELSSR SVSLTCMING FYPSDISVEW EKNGKAEDNY 401
KTTPAVLDSD GSYFLYNKLS VPTSEWQRGD VFTCSVMHEA 441 LHNHYTQKSI
SRSPGK
[0142] A nucleic acid sequence for this M83 020B08H anti-CD83 heavy
chain is provided below (SEQ ID NO:61).
26 1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGTCTCTG GATTCTCCCT CAGCAGCTAC
GACATGACCT 161 GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATGGATCGG 201
AATCATTTAT GCTAGTGGTA CCACATACTA CGCGAACTGG 241 GCGAAAGGCC
GATTCACCAT CTCCAAAACC TCGACCACGG 281 TGGATCTGAA AGTCACCAGT
CCGACAATCG GGGACACGGC 321 CACCTATTTC TGTGCCAGAG AGGGGGCTGG
TGTTAGTATG 361 ACCTTGTGGG GCCCAGGCAC CCTGGTCACC GTCTCCTCAG 401
GGCAACCTAA GGCTCCATCA GTCTTCCCAC TGGCCCCCTG 441 CTGCGGGGAC
ACACCCTCTA GCACGGTGAC CTTGGGCTGC 481 CTGGTCAAAG GCTACCTCCC
GGAGCCAGTG ACCGTGACCT 521 GGAACTCGGG CACCCTCACC AATGGGGTAC
GCACCTTCCC 561 GTCCGTCCGG CAGTCCTCAG GCCTCTACTC GCTGAGCAGC 601
GTGGTGAGCG TGACCTCAAG CAGCCAGCCC GTCACCTGCA 641 ACGTGGCCCA
CCCAGCCACC AACACCAAAG TGGACAAGAC 681 CGTTGCGCCC TCGACATGCA
GCAAGCCCAC GTGCCCACCC 721 CCTGAACTCC TGGGGGGACC GTCTGTCTTC
ATCTTCCCCC 761 CAAAACCCAA GGACACCCTC ATGATCTCAC GCACCCCCGA 801
GGTCACATGC GTGGTGGTGG ACGTGAGCCA GGATGACCCC 841 GAGGTGCAGT
TCACATGGTA CATAAACAAC GAGCAGGTGC 881 GCACCGCCCG GCCGCCGCTA
CGGGAGCAGC AGTTCAACAG 921 CACGATCCGC GTGGTCAGCA CCCTCCCCAT
CGCGCACCAG 961 GACTGGCTGA GGGGCAAGGA GTTCAAGTGC AAAGTCCACA 1001
ACAAGGCACT CCCGGCCCCC ATCGAGAAAA CCATCTCCAA 1041 AGCCAGAGGG
CAGCCCCTGG AGCCGAAGGT CTACACCATG 1081 GGCCCTCCCC GGGAGGAGCT
GAGCAGCAGG TCGGTCAGCC 1121 TGACCTGCAT GATCAACGGC TTCTACCCTT
CCGACATCTC 1161 GGTGGAGTGG GAGAAGAACG GGAAGGCAGA GGACAACTAC 1201
AAGACCACGC CGGCCGTGCT GGACAGCGAC GGCTCCTACT 1241 TCCTCTACAA
CAAGCTCTCA GTGCCCACGA GTGAGTGGCA 1281 GCGGGGCGAC GTCTTCACCT
GCTCCGTGAT GCACGAGGCC 1321 TTGCACAACC ACTACACGCA GAAGTCCATC
TCCCGCTCTC 1361 CGGGTAAA
[0143] In another embodiment, the invention provides a M83 006G05L
light chain that can bind to CD83 polypeptides. The amino acid
sequence for this M83 006G05L light chain is provided below (SEQ ID
NO:62).
27 1 MDMRAPTQLL GLLLLWLPGA RCAYDMTQTP ASVEVAVGGT 41 VAIKCQASQS
VSSYLAWYQQ KPGQPPKPLI YEASMLAAGV 81 SSRFKGSGSG TDFTLTISDL
ECDDAATYYC QQGYSISDID 121 NAFGGGTEVV VKGDPVAPTV LLFPPSSDEV
ATGTVTIVCV 161 ANKYFPDVTV TWEVDGTTQT TGIENSKTPQ NSADCTYNLS 201
STLTLTSTQY NSHKEYTCKV TQGTTSVVQS FSRKNC
[0144] A nucleic acid sequence for M83 006G05L anti-CD83 light
chain is provided below (SEQ ID NO:63).
28 1 ATGGACATGA GGGCCCCCAC TCAACTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC AGATGTGCCT ATGATATGAC 81 CCAGACTCCA GCCTCTGTGG
AGGTAGCTGT GGGAGGCACA 121 GTCGCCATCA AGTGCCAGGC CAGTCAGAGC
GTTAGTAGTT 161 ACTTAGCCTG GTATCAGCAG AAACCAGGGC AGCCTCCCAA 201
GCCCCTGATC TACGAAGCAT CCATGCTGGC GGCTGGGGTC 241 TCATCGCGGT
TCAAAGGCAG TGGATCTGGG ACAGACTTCA 281 CTCTCACCAT CAGCGACCTG
GAGTGTGACG ATGCTGCCAC 321 TTACTATTGT CAACAGGGTT ATTCTATCAG
TGATATTGAT 361 AATGCTTTCG GCGGAGGGAC CGAGGTGGTG GTCAAAGGTG 401
ATCCAGTTGC ACCTACTGTC CTCCTCTTCC CACCATCTAG 441 CGATGAGGTG
GCAACTGGAA CAGTCACCAT CGTGTGTGTG 481 GCGAATAAAT ACTTTCCCGA
TGTCACCGTC ACCTGGGAGG 521 TGGATGGCAC CACCCAAACA ACTGGCATCG
AGAACAGTAA 561 AACACCGCAG AATTCTGCAG ATTGTACCTA CAACCTCAGC 601
AGCACTCTGA CACTGACCAG CACACAGTAC AACAGCCACA 641 AAGAGTACAC
CTGCAAGGTG ACCCAGGGCA CGACCTCAGT 681 CGTCCAGAGC TTCAGTAGGA
AGAACTGTTA A
[0145] In another embodiment, the invention provides a M83 006G05L
heavy chain that can bind to CD83 polypeptides. The amino acid
sequence for this M83 006G05L heavy chain is provided below (SEQ ID
NO:64).
29 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV SPGTPLTLTC 41 TASGFSLSSY
DMSWVRQAPG KGLEYIGIIS SSGSTYYASW 81 AKGRFTISKT STTVDLEVTS
LTTEDTATYF CSREHAGYSG 121 DTGHLWGPGT LVTVSSGQPK APSVFPLAPC
CGDTPSSTVT 161 LGCLVKGYLP EPVTVTWNSG TLTNGVRTFP SVRQSSGLYS 201
LSSVVSVTSS SQPVTCNVAH PATNTKVDKT VAPSTCSKPT 241 CPPPELLGGP
SVFIFPPKPK DTLMISRTPE VTCVVVDVSQ 281 DDPEVQFTWY INNEQVRTAR
PPLREQQFNS TIRVVSTLPI 321 AHQDWLRGKE FKCKVHNKAL PAPIEKTISK
ARGQPLEPKV 361 YTMGPPREEL SSRSVSLTCM INGFYPSDIS VEWEKNGKAE 401
DNYKTTPAVL DSDGSYFLYN KLSVPTSEWQ RGDVFTCSVM 441 HEALHNHYTQ
KSISRSPGK
[0146] A nucleic acid sequence for this M83 006G05L anti-CD83 heavy
chain is provided below (SEQ ID NO:65).
30 1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC TCGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGCCTCTG GATTCTCCCT CAGTAGCTAC
GACATGAGCT 161 GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATACATCGG 201
AATCATTAGT AGTAGTGGTA GCACATACTA CGCGAGCTGG 241 GCGAAAGGCC
GATTCACCAT CTCCAAAACC TCGACCACGG 281 TGGATCTGGA AGTGACCAGT
CTGACAACCG AGGACACGGC 321 CACCTATTTC TGTAGTAGAG AACATGCTGG
TTATAGTGGT 361 GATACGGGTC ACTTGTGGGG CCCAGGCACC CTGGTCACCG 401
TCTCCTCGGG GCAACCTAAG GCTCCATCAG TCTTCCCACT 441 GGCCCCCTGC
TGCGGGGACA CACCCTCTAG CACGGTGACC 481 TTGGGCTGCC TGGTCAAAGG
CTACCTCCCG GAGCCAGTGA 521 CCGTGACCTG GAACTCGGGC ACCCTCACCA
ATGGGGTACG 561 CACCTTCCCG TCCGTCCGGC AGTCCTCAGG CCTCTACTCG 601
CTGAGCAGCG TGGTGAGCGT GACCTCAAGC AGCCAGCCCG 641 TCACCTGCAA
CGTGGCCCAC CCAGCCACCA ACACCAAAGT 681 GGACAAGACC GTTGCGCCCT
CGACATGCAG CAAGCCCACG 721 TGCCCACCCC CTGAACTCCT GGGGGGACCG
TCTGTCTTCA 761 TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCACG 801
CACCCCCGAG GTCACATGCG TGGTGGTGGA CGTGAGCCAG 841 GATGACCCCG
AGGTGCAGTT CACATGGTAC ATAAACAACG 881 AGCAGGTGCG CACCGCCCGG
CCGCCGCTAC GGGAGCAGCA 921 GTTCAACAGC ACGATCCGCG TGGTCAGCAC
CCTCCCCATC 961 GCGCACCAGG ACTGGCTGAG GGGCAAGGAG TTCAAGTGCA 1001
AAGTCCACAA CAAGGCACTC CCGGCCCCCA TCGAGAAAAC 1041 CATCTCCAAA
GCCAGAGGGC AGCCCCTGGA GCCGAAGGTC 1081 TACACCATGG GCCCTCCCCG
GGAGGAGCTG AGCAGCAGGT 1121 CGGTCAGCCT GACCTGCATG ATCAACGGCT
TCTACCCTTC 1162 CGACATCTCG GTGGAGTGGG AGAAGAACGG GAAGGCAGAG 1201
GACAACTACA AGACCACGCC GGCCGTGCTG GACAGCGACG 1241 GCTCCTACTT
CCTCTACAAC AAGCTCTCAG TGCCCACGAG 1281 TGAGTGGCAG CGGGGCGACG
TCTTCACCTG CTCCGTGATG 1321 CACGAGGCCT TGCACAACCA CTACACGCAG
AAGTCCATCT 1361 CCCGCTCTCC GGGTAAA
[0147] In another embodiment, the invention provides a 96G08 light
chain that can bind to CD83 polypeptides and can inhibit
proliferation of human peripheral blood mononuclear cells (PBMCs).
The amino acid sequence for this 96G08 light chain is provided
below (SEQ ID NO:70).
31 1 MDTRAPTQLL GLLLLWLPGA TFAQVLTQTA SPVSAPVGGT 41 VTINCQSSQS
VYNNDFLSWY QQKPGQPPKL LIYYASTLAS 81 GVPSRFKGSG SGTQFTLTIS
DLECDDAATY YCTGTYGNSA 121 WYEDAFGGGT EVVVKRTPVA PTVLLFPPSS
AELATGTATI 161 VCVANKYFPD GTVTWKVDGI TQSSGINNSR TPQNSADCTY 201
NLSSTLTLSS DEYNSHDEYT CQVAQDSGSP VVQSFSRKSC
[0148] The amino acid sequence for this 96G08 light chain with the
CDR regions identified by underlining is provided below (SEQ ID
NO:70).
32 1 MDTRAPTQLL GLLLLWLPGA TFAQVLTQTA SPVSAPVGGT 41 VTINCQSSQS
VYNNDFLSWY QQKPGQPPKL LIYYASTLAS 81 GVPSRFKGSG SGTQFTLTIS
DLECDDAATY YCTGTYGNSA 121 WYEDAFGGGT EVVVKRTPVA PTVLLFPPSS
AELATGTATI 161 VCVANKYFPD GTVTWKVDGI TQSSGINNSR TPQNSADCTY 201
NLSSTLTLSS DEYNSHDEYT CQVAQDSGSP VVQSFSRKSC
[0149] Hence, the CDR regions in the 96G08 light chain include
amino acid sequences QSSQSVYNNDFLS (SEQ ID NO:71), YASTLAS (SEQ ID
NO:72), and TGTYGNSAWYEDA (SEQ ID NO:73).
[0150] A nucleic acid sequence for this 96G08 anti-CD83 light chain
is provided below (SEQ ID NO:74).
33 1 ATGGACACGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC ACATTTGCGC AAGTGCTGAC 81 CCAGACTGCA TCGCCCGTGT
CTGCACCTGT GGGAGGCACA 121 GTCACCATCA ATTGCCAGTC CAGTCAGAGT
GTTTATAATA 161 ACGACTTCTT ATCCTGGTAT CAGCAGAAAC CAGGGCAGCC 201
TCCCAAACTC CTGATCTATT ATGCATCCAC TCTGGCATCT 241 GGGGTCCCAT
CCCGGTTCAA AGGCAGTGGA TCTGGGACAC 281 AGTTCACTCT CACCATCAGC
GACCTGGAGT GTGACGATGC 321 TGCCACTTAC TACTGTACAG GCACTTATGG
TAATAGTGCT 361 TGGTACGAGG ATGCTTTCGG CGGAGGGACC GAGGTGGTGG 401
TCAAACGTAC GCCAGTTGCA CCTACTGTCC TCCTCTTCCC 441 ACCATCTAGC
GCTGAGCTGG CAACTGGAAC AGCCACCATC 481 GTGTGCGTGG CGAATAAATA
CTTTCCCGAT GGCACCGTCA 521 CCTGGAAGGT GGATGGCATC ACCCAAAGCA
GCGGCATCAA 561 TAACAGTAGA ACACCGCAGA ATTCTGCAGA TTGTACCTAC 601
AACCTCAGCA GTACTCTGAC ACTGAGCAGC GACGAGTACA 641 ACAGCCACGA
CGAGTACACC TGCCAGGTGG CCCAGGACTC 681 AGGCTCACCG GTCGTCCAGA
GCTTCAGTAG GAAGAGCTGT 721 TAG
[0151] This nucleic acid sequence for the 96G08 anti-CD83 light
chain with CDR regions identified by underlining is provided below
(SEQ ID NO:99).
34 1 ATGGACACGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC ACATTTGCGC AAGTGCTGAC 81 CCAGACTGCA TCGCCCGTGT
CTGCACCTGT GGGAGGCACA 121 GTCACCATCA ATTGCCAGTC CAGTCACAGT
GTTTATAATA 161 ACGACTTCTT ATCCTGGTAT CAGCAGAAAC CAGGGCAGCC 201
TCCCAAACTC CTGATCTATT ATGCATCCAC TCTGGCATCT 241 GGGGTCCCAT
CCCGGTTCAA AGGCAGTGGA TCTGGGACAC 281 AGTTCACTCT CACCATCAGC
GACCTGGAGT GTGACGATGC 321 GCCACTTACT ACTGTACAGG CACTTATGGT
AATAGTGCTT 361 GGTACGAGGA TGCTTTCGGC GGAGGGACCG AGGTGGTGGT 401
CAAACGTACG CCAGTTGCAC CTACTGTCCT CCTCTTCCCA 441 CCATCTAGCG
CTGAGCTGGC AACTGGAACA GCCACCATCG 481 TGTGCGTGGC GAATAAATAC
TTTCCCGATG GCACCGTCAC 521 CTGGAAGGTG GATGGCATCA CCCAAAGCAG
CGGCATCAAT 561 AACAGTAGAA CACCGCAGAA TTCTGCAGAT TGTACCTACA 601
ACCTCAGCAG TACTCTGACA CTGAGCAGCG ACGAGTACAA 641 CAGCCACGAC
GAGTACACCT GCCAGGTGGC CCAGGACTCA 681 GGCTCACCGG TCGTCCAGAG
CTTCAGTAGG AAGAGCTGTT
[0152] Hence, the CDR regions in the 96G08 light chain include
nucleic acid sequences CAGTCCAGTCAGAGTGTTTATAATA (SEQ ID NO:75),
ATGCATCCACTCTGGCATCT (SEQ ID NO:76), and ACAGGCACTTATGGT AATAGTGCTT
(SEQ ID NO:77).
