U.S. patent application number 11/108257 was filed with the patent office on 2006-02-09 for methods of using human receptor protein 4-1bb.
This patent application is currently assigned to Indiana University Research and Technology Corporation, Indiana University Research and Technology Corporation. Invention is credited to Byoung S. Kwon.
Application Number | 20060029595 11/108257 |
Document ID | / |
Family ID | 21724204 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060029595 |
Kind Code |
A1 |
Kwon; Byoung S. |
February 9, 2006 |
Methods of using human receptor protein 4-1BB
Abstract
Disclosed herein are the methods of using the H4-1BB protein,
ligands to this protein, and various mAbs either directed against
H4-1BB or other molecules that can be used therapeutically. The
nature and importance of the H4-1BB molecule provides the ligands
and related co-stimulatory molecules the ability to enhance or
suppress T-cell activation and proliferation. By treating T-cells
that have expressed receptor protein H4-1BB with one of the four
anti-H4-1BB monoclonal antibodies disclosed herein activation or
inhibition of the immune response is seen. Also disclosed herein is
cDNA for the human receptor H4-1BB. The cDNA of the human receptor
H4-1BB is about 65% homologous to the mouse cDNA 4-1BB and was
isolated by using probes derived from murine cDNA 4-1BB. A fusion
protein for detecting cell membrane ligands to human receptor
protein H4-1BB was developed. It comprises the extracellular
portion of the receptor protein H4-1BB and a detection protein,
alkaline phosphatase, bound to the portion of the receptor protein
H4-1BB. B-cells that have expressed a ligand to receptor protein
H4-1BB can be treated with cells that have expressed receptor
protein H4-1BB and B-cell proliferation may be induced. The use of
H4-1BB to block H4-1BB ligand binding has practical application in
the suppression of the immune system during organ transplantation
or against autoimmune diseases including diabetes, rheumatoid
arthritis, and lupus. Other applications of this technology include
the development of therapeutic methods for the treatment of HIV-1
infected individuals, and the treatment of cancerous tumors.
Inventors: |
Kwon; Byoung S.; (Carmel,
IN) |
Correspondence
Address: |
BARNES & THORNBURG
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
US
|
Assignee: |
Indiana University Research and
Technology Corporation
|
Family ID: |
21724204 |
Appl. No.: |
11/108257 |
Filed: |
April 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09877338 |
Jun 8, 2001 |
6905685 |
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11108257 |
Apr 18, 2005 |
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09007097 |
Jan 14, 1998 |
6303121 |
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09877338 |
Jun 8, 2001 |
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08409851 |
Mar 23, 1995 |
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09007097 |
Jan 14, 1998 |
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08122796 |
Sep 16, 1993 |
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09007097 |
Jan 14, 1998 |
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08012269 |
Feb 1, 1993 |
6362325 |
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08122796 |
Sep 16, 1993 |
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07922996 |
Jul 30, 1992 |
6355476 |
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08012269 |
Feb 1, 1993 |
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07267577 |
Nov 7, 1988 |
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07922996 |
Jul 30, 1992 |
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Current U.S.
Class: |
424/130.1 ;
424/144.1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 29/00 20180101; A61K 2039/505 20130101; C07K 14/52 20130101;
C07K 16/2878 20130101; C07K 16/2818 20130101; A61P 19/02 20180101;
A61P 37/02 20180101; C07K 16/2809 20130101; C07K 2317/74 20130101;
C07K 16/28 20130101; A61P 37/06 20180101; A61P 31/18 20180101; C07K
14/70578 20130101; C07K 2319/00 20130101; C07K 14/715 20130101;
A61P 17/00 20180101; A61P 3/10 20180101; A61K 38/00 20130101 |
Class at
Publication: |
424/130.1 ;
424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of enhancing T-cell activation comprising administering
an effective amount of a first H4-1BB receptor ligand such that
said receptor ligand comes into contact with at least one T-cell,
thereby activating it.
2. The method claim 1 wherein said 144-1BB receptor ligand
administered is an agonistic anti-4-1BB monoclonal antibody.
3. The method claim 1 wherein said H4-1BB receptor ligand
administered is an antagonistic anti-4-1BB monoclonal antibody.
4. The method of claim 1 wherein said first H4-1BB receptor ligand
is a H4-1BB protein.
5. The method of claim 1 wherein said first H4-1BB receptor ligand
is administered at a dosage range equivalent to or greater than
0.20 mmol to 2.0 .mu.mol, one to three times per day.
6. The method of claim 5 wherein the administration of said first
H4-1BB receptor ligand is accomplished through an administration of
a pharmaceutical formulation such as a tablet or intravenous
injection, wherein administration of said first H4-1BB receptor
ligand does not lessen the effectiveness of said first H4-1BB
receptor ligand in activating said at least one T-cell.
7. A method of enhancing T-cell activation of claim 1 further
comprised by administering a second stimulatory molecule, in
conjunction with said first H4-1BB receptor ligand such that each
of these compounds comes into contact with said at least one
T-cell.
8. The method of claim 7, wherein said second stimulatory molecule
is selected from the group consisting essentially of: a) an
anti-CD3 antibody; b) an anti-CD28 antibody; and c) the CD28
protein.
9. The method of claim 5 wherein said first H4-1BB receptor ligand
is administered at a dosage range equivalent to or greater than
0.20 .mu.mol to 2.0 .mu.mol, one to three times per day, and
wherein said second stimulatory molecule is administered at a
dosage range equivalent to or greater than 0.10 .mu.mol to 2.0
.mu.mol, one to three times per day.
10. The method of claim 5 wherein the administration of said first
H4-1BB receptor ligand and said second stimulatory molecule is
accomplished through an administration of a pharmaceutical
formulation such as a tablet or intravenous injection.
11. The method of claim 9, further comprising the use of a third
stimulatory molecule, said second co-stimulatory molecule being an
anti-CD3 antibody and said third stimulatory molecule being an
anti-CD28 antibody.
12. The method of claim 11 wherein the administration of said first
H4-1BB receptor ligand, said second stimulatory molecule, and said
third stimulatory molecule is accomplished through an
administration of a pharmaceutical formulation such as a tablet or
intravenous injection.
13. A method of treating cancerous tumors such that said cancerous
tumors are reduced, comprising the administration of a first
effective amount of H4-1BB receptor ligand such that said compound
comes into contact with at least one T-cell, and wherein said
H4-1BB protein works in conjunction with a second stimulatory
molecule, such that both of these compounds come into contact with
said at least one T-cell.
14. The method of claim 13, wherein said second stimulatory
molecule is selected from the group consisting essentially of: a)
an anti-CD3 antibody; b) an anti-CD28 antibody; and c) the CD28
protein.
15. The method of claim 13 wherein said first H4-1BB receptor
ligand is administered at a dosage range equivalent to or greater
than 2.0 .mu.mol to 8.0 .mu.mol, one to three times per day, and
wherein said second stimulatory molecule is administered at a
dosage range equivalent to or greater than 0.10 .mu.mol to 2.0
.mu.mol, one to three times per day.
16. The method of claim 13 wherein the administration of said first
H4-1BB receptor ligand and said second-stimulatory molecule is
accomplished through an oral administration of a pharmaceutical
formulation such as a tablet or injection.
17. A method of enhancing cytokine production in CD+4 and CD+8
T-cells comprising: a) administering effective amounts of three
compounds contemporaneously, said three compounds comprising: i) an
anti-H4-1BB antibody; ii) an anti-CD3 antibody; iii) an anti-CD28
antibody; and wherein said administration is such that each of said
three compounds comes into contact with said at least one T
cell.
18. The method of claim 17 wherein the cytokine whose production is
enhanced is selected from the group consisting of: a) gamma
interferon (IF); b) interleukin 1 (IL-1); c) interleukin 10
(IL-10); d) B cell growth factor (BCGF); e) B cell differentiating
factor (BCDF); and f) interleukin 2 (IL-2).
19. A method of treating an autoimmune reaction comprising
administering an effective amount of an antagonist to the H4-1BB
protein, said antagonist being capable of preventing the H4-1BB
protein from binding to a H4-1BB receptor, wherein said antagonist
is itself incapable of activating CD4+ or CD8+ T cells.
20. The method of claim 19 wherein said first H4-1BB receptor
ligand is administered at a dosage range equivalent to or greater
than 0.20 .mu.mol to 2.0 .mu.mol, one to three times per day.
21. The method of claim 19 wherein the administration of said first
H4-1BB receptor ligand is accomplished through an administration of
a pharmaceutical formulation such as a tablet or intravenous
injection.
22. The method of claim 19 wherein said autoimmune reaction treated
is one associated with an autoimmune disease, wherein said
autoimmune disease is selected from the group consisting of. a)
Diabetes Melitus; b) Rheumatoid Arthritis; and c) Systemic Lupus
Erthyematosus.
23. The method of claim 19 wherein said method of preventing an
autoimmune reaction is used to suppress an autoimmune response
occurring after an organ transplantation.
24. A method for monitoring the level of progression of Acquired
Immune Deficiency caused by the pathogenic virus HIV-1 is
accomplished by measuring the level of H4-1BB expression in a known
quantity of tissue comprising: a) collecting a sample of CD8+ T
cells; b) fractionating cells and retaining the lysate to test for
the presence of the H4--1BB using a monoclonal antibody(s) directed
against said H4-1BB protein; c) attaching to said antibody(s)
another molecule capable of being detected by a scintillation
counter or fluorescent microscope or other means useful in
measuring the degree of antibody binding; and d) determining the
level of H4-1BB expression in said sample of CD8+ T cells for
comparison with a known measurement that reflects a normal level of
expression of H4-1BB expression in a same size sample of an
equivalent tissue type.
25. A method of preventing an autoimmune reaction comprising
administering an effective amount of an antagonist to the H4-1BB
protein, said antagonist being capable of preventing the H4-1BB
protein from binding to the H4-1BB receptor ligand, wherein said
antagonist is itself incapable of activating CD4+ or CD8+ T
cells.
26. A method of interfering with HIV-1 progression comprising the
step of administering an effective amount of an agent capable of
binding said H4-1BB receptor protein on CD4+ T-lymphocytes, thereby
blocking it.
27. The method of claim 26 wherein said agent is selected from the
group consisting of: a) a 4-1BB-Fc molecule, b) a blocking
anti-4-1BB monoclonal antibody; and c) a fusion protein comprising
a portion of said H4-1BB protein.
28. An antibody that is immuno-reactive with a purified human 4-1BB
polypeptide comprising the N-terminal amino acid sequence
Phe-Glu-Arg-Thr-Arg-Ser-Leu-Gln-Asp-Pro-Cys-Ser-Asn-Cys-Pro-Ala-Gly-Thr.
29. A method of blocking T cell activation comprising administering
an effective amount of an H4-1BB protein antagonist such that said
protein antagonist prevents the activation of the H4-1BB
receptor.
30. A method of treating Human Acquired Immune Deficiency caused by
the viral pathogen HIV-1, comprising administering an effective
amount of a first H4-1BB receptor ligand, such that said receptor
ligand comes into contact with at least one T-cell thereby
activating at least one CD8+ T cell.
31. The method of claim 30 wherein said first H4-1BB receptor
ligand or agonistic mAb is administered at a dosage range
equivalent to or greater than 0.20 .mu.mol to 2.0 .mu.mol, one to
three times per day.
32. The method of claim 30 wherein said at least one CD8+ T cell is
capable of killing HIV-1 infected cells seleceted from the group
consisting of. a) CD4+ cells; b) astrocytes; c) macrophages; d)
dendritic cells; and e) microglial cells.
33. The method of claim 30 wherein the administration of said first
H4-1BB receptor ligand is accomplished through an administration of
a pharmaceutical formulation such as a tablet or intravenous
injection.
34. The method of claim 2 wherein said agonistic anti-4-1BB
monoclonal antibody is an monoclonal antibody designated BBK-1.
35. The monoclonal antibody of claim 34 further comprising a
hybridoma capable of producing said monoclonal antibody designated
BBK-1.
36. The method of using the monoclonal antibody of claim 34 to
enhance T-cell activation comprising the step of treating T-cells
that have expressed receptor protein H4-1BB with said monoclonal
antibody designated BBK-1.
37. The method of claim 3 wherein said antagonistic anti-4-1BB
monoclonal antibody is a monoclonal antibody designated BBK-2.
38. The monoclonal antibody of claim 37 further comprising a
hybridoma capable of producing said monoclonal antibody designated
BBK-2.
39. The method of using the monoclonal antibody of claim 37 to
enhance T-cell activation comprising the step of treating T-cells
that have expressed receptor protein H4-1BB with said monoclonal
antibody designated BBK-2.
40. The method of claim 3 wherein said antagonistic anti-4-1BB
monoclonal antibody is a monoclonal antibody designated BBK-3.
41. The monoclonal antibody of claim 40 further comprising a
hybridoma capable of producing said monoclonal antibody designated
BBK-3.
42. The method of using the monoclonal antibody of claim 40 to
enhance T-cell activation comprising the step of treating T-cells
that have expressed receptor protein H4-1BB with said monoclonal
antibody designated BBK-3.
43. The method of claim 2 wherein said agonistic anti-4-1BB
monoclonal antibody is an monoclonal antibody designated BBK4.
44. The monoclonal antibody of claim 43 further comprising a
hybridoma capable of producing said monoclonal antibody designated
BBK4.
45. The method of using the monoclonal antibody of claim 43 to
enhance T-cell activation comprising the step of treating T-cells
that have expressed receptor protein H4-1BB with said monoclonal
antibody designated BBK-4.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 08/409,851 filed Mar. 23, 1995 which was is a
co-pending application Ser. No. 08/122,796 filed Sep. 16, 1993,
which is a continuation-in-part of co-pending application Ser. No.