[0153] In another embodiment, the invention provides a 96G08 heavy
chain that can bind to CD83 polypeptides and can inhibit
proliferation of human peripheral blood mononuclear cells (PBMCs).
The amino acid sequence for this 96G08 heavy chain is provided
below (SEQ ID NO:78).
35 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGIDLSSD
GISWVRQAPG KGLEWIGIIS SGGNTYYASW 81 AKGRFTISRT STTVDLKMTS
LTTEDTATYF CARVVGGTYS 121 IWGQGTLVTV SSASTKGPSV YPLAPGSAAQ
TNSMVTLGCL 161 VKGYFPEPVT VTWNSGSLSS GVHTFPAVLQ SDLYTLSSSV 201
TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV PRDCGCKPCI 241 CTVPEVSSVF
IFPPKPKDVL TITLTPKVTC VVVDISKDDP 281 EVQFSWFVDD VEVHTAQTQP
REEQFNSTFR SVSELPIMHQ 321 DWLNGKEFKC RVNSAAFPAP IEKTISKTKG
RPKAPQVYTI 361 PPPKEQMAKD KVSLTCMITD FFPEDITVEW QWNGQPAENY 401
KNTQPIMDTD GSYFVYSKLN VQKSNWEAGN TFTCSVLHEG 441 LHNHHTEKSL
SHSPGK
[0154] The amino acid sequence for the 96G08 heavy chain with the
CDR regions identified by underlining is provided below (SEQ ID
NO:78).
36 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGIDLSSD
GISWVRQAPG KGLEWIGIIS SGGNTYYASW 81 AKGRFTISRT STTVDLKMTS
LTTEDTATYF CARVVGGTYS 121 IWGQGTLVTV SSASTKGPSV YPLAPGSAAQ
TNSMVTLGCL 161 VKGYFPEPVT VTWNSGSLSS GVHTFPAVLQ SDLYTLSSSV 201
TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV PRDCGCKPCI 241 CTVPEVSSVF
IFPPKPKDVL TITLTPKVTC VVVDISKDDP 281 EVQFSWFVDD VEVHTAQTQP
REEQFNSTFR SVSELPIMHQ 321 DWLNGKEFKC RVNSAAFPAP IEKTISKTKG
RPKAPQVYTI 361 PPPKEQMAKD KVSLTCMITD FFPEDITVEW QWNGQPAENY 401
KNTQPIMDTD GSYFVYSKLN VQKSNWEAGN TFTCSVLHEG 441 LHNHHTEKSL
SHSPGK
[0155] Hence, the CDR regions in the 96G08 heavy chain include
amino acid sequences SDGIS (SEQ ID NO:79), IISSGGNTYYASWAKG (SEQ ID
NO:80) and VVGGTYSI (SEQ ID NO:81).
[0156] A nucleic acid sequence for the 96G08 anti-CD83 heavy chain
is provided below (SEQ ID NO:82).
37 1 ATGGAGACTG GGCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACACCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGTGTCTG GAATCGACCT CAGTAGCGAT
GGAATAAGCT 161 GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATGGATCGG 201
AATCATTAGT AGTGGTGGTA ACACATACTA CGCGAGCTGG 241 GCAAAAGGCC
GATTCACCAT CTCCAGAACC TCGACCACGG 281 TGGATCTGAA GATGACCAGT
CTGACAACCG AGGACACGGC 321 CACCTATTTC TGTGCCAGAG TTGTTGGTGG
TACTTATAGC 361 ATCTGGGGCC AGGGCACCCT CGTCACCGTC TCGAGCGCTT 401
CTACAAAGGG CCCATCTGTC TATCCACTGG CCCCTGGATC 441 TGCTGCCCAA
ACTAACTCCA TGGTGACCCT GGGATGCCTG 481 GTCAAGGGCT ATTTCCCTGA
GCCAGTGACA GTGACCTGGA 521 ACTCTGGATC CCTGTCCAGC GGTGTGCACA
CCTTCCCAGC 561 TGTCCTGCAG TCTGACCTCT ACACTCTGAG CAGCTCAGTG 601
ACTGTCCCCT CCAGCACCTG GCCCAGCGAG ACCGTCACCT 641 GCAACGTTGC
CCACCCGGCC AGCAGCACCA AGGTGGACAA 681 GAAAATTGTG CCCAGGGATT
GTGGTTGTAA GCCTTGCATA 721 TGTACAGTCC CAGAAGTATC ATCTGTCTTC
ATCTTCCCCC 761 CAAAGCCCAA GGATGTGCTC ACCATTACTC TGACTCCTAA 801
GGTCACGTGT GTTGTGGTAG ACATCAGCAA GGATGATCCC 841 GAGGTCCAGT
TCAGCTGGTT TGTAGATGAT GTGGAGGTGC 881 ACACAGCTCA GACGCAACCC
CGGGAGGAGC AGTTCAACAG 921 CACTTTCCGC TCAGTCAGTG AACTTCCCAT
CATGCACCAG 961 GACTGGCTCA ATGGCAAGGA GTTCAAATGC AGGGTCAACA 1001
GTGCAGCTTT CCCTGCCCCC ATCGAGAAAA CCATCTCCAA 1041 AACCAAAGGC
AGACCGAAGG CTCCACAGGT GTACACCATT 1081 CCACCTCCCA AGGAGCAGAT
GGCCAAGGAT AAAGTCAGTC 1121 TGACCTGCAT GATAACAGAC TTCTTCCCTG
AAGACATTAC 1161 TGTGGAGTGG CAGTGGAATG GGCAGCCAGC GGAGAACTAC 1201
AAGAACACTC AGCCCATCAT GGACACAGAT GGCTCTTACT 1241 TCGTCTACAG
CAAGCTCAAT GTGCAGAAGA GCAACTGGGA 1281 GGCAGGAAAT ACTTTCACCT
GCTCTGTGTT ACATGAGGGC 1321 CTGCACAACC ACCATACTGA GAAGAGCCTC
TCCCACTCTC 1361 CTGGTAAATG A
[0157] The nucleic acid sequence for the 96G08 anti-CD83 heavy
chain with CDR regions identified by underlining is provided below
is provided below (SEQ ID NO:82).
38 1 ATGGAGACTG GGCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACACCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGTGTCTG GAATCGACCT CAGTAGCGAT
GGAATAAGCT 161 GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATGGATCGG 201
AATCATTAGT AGTGGTGGTA ACACATACTA CGCGAGCTGG 241 GCAAAAGGCC
GATTCACCAT CTCCAGAACC TCGACCACGG 281 TGGATCTGAA GATGACCAGT
CTGACAACCG AGGACACGGC 321 CACCTATTTC TGTGCCAGAG TTGTTGGTGG
TACTTATAGC 361 ATCTGGGGCC AGGGCACCCT CGTCACCGTC TCGAGCGCTT 401
CTACAAAGGG CCCATCTGTC TATCCACTGG CCCCTGGATC 441 TGCTCCCCAA
ACTAACTCCA TGGTGACCCT GGGATGCCTG 481 GTCAAGGGCT ATTTCCCTGA
GCCAGTGACA GTGACCTGGA 521 ACTCTGGATC CCTGTCCAGC GGTGTGCACA
CCTTCCCAGC 561 TGTCCTGCAG TCTGACCTCT ACACTCTGAG CAGCTCAGTG 601
ACTGTCCCCT CCAGCACCTG GCCCAGCGAG ACCGTCACCT 641 GCAACGTTGC
CCACCCGGCC AGCAGCACCA AGGTGGACAA 681 GAAAATTGTG CCCAGGGATT
GTGGTTGTAA GCCTTGCATA 721 TGTACAGTCC CAGAAGTATC ATCTGTCTTC
ATCTTCCCCC 761 CAAAGCCCAA GGATGTGCTC ACCATTACTC TGACTCCTAA 801
GGTCACGTGT GTTGTGGTAG ACATCAGCAA GGATGATCCC 841 GAGGTCCAGT
TCAGCTGGTT TGTAGATGAT GTGGAGGTGC 881 ACACAGCTCA GACGCAACCC
CGGGAGGAGC AGTTCAACAG 921 CACTTTCCGC TCAGTCAGTG AACTTCCCAT
CATGCACCAG 961 GACTGGCTCA ATGGCAAGGA GTTCAAATGC AGGGTCAACA 1001
GTGCAGCTTT CCCTGCCCCC ATCGAGAAAA CCATCTCCAA 1041 AACCAAAGGC
AGACCGAAGG CTCCACAGGT GTACACCATT 1081 CCACCTCCCA AGGAGCAGAT
GGCCAAGGAT AAAGTCAGTC 1121 TGACCTGCAT GATAACAGAC TTCTTCCCTG
AAGACATTAC 1161 TGTGGAGTGG CAGTGGAATG GGCAGCCAGC GGAGAACTAC 1201
AAGAACACTC AGCCCATCAT GGACACAGAT GGCTCTTACT 1241 TCGTCTACAG
CAAGCTCAAT GTGCAGAAGA GCAACTGGGA 1281 GGCAGGAAAT ACTTTCACCT
GCTCTGTGTT ACATGAGGGC 1321 CTGCACAACC ACCATACTGA GAAGAGCCTC
TCCCACTCTC 1361 CTGGTAAATG A
[0158] Hence, the CDR regions in the 96G08 anti-CD83 heavy chain
include AGCGATGGAATAAGC (SEQ ID NO:83), ATCATTAGTAGTGGTGGTA
ACACATACTACGCGAGCTGGGCAAAAGGC (SEQ ID NO:84), and G TTGTTGGTGG
TACTTATAGC ATC (SEQ ID NO:85).
[0159] In another embodiment, the invention provides a 95F04 light
chain that can bind to CD83 polypeptides and can inhibit
proliferation of human peripheral blood mononuclear cells (PBMCs).
The amino acid sequence for this 95F04 light chain is provided
below (SEQ ID NO:86).
39 1 MDTRAPTQLL GLLLLWLPGA TFAQAVVTQT TSPVSAPVGG 41 TVTINCQSSQ
SVYGNNELSW YQQKPGQPPK LLIYQASSLA 81 SGVPSRFKGS GSGTQFTLTI
SDLECDDAAT YYCLGEYSIS 121 ADNHFGGGTE VVVKRTPVAP TVLLFPPSSA
ELATGTATIV 161 CVANKYFPDG TVTWKVDGIT QSSGINNSRT PQNSADCTYN 201
LSSTLTLSSD EYNSHDEYTC QVAQDSGSPV VQSFSRKSC
[0160] The amino acid sequence for the 95F04 anti-CD83 light chain
with the CDR regions identified by underlining is provided below
(SEQ ID NO:86).
40 1 MDTRAPTQLL GLLLLWLPGA TFAQAVVTQT TSPVSAPVGG 41 TVTINCQSSQ
SVYGNNELSW YQQKPGQPPK LLIYQASSLA 81 SGVPSRFKGS GSGTQFTLTI
SDLECDDAAT YYCLGEYSIS 121 ADNHFGGGTE VVVKRTPVAP TVLLFPPSSA
ELATGTATIV 161 CVANKYFPDG TVTWKVDGIT QSSGINNSRT PQNSADCTYN 201
LSSTLTLSSD EYNSHDEYTC QVAQDSGSPV VQSFSRKSC
[0161] Hence, the CDR regions in the 95F04 anti-CD83 light chain
include amino acid sequences QSSQSVYGNNELS (SEQ ID NO:87), QASSLAS
(SEQ ID NO:88) and LGEYSISADNH (SEQ ID NO:89).
[0162] A nucleic acid sequence for this 95F04 anti-CD83 light chain
is provided below (SEQ ID NO:90).
41 1 ATGGACACGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC ACATTTGCCC AAGCCGTGGT 81 GACCCAGACT ACATCGCCCG
TGTCTGCACC TGTGGGAGGC 121 ACAGTCACCA TCAATTGCCA GTCCAGTCAG
AGTGTTTATG 161 GTAACAACGA ATTATCCTGG TATCAGCAGA AACCAGGGCA 201
GCCTCCCAAG CTCCTGATCT ACCAGGCATC CAGCCTGGCA 241 TCTGGGGTCC
CATCGCGGTT CAAAGGCAGT GGATCTGGGA 281 CACAGTTCAC TCTCACCATC
AGCGACCTGG AGTGTGACGA 321 TGCTGCCACT TACTACTGTC TAGGCGAATA
TAGCATTAGT 361 GCTGATAATC ATTTCGGCGG AGGGACCGAG GTGGTGGTCA 401
AACGTACGCC AGTTGCACCT ACTGTCCTCC TCTTCCCACC 441 ATCTAGCGCT
GAGCTGGCAA CTGGAACAGC CACCATCGTG 481 TGCGTGGCGA ATAAATACTT
TCCCGATGGC ACCGTCACCT 521 GGAAGGTGGA TGGCATCACC CAAAGCAGCG
GCATCAATAA 561 CAGTAGAACA CCGCAGAATT CTGCAGATTG TACCTACAAC 601
CTCAGCAGTA CTCTGACACT GAGCAGCGAC GAGTACAACA 641 GCCACGACGA
GTACACCTGC CAGGTGGCCC AGGACTCAGG 681 CTCACCGGTC GTCCAGAGCT
TCAGTAGGAA GAGCTGTTAG
[0163] The nucleic acid sequence for the 95F04 anti-CD83 light
chain with CDR regions identified by underlining is provided below
(SEQ ID NO:90).
42 1 ATGGACACGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC 41 TGCTCTGGCT
CCCAGGTGCC ACATTTGCCC AAGCCGTGGT 81 GACCCAGACT ACATCGCCCG
TGTCTGCACC TGTGGGAGGC 121 ACAGTCACCA TCAATTGCCA GTCCAGTCAG
AGTGTTTATG 161 GTAACAACGA ATTATCCTGG TATCAGCAGA AACCAGGGCA 201
GCCTCCCAAG CTCCTGATCT ACCAGGCATC CAGCCTGGCA 241 TCTGGGGTCC
CATCGCGGTT CAAAGGCAGT GGATCTGGGA 281 CACAGTTCAC TCTCACCATC
AGCGACCTGG AGTGTGACGA 321 TGCTGCCACT TACTACTGTC TAGGCGAATA
TAGCATTAGT 361 GCTGATAATC ATTTCGGCGG AGGGACCGAG GTGGTGGTCA 401
AACGTACGCC AGTTGCACCT ACTGTCCTCC TCTTCCCACC 441 ATCTAGCGCT
GAGCTGGCAA CTGGAACAGC CACCATCGTG 481 TGCGTGGCGA ATAAATACTT
TCCCGATGGC ACCGTCACCT 521 GGAAGGTGGA TGGCATCACC CAAAGCAGCG
GCATCAATAA 561 CAGTAGAACA CCGCAGAATT CTGCAGATTG TACCTACAAC 601
CTCAGCAGTA CTCTGACACT GAGCAGCGAC GAGTACAACA 641 GCCACGACGA
GTACACCTGC CAGGTGGCCC AGGACTCAGG 681 CTCACCGGTC GTCCAGAGCT
TCAGTAGGAA GAGCTGTTAG
[0164] In another embodiment, the invention provides a 95F04 heavy
chain that can bind to CD83 polypeptides and can inhibit
proliferation of human peripheral blood mononuclear cells (PBMCs).