08/012,269 filed Feb. 1, 1993, which is a continuation-in-part of
co-pending application Ser. No. 07/922,996 filed Jul. 30, 1992,
which is a continuation-in-part of copending application Ser. No.
07/267,577 filed Nov. 7, 1988, now abandoned.
[0002] The subject matter described herein was in part a subject
invention of NIH Grants Nos. IR23AI23058-03, R01 AI-28175, R01
DE-12156, and P60 KD20542 of which the present inventor was the
Principal Investigator and either the Donald Guthrie Foundation for
Medical Research Inc. of Guthrie Square, Sayre, Penn. 18849-1669 or
Indiana University School of Medicine of Indianapolis, Ind. 46202,
was the Grantee.
FIELD OF THE PRESENT INVENTION
[0003] The present invention relates to the therapeutic and
scientific uses for the human H4-1BB protein, its ligands, and the
development of monoclonal antibodies that recognize and bind the
H4-1BB receptor protein.
BACKGROUND OF THE PRESENT INVENTION
[0004] The immune system of humans and other species require that
white blood cells, which include phagocytes, T lymphocytes and B
cells, be made in the bone marrow. The phagocytes include
macrophage cells which scavenge unwanted materials, such as virus
proteins or bacterial cell walls from the system. The lymphocytes
include helper T cells, killer T cells and B cells, as well as
other types of cells, including those categorized as suppressor T
cells. The B cells produce the antibodies. The killer T cells
physically destroy target cells and the helper T cells facilitate
the whole process. The complexities of the immune system and its
function is facilitated, at least in part, by the lymphokines.
[0005] Lymphokines are signal transduction proteins by which the
immune cells communicate with each other. Scientists have been able
to produce them in sufficient quantities for therapeutic use
against immunologic diseases. There are many known lymphokine
proteins and they include the interferons,
interleukins-1,2,3,4,5,6,7, colony-stimulating factors,
lymphotoxin, tumor necrosis factor and erythropoietin, as well as
others.
[0006] Interleukin 1 (IL-1), secreted from macrophages activates
the helper T cells and acts to raise body temperature, causing
fever, which enhances the activity of the immune cells. The
activated helper T cells produce Interleukin 2 (IL-2), which in
turn stimulates the helper and killer T cells to grow and divide.
The helper T cells also produce another lymphokine, B cell growth
factor (BCGE), which causes B cells to multiply. As the number of B
cells increases, the helper T cells produce another lymphokine
known as the B cell differentiating factor (BCDF), which instructs
some of the B cells to stop replicating and start producing
antibodies.
[0007] T cells also produce gamma interferon (IF), which is similar
to Interleukin 2 in that it has multiple effects. Gamma interferon
helps activate killer T cells, enabling them to attack the invading
organisms. Like BCGF, gamma interferon increases the ability of the
B cells to produce antibodies. IF also keeps the macrophages at the
site of the infection and helps the macrophages digest the cells
they have engulfed. Gathering momentum with each kind of lymphokine
signal between the macrophages and the T cells, the lymphokines
amplify the immune system response such that the virus protein, an
infected cell, or other foreign matter is overwhelmed and removed
from the system. There are many lymphokines, maybe a hundred or
more, which participate in the complex web that is the immune
process. Many lymphokines and their precise effects remain
unknown.
[0008] Lymphokine activities are produced when a certain lymphokine
binds to its specific receptor on the surface of a target cell.
Among scientists there is widespread use of cloned cell lines for
production of lymphokines and their receptors. The isolation of
lymphokine and lymphokine receptor mRNA has become a common
technique. The mouse receptor protein, 4-1BB, was isolated and
identified based on specific expression of the T cell genes using a
technique identified by the present inventor in a prior publication
(Proc. Natl. Acad. Sci. USA. 84, 2896-2900, May 1987, Immunology).
The protocol reported in this publication can be used by scientists
to detect virtually all of the lymphokines. The method is designed
to detect virtually all mRNA expressed differentially. Importantly
the mRNA sequences of immune cells are expressed differentially as
they relate to T cells generally, and to the killer T cells
specifically. Even though the level of expression is low and the
quantity of the lymphokine and its receptor protein is low, this
expressed mRNA can be detected and isolated. The present inventor
believes that the analysis described in the above identified
publication can reveal biologically important molecules such as
lymphokines and their receptors because there are many indications
that biologically important or active molecules are initiated by
cellular signals induced by very scarce message molecules (i.e. IF,
interleukins, Map Kinase Kinase, etc.,).
[0009] Most T cell factors have been classically identified by
recognizing biologic activities in assays, and thereafter purifying
the protein information. An alternative approach is to isolate
putative T cell genes based upon specific expression, insert them
into an appropriate expression vector, and then demonstrate the
function of the unknown isolated protein. Using the aforesaid
modified differential screening procedure, the present inventor
cloned a series of T cell subset-specific cDNAs from cloned helper
T (HTL) L2 cells, and cloned cytolytic T lymphocytes (CTL) L3.
[0010] T cells are critically important in long term acquired
immunity, providing protection against viral, bacterial and
parasitic infection. T cells are activated when they encounter a
peptide from the invading pathogen in context with self-MHC (Major
Histocompatibility Complex) via the T cell's own T cell receptor
(TCR) complex and other co-stimulatory molecule(s), such as CD-28,
or CD-3. Without the engagement of the other co-stimulatory
molecule(s) the T cell is rendered anergic (Vassali et al., 1979
PNAS). To date, the best-characterized co-stimulatory molecule has
been CD-28. More recently, however, other cell surface molecules
have been suggested to play a co-stimulatory role, such as the
molecule 4-1BB. The 4-1BB protein is a -55 kDa homodimeric molecule
expressed on activated T cells in the mouse, and is a member of the
Nerve Growth Factor receptor (NGFR)/Tumor Necrosis Factor receptor
(TNFR) gene super family (Haskins et al., 1983, J. Exp. Med.). This
family is characterized by the presence of cysteine-rich motifs in
the extracellular domains. Other members of this family include
NGFR, B cell activation molecule CD40, the T cell activation
molecule OX-40 in rat and CD27, the two receptors for TNF called
TNFR-1 and TNFR-11, the apoptotic inducing protein Fas, and CD-30
which plays a role in the regulation of cellular growth and
transformation.
[0011] Some of these members have been shown to play important
roles in HIV-1 infection, including CD4+ T cell proliferation,
apoptosis and virus replication. The presence of high serum levels
of CD30 has become a predictor of progression to AIDS, although no
circulating, CD30 cells have been found: in HIV-1 seropositive
individuals. The expression of HIV-1 was induced by triggering CD30
of HIV-1 infected CD4+ T cells through a NF-.kappa.B-dependent
pathway. In HIV-1 individuals, high levels of Fas expression were
observed in peripheral blood lymphocytes. Fas production was found
to trigger or induce marked apoptosis of T lymphocytes, which might
contribute to the CD4+ T cell depletion by HIV-1 infection. The
ability of CD4+ T cells to express the CD40 ligand after in vitro
stimulation is not impaired because of HIV-1 infection, but
CD40/CD40 ligand interaction regulates HIV-1-replication of B cells
in vitro. CD27 signaling enhanced proliferative response of T cells
to the normal extent in HIV-1-infected individuals.
[0012] In the experiments that led to the development of this
invention, a series of T cell subset-specific cDNAs were isolated
from cloned murine T-cells by employing a modified differential
screening procedure. The nucleotide sequence and expression
properties of some of the cDNA species have been reported. One of
the genes not previously characterized, which encodes mouse
receptor protein 4-1BB, was studied further. These studies have led
to the isolation of the human homologue to 4-1BB, H4-1BB, as well
as to a series of monoclonal antibodies capable of binding the
H4-1BB receptor protein and acting thereby as agonists or
antagonists of H4-1BB.
[0013] T cells interact with components of the extracellular matrix
(ECM) through members of the integrin family after transendothelial
migration during homing to sites of inflammation. Integrin
molecules are very late antigens (VLA's) in a family of cell
surface receptors that mediate the adhesion of cells to ECM
proteins as well as other cells. The heterodimeric integrins
comprising of various alpha and beta subunits, act as a transducing
mechanism of extracellular signals. Regulation of integrin function
is utilized by T cells and other leukocytes for rapid adhesion
following activation of the cells.
[0014] The major factors known so far to affect the differentiation
of the T cells are the lymphokines (also referred to as cytokines),
such as IL-2 and L-4. In vitro and in vivo studies with transgenic
mice have demonstrated that IL-2 induces the development of the Th1
subset of T cells by priming them for efficient IF production and
preventing development of IL-4-producing cells. Previously however,
it was unknown how their interactions worked to direct the
amplification of the immune response and development of the Th1 or
Th2 subset of T cells.
[0015] Specific immune responses are governed by the recognition of
antibodies to foreign antigens. Antibodies form a family of
structurally related glycoproteins and confer, generally to the
organism producing them, the protective effect of cell-mediated
immunity. Antibodies are produced by B-lymphocytes and are bound to
the cell membrane, functioning as B cell receptors for antigens.
Antibodies are also secreted by B cell progeny that differentiate
in response to stimulation by antigens. A specific antigen will
trigger the complementary B lymphocyte(s) to proliferate and
differentiate into effector cells, which then eliminate the
antigen. Each lymphocyte produces an antibody of a particular
specificity, and thus immune responses are very specific for
distinct antigens. The portion of the antigen recognized by T and B
lymphocytes are called epitopes or determinants.
[0016] The development of techniques to produce virtually unlimited
amounts of a single (monoclonal) antibody for a specific antigenic
epitope has had an enormous impact on clinical immunology. To
produce a monoclonal antibody of known specificity, a mouse can be
injected with a particular antigen, such as a receptor protein and
the spleen B lymphocytes (that produce the antibody against the
protein) can be fused via somatic cell hybridization to a myeloma
(lymphocyte tumor) to produce an immortal cell line to create a
hybridoma. This is done because normal B-lymphocytes can not grow
indefinitely, yet when fused with the myeloma, the resulting
hybridoma produces a virtually endless supply of a specific
monoclonal antibody. Selection techniques have been developed to
ensure that only the fused cells continue to grow. Each hybridoma
cell is specific for only one antigenic determinant. If several
different antibody producing hybridomas are produced, each
hybridoma clone of an individual B lymphocyte will secrete an
antibody for only one surface antigenic determinant. To determine
which mAbs specifically bind to the protein receptor, or which has
a desired activity (e.g. the mAb acts as an agonist, antagonist, or
has the most specific binding to a critical epitope), the
hybridomas can be screened with an ELISA (enzyme-linked
immunosorbent assay).
[0017] Monoclonal antibodies, have numerous applications: 1) The
hybridoma can produce large quantities of specific antibodies that
are normally either unavailable in small quantities or not
available at all; 2) the hybridoma can be directed to produce
antibodies against a single antigen determinant which, for complex
antigens, may be normally very difficult; 3) pure antibodies can be
obtained against antigens that cannot be purified; 4)
immuno-diagnosis of infectious and systemic diseases by detecting
specific antigens circulating in tissues or using monoclonal
antibodies in immunoassays; 5) characterization of protein
receptors and the role they play in the transition from a naive to:
a memory T cell; and 6) blocking or enhancing immune response or
activation.
[0018] The invention below presents uses for the H4-1BB protein,
its ligands, antibodies thereto and other co-stimulatory molecules
that can be used therapeutically in the treatment of cancer and
HIV-1.
SUMMARY OF THE PRESENT INVENTION
[0019] The present invention includes the human receptor protein
H4-1BB and the cDNA gene encoding for human receptor protein
H4-1BB. The nucleotide sequence of the isolated cDNA is disclosed
herein along with the deduced amino acid sequence. The cDNA gene
identified as pH4-1BB was deposited at the Agricultural Research
Service Culture Collection and assigned the accession number: NRRL
B21131
[0020] The cDNA, including its fragments and derivatives, can be
used as a probe to isolate DNA sequences encoding for proteins
similar to the receptor protein. The cDNA of the human receptor,
H4-1BB, was isolated by using probes derived from cDNA 4-1BB. The
cDNA gene identified as p4-1BB was deposited at the American Type
Culture Collection at 12301 Parklawn Drive, Rockville, Md. 20852
under ATCC No.: 67825.
[0021] The human receptor protein H4-1BB can be produced by: 1)
inserting the cDNA of H4-1BB into an appropriate expression vector,
2) transfecting the expression vector into an appropriate
transfection host, c) growing the transfected hosts in appropriate
culture medial and d) purifying the receptor protein from the
culture media. The protein and fragments and derivatives can be
used: 1) as a probe to isolate ligands to human receptor protein
H4-1BB, 2) to stimulate proliferation of B-cells expressing H4-1BB
ligands, or 3) to block H4-1BB ligand binding.
[0022] B-cell proliferation can be induced by treating B-cells that
have expressed a ligand to receptor protein H4-1BB with cells that
have expressed receptor protein. H4-1BB. The use of H4-1BB protein,
H.sub.41BB ligand protein, or fragments of the proteins, to block
H4-1BB ligand binding has practical application in the suppression
of the immune system during organ transplantation.
[0023] Monoclonal antibodies generated against H4-1BB can be used
to enhance or suppress T-cell proliferation and activation by
treating T-cells that have expressed receptor protein H4-1BB with
an anti-H4-1BB monoclonal antibodies. To enhance immune reaction
antibodies which act as agonists can be generated, to suppress
T-cell proliferation and/or activation antibodies which act as
antagonists can be generated. To this end, four monoclonal
antibodies have been developed for use. The monoclonal antibodies
BBK-1 and BBK4 are agonists to receptor protein H4-1BB, while
monoclonal antibodies BBK-2 and BBK-3 are antagonists to receptor
protein H4-1BB, and can be used to either upregulate the immune
system or suppress its activity. Some tumors are potentially
immunogenic but do not stimulate an effective anti-immune response
in vivo. Tumors may be capable of delivering antigen-specific
signals to T cells, but may not deliver the co-stimulatory signals
necessary for full activation of T cells. A monoclonal antibody
generated against H4-1BB (e.g. an agonist) is capable of
eradicating tumors with low immunogenicity by providing for the
full activation, enhancement, and/or proliferation of T-cells.
Moreover, an anti-H4-1BB mAb agonist has great utility in assessing
the role of the 4-1BB receptor protein in the transition from naive
to memory T-cells. Cross-linking of the 4-1BB with an anti-H4-1BB
mAb agonist, such as BBK-1 or BBK-4 will produce the effects
similar to the binding of the 4-1BB ligand to 4-1BB.
[0024] A mAb aganist H4-1BB can also be used to interfere with
H4-1BB and H4-1BB ligand binding. By interfering with ligand
binding, as with the use of an anti-H4-1BB mAb antagonist BBK-2,
and BBK-3, the immune responses will be suppressed. In this
context, diseases that would benefit from the therapeutic use of
such a mAb include rheumatoid arthritis, systemic lupus
erythematosus, and diabetes. Alternatively this type of molecule is
useful in organ transplantation to suppress immune system mediated
rejection of transplanted tissue.
[0025] A fusion protein can detect cell membrane ligands to human
receptor protein, H4-1BB. A fusion protein of the present invention
comprises the extracellular portion of the receptor protein H4-1BB
and a detection protein (alkaline phosphatase) or Fc portion of an
IgG, bound to the portion of the receptor protein H4-1BB. In
addition, this disclosure demonstrates that co-engagement of CD28
with 4-1BB promoted type 1 effector T cell development. The 4-EBB
signal regulated CD28 mediated cytokine production profiles in two
ways, enhancing type 1 and, at the same time, suppressing type 2
cytokine (lymphokine) production. The 4-1BB-mediated co-stimulation
also induced .gamma.-interferon (IF) production in Th1 cells. The
expression of 4-IBB was subset-specific, being detected
predominantly on IF producing, but not on IL-4-producing cells.
Moreover, it was determined that 4-1BB and CD30 expression were
mutually exclusive, representing type 1 and type 2 subsets,
respectively. The co-engagement of 4-IBB with CD28 enhanced
long-term cell survival for cells susceptible to apoptosis induced
by repeated TCR activation. Therefore, it was demonstrated that
4-IBB and CD30 interplay to regulate the balance between type 1 and
type 2 T cell subsets, and the polarization of the immune response.
Therapeutically they can be used to achieve opposite effects.
[0026] This invention presents data, which demonstrates that there
is a functional correlation between 4-1BB and CD28 in T cell
adhesive responses. The inventors disclose herein that 4-1BB can
induce of cell adhesion. Enhanced cell adhesion through the
presence 4-1BB signal or agonist results in a maximal T cell
activation in response to repeated exposure to sub-optimal
concentrations of anti-CD3 and anti-CD28. Thus, 4-1BB effects were
attributed by its ability in reducing the threshold of anti-CD3
concentration needed to repeatedly activate primary T cells.
Therefore the degree of cell adhesion in response to anti-4-1BR
correlated with the 4-1BB expression levels, and correspondingly
effects the therapeutic use of these molecules.
[0027] The level of 4-1BB expression and the percentage of
4-EBB-expressing T cells was higher in HIV-1 positive individuals
than in the HIV-1 controls (P<0.01). 4-1BB signal cooperated
with CD28 to induce mV-1, and CD4+ T cell proliferation. In
addition, cross-linking 4-1BB with agonistic monoclonal antibodies
enhanced HIV-1 replication both in primary stimulation and
secondary re-stimulation of CD4+ T cells from HIV-1 individuals.
Thus, 4-1BB is involved in the activation of HIV-1 replication from
latently infected CD4+ T cells, and the 4-1BB co-stimulatory
pathway can be the target of therapeutic intervention. If the
pathway of HIV-1 infection is disturbed at the early stages of the
infection, through the use of H4-1BB antibodies a lower virus load
will result. Moreover, the increased 4-1BB expression on CD8+ T
cells is noteworthy because it is correlated with the degree of
immunodeficiency in HIV-1 infection, cross-linking 4-1BB on CD8+ T
cells will induce enhanced cytotoxic activity against mV-1 infected
CD4+ T cells:
[0028] An object of the present invention is to teach a fusion
protein comprising the extracellular portion of H4-1BB and a
detection protein.
[0029] Another object of the present invention is to teach the
method of constructing a monoclonal antibody against H4-1BB.
BRIEF DESCRIPTIONS OF THE FIGURES
[0030] FIG. 1 illustrates the molecules involved in the cognitive
phase of T-cell activation.
[0031] FIG. 2 illustrates the molecules involved in the clonal
expansion of Tcells occurring in the late portion of JT-cell
activation.
[0032] FIG. 3 illustrates the resting portion of a normal T-cell
activation pathway.
[0033] FIG. 4 illustrates a "primed" T-cell during the normal
T-cell activation pathway.
[0034] FIG. 5 illustrates an activated T-cell which was activated
through the presence of a non-self, antigen presenting cell,
representing the conclusion of the normal T-cell activation
pathway.
[0035] FIG. 6 illustrates a anergized T-cell CTLA4-lg alone,
4-1BB/AP and CTLA4-lg together and 4-1BB/AP alone respectively
being used to block steps in the T-cell activation pathway.
[0036] FIG. 7 illustrates 4-1BB/AP and CTLA4-lg together in an
effort to block the T-cell activation pathway.
[0037] FIG. 8 illustrates 4-1BB/AP alone where it is used to block
steps in the T-cell activation pathway.
DETAILED DESCRIPTION
[0038] The following description teaches the isolation of 4-1BB and
its human homologue, H4-1BB, the preparation of the peripheral
blood cells, including the antibodies and reagents used, the
production of fusion protein, immunization, and the production of
monoclonal antibodies acting as either agonists or antagonists of
H4-1BB. Also disclosed is the therapeutic use of: 4-1BB, antibodies
to it, its ligands, and immunoprecipitation studies.
Isolation and Characterization of Mouse Receptor 4-1BB
[0039] SEQ ID NO: 1 shows the nucleotide sequence and the deduced
amino acid sequence of the mouse receptor 4-1BB. The predicted
amino acid sequence is shown below the nucleotide sequence. The
transcript of 4-1BB was inducible by concanavalin A in mouse
splenocytes, T cell clones, and hybridomas. The expression of 4-1BB
transcripts was inhibited by cyclosporin A. The 4-1BB mRNA was
inducible by antigen receptor stimulation but was not inducible by
IL-2 stimulation in the cloned T-cells (1). The 4-1BB cDNA encodes
a peptide of 256 amino acids containing a putative leader sequence,
a potential membrane anchor segment, and other features of known
receptor proteins. Therefore, the expression pattern of 4-1BB
resembles those of lymphokine mRNAs while the sequence appeared
consistent with those of receptor proteins.
[0040] The major species of 4-1BB on the cell surface appears to be
a 55-kDa dimer. 4-1BB also appears to exist as a 30 kDa monomer and
possibly as a 110-kDa tetramer. Since these 4-1BB species were
immunoprecipitated from a homogeneous population of cells (T-cell
clone f1), all forms potentially co-exist on each cell. Peptide
digests from the 4-1BB monomer and dimer are needed to determine
whether COBB exists as a homodimer on the cell surface. A variety
of cell surface receptors such as the insulin receptor (Ebina et
al., 1985), the B cell surface immunoglobin receptor (Vassali et
al.,), the T cell Ag receptor (Haskins et al., 1983), the CD-28
co-stimulatory receptor (Lesslaver et al., 1986), and the CD27
T-ell antigen (Van Lier et al., 1987) are composed of
disulfide-bonded subunits. Receptor dimerization may be required
for ligand binding and subsequent cell signal transduction.
[0041] 4-1BB is not expressed on resting T cells but is inducible
by activators which deliver a complete growth stimulus to the T
cell. The combination of PMA and ionomycin is capable of mimicking
those signals required for T cell proliferation. Although PMA or
ionomycin alone induced 4-1BB mRNA, the combination of PMA and
ionomycin resulted in optimal 4-1BB expression. Furthermore, the
expression of 4-1BB was not transient. When purified splenic
T-cells were stimulated with immobilized anti-CD3, 4-1BB mRNA was
expressed and this expression was maintained for up to 96 hrs
post-stimulation. Cell cycle analysis will be required to confirm
that 4-1BB is expressed throughout cell cycle progression.
[0042] 4-1BB is structurally related to members of the nerve growth
factor receptor super-family. Although these receptors possess
structurally similar ligand-binding properties (cysteine-rich
regions), the cytoplasmic, domains of these proteins are
non-conserved which could allow for diversity in transmembrane
signaling. Some members of this family are involved in the T or B
cell activation process. There are in vitro functional data on the
OX40, CD40 and CD27 antigens. Antibodies against the OX40 augment
the T-cell response in a mixed lymphocyte reaction and antibodies
against CD40 enhance B-cell proliferation in the presence of a
co-activator, such as PMA or CD20 antibodies, and synergizes with
IL-4 in vitro to induce B-cell differentiation and to generate
long-term normal B cell lines. One monoclonal antibody, anti-1A4,
which recognizes an epitope on the CD27 molecule inhibited calcium
mobilzation, IL-2 secretion, helper T cell function, and T-cell
proliferation. On the other hand, CLB-CD27/1, another anti-CD27 mAb
enhanced proliferation of human T cells stimulated with PHA or
anti-CD3 mAb. These results indicate that the CD27 molecule plays
an important role in T cell activation. Except for TNFR's, NCFR and
CD40, the ligands or cell surface molecules to which the members of
the superfamily bind are not yet identified. Identification and
characterization of the ligands to which the receptors bind will be
helpful in better defining the physiologic role of 4-1BB.
[0043] To ascertain whether cell surface 4-1BB could contribute to
T cell activation, the anti-4-1BB 53A2 was used as an antagonist to
4-1BB. The resulting data suggest that 4-1BB does in fact have the
potential to function as an accessory signaling molecule during T
cell activation and proliferation. The addition of soluble 53A2 to
purified splenic T cells stimulated with immobilized anti-CD3
resulted in an amplification of .sup.3H thymidine incorporation
compared to T cells stimulated with anti-CD3 alone. This pattern of
enhancement ranged from 2- to 10-fold in three independent
experiments.
[0044] In the original to signal model of Bretcher and Cohn, they
proposed that signal 1, the occupancy of the T cell antigen
receptor (TCR), resulted inactivation of the T cell in the absence
of signal 2, which is provided by accessory cells. This has since
been confirmed by a variety of studies (Moeller et al., 1989). The
identification of the accessory cell CD28 as a potent
co-stimulatory receptor on T cells was a significant contribution
in beginning to characterize the accessory signal(s) required for
optimal T cell proliferation. It is possible that other cell
surface molecules may contribute to these co-stimulatory activation
requirements.
[0045] The biochemical signals delivered through 4-1BB indicate
that there is a putative p56.sup.lck tyrosine kinase-binding domain
in its cytoplasmic tail. It was later determined that p56.sup.lcs
tyrosinase kinase-binds to 4-1BB. It will also be worthwhile to
determine if 4-1BB-mediated signaling can regulate genes such as
IL-2 and IL-2 receptor, whose expression is required for T cell
activation and subsequent proliferation.
[0046] The precise functions of members of the Nerve Growth Factor
Receptor (NGFR) superfaminly appear to be diverse. An emerging
theme of inquiry concerns the ability of these molecules to
maintain the responsiveness or viability of the particular cell
type in which they are expressed. For instance, NGF is absolutely
required for viability of neurons in vitro and in vivo (Yamori et
al., 1992). The cross-linking of CD40 by soluble antiCD40
monoclonal antibody blocks germinal center centrocytes from
undergoing apoptosis in vitro. Signals delivered through CD40 may
also aid in maintenance of responsiveness to differentiation
factors. The ligation of CD40 with anti-CD40 F(ab').sub.2 fragments
in the presence of IL-4 induced large increases IgE synthesis.
Also, anti-CD40 activated naive B cells treated with IL-10 and
transforming growth factor-.beta. became committed to IgA secretion
(DeFrance et al., 1992). In addition to sharing the molecular
characteristics with the NGFR superfamily, it was noted that the
4-1BB contained a putative zinc finger structure similar to that of
the yeast elF-2b protein. 4-1BB also shares a conserved region with
the sina seven in absentia of Drosophila Melanogaster, which is
required for correct photoreceptor cell development (Carthew and
Rubin, 1990). That particular region is also similar to the protein
product of the DGl7 gene of Dictyostelium, whose expression is
specifically induced during aggregation by cAMP.
[0047] This region forms the pattern of
C-X.sub.2-C-X.sub.9-C-X.sub.3-H-X.sub.3-C-X-C; and the cysteines
and histidine are conserved in a similar space in 4-1BB, sina, and
DGl7 proteins. Ten of 24 amino acids between the 4-1BB and sina
proteins are identical; 3 of 24 are conservative substitutes. The
conserved pattern suggests that these amino acids are functionally
important. The sina protein is localized in the nucleus, suggesting
that it has a regulatory function in cells. The fact that the amino
acid sequence of 4-1BB contains features like a zinc finger motif,
a nuclear protein, and a receptor domain suggests that 4-1BB may
play diverse roles during cellular proliferation and
differentiation.
[0048] In addition, 4-1BB may represent another cell-surface
molecule involved in T cell-APC interactions. The 4-1BB-AP fusion
protein specifically bound to mature B-cell lines,
anti-IF-activated primary B cells, and mature macrophage-cell
lines. 4-1BB-AP bound at low or insignificant levels to immature B-
and macrophage-cell lines, T cell clones, T cell lines, primary
culture T cells, and various non-lymphoid-cell lines. Since
4-1BB-AP binds to mature B cells and macrophages, it is possible
that signals delivered upon COBB binding may modulate APC functions
in some way. This possibility remains to be explored.