The amino acid sequence for this 95F04 heavy chain is provided
below (SEQ ID NO:91).
43 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGIDLSSN
AMIWVRQAPR EGLEWIGAND SNSRTYYATW 81 AKGRFTISRT SSITVDLKIT
SPTTEDTATY FCARGDGGSS 121 DYTEMWGPGT LVTVSSASTK GPSVYPLAPG
SAAQTNSMVT 161 LGCLVKGYFP EPVTVTWNSG SLSSGVHTFP AVLQSDLYTL 201
SSSVTVPSST WPSETVTCNV AHPASSTKVD KKIVPRDCGC 241 KPCICTVPEV
SSVFIFPPKP KDVLTITLTP KVTCVVVDIS 281 KDDPEVQFSW FVDDVEVHTA
QTQPREEQFN STFRSVSELP 321 IMHQDWLNGK EFKCRVNSAA FPAPIEKTIS
KTKGRPKAPQ 361 VYTIPPPKEQ MAKDKVSLTC MITDFFPEDI TVEWQWNGQP 401
AENYKNTQPI MDTDGSYFVY SKLNVQKSNW EAGNTFTCSV 441 LHEGLHNHHT
EKSLSHSPGK
[0165] The amino acid sequence for the 95F04 anti-CD83 heavy chain
with the CDR regions identified by underlining is provided below
(SEQ ID NO:91).
44 1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC 41 TVSGIDLSSN
AMIWVRQAPR EGLEWIGAMD SNSRTYYATW 81 AKGRFTISRT SSITVDLKIT
SPTTEDTATY FCARGDGGSS 121 DYTEMWGPGT LVTVSSASTK GPSVYPLAPG
SAAQTNSMVT 161 LGCLVKGYFP EPVTVTWNSG SLSSGVHTFP AVLQSDLYTL 201
SSSVTVPSST WPSETVTCNV AHPASSTKVD KKIVPRDCGC 241 KPCICTVPEV
SSVFIFPPKP KDVLTITLTP KVTCVVVDIS 281 KDDPEVQFSW FVDDVEVHTA
QTQPREEQFN STFRSVSELP 321 IMHQDWLNGK EFKCRVNSAA FPAPIEKTIS
KTKGRPKAPQ 361 VYTIPPPKEQ MAKDKVSLTC MITDFFPEDI TVEWQWNGQP 401
AENYKNTQPI MDTDGSYFVY SKLNVQKSNW EAGNTFTCSV 441 LHEGLHNHHT
EKSLSHSPGK
[0166] Hence, the CDR regions in the 95F04 anti-CD83 heavy chain
include amino acid sequences SNAMI (SEQ ID NO:92), AMDSNSRTYYATWAKG
(SEQ ID NO:93), and GDGGSSDYTEM (SEQ ID NO:94).
[0167] A nucleic acid sequence for this 95F04 anti-CD83 heavy chain
is provided below (SEQ ID NO:95).
45 1 ATGGAGACTG GGCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGTCTCTG GAATCGACCT CAGTAGCAAT
GCAATGATCT 161 GGGTCCGCCA GGCTCCAAGG GAGGGGCTGG AATGGATCGG 201
AGCCATGGAT AGTAATAGTA GGACGTACTA CGCGACCTGG 241 GCGAAAGGCC
GATTCACCAT CTCCAGAACC TCGTCGATTA 281 CGGTGGATCT GAAAATCACC
AGTCCGACAA CCGAGGACAC 321 GGCCACCTAT TTCTGTGCCA GAGGGGATGG
TGGCAGTAGT 361 GATTATACAG AGATGTGGGG CCCAGGGACC CTCGTCACCG 401
TCTCGAGCGC TTCTACAAAG GGCCCATCTG TCTATCCACT 441 GGCCCCTGGA
TCTGCTGCCC AAACTAACTC CATGGTGACC 481 CTGGGATGCC TGGTCAAGGG
CTATTTCCCT GAGCCAGTGA 521 CAGTGACCTG GAACTCTGGA TCCCTGTCCA
GCGGTGTGCA 561 CACCTTCCCA GCTGTCCTGC AGTCTGACCT CTACACTCTG 601
AGCAGCTCAG TGACTGTCCC CTCCAGCACC TGGCCCAGCG 641 AGACCGTCAC
CTGCAACGTT GCCCACCCGG CCAGCAGCAC 681 CAAGGTGGAC AAGAAAATTG
TGCCCAGGGA TTGTGGTTGT 721 AAGCCTTGCA TATGTACAGT CCCAGAAGTA
TCATCTGTCT 761 TCATCTTCCC CCCAAAGCCC AAGGATGTGC TCACCATTAC 801
TCTGACTCCT AAGGTCACGT GTGTTGTGGT AGACATCAGC 841 AAGGATGATC
CCGAGGTCCA GTTCAGCTGG TTTGTAGATG 881 ATGTGGAGGT GCACACAGCT
CAGACGCAAC CCCGGGAGGA 921 GCAGTTCAAC AGCACTTTCC GCTCAGTCAG
TGAACTTCCC 961 ATCATGCACC AGGACTGGCT CAATGGCAAG GAGTTCAAAT 1001
GCAGGGTCAA CAGTGCAGCT TTCCCTGCCC CCATCGAGAA 1041 AACCATCTCC
AAAACCAAAG GCAGACCGAA GGCTCCACAG 1081 GTGTACACCA TTCCACCTCC
CAAGGAGCAG ATGGCCAAGG 1141 ATAAAGTCAG TCTGACCTGC ATGATAACAG
ACTTCTTCCC 1161 TGAAGACATT ACTGTGGAGT GGCAGTGGAA TGGGCAGCCA 1201
GCGGAGAACT ACAAGAACAC TCAGCCCATC ATGGACACAG 1241 ATGGCTCTTA
CTTCGTCTAC AGCAAGCTCA ATGTGCAGAA 1281 GAGCAACTGG GAGGCAGGAA
ATACTTTCAC CTGCTCTGTG 1321 TTACATGAGG GCCTGCACAA CCACCATACT
GAGAAGAGCC 1361 TCTCCCACTC TCCTGGTAAA TGA
[0168] A related nucleic acid sequence for the 95F04 anti-CD83
light chain is provided below (SEQ ID NO:96).
46 1 ATGGAGACTG GGCTGCGCTG GCTTCTCCTG GTCGCTGTGC 41 TCAAAGGTGT
CCAGTGTCAG TCGGTGGAGG AGTCCGGGGG 81 TCGCCTGGTC ACGCCTGGGA
CACCCCTGAC ACTCACCTGC 121 ACAGTCTCTG GAATCGACCT CAGTAGCAAT
GCAATGATCT 161 GGGTCCGCCA GGCTCCAAGG GAGGGGCTGG AATGGATCGG 201
AGCCATGGAT AGTAATAGTA GGACGTACTA CGCGACCTGG 241 GCGAAAGGCC
GATTCACCAT CTCCAGAACC TCGTCGATTA 281 CGGTGGATCT GAAAATCACC
AGTCCGACAA CCGAGGACAC 321 GGCCACCTAT TTCTGTGCCA GAGGGGATGG
TGGCAGTAGT 361 GATTATACAG AGATGTGGGG CCCAGGGACC CTCGTCACCG 401
TCTCGAGCGC TTCTACAAAG GGCCCATCTG TCTATCCACT 441 GGCCCCTGGA
TCTGCTGCCC AAACTAACTC CATGGTGACC 481 CTGGGATGCC TGGTCAAGGG
CTATTTCCCT GAGCCAGTGA 521 CAGTGACCTG GAACTCTGGA TCCCTGTCCA
GCGGTGTGCA 561 CACCTTCCCA GCTGTCCTGC AGTCTGACCT CTACACTCTG 601
AGCAGCTCAG TGACTGTCCC CTCCAGCACC TGGCCCAGCG 641 AGACCGTCAC
CTGCAACGTT GCCCACCCGG CCAGCAGCAC 681 CAAGGTGGAC AAGAAAATTG
TGCCCAGGGA TTGTGGTTGT 721 AAGCCTTGCA TATGTACAGT CCCAGAAGTA
TCATCTGTCT 761 TCATCTTCCC CCCAAAGCCC AAGGATGTGC TCACCATTAC 801
TCTGACTCCT AAGGTCACGT GTGTTGTGGT AGACATCAGC 841 AAGGATGATC
CCGAGGTCCA GTTCAGCTGG TTTGTAGATG 881 ATGTGGAGGT GCACACAGCT
CAGACGCAAC CCCGGGAGGA 921 GCAGTTCAAC AGCACTTTCC GCTCAGTCAG
TGAACTTCCC 961 ATCATGCACC AGGACTGGCT CAATGGCAAG GAGTTCAAAT 1001
GCAGGGTCAA CAGTGCAGCT TTCCCTGCCC CCATCGAGAA 1041 AACCATCTCC
AAAACCAAAG GCAGACCGAA GGCTCCACAG 1081 GTGTACACCA TTCCACCTCC
CAAGGAGCAG ATGGCCAAGG 1121 ATAAAGTCAG TCTGACCTGC ATGATAACAG
ACTTCTTCCC 1161 TGAAGACATT ACTGTGGAGT GGCAGTGGAA TGGGCAGCCA 1201
GCGGAGAACT ACAAGAACAC TCAGCCCATC ATGGACACAG 1241 ATGGCTCTTA
CTTCGTCTAC AGCAAGCTCA ATGTGCAGAA 1281 GAGCAACTGG GAGGCAGGAA
ATACTTTCAC CTGCTCTGTG 1321 TTACATGAGG GCCTGCACAA CCACCATACT
GAGAAGAGCC 1361 TCTCCCACTC TCCTGGTAAA TGA
[0169] CD83 Modulation of the Immune System
[0170] The invention also provides compositions and methods for
decreasing inappropriate immune responses in animals, including
humans. According to the invention, the CD83 gene has a profound
effect upon T cell activity. In particular, CD4+ T cells require
CD83-related functions. Without CD83, CD4+ T cell activation and/or
proliferation is impaired. The therapeutic manipulation of CD83 may
thus represent a mechanism for the specific regulation of T cell
function in the treatment of T cell mediated diseases, including
autoimmune disorders. For example, antibodies capable of blocking
CD83 function can be used as therapeutics in the treatment of
immune diseases.
[0171] In some embodiments, the CD83-related compositions and
methods of the invention can be used in the treatment of autoimmune
diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against "self tissues" and
that promote the production of cytokines and auto-antibodies
involved in the pathology of the diseases. Modulation of T cell
activity by modulating CD83 can have an effect on the course of the
autoimmune disease.
[0172] Non-limiting examples of autoimmune diseases and disorders
having an autoimmune component that may be treated according to the
invention include diabetes mellitus, arthritis (including
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), psoriasis, Sjogren's Syndrome, including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis.
[0173] As illustrated and provided herein, anti-CD83 antibodies can
inhibit T cell proliferation. The efficacy of anti-CD83-related
compositions for treating autoimmune diseases can be tested in the
animal models provided herein or other models of human diseases
(e.g., EAE as a model of multiple sclerosis and the NOD mice as a
model for diabetes). Such animal models include the mrl/lpr/lpr
mouse as a model for lupus erythematosus, murine collagen-induced
arthritis as a model for rheumatoid arthritis, and murine
experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 840-856). A
CD83-modulatory (e.g., inhibitory) agent of the invention is
administered to test animals and the course of the disease in the
test animals is then monitored by the standard methods for the
particular model being used. Effectiveness of the modulatory agent
is evidenced by amelioration of the disease condition in animals
treated with the agent as compared to untreated animals (or animals
treated with a control agent).
[0174] Similarly, the compositions and methods of the invention
that involve decreasing CD83 function can be used to decrease
transplant rejection and prolong survival of the tissue graft.
These methods can be used both in solid organ transplantation and
in bone marrow transplantation (e.g., to inhibit graft-versus-host
disease). These methods can involve either direct administration of
a CD83 inhibitory agent to the transplant recipient or ex vivo
treatment of cells obtained from the subject (e.g., T cells, Th1
cells, B cells, non-lymphoid cells) with an inhibitory agent
followed by re-administration of the cells to the subject.
[0175] According to the invention, any agent that can modulate CD83
or to further decrease T cell levels can also be used in the
compositions and methods of the invention. In some embodiments,
anti-CD83 antibodies of the invention are used to either activate
or inhibit CD83 activity.
[0176] Stimulating or Inhibiting CD83
[0177] According to the invention, any agent that can inhibit CD83
from performing its natural functions can be used in the
compositions and methods of the invention as a CD83 inhibitory
agent. Indicators that CD83 activity is inhibited include decreased
T cell counts, increased IL-4 cytokine levels, increased IL-10
levels, decreased IL-2 production, and decreased TNF levels
relative to uninhibited levels in wild type CD83 cells.
[0178] Examples of CD83 inhibitors include anti-CD83 antibodies,
CD83 anti-sense nucleic acids (e.g. nucleic acids that can
hybridize to CD83 nucleic acids), organic compounds, peptides and
agents that can mutate an endogenous CD83 gene.
[0179] In some embodiments, the CD83 stimulatory or inhibitory
agents are proteins, for example, CD83 gene products, anti-CD83
antibody preparations, CD83 inhibitors, peptides and protein
factors that can promote CD83 transcription or translation. In
other embodiments, the CD83 stimulatory or inhibitory agents are
peptides or organic molecules. Such proteins, organic molecules and
organic molecules can be prepared and/or purified as described
herein or by methods available in the art, and administered as
provided herein.
[0180] In other embodiments, the CD83 inhibitory agents can be
nucleic acids including recombinant expression vectors or
expression cassettes encoding CD83 anti-sense nucleic acid,
intracellular antibodies capable of binding to CD83 or dominant
negative CD83 inhibitors. Such nucleic acids can be operably linked
to a promoter that is functional in a mammalian cell, and then
introduced into cells of the subject mammal using methods known in
the art for introducing nucleic acid (e.g., DNA) into cells.
[0181] The "promoter functional in a mammalian cell" or "mammalian
promoter" is capable of directing transcription of a polypeptide
coding sequence operably linked to the promoter. The promoter
should generally be active in T cells and antigen presenting cells
and may be obtained from a gene that is expressed in T cells or
antigen presenting cells. However, it need not be a T cell-specific
or an antigen presenting cell specific-promoter. Instead, the
promoter may be selected from any mammalian or viral promoter that
can function in a T cell. Hence the promoter may be an actin
promoter, an immunoglobulin promoter, a heat-shock promoter, or a
viral promoter obtained from the genome of viruses such as
adenoviruses, retroviruses, lentiviruses, herpes viruses, including
but not limited to, polyoma virus, fowlpox virus, adenovirus 2,
bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV),
hepatitis-B virus, Simian Virus 40 (SV40), Epstein Barr virus
(EBV), feline immunodeficiency virus (FIV), and Sra, or are
respiratory synsitial viral promoters (RSV) or long terminal
repeats (LTRs) of a retrovirus, i.e., a Moloney Murine Leukemia
Virus (MoMuLv) (Cepko et al. (1984) Cell 37:1053-1062). The
promoter functional in a mammalian cell can be inducible or
constitutive.
[0182] Any cloning procedure used by one of skill in the art can be
employed to make the expression vectors or expression that comprise
a promoter operably linked to a CD83 nucleic acid, CD83
transcription factor or a nucleic acid encoding an anti-CD83
antibody. See, e.g., Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989;
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y., 2001.
[0183] After constructing an expression vector or an expression
cassette encoding CD83 transcription factors, CD83 anti-sense
nucleic acid, intracellular antibodies capable of binding to CD83
or dominant negative CD83 inhibitors, mammalian cells can be
transformed with the vector or cassette. Examples of such methods
include:
[0184] Direct Injection: Naked DNA can be introduced into cells in
vivo by directly injecting the DNA into the cells (see e.g., Acsadi
et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science
247:1465-1468). For example, a delivery apparatus (e.g., a "gene
gun") for injecting DNA into cells in vivo can be used. Such an
apparatus is commercially available (e.g., from BioRad).