[0049] Chalupny et al., proposed that 4-1BB Rg, a fusion protein
consisting of the extracellular domain of 4-1BB and the Fc region
of human IgG, bound to the extracellular matrix (ECM). The highest
level of 4-1BB Rg binding was to human vitronectin. The inventors
performed an ELISA to test this possibility using 4-1BB-AP and
human vitronectin (Yelios Pharmaceuticals/GIBCO-BRL, Grand Island,
N.Y.) immobilized at 0.007 mg, with 10 mg per well on microtiter
plates. No binding of 4-1BB-AP based on AP activity was observed.
To rule out the possibility that 4-1BB-AP was binding to proteins
extrinsically attached to the cell surface (possible extracellular
matrix components), B-cell lymphomas were washed in acid conditions
prior to the binding assay. 4-1BB-AP still bound specifically to
mature B-cell lymphomas. It is still to be determined whether a
4-1BB-ligand specifically expressed on B cells and macrophages
exists, and whether 4-1BB-AP may bind to the ECM under particular
binding conditions. It is possible that the ECM could facilitate
the binding of 4-1BB to a specific cell-surface ligand.
[0050] B cells and helper T cells interact with each other through
receptors on B cells binding to their specific counter-receptors on
T cells. This interaction-results in a cascade of biochemical
signaling relays between the two cell types. As this interaction
proceeds, these cells become committed to enter the S-phase of the
cell cycle. Initial interactions between TCR and CD4+ on T cells,
and processed antigen-MHC II on B cells, do not result in B cells
capable of entering the cell cycle (Noelle and Snow et al., 1990).
However, studies from in vitro systems suggest that once T cells
are stimulated, they express newly synthesized or modified
cell-surface molecules capable of inducing B cells to enter the
cell cycle. This T cell function is not antigen-specific or
MHC-restricted. In addition, soluble factors are not required for
the Th induction of B-cell activation. Once B cells enter the cell
cycle, IL-4 induces B cells to progress from G.sub.1 to S phase.
The ability of activated T cells or T-cell membranes to promote the
entry of B cells into the cell cycle can be blocked by either
cycloheximide or cyclosporin A treatment. These newly expressed
membrane proteins appear to be "lymphokine-like" in their induction
characteristics.
[0051] 4-1BB has expression properties which meet the requirements
of a B-cell co-stimulator. 4-1BB is inducible by anti-CD3 or
TCR-mediated T-cell stimulation, and its expression is sensitive to
cyclosporin A as well as cycloheximide treatment. Interestingly,
paraformaldehyde-fixed SF21-4-1BB cells, synergized anti-.mu. and
induced B-cell proliferation. The co-stimulation of splenic B cells
by SF21-4-1BB occurred at optimal (10 .mu.g/ml) and sub-optimal
(1.0-0.1 m/ml) doses of anti-.mu.. The addition of SF21-4-1BB cells
to resting B cells, did not result in significant B-cell
proliferation. SF21-4-1BB cells did not synergize with TPA or
ionomycin, or sub-optimal concentrations of LPS in inducing B-cell
proliferation.
[0052] Although the baculovirus system has been used to express
large amounts of recombinant soluble proteins, this system may be
utilized for the expression of recombinant cell-surface proteins.
The baculovirus infection provides a convenient means to express
uniformity high levels of recombinant protein on a per cell basis.
It is noteworthy, that the addition of SF21 cells alone did not
result in significant levels of co-stimulation. This can be a
potential problem when using COS- or L- cell lines which can
exhibit strong co-stimulation activity on their own.
[0053] Another member of the NGFR superfamily, CD40, is expressed
on B cells and interacts with gp39, a molecule expressed on
activated T-cells. The cDNAs encoding the murine and human gp39
proteins have been cloned; this cell surface molecule is a type II
membrane protein with homology to tumor necrosis factor. Noelle et
al., found that a CD40-immunoglobulin fusion protein, is capable of
blocking T cell-induced B-cell proliferation and differentiation in
a dose-dependent manner. Armitage et al. have isolated a cDNA for
murine gp39 and showed that gp39 could induce B-cell proliferation
in the absence of co-stimuli, and result in IgE production in the
presence of IL-4-. Hollenbaugh et al., have shown that COS cells
transfected with human gp39 can synergize with either TPA or
anti-CD20 in inducing human B-cell proliferation and is able to
stimulate B cells without a co-stimulator only at low levels. These
data indicate that CD40 may be one of the B-cell-surface molecules
that transmit signals during physical contact with T cells.
[0054] Cell-surface receptors communicate with their external
milieu by interacting either with soluble factors or other cell
surface-molecules expressed on neighboring cells. The role of
biochemical signals delivered by cell-cell contact versus those
delivered by soluble factors interacting with cell surface
receptors is not clear. The NGFR superfamily is unusual for the
TNRF I and II as well as the NGFR bind to more than one ligand. The
TNFRs I and II both bind to TNF-a and TNF-R. The NGFR binds to NGF,
brain-derived neurotrophic factor, and neurotrophin-3.
[0055] In addition, one ligand may function as both a cell surface
and soluble ligand. Recent evidence on the CD40 ligand, gp39,
suggests that this ligand can exist as a membrane bound as well as
a soluble ligand. It may be possible that 4-1BB is secreted and
interacts with B cells in a soluble form as well as a membrane
bound form. A member of the NGFR receptor family, CD27, which is
expressed on T cells, is secreted in addition to being expressed on
the cell surface (Hintzen et al., 1991). It is also possible that
more than one 1 ligand, if soluble and on the cell surface, may
bind to 4-1BB.
Isolation of the Human Homologue, H4-1BB
[0056] In order to isolate the human homologue (H4-1BB) of mouse
4-1BB two sets of polymerase chain reaction (PCR) primers were
designed. To design the PCR primers, the amino acid sequence among
the members of nerve growth factor receptor (NGFR) superfamily were
compared because 4-1BB is a member of the superfamily (Mallett and
Barclay, 1991). The amino acid sequences employed were mouse 4-1BB,
human NGFR, human tumor necrosis factor receptors, human CD40, and
human CD27. The areas of sequence conservation among the NGFR
superfamily were chosen.
Materials and Methods
[0057] Peripheral blood lymphocytes from normal healthy individuals
were isolated and activated with PMA (10 ng/ml) and ionomycin (1
mM). Messenger RNA from the lymphocytes was isolated. Using reverse
transcriptase the human lymphocyte mRNA was converted to
single-stranded cDNA. The cDNA was then amplified with Taq
polymerase with combination of the primers. A combination of
primers was used-and produced a specific band of .about.240 bp. The
240 bp is an expected size of human 4-1BB if the human homologue
protein is similar to mouse 4-1BB in size. The PCR product (240 bp)
was cloned in PGEM3 vector and sequenced. One open reading frame of
the PCR product was .about.65% identical to mouse 4-1BB. Therefore,
it was concluded that the 240-bp PCR product is the human homologue
of mouse 4-1BB. The 240-bp PCR product was used to screen
.lamda.gt11 cDNA library of activated human T lymphocytes. An 0.85
kb cDNA was isolated. The sequence of the cDNA is shown in SEQ. ID
NO: 7 and the predicted amino acid sequence is shown in SEQ. ID NO:
8.
[0058] An expression plasmid to produce H4-1BB-AP fusion protein
was constructed. The 5' portion of the H4-1BB cDNA including
sequences encoding the signal sequence and the entire extracellular
domain, was amplified by PCR. For correctly oriented cloning, a
Hind III site on the 5' end of the forward primer and a Bg1 II site
on the 5' end of the reverse primer were created.
[0059] The Hind III-Bg1 II H4-1BB fragment was inserted into the
mammalian expression vector APtaq-1, upstream of the coding
sequence for human placental alkaline phosphatase (AP).
[0060] H4-1BB-AP will be used to identify cells and tissues that
express, ligand for human 4-1BB (i.e. H4-1BBL). The studies with
mouse 4-1BB indicated that the ligand for 4-1BB is on the cell
surface. B cells and macrophages were major cells that express
4-1BBL. It is expected that H4-1BBL be expressed on human B cells
and macrophages.
Results
[0061] A mammalian expression cDNA library was generated from human
cell lines that express H4-1BBL. The library was screened by Iodine
labeled H4-1BB-AP. cDNA for H4-1BBL was then be isolated and
characterized. Soluble recombinant H4-1BBL was then be produced.
The generated antibodies were then used to suppress or enhance
immune responses as described below. Monoclonal antibodies to
H4-1BBL were produced and are discussed below.
[0062] According to studies completed by the inventor, 4-1BB acts
as a co-stimulatory signal. It is expected then that H4-1BB acts as
a co-stimulatory signal for T cell activation. Mouse 4-1BB helped B
cells with proliferation and differentiation. H4-1BB has been found
to do the same. H4-1BB-AP, H4-1BBL and various monoclonal
antibodies disclosed below can be used to suppress or enhance human
immune responses.
EXAMPLE 1
Production of a Monoclonal Antibody to H4-1BB
[0063] The 4-1BB molecule is expressed on activated but not resting
murine T cells, while cross-linking of 1 AH2 mAb directed against
murine 4-1BB has been shown to enhance-anti-CD3-induced T cell
proliferation. Normal splenic cell antigen presentation and T cell
activation can be blocked by inhibiting the binding of 4-1BB on T
cells to its ligand on B cells and macrophages with 4-1BB/AP. This
protein 4-1BB/AP is a fusion protein containing the extracellular
domains of 4-1BB and alkaline phosphatase. Human 4-1BB mAbs were
characterized, and then isolated.
Materials and Methods
Production of Recombinant Human 4-1BB
[0064] The PGEX-3 expression vector (Pharmacia) containing the full
length cDNA sequence encoding 4-1BB and the: GST-binding domain of
glutathionine S-transferase (GST) was constructed and the fusion
protein expressed in bacteria. Fusing H4-1BB with GST, allowed for
efficient purification of a rH4-1BB when isolated by GST-sepharose
and a Sepharose-4B column chromatographies. The GST-binding domain
is cleaved prior to immunization. The rH4-1BB fraction is purified
by GST-sepharose column and Sepharose 4B column chromatographies
and subsequently cleaved with factor Xa to release the H4-1BB
portion prior to immunization.
[0065] BALB/c animals are immunized with a rH4-1BB protein and the
splenocytes fused with the Sp2/0 fusion partner. BALB/c mice should
be immunized with 50 .mu.g of sH4-1BB emulsified in Titermax
(Cytkx) or "Complete Friends" adjuvant. Three intraperitoneal (ip)
injections should be administered 2-weeks apart. Three days
following the last injection, the mouse host is sacrificed and
their spleens are removed. Spleen cells were fused with Sp2/0
myeloma cells. Spleen cells and Sp2/0 are mixed at 5:1 ratio and
fused using 50% PEG. Cells are then washed, re-suspended in OptiMEM
(Gibco), 10% FCS, 5 mM hypoxanthine, 1% aminopterin, and 0.8 mM
thymidine (HAT) and cultured in 96 well U-bottom plates (Corning).
Resulting cell supernatants were screened by ELISA for rh4-1BB
reactivity. Clones were isolated and subcloned.
Activated: T cells Co-express 4-1BB+ and CD45RA and CD45RO
[0066] It has been shown previously that murine 4-1BB is associate
with p56.sup.lck by a series of immune-precipitation studies and
peptide mapping study. The data gathered indicates that 4-1BB forms
a multi-peptide complex with CD45 and p56.sup.lck on activated T
cells. To better assess the association of 4-1BB and CD45 in
humans, PBMCs stimulated with PHA for 48 hrs were analyzed for
expression of CD45RA and CD45RO isoforms by multi-color FCM.
Sixteen to 19% of cells expressed 4-1BB, and nearly all (except 1%)
expressed CD45RA and nearly all express CD45RO after correcting for
non-specific binding of the antibodies.
Uses for an Anti-H4-1BB mAb
[0067] Although some mAbs specifically recognize 4-1BB expressed on
SF-21 cells, they do not recognize 4-1BB expressed on activated T
cells. This is likely due to the mAb having specificity for a
cryptic or unique bindings site(s) that is not exposed or present
on T-cells but is accessible or present on SF-21 cells due to
slight differences in glycosylation and processing between human T
cells and insect cells (SF-21).
[0068] In mice, neither 4-1BB mRNA nor surface expression is
detectable on resting splenocytes or unstimulated cloned T cells.
But upon activation of T cells by anti-CD3, anti-TNF.alpha. or
anti-TNF-.beta., 4-1BB mRNA is detected within 3 hrs of stimulation
and is first detectable on the cell surface 2-3 days following
stimulation. Maximum surface expression is reached about 6 days
following stimulation. As in the mouse, 4-1BB is not detected on
the surface of freshly isolated peripheral blood T cells in man,
but is readily detected following PHA-stimulation. Unlike in the
mouse, 4-1BB is expressed much more rapidly in humans, reaching a
peak expression level within 12-48 hrs. 4-1BB expression begins to
decrease within 72 hrs., post-stimulation, as do the number of
cells expressing 4-1BB on their cell surface. In both mouse and
humans, 4-1BB, is expressed on CD4+ and CDS+ T cell subsets.