[0185] Receptor-Mediated DNA Uptake: Naked DNA can also be
introduced into cells in vivo by complexing the DNA to a cation,
such as polylysine, which is coupled to a ligand for a cell-surface
receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol.
Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967;
and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to
the receptor facilitates uptake of the DNA by receptor-mediated
endocytosis. A DNA-ligand complex linked to adenovirus capsids that
naturally disrupt endosomes, thereby releasing material into the
cytoplasm can be used to avoid degradation of the complex by
intracellular lysosomes (see for example Curiel et al. (1991) Proc.
Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.
Acad. Sci. USA 90:2122-2126).
[0186] Retroviruses: Defective retroviruses are well characterized
for use in gene transfer for gene therapy purposes (for a review
see Miller, A. D. (1990) Blood 76:271). A recombinant retrovirus
can be constructed having nucleotide sequences of interest
incorporated into the retroviral genome. Additionally, portions of
the retroviral genome can be removed to render the retrovirus
replication defective. The replication defective retrovirus is then
packaged into virions that can be used to infect a target cell
through the use of a helper virus by standard techniques. Protocols
for producing recombinant retroviruses and for infecting cells in
vitro or in vivo with such viruses can be found in Current
Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene
Publishing Associates, (1989), Sections 9.10-9.14 and other
standard laboratory manuals. Examples of suitable retroviruses
include pLJ, pZIP, pWE and pEM which are available to those skilled
in the art. Examples of suitable packaging virus lines include ?
Crip, ? Cre, ? 2 and ? Am. Retroviruses have been used to introduce
a variety of genes into many different cell types, including
epithelial cells, endothelial cells, lymphocytes, myoblasts,
hepatocytes, bone marrow cells, in vitro and/or in vivo (see for
example Eglitis, et al. (1985) Science 230:1395-1398; Danos and
Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et
al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et
al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991)
Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991)
Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl.
Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy
3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci USA
89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S.
Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89/07136; PCT
Application WO 89/02468; PCT Application WO 89/05345; and PCT
Application WO 92/07573). Retroviral vectors require target cell
division in order for the retroviral genome (and foreign nucleic
acid inserted into it) to be integrated into the host genome to
stably introduce nucleic acid into the cell. Thus, it may be
necessary to stimulate replication of the target cell.
[0187] Adenoviruses: The genome of an adenovirus can be manipulated
such that it encodes and expresses a gene product of interest but
is inactivated in terms of its ability to replicate in a normal
lytic viral life cycle. See, for example, Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434;
and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral
vectors derived from the adenovirus strain Ad type 5 dl 324 or
other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are
available to those skilled in the art. Recombinant adenoviruses are
advantageous in that they do not require dividing cells to be
effective gene delivery vehicles and can be used to infect a wide
variety of cell types, including airway epithelium (Rosenfeld et
al. (1992) cited supra), endothelial cells (Lemarchand et al.
(1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz
and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and
muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA
89:2581-2584). Additionally, introduced adenoviral DNA (and foreign
DNA contained therein) is not integrated into the genome of a host
cell but remains episomal, thereby avoiding potential problems that
can occur as a result of insertional mutagenesis in situations
where introduced DNA becomes integrated into the host genome (e.g.,
retroviral DNA). Moreover, the carrying capacity of the adenoviral
genome for foreign DNA is large (up to 8 kilobases) relative to
other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand
and Graham (1986) J. Virol. 57:267). Most replication-defective
adenoviral vectors currently in use are deleted for all or parts of
the viral E1 and E3 genes but retain as much as 80% of the
adenoviral genetic material.
[0188] Adeno-Associated Viruses: Adeno-associated virus (AAV) is a
naturally occurring defective virus that requires another virus,
such as an adenovirus or a herpes virus, as a helper virus for
efficient replication and a productive life cycle. (For a review
see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)
158:97-129). It is also one of the few viruses that may integrate
its DNA into non-dividing cells, and exhibits a high frequency of
stable integration (see for example Flotte et al. (1992) Am. J.
Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J.
Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol.
62:1963-1973). Vectors containing as little as 300 base pairs of
AAV can be packaged and can integrate. Space for exogenous DNA is
limited to about 4.5 kb. An AAV vector such as that described in
Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to
introduce DNA into cells. A variety of nucleic acids have been
introduced into different cell types using AAV vectors (see for
example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA
81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;
Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.
(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol.
Chem. 268:3781-3790).
[0189] Transformed mammalian cells can then be identified and
administered to the mammal from whence they came to permit
expression of a CD83 transcription factor, CD83 anti-sense nucleic
acid, intracellular antibody capable of binding to CD83 proteins,
or dominant negative CD83 inhibitors. The efficacy of a particular
expression vector system and method of introducing nucleic acid
into a cell can be assessed by standard approaches routinely used
in the art. For example, DNA introduced into a cell can be detected
by a filter hybridization technique (e.g., Southern blotting). RNA
produced by transcription of an introduced DNA can be detected, for
example, by Northern blotting, RNase protection or reverse
transcriptase-polymerase chain reaction (RT-PCR). The CD83 gene
product can be detected by an appropriate assay, for example, by
immunological detection of a produced CD83 protein, such as with a
CD83-specific antibody.
[0190] Anti-sense Nucleic Acids
[0191] Anti-sense nucleic acids can be used to inhibit the function
of CD83. In general, the function of CD83 RNA is inhibited, for
example, by administering to a mammal a nucleic acid that can
inhibit the functioning of CD83 RNA. Nucleic acids that can inhibit
the function of a CD83 RNA can be generated from coding and
non-coding regions of the CD83 gene. However, nucleic acids that
can inhibit the function of a CD83 RNA are often selected to be
complementary to CD83 nucleic acids that are naturally expressed in
the mammalian cell to be treated with the methods of the invention.
In some embodiments, the nucleic acids that can inhibit CD83 RNA
functions are complementary to CD83 sequences found near the 5' end
of the CD83 coding region. For example, nucleic acids that can
inhibit the function of a CD83 RNA can be complementary to the 5'
region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID
NO:10.
[0192] A nucleic acid that can inhibit the functioning of a CD83
RNA need not be 100% complementary to SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5 or SEQ ID NO:10. Instead, some variability the sequence of
the nucleic acid that can inhibit the functioning of a CD83 RNA is
permitted. For example, a nucleic acid that can inhibit the
functioning of a CD83 RNA from a human can be complementary to a
nucleic acid encoding either a human or a mouse CD83 gene
product.
[0193] Moreover, nucleic acids that can hybridize under moderately
or highly stringent hybridization conditions to a nucleic acid
comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:10
are sufficiently complementary to inhibit the functioning of a CD83
RNA and can be utilized in the methods of the invention.
[0194] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization are somewhat sequence dependent, and may differ
depending upon the environmental conditions of the nucleic acid.
For example, longer sequences tend to hybridize specifically at
higher temperatures. An extensive guide to the hybridization of
nucleic acids is found in Tijssen, Laboratory Techniques in
Biochemistry and Molecular biology-Hybridization with Nucleic Acid
Probes, page 1, chapter 2 "Overview of principles of hybridization
and the strategy of nucleic acid probe assays" Elsevier, New York
(1993). See also, J. Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, N.Y., pp 9.31-9.58
(1989); J. Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, N.Y. (3rd ed. 2001).
[0195] Generally, highly stringent hybridization and wash
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific double-stranded
sequence at a defined ionic strength and pH. For example, under
"highly stringent conditions" or "highly stringent hybridization
conditions" a nucleic acid will hybridize to its complement to a
detectably greater degree than to other sequences (e.g., at least
2- fold over background). By controlling the stringency of the
hybridization and/or washing conditions nucleic acids that are 100%
complementary can be hybridized.
[0196] For DNA-DNA Hybrids, the T.sub.m can be Approximated from
the Equation of Meinkoth and Wahl Anal. Biochem. 138:267-284
(1984):
T.sub.m81.5.degree.C.+16.6 (log M)+0.41 (% GC)-0.61 (%
form)-500/L
[0197] where M is the molarity of monovalent cations, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, % form
is the percentage of formamide in the hybridization solution, and L
is the length of the hybrid in base pairs. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe.
[0198] Very stringent conditions are selected to be equal to the
T.sub.m for a particular probe.
[0199] Alternatively, stringency conditions can be adjusted to
allow some mismatching in sequences so that lower degrees of
similarity can hybridize. Typically, stringent conditions will be
those in which the salt concentration is less than about 1.5 M Na
ion, typically about 0.01 to 1.0 M Na ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide.
[0200] Exemplary low stringency conditions include hybridization
with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS
(sodium dodecyl sulphate) at 37.degree. C., and a wash in 1X to 2X
SSC (20X SSC=3.0 M NaCl and 0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5X to 1X SSC at 55 to 60.degree. C.
Exemplary high stringency conditions include hybridization in 50%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1X
SSC at 60 to 65.degree. C.
[0201] The degree of complementarity or sequence identity of
hybrids obtained during hybridization is typically a function of
post-hybridization washes, the critical factors being the ionic
strength and temperature of the final wash solution. The type and
length of hybridizing nucleic acids also affects whether
hybridization will occur and whether any hybrids formed will be
stable under a given set of hybridization and wash conditions.
[0202] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids that have more than
100 complementary residues on a filter in a Southern or Northern
blot is 50% formamide with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of highly
stringent conditions is 0.1 5 M NaCl at 72.degree. C. for about 15
minutes. An example of stringent wash conditions is a 0.2x SSC wash
at 65.degree. C. for 15 minutes (see also, Sambrook, infra). Often,
a high stringency wash is preceded by a low stringency wash to
remove background probe signal. An example of medium stringency for
a duplex of, e.g., more than 100 nucleotides, is 1x SSC at
45.degree. C. for 15 minutes. An example low stringency wash for a
duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at
40.degree. C. for 15 minutes. For short probes (e.g., about 10 to
50 nucleotides), stringent conditions typically involve salt
concentrations of less than about 1.0M Na ion, typically about 0.01
to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3,
and the temperature is typically at least about 30.degree. C.
[0203] Stringent conditions can also be achieved with the addition
of destabilizing agents such as formamide. In general, a signal to
noise ratio of 2x (or higher) than that observed for an unrelated
probe in the particular hybridization assay indicates detection of
a specific hybridization. Nucleic acids that do not hybridize to
each other under stringent conditions are still substantially
identical if the proteins that they encode are substantially
identical. This occurs, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code.
[0204] The following are examples of sets of hybridization/wash
conditions that may be used to detect and isolate homologous
nucleic acids that are substantially identical to reference nucleic
acids of the present invention: a reference nucleotide sequence
preferably hybridizes to the reference nucleotide sequence in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2X SSC, 0.1% SDS at 50.degree. C.,
more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1X SSC, 0.1%
SDS at 50.degree. C., more desirably still in 7% sodium dodecyl
sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with
washing in 0.5X SSC, 0.1% SDS at 50.degree. C., preferably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 0.1X SSC, 0.1% SDS at 50.degree. C.,
more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1X SSC,
0.1% SDS at 65.degree. C.
[0205] In general, T.sub.m is reduced by about 1.degree. C. for
each 1% of mismatching. Thus, T.sub.m, hybridization, and/or wash
conditions can be adjusted to hybridize to sequences of the desired
sequence identity. For example, if sequences with >90% identity
are sought, the T.sub.m can be decreased 10.degree. C. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
and its complement at a defined ionic strength and pH. However,
severely stringent conditions can utilize a hybridization and/or
wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting
point (T.sub.m); moderately stringent conditions can utilize a
hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower
than the thermal melting point (T.sub.m); low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or
20.degree. C. lower than the thermal melting point (T.sub.m).
[0206] If the desired degree of mismatching results in a T.sub.m of
less than 45.degree. C. (aqueous solution) or 32.degree. C.
(formamide solution), it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes, Part 1, Chapter 2
(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols
in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.). Using these references and the teachings
herein on the relationship between T.sub.m, mismatch, and
hybridization and wash conditions, those of ordinary skill can
generate variants of the present homocysteine S-methyltransferase
nucleic acids.
[0207] Precise complementarity is therefore not required for
successful duplex formation between a nucleic acid that can inhibit
a CD83 RNA and the complementary coding sequence of a CD83 RNA.
Inhibitory nucleic acid molecules that comprise, for example, 2, 3,
4, or 5 or more stretches of contiguous nucleotides that are
precisely complementary to a CD83 coding sequence, each separated
by a stretch of contiguous nucleotides that are not complementary
to adjacent CD83 coding sequences, can inhibit the function of CD83
RNA. In general, each stretch of contiguous nucleotides is at least
4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary
intervening sequences are preferably 1, 2, 3, or 4 nucleotides in
length. One skilled in the art can easily use the calculated
melting point of an anti-sense nucleic acid hybridized to a sense
nucleic acid to determine the degree of mismatching that will be
tolerated between a particular anti-sense nucleic acid and a
particular CD83 RNA.
[0208] Nucleic acids that complementary a CD83 RNA can be
administered to a mammal or to directly to the site of the
inappropriate immune system activity. Alternatively, nucleic acids
that are complementary to a CD83 RNA can be generated by
transcription from an expression cassette that has been
administered to a mammal. For example, a complementary RNA can be
transcribed from a CD83 nucleic acid that has been inserted into an
expression cassette in the 3' to 5' orientation, that is, opposite
to the usual orientation employed to generate sense RNA
transcripts. Hence, to generate a complementary RNA that can
inhibit the function of an endogenous CD83 RNA, the promoter would
be positioned to transcribe from a 3' site towards the 5' end of
the CD83 coding region.
[0209] In some embodiments an RNA that can inhibit the function of
an endogenous CD83 RNA is an anti-sense oligonucleotide. The
anti-sense oligonucleotide is complementary to at least a portion
of the coding sequence of a gene comprising SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5 or SEQ ID NO:10. Such anti-sense oligonucleotides
are generally at least six nucleotides in length, but can be about
8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Longer
oligonucleotides can also be used. CD83 anti-sense oligonucleotides
can be provided in a DNA construct and introduced into cells whose
division is to be decreased, for example, into CD4.sup.+ T cells,
Th-1 cells, Th-2 cells or lymphocyte precursor cells.
[0210] Anti-sense oligonucleotides can be composed of
deoxyribonucleotides, ribonucleotides, or a combination of both.
Oligonucleotides can be synthesized endogenously from transgenic
expression cassettes or vectors as described herein. Alternatively,
such oligonucleotides can be synthesized manually or by an
automated synthesizer, by covalently linking the 5' end of one
nucleotide with the 3' end of another nucleotide with
non-phosphodiester internucleotide linkages such alkylphosphonates,
phosphorothioates, phosphorodithioates, alkylphosphonothioates,
alkylphosphonates, phosphoramidates, phosphate esters, carbamates,
acetamidate, carboxymethyl esters, carbonates, and phosphate
triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux,
1994, Meth. Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev.
90:543-583.
[0211] CD83 anti-sense oligonucleotides can be modified without
affecting their ability to hybridize to a CD83 RNA. These
modifications can be internal or at one or both ends of the
anti-sense molecule. For example, internucleoside phosphate
linkages can be modified by adding peptidyl, cholesteryl or diamine
moieties with varying numbers of carbon residues between these
moieties and the terminal ribose. Modified bases and/or sugars,
such as arabinose instead of ribose, or a 3',5'-substituted
oligonucleotide in which the 3' hydroxyl group or the 5' phosphate
group are substituted, can also be employed in a modified
anti-sense oligonucleotide. These modified oligonucleotides can be
prepared by methods available in the art. Agrawal et al., 1992,
Trends Biotechnol. 10: 152-158; Uhlmann et al., 1990, Chem. Rev.