[0069] 4-1BB is associated with p56.sup.lck. A 56 kDa protein is
detected when [.sup.32]PO.sub.4 was transferred from gamma-labeled
ATP onto the p56 protein in ConA activated thymocytes that were
subjected to immunoprecipitated with anti-4-1BB mAb, 1AH2. By
peptide mapping, this 56 kDa phosphoprotein was identified as
p56.sup.lck. The p56.sup.lck and 4-1BB molecules were also found to
be co-immunoprecipitated in insect cells studies (SF-21) and in
HeLa cells transfected with 4-1BB and p56.sup.lck. Furthermore,
cross-linking of 4-1BB activated p56.sup.lck. Cysteine residues
critical for p56.sup.lck --CD4/CD8 complex formation were also
critical for p56.sup.lck-4-1BB interaction. In preliminary results,
it was noted that anti-4-1BB also immunoprecipitated a protein of
-200 kDa from biotin-surface labeled ConA activated thymocytes.
When anti-CD45 mAb was used for immunoprecipitation, a .about.30
kDa protein, of similar size to murine 4-1BB, was detected. Other
have previously shown that CD45 mediates the dephosphorylation of
certain proteins such as p56.sup.lck (Biffen et al., 1994). Perhaps
4-1BB plays a role in bringing CD45 and p56.sup.lck together and
facilitates the dephosphorylation of p56.sup.lck by the CD45
phosphatase.
[0070] To further assess the association of 4-1BB and CD45,
PHA-stimulated PBMCs were analyzed by multicolor FCM. Approximately
16-19% of PBMCs cultured in PHA for 48 hrs express 4-1BB. If all
4-1BB+ cells express CD45RA and express CD45RO, the .about.17.5% of
4-1BB+cells must co-express both CD45RA and CD45RO on their cell
surface. Of the PHA-stimulated CD45RA.sup.hiRO.sup.hi cells,
approximately 50% express 4-1BB. This data further supports the
hypothesis that CD45 and 4-1BB share an association. More
importantly, it suggests that 4-1BB may play a role in T cell
transition from a naive phenotype (CD45RA.sup.hiRO.sup.hi) to a
memory phenotype (CD45RA.sup.10RO.sup.hi). Picker et al.,
previously demonstrated through multi-color FCM, that naive T cells
undergo a "stepwise, unidirectional progression" from a naive
(CD45RA.sup.loRO.sup.hi) to a memory (CD45RA.sup.10RO.sup.hi)
phenotype through a distinct CD45RA.sup.hiRO.sup.hi intermediate
cell type. Peripheral blood few cells that express this
intermediate phenotype are detectable. However, in secondary
lymphoid tissue, such as tonsil, 2-10% of T cells were found to be
CD45RA.sup.hiRO.sup.hi. Much is know about the naive and memory T
cells, but little is known about the CD45RA.sup.hiRO.sup.hi in
transitional cells. Nor is it known what events occur during this
transition phase that result in memory T cell development.
Therefore, it will be necessary to assess the role of 4-1BB in the
transition from a naive to that of a memory T cell and the apparent
association of 4-1BB and CD45. In this regard, anti-H4-1BB mab's
are invaluable.
Results
[0071] The anti-H4-1BB mAb can be used to enhance T-cell
cross-linking and therefore induce T-cell activation against
certain types of cancer cells (e.g. melanoma). By using the mAb in
experiments with various cancer cells in the presence of T-cells
dosages and proper formulations of initiating T-cell activation
against the cancer cells can be determined. The formulations are
tested in animal models with the same type of cancer and the
formulations and dosages are refined for testing in humans.
[0072] Sinovial T-lymphocytes in patients with rheumatoid arthritis
express H4-1BB, but 4-1BB is not expressed in sinovial
T-lymphocytes, of patients: without this disease. This disease
involves an undesired immune response against the patient's own
tissue. Therefore, blocking the undesired immune response would
provide relief for the arthritis sufferer. By injecting the patient
with an anti-H4-1BB mAb or the fusion protein, the binding between
H4-1BB and its ligand would be blocked. If the binding of an mAb
and H4-1BB did not enhance activation of the immune system, then
the anti-H4-1BB mAb interference with binding would have the
desired effect, otherwise the fusion protein would be used for
blocking binding. The fusion protein (monomeric) should not
stimulate H4-1BB or its ligand but is a good ligand binding blocker
because it binds to the H4-1BB ligand thereby preventing H4-1BB
from binding and stimulating the ligand. A similar method of
blocking ligand binding would be useful for treating patients with
systemic lupus erythematosus. For patients with Type I
diabetes--T-cells attack their own insulin producing cells,
pancreatic Beta cells. By injecting the mAb or fusion protein this
destruction can be blocked.
[0073] Peripheral blood T cells in patients with AIDS or certain
types of viral flu are expressing H4-1BB, whereas the same cells in
normal patients are not expressing H4-1BB. Therefore, 4-1BB is
important in this immune response. The enhancement or blocking of
H4-1BB ligand binding or cross-linking will be important in
regulating the T-cells in patients with these diseases.
EXAMPLE 2
Uses of H.sub.41BB Antibodies in the Suppression of Immune
Responses
[0074] FIGS. 1 and 2 illustrate the molecules involved in
T-cell-activation. During early T-cell activation (cognitive
phase), resting T cells express the TCR/CD3 complex and other
"accessory" molecules. Among these constitutively expressed
molecules, CD4+ (or CD8+), LFA-1, and CD28 are probably the ones to
receive co-stimulatory signals. Initial interaction with the
TCR/CD3 complex in combination with these `accessory`
co-stimulatory signals leads to subsequent expression of additional
receptor molecules such as CD28, CTTA4, and 4-1BB. These newly
expressed molecules-will receive additional important
co-stimulatory signals at later stages of T-cell activation, such
as during clonal expansion.
[0075] FIGS. 3-5 illustrate a normal T-cell activation pathway.
FIGS. 6-8 illustrate the blocking of immune responses with soluble
chimera of 4-1BB. If 4-1BB plays a role in T-cell activation,
blocking of the interaction to its ligand on antigen-presenting
cells will result in suppression of T-cell dependent immune
responses. It is well documented that blocking of the interaction
of CD28 to its counter-receptor B7 suppresses in varying degrees,
both in vivo antibody production and cell-mediated immune
responses. Blocking of both interactions should result in a more
effective immunosuppression; since 4-1BB is induced during T-cell
activation. Blocking of the interaction of 4-1BB to its ligand is
of importance at later stages of the activation process where the
CD28AB7 interaction is no longer be of relevance.
[0076] As illustrated with mouse receptor 4-1BB and mouse ligand
4-1BBL above, addition of H4-1BB-AP will coat the H4-1BBL
expressing cells and block the normal interaction between
H.sub.41BB and H4-1BBL. This will lead to immunosuppression. This
type of immunosuppression is antigen-specific. Therefore, it avoids
the generalized immunosuppression produced by anti-CD3 or
cyclosporin A treatments. H4-1BB-AP treatment can be used to treat
certain autoimmune diseases and to facilitate organ
transplantation.
Binding Activity
[0077] The portion of the receptor protein H4-1BB binds to the cell
membrane ligands and binding can be detected by relative activity
assays for the detection protein. The fusion protein is placed in
the presence of a cell suspected to express the receptor protein
H4-1BB. Then the cell is washed of any fusion protein not bound to
the cell membrane ligands. Once the washed cells are placed in the
presence of a substrate for the detection protein and the relative
activity of the detection protein can be measured.
Enhancement of Immune Reaction
[0078] H4-1BB may function at the late stage of T cell activation
and may be a critical molecule for completion of T cell activation.
Most tumors display tumor-specific antigens. One reason, however,
why immunogenic tumors can escape host immunity is that
tumor-reactive T cells receive inadequate co-stimulation. The
introduction of the co-stimulatory molecules, such as H4-1BB into
the tumor, therefore, could enhance the antitumor immunity of
cytotoxic T cells (CTL) by upregulating activity. H4-1BBL can be
expressed in cell-specific fashion. For example, the H4-1BBL can be
expressed in melanoma using melanocyte-specific promoter such as
tyrosinase promoters. The H4-1BBL-expressing melanoma will
stimulate cytotoxic T cells through H4-1BB and activate the
melanoma-specific CTL. The activated melanoma-specific CTL can then
destroy the target cancer (e.g. melanoma).
EXAMPLE 3
Co-Stimulation of CD28 with Human 4-1BB to Promote Type 1
Cytokines
[0079] An investigation was made to determine the role of H4-1BB in
CD28 stimulation. Also studied was whether CD28 requires an
additional co-stimulatory signal to promote human effector T cell
development. In that effort it was known that the cytokine IL-2
cooperates with CD28 in inducing IF production in Th1 cells. in
vitro, it was determined that IL-2 is not an absolute requirement
for antigen-induced priming of a Th1 response, although its
presence during priming enhances the ability of antigen-primed Th1
cells to produce IF (gamma interferon). IF produced by Th1 cells
amplifies Th1 development and inhibits proliferation at Th2 cells,
whereas IL-4 produced by Th2 cells blocks development of Th1 cells.
Once a T cell immune response begins to develop to Th1 or Th2 it
tends to become progressively polarized in that direction. It
remains unclear whether initial cytokine secretion of T cells is
determined by an independent regulatory process.
[0080] T cell activation requires a signal delivered through TCR
and a second signal, mostly through CD28, referred to as
co-stimulation. T cells activated in vitro in the absence of a CD28
signal are defective in their response to forthcoming antigenic
stimulus and are characterized as anergetic (e.g. "anergized").
When human T cells were repeatedly reactivated in vitro with
anti-CD28, the process reduced IL-2 production, and induced IL-4 to
produce Th2-life cells. Studies of response to antigen specific
cytokine production in CD28-deficient mice demonstrated that IL-4
and, to a lesser extent, IF production are augmented by
CD28-mediated signals.
[0081] CD28 signaling has recently been shown to prevent apoptosis,
a phenomenon known as activation-induced cell death (AICD) both in
anti-CD3-activated murine and human T cells. Repeated TCR
engagement results in an accumulation of cells that express Fas or
a prolonged unresponsiveness to CD28 signaling. The loss of CD28
responsiveness and acquisition of Fas might result normally after
prolonged stimulation, providing a mechanism to prevent the
reactivation of effector T cell populations. Therefore,
co-engagement of additional co-stimulatory factors to CD28 should
be required to protect the cells from AICD for long-term T cell
maintenance and effector T cell differentiation.
[0082] The CD30 molecule is preferentially expressed by human CD4+
and CD8+ clones with Th2-type cytokine profile. There is an inverse
correlation between CD30 expression and production of IF. The
expression of 4-1BB and CD30 is equally activation-dependent and is
confined predominantly to CD45R0+cells. Exposure to 4-1BB supported
IL-2 production and proliferation of murine splenic T cells. The
4-1BB molecule is expressed both on CD4+ and CD8+ T cells and is
associated with p56.sup.lck.
[0083] It is disclosed herein that 4-1BB plays regulatory roles
with CD28-mediated co-stimulation to specifically promote type 1
cell development, as well as to prevent AICD. 4-1BB-mediated type 1
responses were not observed in CD30-positive cells. The current
results indicate that 4-1BB and CD30, whose expressions are
mutually exclusive, may counteract to regulate the balance of types
1 and type 2 development.
Materials and Methods
Antibodies and Reagents
[0084] Monoclonal anti-4-1BB, was used to ligate 4-1BB and stain
4-1BB on T cells for flow cytometric analysis. Anti-CD28, mAb 9.3
(mouse IgG2.) was a kind gift from Dr. Carl H. June, and CD28.2
(mouse IgG.sub.1) was purchased from PharMingen (San Diego,
Calif.). Monoclonal antibody to human CD3 (OKT3, mouse IgG.sub.1)
was purchased from Ortho Diagnostic (Westwood, Mass.). Secondary
cross-linking goat anti-mouse IgG (H+L), and anti-mouse
IgG.sub.1-FITC for flow cytometry were purchased from Zymed (South
San Francisco, Calif.) and Southern Biotechnology Associates
(Birmingham, Ala.), respectively. Anti-IF-Phycoerythrin (PE),
anti-IL-4-PE, anti-CD4-Cy-Chrome, and PE labeled mouse IgG.sub.1
isotype control (NOPC-21) were purchased from PharMingen. 4-1BBFc,
a fusion protein consisting of the extracellular portion of human
4-1BB coupled with Fc region of human IgG, (38), was obtained from
Immunex (Seattle, Wash.). An isotope control mouse IgG, was
purchased from Sigma (St. Louis, Mo.). A premixed cocktail of
monoclonal antibodies, and complement (LymphoKwik) to isolate T
helper cells was purchased from One Lambda (Canoga Park,
Calif.).
Cells
[0085] Human PBMC were isolated from buffy coats of healthy donors
by Histopaque-1077 (Sigma) density centrifugation. CD4+ T cells
were purified from PBMC by depleting CD8+ T cells, B, and
macrophage cells by the corresponding monoclonal antibodies and
complement. The purity of CD4+ T cells was about 85%, as determined
by flow cytometric analysis. The purified T cells or PBMC at
1.times.10.sup.6 cells/ml were activated by phytohemagglutinin
(PHA, Calbiochem) at 5 .mu.g/ml for 4 days and, after washing to
remove PHA, were subsequently expanded in the presence of IL-2 at
100 .mu.l/ml from 3 to 10 days depending on the following
experiments with replacement of fresh IL-2 every 3 days. The
resulting cells with >95% T cells are hereinafter referred to as
"PHA/IL-2 cells." For the proliferation assays, cells were cultured
for 10 days in IL-2 until all the clustered PHA blasts became
completely dispersed, with reduced cell sizes before
reactivation.
Reactivation of T Cells
[0086] The PHA/IL-2 cells from purified CD4+ T cells or PBMC were
reactivated with 1 .mu.g/ml soluble anti-CD3 mAb in the presence of
5-fold excess goat anti-mouse IgG in the plates (Costar, Cambridge,
Mass.) coated with isotype control mouse IgG.sub.1, (MOPC-21), or
antibodies to CD28 in the presence of additional antibodies to
4-1BB, CD30 or a combination of the two antibodies, 10 .mu.g/ml
each at 4.degree. C. overnight. After 3- to 5-day anti-CD3
activation, the cells were transferred to new plates coated with
antibodies in the same way to repeat another cycle of reactivation,
if necessary.
Cytokine Production
[0087] Following anti-CD3 reactivation of PHA/IL-2 cells in 24-well
plates coated with an isotype control mouse IgG.sub.1, anti-4-1BB,
anti-CD28, and a combination of anti-4-1BB and anti-CD28 at 10
.mu.g/ml each antibody, conditioned media were collected in order
to measure IL-2, IF, TNF-.alpha., IL-4, and TGF-.beta.. The
cytokine IL-2 was assayed by an IL-2-dependent cell line, CTLL-2
and the rest of cytokines were measured by commercially available
ELISA kits; (Endogen), TNF-.alpha. (Genzyme), IL-4 and TGF-.beta. R
and D Systems).
Cell Proliferation Assay
[0088] The primary T cells were repeatedly activated by three
consecutive cycles as described above. After each three cycles of
anti-CD3 activation, cells were collected and reactivated by
antiCD3, 1 .mu.g/m and cross-linking anti-mouse IgG, 5 .mu.g/ml to
measure the responsiveness to the immobilized anti-CD28 or
anti-4-1BB or both antibodies in anti-CD3-mediated TCR activation
in 96-well microtiter plates (5.times.10.sup.4 cells/200 ul/well).
Anti-CD28 at designated concentrations with or without additional
anti-4-1BB (10 .mu.g/ml) was coated onto 96-well plates in PBS at
4.degree. C. overnight. Incorporation of [.sup.3H] thymidine was
measured for the last 6 hours of the 3-day culture.
Flaw Cytometry
[0089] For measuring 4-1BB expression, approximately
2.times.10.sup.6 cells reactivated in the different co-stimulatory
conditions were washed, suspended in a 100 .mu.l of 2 .mu.g/1 ml
anti-4-1BB in staining solution (PBS containing 1% BSA), and
incubated at 4.degree. C. for 30 minutes. The cells-were
subsequently washed three times, re-suspended in 200 .mu.d of
FITC-conjugated anti-mouse IgG.sub.1, 1 .mu.g/ml, and incubated for
30 minutes. After being washed, the samples were fixed with 1%
paraformaldehyde prior to flow cytometric analysis on the FACScan
(Becton Dickinson, Mountainview, Calif.). Gates were set on live
cells only, based on forward-versus-side scatter profiles. In every
case at least 10,000 events were collected for each sample. For
measuring intracellular IF and IL-4 levels, the protocols
recommended by the manufacturer were followed (incorporated herein
by reference).
[0090] PBMC activated with PHA were repeatedly activated for 3 days
by anti-CD3 and anti-CD28 with or without anti-4-1BB, collected,
washed, and stained for surface 4-1BB, and CD30 with
anti-4-1BB-FITC and anti-CD30-FITC, respectively as described
above. To identify 4-1BB and CD30, two-color staining with
anti-4-1BB-biotin and streptavidin-PE for 4-1BB and anti-CD30-FITC
for CD30 was used. Following the fixing of the cells with 4%
paraformaldehyde and permeabilization with 0.1% saponin, the cells
were further stained with IF-PE or IL-4-PE at 1 .mu.g/ml, in 0.1%
saponin. Cells were finally re-suspended in PBS containing 1% BSA
and analyzed for two-color stained surface markers and
intracellular cytokines by FACS can. In some cases, the cells were
stained for CD4 in addition to 4-1BB or CD30 with
anti-CD4-Cy-Chrome before intracellular staining.
Results
[0091] The maximal expression of 4-1BB was seen after repeated
activation by its own signal (e.g. positive feedback loop). CD28 is
expressed on the majority of naive and memory T cells. In contrast
to CD28, 4-1BB, another co-stimulatory molecule, were not detected
in naive T cells freshly prepared from healthy volunteers. The
activation conditions for the maximum 4-1BB expression on human T
cells were also investigated. Expression of 4-1BB was induced by
PHA or anti-CD3 stimulation with maximum plateau levels after 3 to
4 days. At peak, less than 20% of T cells were 4-1BB-positive.
[0092] Upon resting in IL-2 (100 u/ml) for 10 days, following PHA
stimulation (referred to as PHA/IL-2 cells), 4-1BB expression
declined to less than 5% of T cells positive. Reactivation of
PHA/IL-2 cells by anti-CD3 and anti-CD28 in the presence or absence
of anti-4-1BB gave rise to a remarkable increase of 4-1BB. More
than 50% of T cells became 4-1BB-positive in 3 days. The high 4-1BB
expression was transient, and continuous anti-CD3 reactivation with
anti-CD28 alone for the next cycle of reactivation did not maintain
4-1BB expression resulting in less than 10% 4-1BB positive cells.
In contrast, 4-1BB co-engagement with anti-CD28 co-stimulation
resulted in a dramatic difference, leading to a continuous increase
in 4-1BB expression to higher than 60% with repeated activation.
This finding indicates that high 4-1BB expression requires repeated
TCR activation with co-stimulatory signals from both CD28 and
4-1BB. Anti-CD3 activation with 4-1BB co-stimulation without CD28
involvement resulted in only modest effects on the 4-1BB
induction.
[0093] It was therefore determined that the 4-1BB signal plays an
important role in its own expression, with CD28 remaining
essential. The high 4-1BB expression caused by co-stimulation of
its own signal with CD28 may provide the means for polarizing to
4-1BB-expressing T cells through positive feedback loops during
repeated antigen challenge in vivo. The 4-1BB signal regulated CD28
co-stimulation to enhance type 1 cytokine but to suppress type 2
cytokine production.
[0094] PHA/L-2 cells were reactivated by anti-CD3 with
co-stimulatory antibodies to 4-1BB, or CD28, or a combination of
both 4-1BB and CD28. The cytokines secreted into the conditioned
media following 3-day-reactivation were assayed. Since 4-1BB is
induced both in CD4+ and CD8+ cells. The designation given the
effector phenotypes for CD4+ and CD8+ T cells was collectively type
1 and type 2, respectively, instead of Th1 or Th2. The 4-1BB signal
enhanced IL-2, TNF-.alpha., and IF, which are largely classified as
type 1 cytokines, several times higher than the levels induced by
CD28 co-stimulation alone. Among the induced cytokines, IF (gamma
interferon) was most significantly enhanced by 4-1BB (7.3-fold).
The 4-1BB alone, without CD28 co-stimulation, did not show
significant effect. It was surprising that additional 4-1BB signal
to CD28 instead suppressed IL-4, a type 2 cytokine and TGF-.beta.,
to a lesser degree, below the levels induced by CD28 co-stimulation
alone. The 4-1BB signal suppressed CD28-mediated IL-4 production in
a dose-dependent manner. These results indicate that 4-1BB plays a
regulatory role in inducing specifically type 1 and suppressing
type 2 cytokine production as well when co-engaged with CD28
co-stimulation.
[0095] The 4-1BB signal expanded IF-producing cell population
exclusively in CD30-negative cells. Concurrent enhancement of type
1 and suppression of type 2 cytokine production by 4IBB signaling
spurred the examination as to whether 4-1BB co-engagement with CD28
in co-stimulation was actually involved in the induction of type 1
subset T cell development. The IF-producing cells were identified
at the single-cell level by detecting intracellular IF by flow
cytometry.
[0096] PHA/IL-2 cells produced IF-producing cells in 3% of the
total population upon reactivation with anti-CD3 and anti-CD28. The
same cells reactivated by anti-CD3 and anti-CD28 with additional
anti-4-1BB, increased IF-producing cells to more than 15% of the
population. Therefore, enhanced IF in the culture medium as a
result of 4-1BB co-engagement to CD28 in co-stimulation may be
attributed to an increase of IF-producing cells. It is not known
whether IF-producing cells were developed directly by the 4-1BB
signal or indirectly by the promoted cytokines or a combination of
both.
[0097] The IF-producing cells generated by anti-4-1BB were further
identified by two surface markers, 4-1BB and CD30, another
inducible co-stimulatory molecule with structural homology to 4-1BB
as members of TNF receptor superfamily. Other investigators have
proposed CD30 as a potential Th2 marker. The majority of
IF-producing cells were 4-1BB-positive. Conversely to 4-1BB, the
same IF-producing cells appeared to be predominantly CD30 negative.
These results indicate that the 4-1BB signal is involved in
expanding specifically IF-producing but CD30-negative cells
possibly due to type 1 subset-specific 4-1BB expression. A positive
correlation between the intensities of intracpellular IF and
surface 4-1BB expression, as manifested in the dot plot by tilted
cone-shaped patterns was also found. The positive correlation of IF
and 4-1BB expression supports the view that the 4-1BB signal is
directly involved in de novo IF induction, as well as expansion of
IF-producing cells. The 4-1BB protein was expressed preferentially
on IF-producing, not on IL-4-producing T cells
[0098] Because 4-1BB-induced IF-producing cells lacked CD30, next
examined was whether 4-1BB expression was exclusively limited to
the cells with type 1 phenotype. For the purposes of this
disclosure gamma interferon and IL-4 cytokines were chosen as
representative marker cytokines for types 1 and 2 phenotypes,
respectively, and compared the level of 4-1BB expression in two
subset populations.
[0099] The PHA/IL-2 cells were reactivated by anti-CD3 with
anti-CD28 and anti-4-IBB for 3 days, and were stained for surface
4-1BB and, subsequently identified by marker cytokines, IF or IL.
The level of 4-1BB expression in the populations gated out for IF-
and IL-4-positive cells were compared. A significant difference in
4-1BB expression levels between the IF and IL-4-producing cells was
found. The 4-1BB expression was highly enriched in IF-positive
cells (61.7%) compared to IL-4-producing cells (22.7%). These
results indicate that 4-1BB is not randomly, but preferentially
expressed on type 1 subset cells. 4-1BB and CD30 are mutually
exclusive in expression.
[0100] The results indicate that 4-1BB meditates type 1 T cell
differentiation based on the preferential 4-1BB expression on the
IF-producing cells and its ability in inducing IF production. On
the other hand, other groups have demonstrated that CD30 is a Th2
marker or a marker for the IL-4 response. Also examined was whether
4-1BB and CD30 expression were exclusively divided into two
different subsets of T cell populations. The surface expression of
4-1BB and CD30 by flow cytometry in CD4+ T cells using two-color
staining was analyzed. Both 4-1BB and CD30 were expressed on CD4+
and CD4- cells that were more than 95% CD8+. The correlation of
4-1BB and CD30 expression in CD8+ cells was analyzed because 4-1BB
expressed predominantly on CD8+ cells. 4-1BB and CD30-expressing
cells were largely divided into two separate groups with only a
minor cell population shared with both 4-1BB and CD30. These CD4+
cells also gave rise to similar 4-1BB and CD30 expression patterns
with mutually exclusive expression with one another. The results
demonstrated that 4-1BB and CD30 were mutually exclusive in
expression on T cells, and this mutual exclusiveness of the two
molecules did not result from their differential dominance in
expression between CD4+ and CD8+ cells.
4-1BB Signaling Down-Regulated CD30-Positive Cell Population
[0101] Considering that both 4-1BB and CD30 are co-stimulatory
molecules, the possibility of cross-regulation between 4-1BB and
CD30 in developing CD30-producing cell populations was examined. To
do this the levels of CD30-expressing cell development were
measured by flow cytometry during early activation in response to
4-1BB and CD30 signaling. PHA-activated (2 days)
PBMC-were-immediately reactivated for 2 days with anti-CD3 and
anti-CD28 plus additional antibodies to 4-1BB or CD30, or both
4-1BB and CD30. It was determined that the 4-1BB and CD30 signaling
counteracted one another in the levels of CD30-producing cell
development. 4-1BB signaling clearly down-regulated the CD30 levels
below the levels of the cells reactivated with CD28 alone. CD30
signaling increased CD30-positive cell population in exactly the
same manner that 4-1BB-induced 4-1BB expression. The 4-1BB signal
reversed the effect of CD30 when both 4-1BB and CD30 signals were
present. 4-1BB and CD30 signaling might cross-regulate each other's
functions, by down-regulating the cell population with counterpart
molecules through the release of antagonistic cytokines, for
example, IF and IL-4. The results support that 4-1BB and CD30 may
be primarily responsible for the maintenance of the subset cells in
expressing their own molecules. Regulation in co-engagement of
4-1BB and CD30 with CD28 during repeated TCR activation may provide
another controlling mechanism for polarizing type 1 and 2
development. Co-engagement of 4-1BB signal with CD28 co-stimulation
was critical to maintain proliferation during repeated anti-CD3
activation
[0102] It was also discovered that after multiple cycles of
reactivation of previously activated cells by a high dose of
soluble anti-CD3, significant cell death was seen. This was true
even in the presence of substantial amounts of anti-CD28. These
results are consistent with recent reports by others that TCR
reengagement induced apoptosis after a strong initial proliferative
response to antigen, and large concentrations of IL-2 for cell
cycle progression. One of the consequences of repeated in vitro
activation, especially following an exposure to high dose IL-2, is
the gradual loss of responsiveness to CD28 co-stimulation. The
repeated TCR activation, a condition for cell death, instead,
induced 4-1BB expression. It is therefore, believed that continuous
reactivation may cause irreversible damage, leading to apotosis,
even in the presence of CD28 signaling, and that co-engagement of
4-1BB with CD28 could prevent the anti-CD3 reactivation driven
apoptosis. The effects of 4-1BB on cell proliferative activity
during in vitro repeated activity by anti-CD3 and anti-CD28 was
studied. The PHA-activated T cells, after IL-2 stimulation for 10
days until the T cell blasts returned to smaller cell sizes, were
continuously reactivated by anti-CD3 and anti-CD28 with or without
4-1BB co-engagement. After each of three cycles of reactivation,
the cells were reactivated again by anti-CD3 in 96-well plates
coated with serially diluted anti-CD28 from 0.01 to 10 .mu.g/1 ml
with or without an additional 10 .mu.g/ml of anti-4-1BB.