90:543-584; Uhlmann et al., 1987, Tetrahedron. Lett.
215:3539-3542.
[0212] In one embodiment of the invention, expression of a CD83
gene is decreased using a ribozyme. A ribozyme is an RNA molecule
with catalytic activity. See, e.g., Cech, 1987, Science 236:
1532-1539; Cech, 1990, Ann. Rev. Biochem. 59:543-568; Cech, 1992,
Curr. Opin. Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996,
Trends Genet. 12: 510-515. Ribozymes can be used to inhibit gene
function by cleaving an RNA sequence, as is known in the art (see,
e.g., Haseloff et al., U.S. Pat. No. 5,641,673).
[0213] CD83 nucleic acids complementary to SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5 or SEQ ID NO:10 can be used to generate ribozymes
that will specifically bind to mRNA transcribed from a CD83 gene.
Methods of designing and constructing ribozymes that can cleave
other RNA molecules in trans in a highly sequence specific manner
have been developed and described in the art (see Haseloffet al.
(1988), Nature 334:585-591). For example, the cleavage activity of
ribozymes can be targeted to specific RNAs by engineering a
discrete "hybridization" region into the ribozyme. The
hybridization region contains a sequence complementary to the
target RNA and thus specifically hybridizes with the target (see,
for example, Gerlach et al., EP 321,201). The target sequence can
be a segment of about 10, 12, 15, 20, or 50 contiguous nucleotides
selected from a nucleotide sequence shown in SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5 or SEQ ID NO:10. Longer complementary sequences
can be used to increase the affinity of the hybridization sequence
for the target. The hybridizing and cleavage regions of the
ribozyme can be integrally related; thus, upon hybridizing to the
target RNA through the complementary regions, the catalytic region
of the ribozyme can cleave the target.
[0214] Other CD83 Modulating Molecules
[0215] A wide variety of molecules may be used to modulate CD83
activity or function. Such molecules can also be used to modulate
the immune system independent of CD83. Compositions and methods for
modulating CD83 activity or expression can include these molecules
as well as other components. Representative examples that are
discussed in more detail below include transcription factors,
RNA-binding factors, organic molecules, or peptides.
[0216] RNA-Binding Factors:
[0217] One class of molecules that can be used to modulate the CD83
gene is the RNA binding factors. Such factors include those
described in PCT/EP01/14820 and other sources.
[0218] For example, the HuR protein (Genbank accession number
U38175) has the ability to specifically bind to CD83 RNA at AU-rich
elements or sites. Such AU-rich elements comprise sequences such as
AUUUA (SEQ ID NO:49), AUUUUA (SEQ ID NO:50) and AUUUUUA (SEQ ID
NO:51). Binding by such HuR proteins to CD83 mRNA is thought to
increase the stability, transport and translation of CD83 mRNA, and
thereby increase the expression of CD83 polypeptides. Hence, CD83
expression may be increase by administering HuR proteins or nucleic
acids to a mammal.
[0219] Conversely, CD83 expression may be decreased by
administering factors that block HuR binding to CD83 mRNA. Factors
that block HuR binding include proteins or nucleic acids that can
bind to the AU-rich elements normally bound by HuR, for example,
nucleic acids or anti-sense nucleic acids that are complementary to
AU-rich elements.
[0220] Organic Molecules:
[0221] Numerous organic molecules may be used to modulate the
immune system. These compounds include any compound that can
interact with a component of the immune system. Such compounds may
interact directly with CD83, indirectly with CD83 or with some
other polypeptide, cell or factor that plays a role in the function
of the immune system. In some embodiments, the organic molecule can
bind to a CD83 polypeptide or a CD83 nucleic acid.
[0222] Organic molecules can be tested or assayed for their ability
to modulate CD83 activity, CD83 function or for their ability to
modulate components of the immune system. For example, within one
embodiment of the invention suitable organic molecules may be
selected either from a chemical library, wherein chemicals are
assayed individually, or from combinatorial chemical libraries
where multiple compounds are assayed at once, then deconvoluted to
determine and isolate the most active compounds.
[0223] Representative examples of such combinatorial chemical
libraries include those described by Agrafiotis et al., "System and
method of automatically generating chemical compounds with desired
properties," U.S. Pat. No. 5,463,564; Armstrong, R. W., "Synthesis
of combinatorial arrays of organic compounds through the use of
multiple component combinatorial array syntheses," WO 95/02566;
Baldwin, J. J. et al., "Sulfonamide derivatives and their use," WO
95/24186; Baldwin, J. J. et al., "Combinatorial dihydrobenzopyran
library," WO 95/30642; Brenner, S., "New kit for preparing
combinatorial libraries," WO 95/16918; Chenera, B. et al.,
"Preparation of library of resin-bound aromatic carbocyclic
compounds," WO 95/16712; Ellman, J. A., "Solid phase and
combinatorial synthesis of benzodiazepine compounds on a solid
support," U.S. Pat. No. 5,288,514; Felder, E. et al., "Novel
combinatorial compound libraries," WO 95/16209; Lemer, R. et al.,
"Encoded combinatorial chemical libraries," WO 93/20242; Pavia, M.
R. et al., "A method for preparing and selecting pharmaceutically
useful non-peptide compounds from a structurally diverse universal
library," WO 95/04277; Summerton, J. E. and D. D. Weller,
"Morpholino-subunit combinatorial library and method," U.S. Pat.
No. 5,506,337; Holmes, C., "Methods for the Solid Phase Synthesis
of Thiazolidinones, Metathiazanones, and Derivatives thereof," WO
96/00148; Phillips, G. B. and G. P. Wei, "Solid-phase Synthesis of
Benzimidazoles," Tet. Letters 37:4887-90, 1996; Ruhland, B. et al.,
"Solid-supported Combinatorial Synthesis of Structurally
Diverse-Lactams," J. Amer. Chem. Soc. 111:253-4, 1996; Look, G. C.
et al., "The Indentification of Cyclooxygenase-1 Inhibitors from
4-Thiazolidinone Combinatorial Libraries," Bioorg and Med. Chem.
Letters 6:707-12, 1996.
[0224] Peptides:
[0225] Peptide molecules that modulate the immune system may be
obtained through the screening of combinatorial peptide libraries.
Such libraries may either be prepared by one of skill in the art
(see e.g., U.S. Pat. Nos. 4,528,266 and 4,359,535, and Patent
Cooperation Treaty Publication Nos. WO 92/15679, WO 92/15677, WO
90/07862, WO 90/02809, or purchased from commercially available
sources (e.g., New England Biolabs Ph.D..TM. Phage Display Peptide
Library Kit).
[0226] Methods of Using the CD83 Mutant Mouse
[0227] In one embodiment, the invention provides a method for
identifying ligands, receptors, therapeutic drugs and other
molecules that can modulate the phenotype of the mutant CD83 in
vivo. This method involves administering a test compound to the
mutant CD83 mouse of the invention and observing whether the
compound causes a change in the phenotype of the mutant mouse.
Changes in phenotype that are of interest include increases or
decreases in T cells (especially CD4+ T cells), increases or
decreases in GMCSF, IL-2, IL-4 or IL-10 cytokine production,
increases or decreases in inflammation, increases or decreases in
dendritic cell function and other T cell responses known to one of
skill in the art.
[0228] Test compounds can be screened in vitro to ascertain whether
they interact directly with CD83. In vitro screening can, for
example, identify whether a test compound or molecule can bind to
the cytoplasmic tail or the membrane-associated portions of CD83.
Such information, combined with observation of the in vivo
phenotype before and after administration of the test compound
provides further insight into the function of CD83 and provides
targets for manipulation T cell activation and other functions
modulated by CD83.
[0229] The invention is not limited to identification of molecules
that directly associate with CD83. The in vivo screening methods
provided herein can, also identify test compounds that have an
indirect effect on CD83, or that partially or completely replace a
function of CD83.
[0230] Increases or decreases in T cell numbers can be observed in
blood samples or in samples obtained from thymus, spleen or lymph
node tissues. In order to observe the activation of T cells and/or
the interaction of T cells and dendritic cells, dendritic cells can
be pulsed with antigens ex vivo and then injected into mice to
prime CD4+ T cells in draining lymphoid organs. See Inaba et al.,
J. Exp. Med. 172: 631-640, 1990; Liu, et al., J. Exp. Med. 177:
1299-1307, 1993; Sornasse et al., J. Exp. Med. 175: 15-21, 1992.
Antigens can also be deposited intramuscularly and dendritic cells
from the corresponding afferent lymphatics can carry that antigen
in a form stimulatory for T cells. Bujdoso et al., J. Exp. Med.
170: 1285-1302, 1989. According to the invention, factors
stimulating the interaction of dendritic cells with T cells in vivo
can be identified by administering antigens in this manner and then
observing how T cell respond, e.g. by observing whether T cell
activation occurs.
[0231] Increases or decreases in cytokine levels can be observed by
methods provided herein or by other methods available in the
art.
[0232] Compositions
[0233] The CD83 nucleic acids, polypeptides and antibodies of the
invention, including their salts, are administered so as to achieve
a reduction in at least one symptom associated with an infection,
indication or disease.
[0234] To achieve the desired effect(s), the nucleic acid,
polypeptide or antibody, a variant thereof or a combination
thereof, may be administered as single or divided dosages, for
example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of
at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about
0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to
about 50 to 100 mg/kg of body weight, although other dosages may
provide beneficial results. The amount administered will vary
depending on various factors including, but not limited to, the
nucleic acid, polypeptide or antibody chosen, the disease, the
weight, the physical condition, the health, the age of the mammal,
whether prevention or treatment is to be achieved, and if the
nucleic acid, polypeptide or antibody is chemically modified. Such
factors can be readily determined by the clinician employing animal
models or other test systems that are available in the art.
[0235] Administration of the therapeutic agents in accordance with
the present invention may be in a single dose, in multiple doses,
in a continuous or intermittent manner, depending, for example,
upon the recipient's physiological condition, whether the purpose
of the administration is therapeutic or prophylactic, and other
factors known to skilled practitioners. The administration of the
CD83 nucleic acids, polypeptides and antibodies of the invention
may be essentially continuous over a preselected period of time or
may be in a series of spaced doses. Both local and systemic
administration is contemplated.
[0236] To prepare the composition, CD83 nucleic acids, polypeptides
and antibodies are synthesized or otherwise obtained, purified as
necessary or desired and then lyophilized and stabilized. The
nucleic acid, polypeptide or antibody can then be adjusted to the
appropriate concentration, and optionally combined with other
agents. The absolute weight of a given nucleic acid, polypeptide or
antibody included in a unit dose can vary widely. For example,
about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least
one nucleic acid, polypeptide or antibody of the invention, or a
plurality of CD83 nucleic acid, polypeptides and antibodies
specific for a particular cell type can be administered.
Alternatively, the unit dosage can vary from about 0.01 g to about
50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25
g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g,
from about 0.5 g to about 4 g, or from about 0.5 g to about 2
g.
[0237] Daily doses of the CD83 nucleic acids, polypeptides or
antibodies of the invention can vary as well. Such daily doses can
range, for example, from about 0.1 g/day to about 50 g/day, from
about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12
g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day
to about 4 g/day, and from about 0.5 g/day to about 2 g/day.
[0238] Thus, one or more suitable unit dosage forms comprising the
therapeutic CD83 nucleic acids, polypeptides or antibodies of the
invention can be administered by a variety of routes including
oral, parenteral (including subcutaneous, intravenous,
intramuscular and intraperitoneal), rectal, dermal, transdermal,
intrathoracic, intrapulmonary and intranasal (respiratory) routes.
The therapeutic CD83 nucleic acids, polypeptides or antibodies may
also be formulated for sustained release (for example, using
microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091).
The formulations may, where appropriate, be conveniently presented
in discrete unit dosage forms and may be prepared by any of the
methods well known to the pharmaceutical arts. Such methods may
include the step of mixing the therapeutic agent with liquid
carriers, solid matrices, semi-solid carriers, finely divided solid
carriers or combinations thereof, and then, if necessary,
introducing or shaping the product into the desired delivery
system.
[0239] When the therapeutic CD83 nucleic acids, polypeptides or
antibodies of the invention are prepared for oral administration,
they are generally combined with a pharmaceutically acceptable
carrier, diluent or excipient to form a pharmaceutical formulation,
or unit dosage form. For oral administration, the CD83 nucleic
acids, polypeptides or antibodies may be present as a powder, a
granular formulation, a solution, a suspension, an emulsion or in a
natural or synthetic polymer or resin for ingestion of the active
ingredients from a chewing gum. The active CD83 nucleic acids,
polypeptides or antibodies may also be presented as a bolus,
electuary or paste. Orally administered therapeutic CD83 nucleic
acids, polypeptides or antibodies of the invention can also be
formulated for sustained release, e.g., the CD83 nucleic acids,
polypeptides or antibodies can be coated, micro-encapsulated, or
otherwise placed within a sustained delivery device. The total
active ingredients in such formulations comprise from 0.1 to 99.9%
by weight of the formulation.
[0240] By "pharmaceutically acceptable" it is meant a carrier,
diluent, excipient, and/or salt that is compatible with the other
ingredients of the formulation, and not deleterious to the
recipient thereof.
[0241] Pharmaceutical formulations containing the therapeutic CD83
nucleic acids, polypeptides or antibodies of the invention can be
prepared by procedures known in the art using well-known and
readily available ingredients. For example, the nucleic acid,
polypeptide or antibody can be formulated with common excipients,
diluents, or carriers, and formed into tablets, capsules,
solutions, suspensions, powders, aerosols and the like. Examples of
excipients, diluents, and carriers that are suitable for such
formulations include buffers, as well as fillers and extenders such
as starch, cellulose, sugars, mannitol, and silicic derivatives.
Binding agents can also be included such as carboxymethyl
cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose
and other cellulose derivatives, alginates, gelatin, and
polyvinyl-pyrrolidone. Moisturizing agents can be included such as
glycerol, disintegrating agents such as calcium carbonate and
sodium bicarbonate. Agents for retarding dissolution can also be
included such as paraffin. Resorption accelerators such as
quaternary ammonium compounds can also be included. Surface active
agents such as cetyl alcohol and glycerol monostearate can be
included. Adsorptive carriers such as kaolin and bentonite can be
added. Lubricants such as talc, calcium and magnesium stearate, and
solid polyethyl glycols can also be included. Preservatives may
also be added. The compositions of the invention can also contain
thickening agents such as cellulose and/or cellulose derivatives.
They may also contain gums such as xanthan, guar or carbo gum or
gum arabic, or alternatively polyethylene glycols, bentones and
montmorillonites, and the like.
[0242] For example, tablets or caplets containing the CD83 nucleic
acids, polypeptides or antibodies of the invention can include
buffering agents such as calcium carbonate, magnesium oxide and
magnesium carbonate. Caplets and tablets can also include inactive
ingredients such as cellulose, pregelatinized starch, silicon
dioxide, hydroxy propyl methyl cellulose, magnesium stearate,
microcrystalline cellulose, starch, talc, titanium dioxide, benzoic
acid, citric acid, corn starch, mineral oil, polypropylene glycol,
sodium phosphate, zinc stearate, and the like. Hard or soft gelatin
capsules containing at least one nucleic acid, polypeptide or
antibody of the invention can contain inactive ingredients such as
gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch,
talc, and titanium dioxide, and the like, as well as liquid
vehicles such as polyethylene glycols (PEGs) and vegetable oil.
Moreover, enteric-coated caplets or tablets containing one or more
CD83 nucleic acids, polypeptides or antibodies of the invention are
designed to resist disintegration in the stomach and dissolve in
the more neutral to alkaline environment of the duodenum.