[0103] The cells were examined for proliferative activity by
measuring [.sup.3H] thymidine incorporation. As the number of
reactivation cycles proceeded, cells became less responsive to
anti-CD28, resulting in a lower maximum proliferation plateau, even
with saturated anti-CD28 concentrations. After the third cycle
re-activation, T cells barely responded to anti-CD28 alone. The
4-1BB co-engagement with CD28, however, exerted a dramatic effect
in overcoming the defective proliferation, as well as fully
restoring maximal plateaus of expression. The 4-1BB signal alone
failed to demonstrate a strong level of expression. The 4-1BB
effects were blocked by the functional antagonist, 4-1BBFc, the
soluble form of 4-1BB when it was included in reactivation,
indicating the specificity of anti-4-1BB.
[0104] Because the most significant 4-1BB effects were observed
after the response of anti-CD28 has been weakened by anti-CD28
repeated activation, one of the primary functions of 4-1BB
response, is likely to maintain clonal expansion by synergistic
cooperation with CD28 during continuous high-dose antigen
re-challenge.
Effects of the 4-1BB on Progression of the Cell Cycle in Repeatedly
Activated T Cells
[0105] Since both cell proliferation and death events occurred
simultaneously during repeated in vitro CD-3 activation, the
effects of 4-1BB signal on cell cycle progression and apoptotic
events was determined. This was accomplished by measuring DNA
stained with propidium iodide. Staining was done during
reactivation.
[0106] In order to examine the effect of 4-1BB signal transduction
on cell cycle and apoptotic status during AICD, the PHA/IL-2 cells
were reactivated with anti-CD3, anti-CD28, or anti-4-IBB alone, or
with a combination of both antibodies for 3 days in two cycles. The
DNA content of cells stained with propidium iodide were analyzed by
the ModFit software program.
[0107] Consistent with previous proliferation results, neither
anti-CD28-nor anti-4-IBB ligation alone gave rise to a significant
cell population in S-phase (less than 10%), but a relatively high
fraction was in sub-G-phase supposedly representing apoptotic
cells. Simultaneous ligation of 4-1BB and CD28 resulted in a
dramatic increase of the cell population in the S phase (higher
than 40%), and a concomitant decrease in sub-G-phase cells. These
results indicate that additional co-stimulation between 4-1BB and
CD28 was essential, not only for enhancing the progression of cells
through the G1/S phase transition, but also for preventing
apoptotic cell death during chronic stimulation. The 4-1BB molecule
is a regulatory co-stimulatory molecule for promoting type 1 subset
development in cooperation with CD28.
[0108] The data disclosed herein support a two-step model of
activation of T cells. In the first step, T cell activation
involves CD28-B7 recognition as a co-stimulatory signal with
antigenic signal via TCR. As a result of primary T cell activation,
the activated T cells can enter into secondary cognate dependent
recognition. The new regulatory co-stimulatory molecules and their
ligands, provided by activated APCs, will interact by sequential
cross-talk between T cells and APCs in a way specific for the
different circumstances. In the secondary activation phase, cells
will require additional-signals besides CD28-B7 recognition in
order to prevent TCR activation-driven cell death, as well as to
promote effector cell differentiation, especially after cells
progress through many cycles of division. In this model, the
cognate-dependent recognition of new regulatory molecules must be
tightly regulated to deliver either the survival or death
signal.
[0109] To delineate the role of 4-1BB, an experimental model with
in vitro repeatedly activated cells was adopted. CD28 alone
appeared to be incapable of maintaining cell cycling after cells
were repeatedly activated. The results indicate that the
co-engagement of 4-1BB and CD28 maintained long-term proliferative
activity. Therefore, 4-1BB may be able to inhibit complex
regulatory networks by CD30 or Fas which promotes cell death. In
this context, 4-1BB is a survival factor specifically directed to
type 1cells in the secondary activation phase. Moreover, it has
been shown that 4-1BB co-engagement with CD28 is critical to the
promotion of type 1 phenotype responses, and in the specific
expansion of the cell populations expressing 4-1BB.
[0110] Delayed type hypersensitivity (DTH) is mediated primarily by
Th1 populations that produce IF. Alteration of the Th1/Th2
regulatory networks may be important in determining the local
immune responses. The production of Th2 cells provides a strong
foundation for their anti-inflammatory effects in vivo. Recently,
it has been reported that the CD28/B7 pathway selectively promotes
Th2 development for autoantigen-specific T cells in the non-obese
diabetic mouse. Blockade of 4-1BB-mediated Th1 responses to induce
anti-autoimmune Th2 cells is thus a good approach for
antigen-specific therapies for the harmful DTH reaction involved in
autoimmune diseases. The pathogenic progression of HIV infection
also has been associated with diminished type 1 and enhanced type 2
cytokine production. Therefore, enhancing the activity of type 1
cytokines would have the benefit of offering a way to intervene in
HIV-infected individuals. CD30 signal enhances both HIV
transcription and IL-4 production, in response to CD3 antibody.
Therefore continuous use of anti-CD28 co-stimulation would prevent
or hinder HIV-1 promotion.
EXAMPLE 4
Use of Anti-H4-1BB Antibodies Against HIV-1 Infected Cells
[0111] 4-1BB is not detected in human peripheral blood T
lymphocytes, but it is induced by stimulation with mitogen and/or
cross-linking of CD3, ranging from 5% to 20%, followed by a gradual
decrease after prolonged, activation. Signaling through 4-1BB is
involved in the regulation of proliferation and survival of T
lymphocytes. 4-1BBFc fusion protein prevented anti-CD3-induced
proliferation and caused cell death, not only in murine
splenocytes, but also in human PBMC. Recently, it was determined
that the CD28 molecule co-stimulation was essential for the
induction of 4-1BB, which in turn enhanced its own expression as a
positive feedback loop upon continuous stimulation. The 4-1BB
signal resulting from combined co-stimulation from both CD28 and
4-1BB subsequently facilitated cell survival and effector cell
development. In HIV-1 infection, CD28 expression is down-regulated,
as manifested by a significant correlation observed between the
number of CD28 and CD8+ T cells and the presence of HIV-1-related
disease. Previous studies by others indicated that co-stimulation
of CD4+ T cell via soluble monoclonal antibodies against CD28
promoted HIV-I infection and replication in vitro. In contrast to
these results, when CD4+ T cells from HIV-1 individuals were
stimulated with immobilized anti-CD3 plus immobilized anti-CD28
mAb, there was an increased number of polyclonal CD4+ T cells with
a declined HIV-1 viral load.
The Role of 4-1BB in T Lymphocytes from HIV-1 Infected
Individuals
[0112] The levels of 4-1BB expression on T cells from 40 HIV-1
positive and 12 HIV-1 negative individuals were compared. Also
examined was whether T cells from HIV-1 positive individuals,
impaired in their response to TCR/CD3-mediated signal, could be
restored by 4-1BB co-stimulation, and the effects of 4-1BB ligation
on HIV-1 viral load.
Materials and Methods
Patients
[0113] The HIV-1-infected patients' blood samples were from the
outpatient clinic at Wishard Hospital Department of Medicine,
Division of Infectious Diseases. The study covered 55 HIV-1
positive individuals, of which 47 were classified as CD stage
II/III and 18 were classified as having HIV-1 related diseases
(stage 1V). Twelve sex- and age-comparable healthy donors were
studied as controls.
Antibodies and Reagents
[0114] The anti-4-1BB monoclonal antibodies (mAbs), BBK-1, BBK-2,
BBK-3 and BBK-4 were produced as described previously. BBK-1 and
BBK-4 are agonistic for T cell activation and were used in the
present studies. BBK-2 and BBK-3 are H4-1BB antagonists, and were
also developed and used for the present studies. FITC-conjugated
anti-4-IBB mAb and was generated by incubating the purified BBK-1
with FITC (Pierce, Rockford, EL) according to the manufacturer's
instructions. Anti-CD28 mAb, 9.3, was a gift from Dr. Carl June
(Naval Medical Research Institute, Bethesda, Md.). Anti-CD3 mAb was
purchased from Ortho (Raritan, N.J.). Anti-CD4 mAb/Cychrome,
anti-CD8 mAb/Cychrome and isotype control mAbs were obtained from
Pharmingen (San Diego, Calif.). Magnetic beads that were conjugated
with Goat anti-mouse IgG(M-450) were purchased from Dynal (Lake
Success, N.J.).
Cells and Cell Stimulation
[0115] Human peripheral blood mononuclear cells (PBMC's) were
prepared from EDTA anti-coagulated blood by Histopaque 1077 (Sigma,
St. Louis, Mo.) density centrifugation. CD4+ T cells were prepared
from PBMC by negative depletion using Lymphokwik (One Lambda Inc.,
Canoga Park, Calif.) according to manufacturer's instruction.
Briefly, PBMC were treated with Lymphokwik Th isolation solution
for 45 min at 37.degree. C. followed by a 5 minute centrifugation.
The purity of CD4+ T cells were around 85-90% with less than 5%
CD8+ T cells by flow-cytometric analysis. The culture medium was
RPMI 1640 (Life Technologies. Inc.) supplemented with 10% fetal
bovine serum (Elyclone, Utah), penicillin (50 u/ml), streptomycin
(50 .mu.g/ml), and 2 mM glutamine (Sigma) (RPMI-CM). PBMC were used
before or after PHA (Calbiochem, 5 .mu.g/ml) activation for 3 days
in RPMI-CM. Freshly-isolated CD4+ T cell (2.5.times.10.sup.4/well)
were added into a flat-bottom 96-well microtiter plate (Costar
Corporation, Cambridge, Mass.) coated with anti-CD3 mAb in
combination with 4B4 or 9.3 or both as indicated concentration.
[0116] Antibody immobilization to culture plates was carried out in
phosphate-buffered saline, pH 7.3 (PBS) overnight at 4.degree. C.
In some experiments, mAbs were conjugated to magnetic beads M450,
Dynal) by adding 150 fg of each antibody per bead and added at a
ratio of three beads per CD4+ T cell in virus induction studies.
Polyclonal CD4+ T cell lines were generated by culturing fresh CD4+
T cells with PHA-(5 .mu.g/ml) and recombinant IL-2 (20 u/ml)
(Boehringer, Mannheim, Indianapolis, Ind.) 10 days with replacement
of fresh IL-2 every 3 days. CD4+ T cell blasts
(5.times.10.sup.4/well) from polyclonal T cell lines were further
cultured for 3 days in a 96-well plate in the presence of
co-stimulatory antibodies as described above. Human 4-1BB cDNA
transfected Jurkat cells (J8-1) were prepared, and maintained in
RPU-CM.
Flow-Cytometry Analysis
[0117] Cells were stained and analyzed on FACScan (Becton
Dickinson), as described previously (21). Briefly, fresh cells were
washed once and cultured cells 3 times in PBS containing 1%. BSA.
Approximately 2.5-5.times.10.sup.5 cells were re-suspended in 200
.mu.l PBS-1% BSA with diluted mAbs and incubated on ice for 30
minutes. After washing twice, cells were fixed with 1%
paraformaldehyde. Flow cytometry analysis was carried out on
lymphocyte-gated cells based on forward-versus-side scatter
profiles.
Proliferation Assay
[0118] The cells in quadruplicate wells were stimulated for 5 days
and then pulsed for 6 hrs with [.sup.3H] thymidine (TdR) (NEN,
Boston, Mass.) at 1.0 .mu.ci/well. The stimulation index was
calculated by dividing the counts per minute (CPM) of [3H] of
stimulated cells by those of unstimulated cells.
RT Activity Assay
[0119] Virion-associated reverse transcriptase (RT) activity was
measured as described by Willey et al., (J. Virol. 1988, 62:13947),
with modification as follows: 5 .mu.l of 7-day culture supernatants
were added in triplicate to 25 .mu.l of a mixture which contained a
template primer Poly(A) (5 .mu.g/ml), Oligo(dT) (1.57 .mu.g/ml)
(Pharmacia) and 10 .mu.g [.sup.3H] dTTP (Amersham Corp., Arlington
Heights, Ill.) in 50 mM Tris, pH 7.8, 7.5 mM MgCl.sub.2, 2 mM DTT.
After incubation for 2 hrs at 37.degree. C., 6 .mu.l of the mixture
was spotted onto DE81 paper, air-dried and washed five times in
2.times.SSC buffer and two additional times in 95% ethanol. The
papers were dried, cut and counted on a scintillation counter.
Gene Transfection and Chlorampenicol Acetyltransferase (CAT)
Activity Assay
[0120] Human Jurkat T lymphocytes and 4-IBB-transfected subline,
J8-1, were transiently transfected with PHIV-1-LTR-CAT plasmid (20
.mu.g/10.sup.7 cells) by DEAE-Dextran method as described by
Bressler, P. (J. Immunol. 1991; 147:2290-94). Luciferase genes were
co-transfected for normalization. After 24 hr post-transfection,
the cells were stimulated with immobilized mAbs as indicated or PHA
(5 .mu.g/ml) plus PMA (10 ng/ml) for 24 hr prior to harvest.
Whole-cell extracts were prepared from transfectants and CAT
activity were performed in a final volume of 150 .mu.l containing
0.1 .mu.ci (3.7 KBq) of .sup.14C-chloramphenicol, 4 mM
butyl-coenzyme A and 0.25M Tris-HCl pH 7.4 at 37.degree. C.
overnight. The results are given in percent conversion of
chloramphenicol to its monoacetylated forms. Values were obtained
from 4 independent transfections following normalization with
luciferase activities.