[0243] The therapeutic CD83 nucleic acids, polypeptides or
antibodies of the invention can also be formulated as elixirs or
solutions for convenient oral administration or as solutions
appropriate for parenteral administration, for instance by
intramuscular, subcutaneous, intraperitoneal or intravenous routes.
The pharmaceutical formulations of the therapeutic CD83 nucleic
acids, polypeptides or antibodies of the invention can also take
the form of an aqueous or anhydrous solution or dispersion, or
alternatively the form of an emulsion or suspension or salve.
[0244] Thus, the therapeutic CD83 nucleic acids, polypeptides or
antibodies may be formulated for parenteral administration (e.g.,
by injection, for example, bolus injection or continuous infusion)
and may be presented in unit dose form in ampoules, pre-filled
syringes, small volume infusion containers or in multi-dose
containers. As noted above, preservatives can be added to help
maintain the shelve life of the dosage form. The active CD83
nucleic acids, polypeptides or antibodies and other ingredients may
form suspensions, solutions, or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Alternatively, the active
CD83 nucleic acids, polypeptides or antibodies and other
ingredients may be in powder form, obtained by aseptic isolation of
sterile solid or by lyophilization from solution, for constitution
with a suitable vehicle, e.g., sterile, pyrogen-free water, before
use.
[0245] These formulations can contain pharmaceutically acceptable
carriers, vehicles and adjuvants that are well known in the art. It
is possible, for example, to prepare solutions using one or more
organic solvent(s) that is/are acceptable from the physiological
standpoint, chosen, in addition to water, from solvents such as
acetone, ethanol, isopropyl alcohol, glycol ethers such as the
products sold under the name "Dowanol," polyglycols and
polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain
acids, ethyl or isopropyl lactate, fatty acid triglycerides such as
the products marketed under the name "Miglyol," isopropyl
myristate, animal, mineral and vegetable oils and
polysiloxanes.
[0246] It is possible to add, if necessary, an adjuvant chosen from
antioxidants, surfactants, other preservatives, film-forming,
keratolytic or comedolytic agents, perfumes, flavorings and
colorings. Antioxidants such as t-butylhydroquinone, butylated
hydroxyanisole, butylated hydroxytoluene and a-tocopherol and its
derivatives can be added.
[0247] Also contemplated are combination products that include one
or more CD83 nucleic acids, polypeptides or antibodies of the
present invention and one or more other anti-microbial agents. For
example, a variety of antibiotics can be included in the
pharmaceutical compositions of the invention, such as
aminoglycosides (e.g., streptomycin, gentamicin, sisomicin,
tobramycin and amicacin), ansamycins (e.g. rifamycin), antimycotics
(e.g. polyenes and benzofuran derivatives), .beta.-lactams (e.g.
penicillins and cephalosporins), chloramphenical (including
thiamphenol and azidamphenicol), linosamides (lincomycin,
clindamycin), macrolides (erythromycin, oleandomycin, spiramycin),
polymyxins, bacitracins, tyrothycin, capreomycin, vancomycin,
tetracyclines (including oxytetracycline, minocycline,
doxycycline), phosphomycin and fusidic acid.
[0248] Additionally, the CD83 nucleic acids, polypeptides or
antibodies are well suited to formulation as sustained release
dosage forms and the like. The formulations can be so constituted
that they release the active nucleic acids, polypeptide or
antibody, for example, in a particular part of the intestinal or
respiratory tract, possibly over a period of time. Coatings,
envelopes, and protective matrices may be made, for example, from
polymeric substances, such as polylactide-glycolates, liposomes,
microemulsions, microparticles, nanoparticles, or waxes. These
coatings, envelopes, and protective matrices are useful to coat
indwelling devices, e.g., stents, catheters, peritoneal dialysis
tubing, draining devices and the like.
[0249] For topical administration, the therapeutic agents may be
formulated as is known in the art for direct application to a
target area. Forms chiefly conditioned for topical application take
the form, for example, of creams, milks, gels, dispersion or
microemulsions, lotions thickened to a greater or lesser extent,
impregnated pads, ointments or sticks, aerosol formulations (e.g.,
sprays or foams), soaps, detergents, lotions or cakes of soap.
Other conventional forms for this purpose include wound dressings,
coated bandages or other polymer coverings, ointments, creams,
lotions, pastes, jellies, sprays, and aerosols. Thus, the
therapeutic CD83 nucleic acids, polypeptides or antibodies of the
invention can be delivered via patches or bandages for dermal
administration. Alternatively, the nucleic acid, polypeptide or
antibody can be formulated to be part of an adhesive polymer, such
as polyacrylate or acrylate/vinyl acetate copolymer. For long-term
applications it might be desirable to use microporous and/or
breathable backing laminates, so hydration or maceration of the
skin can be minimized. The backing layer can be any appropriate
thickness that will provide the desired protective and support
functions. A suitable thickness will generally be from about 10 to
about 200 microns.
[0250] Ointments and creams may, for example, be formulated with an
aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or
oily base and will in general also contain one or more emulsifying
agents, stabilizing agents, dispersing agents, suspending agents,
thickening agents, or coloring agents. The active CD83 nucleic
acids, polypeptides or antibodies can also be delivered via
iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122;
4,383,529; or 4,051,842. The percent by weight of a therapeutic
agent of the invention present in a topical formulation will depend
on various factors, but generally will be from 0.01% to 95% of the
total weight of the formulation, and typically 0.1-85% by
weight.
[0251] Drops, such as eye drops or nose drops, may be formulated
with one or more of the therapeutic CD83 nucleic acids,
polypeptides or antibodies in an aqueous or non-aqueous base also
comprising one or more dispersing agents, solubilizing agents or
suspending agents. Liquid sprays are conveniently delivered from
pressurized packs. Drops can be delivered via a simple eye
dropper-capped bottle, or via a plastic bottle adapted to deliver
liquid contents dropwise, via a specially shaped closure.
[0252] The therapeutic nucleic acids, polypeptide or antibody may
further be formulated for topical administration in the mouth or
throat. For example, the active ingredients may be formulated as a
lozenge further comprising a flavored base, usually sucrose and
acacia or tragacanth; pastilles comprising the composition in an
inert base such as gelatin and glycerin or sucrose and acacia; and
mouthwashes comprising the composition of the present invention in
a suitable liquid carrier.
[0253] The pharmaceutical formulations of the present invention may
include, as optional ingredients, pharmaceutically acceptable
carriers, diluents, solubilizing or emulsifying agents, and salts
of the type that are available in the art. Examples of such
substances include normal saline solutions such as physiologically
buffered saline solutions and water. Specific non-limiting examples
of the carriers and/or diluents that are useful in the
pharmaceutical formulations of the present invention include water
and physiologically acceptable buffered saline solutions such as
phosphate buffered saline solutions pH 7.0-8.0.
[0254] The CD83 nucleic acids, polypeptides or antibodies of the
invention can also be administered to the respiratory tract. Thus,
the present invention also provides aerosol pharmaceutical
formulations and dosage forms for use in the methods of the
invention. In general, such dosage forms comprise an amount of at
least one of the agents of the invention effective to treat or
prevent the clinical symptoms of a specific infection, indication
or disease. Any statistically significant attenuation of one or
more symptoms of an infection, indication or disease that has been
treated pursuant to the method of the present invention is
considered to be a treatment of such infection, indication or
disease within the scope of the invention.
[0255] Alternatively, for administration by inhalation or
insufflation, the composition may take the form of a dry powder,
for example, a powder mix of the therapeutic agent and a suitable
powder base such as lactose or starch. The powder composition may
be presented in unit dosage form in, for example, capsules or
cartridges, or, e.g., gelatin or blister packs from which the
powder may be administered with the aid of an inhalator,
insufflator, or a metered-dose inhaler (see, for example, the
pressurized metered dose inhaler (MDI) and the dry powder inhaler
disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W.
and Davia, D. eds., pp. 197-224, Butterworths, London, England,
1984).
[0256] Therapeutic CD83 nucleic acids, polypeptides or antibodies
of the present invention can also be administered in an aqueous
solution when administered in an aerosol or inhaled form. Thus,
other aerosol pharmaceutical formulations may comprise, for
example, a physiologically acceptable buffered saline solution
containing between about 0.1 mg/ml and about 100 mg/ml of one or
more of the CD83 nucleic acids, polypeptides or antibodies of the
present invention specific for the indication or disease to be
treated. Dry aerosol in the form of finely divided solid nucleic
acid, polypeptide or antibody particles that are not dissolved or
suspended in a liquid are also useful in the practice of the
present invention. CD83 nucleic acids, polypeptides or antibodies
of the present invention may be formulated as dusting powders and
comprise finely divided particles having an average particle size
of between about 1 and 5 .mu.m, alternatively between 2 and 3
.mu.m. Finely divided particles may be prepared by pulverization
and screen filtration using techniques well known in the art. The
particles may be administered by inhaling a predetermined quantity
of the finely divided material, which can be in the form of a
powder. It will be appreciated that the unit content of active
ingredient or ingredients contained in an individual aerosol dose
of each dosage form need not in itself constitute an effective
amount for treating the particular infection, indication or disease
since the necessary effective amount can be reached by
administration of a plurality of dosage units. Moreover, the
effective amount may be achieved using less than the dose in the
dosage form, either individually, or in a series of
administrations.
[0257] For administration to the upper (nasal) or lower respiratory
tract by inhalation, the therapeutic CD83 nucleic acids,
polypeptides or antibodies of the invention are conveniently
delivered from a nebulizer or a pressurized pack or other
convenient means of delivering an aerosol spray. Pressurized packs
may comprise a suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. Nebulizers include, but are not limited to, those
described in U.S. Pat. Nos. 4,624,251; 3,703,173; 3,561,444; and
4,635,627. Aerosol delivery systems of the type disclosed herein
are available from numerous commercial sources including Fisons
Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, N.J.) and
American Pharmoseal Co., (Valencia, Calif.). For intra-nasal
administration, the therapeutic agent may also be administered via
nose drops, a liquid spray, such as via a plastic bottle atomizer
or metered-dose inhaler. Typical of atomizers are the Mistometer
(Wintrop) and the Medihaler (Riker).
[0258] Furthermore, the active ingredients may also be used in
combination with other therapeutic agents, for example, pain
relievers, anti-inflammatory agents, antihistamines,
bronchodilators and the like, whether for the conditions described
or some other condition.
[0259] The present invention further pertains to a packaged
pharmaceutical composition for controlling microbial infections
such as a kit or other container. The kit or container holds a
therapeutically effective amount of a pharmaceutical composition
for modulating immune responses and instructions for using the
pharmaceutical composition for control of the immune response. The
pharmaceutical composition includes at least one nucleic acid,
polypeptide or antibody of the present invention, in a
therapeutically effective amount such that the selected disease or
immunological condition is controlled.
[0260] The invention will be further described by reference to the
following detailed examples, which are given for illustration of
the invention, and are not intended to be limiting thereof.
EXAMPLE 1
Mouse Mutation and Characterization Mutant Generation
[0261] Male C57BL6 mice received 3 weekly injections of
N-ethyl-N-nitrosourea (ENU) at a concentration of 100mg/kg.
N-Ethyl-N-nitrosourea was quantified prior to injection by
spectrophotometry. Mice that regained fertility after a minimum
period of 12 weeks were then used to generate pedigree founder G1
animals. G1 male mice were crossed to C57BL6J females and their
female progeny (G2 animals) crossed back to their fathers to
generate G3 animals for screening.
[0262] G3 mice were weaned at 3 weeks of age. Each animal then
underwent a series of screens designed to assess a number of
parameters, including immune function, inflammatory response and
bone development. In the initial screen, conducted at 6 weeks of
age, 150-200 .mu.l of whole blood was collected by retro-orbital
bleed into heparinized tubes. Cells were pelleted and red blood
cells lysed. Samples were then stained with antibodies to cell
surface markers expressed on distinct lymphoid and myeloid
sub-populations. These samples were analyzed by flow-cytometry.
[0263] Mutant Identification
[0264] A group of 27 G3 mice from 2 different pedigrees, pedigree 9
and pedigree 57 (i.e. derived from 2 distinct G1 fathers) were
analyzed in this screen. Two animals from pedigree 9 were
identified as having a reduced (>2 standard deviation from
normal) percentage of CD4+ T cells in peripheral blood (FIG. 1).
Both animals were descended from the same G1 and shared the same
mother. All other animals screened on that day had a normal
percentage of CD4+ T cells. The number of phenodeviants identified
(2 from a litter of 9 animals) was suggestive of a trait controlled
by a single gene and inherited in a Mendelian fashion.
[0265] A second litter generated from Pedigree 9 bred to G2
daughter #4 exhibited an identical phenotype with reduced numbers
of CD4+ T cells, further suggesting that the trait had a genetic
basis. The phenotype was designated LCD4.1 (Low CD4 Mutant # 1) and
was used for mapping experiments.
[0266] Mutation Mapping
[0267] In order to map the LCD4.1 mutant phenotype, affected G3
male mice (presumptive homozygous for the mutation) were bred to
female animals from the C3HeB/FeJ strain to generate F 1 progeny.
These F 1 females (presumptively heterozygous for the mutation)
were then mated back to their affected father to generate N2
progeny.
[0268] Blood was collected from N2 animals and flow cytometric
analysis was performed to identify CD4+ T cells. For a phenotype
controlled by a single gene, breeding homozygous fathers to
heterozygous daughters should yield 50% normal N2 animals and 50%
affected N2 animals. This ratio of normal to affected animals was
observed in the N2 generation: Multiple N2 animals exhibited a
reduced percentage of CD4+ T cells, indicating that the phenotype
was heritable (FIG. 2).
[0269] DNA samples were prepared from samples of tail tissue
collected from these N2 mice and used for a genome scan, using a
collection of assembled markers, and performed on the ABI 3100 DNA
analyzer. Initial genetic linkage was seen to the tip of chromosome
13, where the closest microsatellite marker was D 13Mit139 with a
LOD score of 8.2. By calculating upper and lower confidence limits,
the mutant gene was located between 13.4 and 29.6 cM on chromosome
13. Through additional genotyping, this region was reduced to an 11
cM interval on chromosome 13. No significant linkage to other
chromosomal regions was seen.
[0270] Mutation Identification
[0271] A candidate gene, CD83, was identified for gene-testing
based upon its reported position within the interval. CD83 has
previously been used as a marker of dendritic cell activation,
suggesting that it might play a role in dendritic cell function and
hence in regulating T cell development and function.
[0272] Sequence analysis of the mutant DNA revealed a mutation in
the stop codon of CD83. All affected animals were homozygous for
this mutation while non-affected animals carried one wild-type
allele and one mutant allele (FIG. 3 and FIG. 4). The mutation
destroyed the stop codon and resulted in the addition of a unique
55 amino acid tail to the C-terminus of CD83 (FIG. 5).
[0273] Additional Functional Data
[0274] A reduction in CD4+ T cells was seen in peripheral blood,
spleen tissues and lymph nodes from homozygous LCD4.1 mice.
Although there were a reduced number of CD4+ T cells in the thymus
there is no overt block in the developmental process and there was
substantially no alteration in B cell development in the bone
marrow. Histological evaluation of thymus, spleen and lymph nodes
from affected mice revealed no gross alteration in tissue
architecture.
[0275] Dendritic cells can be differentiated from bone marrow of
wild type mice by culture in GM-CSF. These cells can be
characterized by the surface expression of dendritic cell markers,
including CD86 and CD 11 c. Both LCD4.1 affected and normal animals
were capable of giving rise to CD86+CD11c+cells under these culture
conditions. LCD4.1 mutant mice thus were capable of generating
dendritic cells under in vitro culture conditions. These data
suggest that the phenotype seen in LCD4.1 mice is not due to a
failure of dendritic cells to develop but rather may reflect a
defect in function.
[0276] To track dendritic cells, the sensitizing agent FITC was
applied to the dorsal surface of the ears of LCD4.1 affected and
wild-type mice. FITC was picked up by dendritic cells that then
migrated to the draining auricular lymph nodes, where the presence
of the FITC label on the dendritic cell surface permitted detection
by flow-cytometry. FITC labeled cells expressing CD86 were detected
in equal proportions in draining lymph node from normal and
affected LCD4.1 mice. These data indicate that LCD4.1 mutant
animals are capable of generating dendritic cells in vivo and that
these cells are able to pick up antigen in the ear and travel to
the draining lymph node.
EXAMPLE 2
CD83 and CD4.sup.+ T Cell Function Materials and Methods
[0277] Spleens were removed from wild type and mutant mice and
digested with collagenase to liberate dendritic cells. Spleens were
stained for surface expression of CD4 (helper T cells) and CD 11c
(dendritic cells). Cells expressing these markers were purified by
fluorescence activated cell sorting (FACS sorting). CD 11c and
CD4+positive cells were also purified from an allogeneic mouse
strain, BALBc.
[0278] Mixed lymphocyte cultures were set up using purified cell
populations. Dendritic cells from BALBc animals were used to
stimulate CD4+ T cells from wild type and mutant mice. In a
reciprocal experiment dendritic cells prepared from wild type and
mutant mice were used to stimulate BALBc CD4+ T cells. After 5 days
in culture proliferative responses were measured by incorporation
of tritiated thymidine.
[0279] Dendritic cells from wild type and mutant mice were both
capable of activating allogeneic T cells, suggesting that dendritic
cell function was unimpaired in the mutant animal (FIG. 6a). In
contrast CD4+ T cells from mutant animals exhibited a diminished
response after 5 days of stimulation (FIG. 6b).
[0280] These data suggest that the mutation in the CD83 gene has
minimal effect on dendritic cells intrinsic function but rather has
a profound effect upon T cell activity. The CD4+ T cell therefore
may have a novel requirement for CD83 functionality on T cells
during allogeneic activation. CD83 may be influencing the extent of
CD4+ T cell activation or altering the duration of the CD4+ T cell
proliferative response. The therapeutic manipulation of CD83 may
thus represent a mechanism for the specific regulation of T cell
function in the treatment of T cell mediated diseases, including
autoimmune disorders. Antibodies capable of blocking CD83 function
may be used as therapeutics in the treatment of immune diseases
whilst the activation of CD83 may-have utility in enhancing immune
responses in cancer and other circumstances.
CONCLUSION
[0281] Although CD83 has been described as a marker of dendritic
cell activation there has previously been little data describing
its function in vivo. However, the mutation provided by the
invention destabilizes or inactivates the protein and leads to
impaired surface expression. As a consequence, CD4+ T cell function
is impaired. However, the development of dendritic cells is not
inhibited and mutant dendritic cells retain functionality.
Nonetheless, the result is impaired development of CD4+ T cells.
This impaired ability to activate T cells is also seen in a slight
decrease in contact sensitivity responses in LCD4.1 mutant
mice.
EXAMPLE 3
Mutant CD83 Have Different Cytokine Levels than Wild Type Mice
[0282] This Example demonstrates that CD4.sup.+ T-cells from CD83
mutant animals express higher levels of IL-4 and lower levels of
IL-2 compared to CD4.sup.+ T-cells from CD83 wild type animals.
[0283] Methods for Cell Activation and Cytokine Measurements
[0284] Spleens cells from 6-8-week-old homozygous CD83 wild type or
CD83 mutant (LCD4.1) mice were used to isolate CD4.sup.+ T-cells by
positive selection using magnetic beads (Miltenyi Biotec). A 96
round bottom plate was coated with 50 .mu.L per well of a solution
containing either 1 or 10 .mu.g/mL of anti-CD3 and 0.1 or 0.2
.mu.g/mL of anti-CD28 antibodies (both from Pharmingen) in PBS
overnight. This plate was then washed using 150 .mu.L of PBS three
times. To this pre-coated plate, 20,000 CD4.sup.+ T-cells (either
wild type or CD83 mutant) were added in a 200 .mu.L final volume of
RPMI containing 10% FBS, 55 .mu.M .beta.-mercaptoethanol and
antibiotics. The plates were then incubated in a CO.sub.2 incubator
at 37.degree. C. for 44 to 72 hours. For determination of cytokine
levels, supernatants were harvested and cytokines were measured
using either a Cytometric Bead Array system (Pharmingen) or ELISA
(R&D). For RNA measurements, the cells were harvested and RNA
was isolated using Tri reagent (Sigma). IL-10 and IL-4 mRNA levels
were measured by reverse transcription and TaqMan (Applied
Biosystems) analysis.
[0285] Results:
[0286] FIG. 7 shows the IL-2, IL-4, IL-5, TNFa and IFN? levels
produced by either wild type or CD83 mutant CD4.sup.+ T-cells.
Purified cells were incubated as described above in the presence of
1 .mu.g/mL of anti-CD3 and 0.2 .mu.g/mL of anti-CD28 antibodies for
72 hours. The supernatants were then simultaneously analyzed for
production of IL-2, IL-4, IL-5, TNFa and IFN? using the cytometric
bead array system from Pharmingen.
[0287] FIG. 7 demonstrates that CD4.sup.+ T-cells from CD83 mutant
animals expressed higher levels of IL-4 and lower levels of IL-2
compared to CD4.sup.+ T-cells from CD83 wild type animals. Other
cytokines and a new set of stimulation assays were analyzed
including the production levels of IL-10 and GMCSF by these cells
(FIGS. 8 and 9). In both cases, cells from mutant animals produce
larger amounts of IL-10 and GMCSF than did wild type animals. FIG.
10 shows that mRNA levels for both IL-4 and IL-10 were increased in
cells from activated mutant CD83, CD4.sup.+ T-cells compared with
cells from wild type animals.
EXAMPLE 4
Anti-CD83 Antibodies Mimic the Effects of the CD83 Mutation
[0288] Methods for antibody testing:
[0289] For modulation of cytokine production by anti-CD83
antibodies, CD4.sup.+ T-cells were isolated and activated as
described above. Activation was performed in the presence of
increasing concentrations of anti-CD83 antibodies. For
proliferation assays, CD4.sup.+ T-cells were isolated from an OT2tg
mouse. OT2tg mice are transgenic mice with a T-cell receptor
specific for chicken ovalbumin (OVA) 323-339 peptide. Dendritic
cells were isolated from a C57BL6 mouse by a negative selection
using B220 magnetic beads (Miltenyi Biotec) followed by positive
selection using CDl 1-c magnetic beads (Milteny Biotec). Five
thousand CD4.sup.+ T-cells were then mixed with five thousand
dendritic cells in a 96 well plate in the presences of 1 .mu.M OVA
peptide using RPMI (55 .mu.M BME, 10% FBS plus antibiotics) in a
final 200uL volume. These cells were then incubated for 48 to 72
hours in a CO.sub.2 incubator at 37.degree. C. and pulsed using
[.sup.3H] thymidine for 8 hours. Cells were then harvested and
[.sup.3H] thymidine incorporation was quantified using a top
counter.
[0290] Results:
[0291] In some assays, anti-CD83 antibodies decreased production of
IL-4 by activated CD4.sup.+ T-cells in a dose dependent manner.
Different antibody preparations did provide somewhat different
degrees of inhibition of IL-4 production (FIG. 11). Accordingly,
the epitope and/or degree of affinity of the antibodies for the
CD83 antigen may influence whether or not IL-4 production is
significantly inhibited.
[0292] The effects of anti CD83 antibodies on proliferation of a
peptide specific T-cell proliferation assay using the OT2 T-cell
receptor (TCR) transgenic system were also observed. CD4.sup.+
T-cells derived from these TCR transgenic animals express high
levels of a T-cell receptor specific for chicken ovalbumin (OVA)
323-339 peptide and thus have high levels of proliferation when
mixed with antigen presenting cells (dendritic cells were used) in
the presence of the OVA peptide. In such assays, anti-CD83
antibodies were able to decrease proliferation of CD4.sup.+ T-cells
in this system (FIG. 12). However, different antibody preparations
had somewhat different effects on the proliferation of CD4.sup.+
T-cells. Accordingly, the CD83 epitope and/or degree of affinity of
the antibodies for the CD83 antigen may influence whether or not
CD4.sup.+ T-cell proliferation is significantly inhibited.
EXAMPLE 5
Increased T-Cell Proliferation by Transgenic Expression of CD83
[0293] This Example illustrates that over expression of CD83 in
transgenic mice leads to increased T-cell proliferation.
[0294] Materials and Methods
[0295] A 34.3 kb fragment of normal mouse genomic DNA, including
the .about.18 kb coding region of the CD83 gene, as well as
.about.10.6 kb of upstream flanking sequences and .about.5.7 kb of
downstream sequences was microinjected into normal mouse one-cell
embryos. Four individual founder animals were generated. Transgenic
mice were then crossed to a male OT2tg mouse. Male offspring
carrying both the CD83 and OT2 transgene were used to analyze
peptide specific T-cell proliferation.
[0296] For proliferation assays, CD4.sup.+ T-cells and dendritic
cells were isolated from either OT2tg [transgenic mice with a
T-cell receptor specific for chicken ovalbumin (OVA) 323-339
peptide] CD83 wild type or from OT2tg CD83 transgenic mice as
described above (Example 4). Five thousand OT2tg CD4.sup.+ T-cells
from either wild type or CD83 transgenic animals were then mixed
with five thousand wild type dendritic cells or five thousand CD83
transgenic dendritic cells in a 96 well plate in the presence of
increasing concentrations of OVA peptide using RPMI (55 .mu.M BME,
110% FBS plus antibiotics) in a final 200uL volume. These cells
were then incubated for 48 to 72 hours in a CO.sub.2 incubator at
37C and pulsed using [.sup.3H] thymidine for 8 hours. Cells were
then harvested and [.sup.3H] thymidine incorporation was quantified
using a top counter.
[0297] Results:
[0298] OT2tg CD4.sup.+ T-cells derived from CD83 transgenic mice
proliferated at higher rates than the same cell population derived
from a CD83 wild type animal (FIG. 13). This increased
proliferation was seen at all the concentrations of OVA peptide
tested. Whereas OT2tg CD4.sup.+ T-cells derived from CD83
transgenic animals exhibited increased proliferation, dendritic
cells from CD83 transgenic animals did not exhibit a substantial
increase in proliferation. Therefore, it appears that transgenic
expression in the CD4.sup.+ T-cell, and not in dendritic cells is
what led to the increased proliferation of CD4.sup.+ T-cells.
EXAMPLE 6
Inhibition of Proliferation of PHA Activated Human PBMCs by Protein
A Purified Rabbit Anti-Mouse CD83 Antibodies
[0299] This Example shows that antibodies raised against the CD83
protein can inhibit proliferation of human peripheral blood
mononuclear cells.
[0300] Materials and Methods
[0301] Rabbit polyclonal sera was raised against mouse CD83 protein
by immunizing rabbits using a mouse CD83 external domain protein
fused to a rabbit Ig domain (FIG. 14). Pre-immune sera and
anti-mouse polyclonal sera were then purified using a protein A
column (Pharmacia Biotech) as described by the manufacturer, then
dialyzed against PBS and stored at 4.degree. C. To monitor the
recognition of mouse CD83 protein by the polyclonal sera, which was
obtained at different dates post immunization, a titer was obtained
using an antigen specific ELISA (FIG. 15). As illustrated by FIG.
15, a good polyclonal response was obtained against the mouse CD83
protein.
[0302] Human peripheral blood mononuclear cells (PBMCs) were
isolated using a Ficoll gradient (Ficoll Paque Plus, Pharmacia) and
washed with PBS buffer. For activation and proliferation studies,
five thousand cells were incubated in 200 .mu.L of media (RPMI, 10%
FBS, antibiotics) and 5 .mu.g/mL of Phaseolus vulgaris
leucoagglutinin (PHA) in the presence or absence of increasing
concentrations of Protein A purified pre-immune sera or with
similarly purified anti-CD83 polyclonal antibodies. After 48 hours
at 37.degree. C. in a CO.sub.2 incubator the cells were pulsed with
[.sup.3H] thymidine for 8 hours and harvested. Thymidine
incorporation into the PBMCs was measured using a top counter for
analysis.
[0303] A Selected Lymphocyte Antibody Method (SLAM) procedure was
used to establish monoclonal antibody cell lines from the rabbits
used to generate the anti-CD83 antibodies. Antibody forming cells
were isolated from the immunized rabbits that produced antibodies
capable of binding CD83, the genes encoding antibodies that
recognized CD83 and inhibited proliferation of lymphocytes were
then cloned by PCR amplification and sequenced. Separate lines of
monoclonal antibody producing cells were then established and
expanded in culture. Antibodies were purified using Protein A
chromatography according to manufacturer's instructions and tested
for their ability to recognize CD83 proteins and to inhibit
proliferation of PHA stimulated human PBMCs.
[0304] Results
[0305] FIG. 16 illustrates that proliferation of PHA-activated
human PBMCs was inhibited by polyclonal antibodies raised against
the external region of the mouse CD83 protein. Proliferation of
PHA-activated human PBMCs was not affected by addition of
increasing concentrations of protein A purified rabbit pre-immune
sera. When increasing concentrations of protein A purified rabbit
polyclonal sera raised against the mouse CD83 protein was added, a
concentration dependent decrease in proliferation was observed.
[0306] These data indicate that antibodies raised against the mouse
protein are able to cross-react with the human protein. Moreover,
antibodies raised against the mouse protein are able to inhibit
proliferation of PHA-activated human PBMCs.
[0307] A summary of the characteristics of two monoclonal antibody
preparations having functional activity is shown in Table 1.
Isolated recombinant mouse and human CD83 protein preparations were
used for the BIACORE and ELISA assays. Endogenous human CD83
protein expressed in a human KMH2 cell line was used for FACS
assays.
47TABLE 1 Monoclonal Antibody Functionality and Reactivity with
Mouse and Human CD83 Assay 95F04 Antibodies 96G08 Antibodies
Inhibition of human PBMC ++ +++ proliferation Biacore - mouse CD83
+++ +++ Biacore - human CD83 ++ - ELISA - mouse CD83 +++ +++ ELISA
- human CD83 ++ - FACS - human CD83 ND ++ ND: not determined
[0308] While the 96G08 antibodies appeared to have reduced affinity
for human CD83 protein via the Biacore and ELISA assays, the FACS
assay indicated that this antibody preparation could bind to
endogenously produced human CD83 (FIGS. 18 and 19). Moreover, the
96G08 antibodies were able to inhibit proliferation of human
peripheral blood mononuclear cells (PBMCs), as illustrated in FIG.
20. Hence, some aspect of either the purification or the structure
of the isolated recombinant human protein may have influenced the
in vitro binding of 96G08 antibodies to the recombinant human CD83.
For example, the recombinant human CD83 protein employed for the
Biacore and ELISA assays is a chimeric protein that is joined to a
portion of an immunoglobulin Fc fragment. Removal of this Fc
fragment may improve in vitro binding to the human CD83
protein.
[0309] FIG. 20 illustrates that the 95F04 and 96G08 antibody
preparations can inhibit proliferation of PHA activated human
peripheral blood mononuclear cells as detected by incorporation of
[.sup.3H] thymidine. As shown, when no antibody was present about
10,000 cpm of [.sup.3H] thymidine was incorporated into human
peripheral blood mononuclear cells. However, when 30 .mu.g/ml of
the 95F04 antibody preparation was present, incorporation of
[.sup.3H] thymidine dropped to about 2000 cpm. The 96G08 antibody
preparation had an even greater effect on [.sup.3H] thymidine
incorporation. When 30 .mu.g/ml 96G08 antibody preparation was
added to human peripheral blood mononuclear cells, [.sup.3H]
thymidine incorporation was reduced to about 300 cpm. These data
indicate that the 95F04 and 96G08 antibody preparations can alter
the function of human CD83 in vitro.
EXAMPLE 7
Multimerized Anti-CD83 Antibodies Inhibit Proliferation of Immune
Cells
[0310] This Example shows that antibodies raised against the CD83
protein as described in the previous example are particularly
effective at inhibiting proliferation of immune cells after the
antibodies are multimerized or multimerized by binding the
antibodies to a solid support or by cross-linking in solution.
[0311] Materials and Methods
[0312] Round bottom microtiter plates were coated with different
preparations of anti-CD83 antibody preparations by incubating the
plates with 50 .mu.l of 50 .mu.g/ml antibody preparation per well
either for 2 hours at 37.degree. C. or overnight at 4.degree. C. As
a positive control, some wells were coated with anti-LFA antibodies
that are known to inhibit proliferation of lymphocytes. After
coating, the wells were then washed thoroughly with PBS.
[0313] Mouse (C57B 16) spleen cells were isolated and plated in the
antibody or control treated wells at 30,000 cells per well. For
activation, Concavalin A was added to a final concentration of 1.0
.mu.g/ml. Cellular proliferation was assessed by measuring the
incorporation of tritiated thymidine during the last 6 to 8 hours
of a 48 hour incubation. In another experiment, the specificity of
the observed antibody-induced inhibition of lymphocyte
proliferation was tested by repeating this experiment with addition
of mouse CD83 protein before adding the lymphocytes to the antibody
coated microtiter wells.
[0314] As described in more detail below, the 6G05 antibody
preparation was identified as a good inhibitor of lymphocyte
proliferation. In contrast, the 112D08 antibody preparation was
identified as having little or no inhibitory activity when bound to
microtiter wells. The 112D08 antibody preparation was used as a
negative control in some of the subsequent experiments.
[0315] The inhibitory activities of plate-bound versus soluble,
cross-linked 6G05 antibodies were compared in another experiment.
Plate-bound 6G05 antibodies were prepared as described above.
Approximately 30,000 activated lymphocytes were added per well to
antibody coated plates or to non-coated plates containing 1.0 or
5.0 .mu.g/ml soluble 6G05 antibody preparation. A secondary rabbit
anti-mouse antibody (10 .mu.g/ml or 25 .mu.g/ml) was added to the
wells containing the soluble 6G05 antibody preparation to act as a
cross-linking reagent for the 6G05 antibodies. Cellular
proliferation was assessed by incorporation of tritiated thymidine
as described above.
[0316] Results
[0317] The results of one screen for anti-CD83 antibody
preparations that can inhibit lymphocyte proliferation are shown in
FIGS. 25A-B. As illustrated in FIG. 25A many anti-CD83 antibody
preparations inhibit proliferation of activated lymphocytes,
including the 94c09, 98a02, 94d08, 98d11, 101b08, 6g05, 20d04,
14c12, 11g05, 12g04, 32f12 and 98b11 preparations. Note that some
variation in the degree of inhibition obtained is observed. For
example, while the 98b 1 preparation is not so effective, the 6g05
antibody preparation is a highly effective inhibitor of lymphocyte
proliferation.
[0318] FIG. 25B further illustrates that some antibody preparations
are highly effective inhibitors (e.g. 117G12) but others are not
(e.g. 98g08). The 824pb antibody refers to rabbit polyclonal
antisera; as shown this polyclonal antisera was not particularly
effective at inhibiting lymphocyte proliferation
[0319] FIG. 26 illustrates that the inhibitory activity of the 6g05
antibody preparation is quenched by soluble mouse CD83 protein. In
this assay, mouse CD83 protein was added to anti-CD83
antibody-coated wells before activated lymphocytes were introduced.
Both a highly effective proliferation inhibitor (6g05) and an
antibody preparation with little or no inhibitory activity (98g08)
were tested. A control having no antibody and no mouse CD83 protein
as well as a control with added mouse CD83 and no antibody was
included. Cellular proliferation of the activated lymphocytes was
assessed by observing the incorporation of tritiated thymidine as
described above. As shown in FIG. 26, the 6g05 antibody strongly
inhibits lymphocyte proliferation when no mouse CD83 is present.
However, when mouse CD83 is added before the lymphocytes, the 6g05
antibody exhibits little or no inhibition of lymphocyte
proliferation. These data indicate that the inhibitory activity of
the 6g05 antibody preparation operates through the CD83 gene
product, rather than through some non-specific interaction with
lymphocytes.
[0320] FIGS. 27 and 28 illustrate that anti-CD83 antibodies that
are multimerized by use of a rabbit anti-mouse antibody have
inhibitory activity that is like that of plate-bound anti-CD83
antibodies. The proliferation of lymphocytes was measured by
observing the incorporation of tritiated thymidine with and without
anti-CD83 antibodies as described above. In one set of assays
plate-bound 6g05 antibodies were used and in another soluble 6g05
antibodies were employed. The soluble 6g05 antibodies were
cross-linked by addition of rabbit anti-mouse antibodies that bind
to the Fc region of the 6g05 antibodies. For comparison, a soluble
and plate-bound antibody preparation with no inhibitory activity
(the 112D08 antibody preparation was also tested. A similar series
of assays were set up using a panel of soluble anti-CD83
antibodies.
[0321] As shown in FIG. 27, both plate-bound and crosslinked 6g05
antibodies were highly effective inhibitors of lymphocyte
proliferation. These data indicate that the method of aggregating
anti-CD83 antibodies is not particularly important. In other words
the multimerization can be achieved by adhering or attaching
antibodies to a solid support or by crosslinking the anti-CD83
antibodies through their Fc regions using a rabbit anti-mouse
secondary antibody. So long as the anti-CD83 antibodies are in
close proximity, they are effective inhibitors of lymphocyte
proliferation.
[0322] FIG. 28 shows that many soluble anti-CD83 antibodies exhibit
good inhibition of lymphocyte proliferation when they are
cross-linked with the rabbit anti-mouse secondary antibody. For
example, the 6g05, 11g04, 12g04, 14c12, 20d04, 32f12, 94c09, 94d08,
98a02, 98d11(3), 101B08(2.7) and 117g12 antibody preparations
strongly inhibit lymphocyte multimerization when cross-linked with
the rabbit anti-mouse antibodies.
EXAMPLE 8
Multimerized Anti-CD83 Antibodies Inhibit Proliferation of Immune
Cells in a Mixed Lymphocyte Reaction
[0323] This Example shows that multimerized anti-CD83 antibodies
inhibit proliferation of lymphocytes in a mixed lymphocyte reaction
(MLR) assay.
[0324] Materials and Methods
[0325] The MLR assay employed was a modification of the procedure
described in Bradley, pp 162-166 in Mishell et al., eds. Selected
Methods in Cellular Immunology (Freeman, San Francisco, 1980); and
Battisto, et al., Meth, in Enzymol. 150:83-91 (1987).
[0326] Spleens were removed from BALBc and C57B 16 mice and
digested with collagenase to liberate dendritic and CD4.sup.+
cells, respectively. Spleens were stained for surface expression of
CD4 (helper T cells) or CD11c (dendritic cells). Cells expressing
these markers were purified by using magnetic beads (Miltenyi)
according to the manufacturer's instructions.
[0327] Mixed lymphocyte cultures were set up using purified cell
populations. Plates with different anti-CD83 antibody preparations
bound thereto were prepared as described in the previous examples.
Approximately 1250 CD11c dendritic cells were used to stimulate
approximately 20,000 CD4+ T cells. After 4 days in culture,
proliferative responses were measured by incorporation of tritiated
thymidine. A positive control antibody, the anti-LFA antibody, was
also used for comparison purposes in this assay because it is known
to inhibit lymphocyte proliferation in MLR assays.
[0328] A similar experiment was performed to assess the recall
response of lymphocytes exposed to 100 .mu.g/ml anti-CD83
antibodies. Prior to spleen removal and CD 11 c and CD4+ cell
isolation, BALBc mice were first immunized with keyhole limpet
hemocyanin (KLH) in a 1:1 ratio with complete Freund's adjuvant
close to the lymph node area. Lymph nodes were harvested and
challenged in vitro with KLH at a final concentration of 2.5
.mu.g/ml and the proliferative response of the cells was assayed as
described above by observing incorporation of tritiated
thymidine.
[0329] Results
[0330] FIG. 29 shows that the conditions employed several
monoclonal anti-CD83 antibodies can inhibit lymphocyte
proliferation in a mixed lymphocyte reaction assay. For example,
the 98a02, 98d11, 20d04, 14c12, 12g04, and 117g12 inhibit
lymphocyte proliferation in this assay.
[0331] FIG. 30 shows that many anti-CD83 antibody preparations can
inhibit the recall response of lymphocytes. For example, 94c09,
98a02, 6g05, 20d04, and 117104 antibody preparations inhibited
proliferation of activated lymphocytes exposed to an antigen (KLH)
to which they had been immunized.
[0332] These data suggest that anti-CD83 antibodies can quiet the
proliferative response of CD4+ T cells after stimulation by
allogenic CD11 cells and/or antigen.
EXAMPLE 9
[0333] Exposure to Anti-CD83 Antibodies Does Not Cause Apoptosis of
Activated Lymphocytes
[0334] This Example shows that exposure to anti-CD83 antibodies
does not lead to apoptosis of activated lymphocytes.
[0335] Materials and Methods
[0336] Mouse (C57B 16) spleen cells were isolated and activated by
incubation for 24 hours with 1.0 .mu.g/ml Concavalin A in the
presence or absence of anti-CD83 antibodies and rabbit anti-mouse
antibodies as a crosslinking reagent as described above. Cells were
incubated for 48 hours at 37.degree. C. Proliferative responses
were measured by incorporation of tritiated thymidine. Total
caspase activity and annexinV expression levels were used as a
measure of apoptosis.
[0337] Homogeneous total caspase activity was measured using a kit
(Roche( following the manufacturer's instructions.
[0338] To test for apoptosis using annexinV expression, cells were
incubated with annexin-FITC and propidium iodide (AnnexinV-FITC
kit, Calbiochem) and the percentage of positive Annexin V-FITC
labeled cells was determined by Fluorescence Activated Cell sorting
(FACs).
[0339] Results
[0340] FIGS. 31A-B shows that soluble but cross-linked 6g05 and
14c12 anti-CD83 antibody preparations not only inhibit activated
lymphocyte cell proliferation (FIG. 31B) but also have very low
caspase activity (FIG. 31A). Similarly, FIG. 32 shows that the
percentage of activated lymphocytes that express annexinV is
reduced after treatment with soluble but cross-linked 6g05 and
14c12 anti-CD83 antibody preparations.
[0341] These data indicate that while anti-CD83 antibodies inhibit
proliferation of ConA activated splenocytes, they do not induce
apoptosis of immune cells. Instead, anti-CD83 antibodies actually
depress the expression of apoptosis markers. Hence, the reduction
in cell proliferation observed when activated lymphocytes are
exposed to anti-CD83 antibodies is not due to increased programmed
cell death.
EXAMPLE 10
[0342] : Exposure to Anti-CD83 Antibodies Does Not Inhibit
Activation of Lymphocytes
[0343] This Example shows that exposure to anti-CD83 antibodies
does not inhibit activation of lymphocytes.
[0344] Materials and Methods
[0345] Mouse (B6) spleen cells were isolated and activated using
Concavalin A as described above in the presence or absence of
anti-CD83 antibodies and the secondary anti-mouse crosslinking
antibodies. The anti-CD83 antibody preparations employed included
the 6g05, 14c12, 98b11 and 112d08 preparations. Activation of the
cells was assessed using CD69 expression as a marker of cell
activation.
[0346] Results
[0347] FIG. 33 illustrates that splenocytes activated with
Concavalin A express the CD69 activation marker even though they
were incubated with anti-CD83 antibodies. In particular, the star
or asterisks in the lower right hand corner of the graph shows the
level of CD69 expression observed when splenocytes are not
activated with Concavalin A. However, when splenocytes were
activated with Concavalin A they expressed high levels of CD69 even
after incubation with any of the 6g05, 14c12, 98b11 or 112d08
anti-CD83 antibody preparations.
[0348] These results indicate that while cellular proliferation of
lymphocytes exposed to anti-CD83 antibodies is arrested, the
lymphocytes still undergo activation.
EXAMPLE 11
Anti-CD83 Antibodies Arrest the Lymphocyte Cell Cycle in the G0/G1
Stage
[0349] This Example shows that exposure to anti-CD83 antibodies
arrests activated lymphocytes in the G0/G 1 stage of the cell
cycle.
[0350] Materials and Methods
[0351] Mouse (B6) spleen cells were isolated and activated by
incubation for 48 hours with 1.0 .mu.g/ml Concavalin A in the
presences of anti-CD83 antibodies with the crosslinking antibodies
as described above. To analyze cell cycle distribution, cells were
fixed and DNA was stained with propidium iodine according to the
protocol described for the flowcytometer (Cold Spring Harbor,
N.Y.). WinMDI software was used for background subtraction caused
by debris in the DNA histogram. Each histogram was further analyzed
by cycle red software to obtain the distribution of cells therein.
In addition, the size and shape of the activated cells was assessed
by their forward (FSC) and side (SSC) scatter during this
experiment.
[0352] The anti-CD83 antibody preparations employed were the 6g05
and 14c12 preparations that had been shown to inhibit cellular
proliferation and the 112d08 preparation that had little or no
effect on cellular proliferation. Cells having 2N complement of DNA
were assumed to be in the G 1/G0 phase of the cell cycle; cells
having 3N complement of DNA were assumed to be in the G2/M phase of
the cell cycle; and cells having 4N complement of DNA were assumed
to be in the S phase of the cell cycle. The percentage of cells
having G1/G0, G2/M or S phase of the cell cycle was determined and
plotted in FIGS. 35A-C.
[0353] Results
[0354] FIG. 34 shows that a population of activated splenocytes
mixed with anti-CD83 antibody preparations have lost the blasting
(dividing) cells as detected by FACS sorting. Almost all cells sort
as small cells with a 2N content of DNA as illustrated by the high
proportion of cells towards the left (smaller) side of the
population distribution in FIG. 34.
[0355] FIGS. 35A-C show that treatment of Concavalin A activated
lymphocytes with either of 6g05 and 14c12 antibody preparations
leads to a cellular population that was enriched in cells in the G
1/G0 stage of the cell cycle. Treatment with either the rabbit
anti-mouse antibody or the 112d08 antibody preparation that has
little or no effect on cell proliferation did not lead to a
cellular population that was enriched in cells in the G1/G0 stage
of the cell cycle.
[0356] These data indicate that exposure to anti-CD83 antibodies
arrests lymphocytes in the G1/G0 stage. Taken together with the
data in preceding Examples, these data indicate that anti-CD83
antibodies can cause lymphocytes to enter a state of antigen
specific unresponsiveness or anergy.
[0357] All patents and publications referenced or mentioned herein
are indicative of the levels of skill of those skilled in the art
to which the invention pertains, and each such referenced patent or
publication is hereby incorporated by reference to the same extent
as if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Applicants
reserve the right to physically incorporate into this specification
any and all materials and information from any such cited patents
or publications.
[0358] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims. As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "a host cell" includes a plurality (for example, a culture or
population) of such host cells, and so forth. Under no
circumstances may the patent be interpreted to be limited to the
specific examples or embodiments or methods specifically disclosed
herein. Under no circumstances may the patent be interpreted to be
limited by any statement made by any Examiner or any other official
or employee of the Patent and Trademark Office unless such
statement is specifically and without qualification or reservation
expressly adopted in a responsive writing by Applicants.
[0359] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
[0360] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0361] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
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