Statistical Analysis
[0121] Data are presented as means.+-.SD. Two-tailed Student's
t-test was used to determine the significance of the differences
between groups. Correlation was calculated using a "r" linear
correlation coefficient.
Results
Expression of 4-1BB on CD4+ and CD8+ T Cells from HIV-I PBMC
[0122] Clinical correlation. Forty HIV-1-infected subjects and
twelve-seronegative controls were examined for 4-1BB expression on
T cells by immunofluorescent cytometry.
[0123] The expression levels of 4-1BB were not detectable on
unstimulated T lymphocytes either from HIV-1-infected or control
individuals prior to in vitro stimulation. After PHA stimulation,
the percentage of 4-1B expressing cells was significantly higher in
HIV positive individuals than in the HIV-1 negative control
individuals. It should be noted that not only the level of 4-1BB
expression but also the population of 4-1BB T cells were increased
in HIV-1 positive individuals. The distribution of 4-1BB expression
on CD4+ and CD8+ T cells-within the 3 groups was expressed on 10.9%
of CD4+ T cells in HIV-1-controls, whereas 4-1BB was expressed on
28.9% of CD4+ T cells among asymptomatic HIV-1 positive individuals
(P<0.01). 4-1BB was expressed on 30.9% of CD4+ T cells in stage
1V individuals. The difference in 4-1BB expression between control
and stage II/III patient T cells was more profound in CD8+ T cells
than in CD4+ T cells. Furthermore, a significant increase of 4-1BB
CD8+ T cells was found in stage IV patients (median 47.9%),
compared to asymptomatic individuals (P<0.05). In stage II/III
individuals, there was a significant correlation in 4-1BB
expression between CD4+ and CD8+ T cells (r 0.72 P<0.01). There
was also a reverse correlation between absolute CD4+ cell counts
and percentage of 4-MM-expressing CD8+ T cells (r=0.63) P<0.05)
in all HIV-1 positive individuals.
[0124] The Proliferative Response of CD4+ T Cells to 4-1BB
Co-Stimulation
[0125] 4-1BB signal alone with CD3 stimulation was not sufficient
for the proliferation of the primary T cells. Therefore, the
synergistic effects of anti-4-1BB with anti-CD28 mAb were tested,
and the proliferation of CD4+ T cells from 3 V-1-healthy donors was
measured through the use of serially diluted anti-CD28 (0 to 10
.mu.g/ml) with or without anti-4-1BB (10 .mu.g/ml) were immobilized
on tissue culture plates, with anti-CD3-mAb (1 .mu.g/ml). The CD4+
T cells were cultured with the mAbs for 5 days and cell
proliferation was measured by [.sup.3H] TdR uptake. It was found
that 1 .mu.g/ml of anti-CD28 mAb was able to synergize with 4-1BB
co-stimulatory activity. Based on these results, the combined
anti-CD28 (1 .mu.g/ml) and anti-4-1BB mAb (10 .mu.g/ml) were used
to investigate the 4-1BB co-stimulatory function on CD4+ T cell
proliferation from HIV-1 positive individuals. The CD4+ T cells
purified from PBMC from 9 HIV-1positive individuals (CD4+ counts:
468.+-.142) were examined for proliferation after 5-day cultures.
4-1BB signal alone showed nearly no stimulatory activity in CD4+ T
cells of HIV-1 positive individuals. However, CD4+ T cell
proliferation occurred with 4-1BB cross-linking when sub-optimal
stimulation through CD28 was added. In addition, it was determined
that the CD4+ T cell proliferative responses were lower in HIV-1
infected individuals-compared with HIV-1individuals. Furthermore,
the lower proliferation corresponded to lower. CD4+ T cell
counts.
H4-1BB Co-Stimulation and HIV-1 Production
[0126] To study the effect of 4-1BB co-stimulation on HIV-1
production in CD4+ T cell, the CD4+ T cells by plate-bound or bead
conjugated mAbs, as described in Materials and Methods were
stimulated. HIV-1 production was measured by reverse transcriptase
(RT) activity and cell proliferation was measured by [.sup.3H] TdR
uptake as designated by a stimulation index (SI). The positive
threshold of RT activity was set by the values higher than the
median of negative controls by 3 standard deviations. Table 1
summarizes the results: from the 6 HIV-1+ patients who had a
positive virus replication (from at least one of the stimulation
groups) among 10 tested individuals. The proliferation index of
some groups was not determined, because nearly no cells survived in
these groups after 7-day culture. In two patients, RT activity was
detected only from the combined CD28 and 4-1BB co-stimulation
group, but not from CD28 alone group, although both groups showed a
similar stimulation index. In three patients, RT activity was
similar or slightly higher in the combined CD28 and 4-1BB
co-stimulation groups, compared with CD28 co-stimulation alone. In
all these 6 patients, no virus was detected from CD3 stimulation or
CD3 stimulation with 4-1BB co-stimulation groups.
[0127] It was also found that most of the CD4+T cells which
produced virus after stimulation were from patients of lower CD4
counts. Table 2 shows data from 6 of 9 patients whose T cells were
stimulated with mAbs conjugated to Dynal M450 beads and produced RT
activity from at least one of the stimulation groups. Compared with
plate-bound mAb stimulation, the stimulation mediated by
bead-conjugated mAbs was higher in levels of RT activities, perhaps
indicating that mAbs immobilized on beads gave stronger signals.
Bead-conjugated mAb stimulation could make cells proliferate even
in CD3 and CD3 plus. 4-IBB stimulation group. It became, therefore,
possible to observe the function of 4-1BB co-stimulation alone in
virus replication. As shown in Table 2 the combined CD28 and 4-1BB
co-stimulation produced virus from CD4+ T cell cultures from all
donors. The RT activity increased from 1.2-fold to 1 I-fold by
combined co-stimulation of CD28 and 4-1BB compared to CD28
co-stimulation alone. Similar results were obtained when another
anti-4-1BB mAb (BBK4) were used in 2 donors (data not shown).
[0128] Importantly, the virus was also detected in co-stimulation
with anti-4-1BB mAb alone, although the stimulation index was very
low after 7-day culture in these groups. The data summarized in
Tables 1 and 2 suggest that co-stimulation via 4-1BB results in
CD4+ T cell activation and subsequently, enhances virus production.
These activation signals for HIV-1 production may coincide with
those that mediate the proliferative response.
4-1BB Co-Stimulation and Virus Replication in Polyclonal CD4+ T
Cell Lines from HIV-1+ Individuals
[0129] To further confirm the findings that 4-1BB signal enhanced
HIV-1 replication in CD4+ T cells from HIV-1-infected individuals,
polyclonal CD4+ T cell lines were generated from 6
HIV-1+asymptomatic individuals by stimulation with PHA and IL-2 for
10 days. No, or very low RT activities were detected from the
culture supernatants. These cells were subsequently stimulated with
immobilized anti-CD3 mAb and additional co-stimulatory antibodies
for 3 days. The virus level was significantly higher in 4-1BB
co-stimulation than anti-CD3 mAb stimulation alone (P<0.05). In
contrast, although the virus level was higher in CD28
co-stimulation group than in CD3 stimulation group, no statistical
significance was found between these two groups. In this study, the
proliferation Index were almost the same in all of these groups.
These results from polyclonal CD4+ T cell lines are consistent with
those obtained from primary CD4+ T cell stimulation which presented
in Tables 1 and 2. Taken together, the data suggest that 4-1BB
co-stimulation enhances virus production in HIV-1-infected CD4+ T
cell cultures.
4-IBB Ligation Enhances LTR-Driven Transcription in Jurkat Cell
Line
[0130] The J8-1 cell line is a subline of Jurkat, and is a
4-IBB-transfectant that expresses a high level of 4-1BB
constitutively. J8-1 was transiently transfected with
pHIV-1+-LTR-CAT and subsequently activated with immobilized
co-stimulatory mAb or PHA plus PMA. The parental Jurkat cells which
express-no detectable 4-1BB by flow cytometry were used as negative
controls. The level of CAT activities in stimulation groups was
shown as fold over unstimulated control. A 1.9-fold increase in CAT
activity was observed in immobilized anti-CD3 mAb stimulation in
J8-1 compared with isotype control. Anti-4-IBB mAb by itself did
not increase the CAT activity in JS-1. However, when combined with
immobilized anti-CD3 mAb, anti-4-IBB mAb gave a 5.8-fold increase
in CAT activity compared with isotype control or a 3.2-fold
increase compared with anti-CD3 mAb stimulation alone. 4-1BB
stimulation had an additional effect on CD3 plus CD28 combined
stimulation. In the parental Jurkat cell, 4-1BB co-stimulation
produced 1.2-fold more CAT activity compared with anti-CD3 mAb
alone. These slight increases of CAT activity from 4-1BB
co-stimulation in parental Jurkat cells may come from the small
amount of 4-1BB expression induced during activation. From these
data, it was shown that 1) H4-1BB co-stimulation with TCR/CD3
enhances transcription of the HIV-1 LTR; 2) 4-1BB provides
additional stimulatory effect on HIV-1-LTR to anti-CD3 plus
anti-CD28 stimulation.
[0131] The results of this data indicate that the relative
proportion of T cells expressing 4-1BB both in CD4+ and CD8+ T
cells is increased from HIV-1-seropositive individuals after PHA
stimulation, and relative expression of 4-1BB on CD8+ T cells is
correlated to CD4+ T cell counts, which may be related to disease
severity and progress. The 4-1BB molecule was expressed rapidly and
reached its peak between 48-72 hrs. The levels of expressed H4-1BB
began to decrease by 72 hrs post-stimulation and eventually went
back to normal levels. Once these cells were reactivated, it was
determined that the level of 4-1BB on each cell and the number of
4-1BB expressing cells increased.
[0132] During HIV-1 infection, at an early stage after HIV antigen
stimulation, T cells are primed, but after several weeks, some of
them acquire characteristics of memory cells, and continue to
express the same set of activation markers. This set of markers
includes: HLA-DR and CD38. These may account for the finding that
no 4-1BB expression was found on resting T cells, which is itself
related to the continuous activation and death of CD4+ 4-IBB+T
cells in response to HIV-1 antigen in vivo. The increased 4-1BB
expression on T lymphocytes of HIV-1-infected individuals after in
vitro stimulation would then reflect not only the current state of
immune activation in HIV-1 infection, but also the state of
memory-primed cells challenged by HIV-1 antigen during acute
infection in vivo. The increased 4-1BB expression on CD4+ T cells
after re-stimulation is related to the increase of virus
replication. In HIV-1 infection, CD8+ T cells, play a role in
suppressing HIV-1 replication through the classical HLA-restricted
cytolysis of infected cells and non-cytolytic mechanism that
involves some secreted CD8-cell antiviral factors. 4-1BB, as a
co-stimulatory molecule, with its increased expression on CD8+
cells in HIV-1 infection, may relate to CD8+ T cell proliferation
and antiviral function. In HIV-1 patients, CD8+ T cells generally
proliferate more vigorously than CD4+ T cells when the cells were
stimulated with anti-4-1BB mAb.
[0133] Ligation of CD28 with soluble mAb in the presence or absence
of soluble anti-CD3 has been reported to induce virus from CD4+ T
cells prepared from HIV-1-infected donors. Activation of CD4+ T
cells from HIV-1 positive donors with immobilized anti-CD3 and
anti-CD28 mAb, however, induced a virus-resistant state. This
effect was specific for macrophage-tropic HIV-1 and appears to be
the result of down-regulation of CCR5, the fusion cofactor.
Although no statistically significant difference was found, the
ligation of CD28 with immobilized 9.3 did increase HIV-1
replication from some HIV-1 infected donors. This observation,
which differs from other reports, may be because of the methods of
immobilization of anti-CD3 or anti-CD28 mAb on the beads. Studies
using bead-immobilized mAbs showed that the additional signal from
4-1BB enhanced virus replication from primary CD4+ T cells from
HIV-1 positive individuals. Because the exact amount of anti-CD28
mAb and anti-CD3 mAb were used in combined CD28 and 4-1BB
co-stimulation and CD28 co-stimulation groups, the enhancement of
virus production should be considered from additional 4-1BB
signaling.
[0134] Furthermore, virus was induced by 4-1BB co-stimulation
alone. In some donors, the virus levels were even higher than that
by CD28 co-stimulation, although the stimulation index of primary
CD4+ T cells in response to 4-1BB co-stimulation was very low. In
polyclonal CD4+ T cell lines, a statistically significant
difference of virus replication was found between 4-1BB
co-stimulation and CD3 stimulation alone. This comparison was based
on the similar stimulation index (SI) within each group. From the
present studies, it was shown that cross-linking of 4-1BB and CD3
in CD4+ T cells from HIV-1+individuals induced virus production.
The function of 4-1BB co-stimulation on virus production was not
correlated with the function on T cell proliferation, perhaps
suggesting that the cellular pathways that mediate HIV-1 induction
might be similar, but not identical, to those of mitogenic
stimulation.
[0135] A number of factors have been reported to up-regulate HIV-1
expression in vitro. Many of these agents including TNF-.alpha.,
anti-CD30 mAb, HIV Tat protein, activate HIV-1 transcription
through the NF-.kappa.B enhancer present in the HIV-1-LTR In the
experiments done it was shown that, HIV-1-LTR transactivation had
been obtained by a combination of anti-CD3 and anti-4-1BB mAb.
These results suggest several potential roles of 4-1BB in HIV-1
pathogenesis: (1) 4-1BB directly upregulates the transcription of
the viral genome in latently infected cells; (2) 4-1BB and 4-1BB
ligand interaction may activate virus replication of CD4+ T cells
in the presence of antigenic stimuli; (3) 4-1BB co-stimulation may
activate resting CD4+ T cells and as a consequence, promote an
efficient propagation of newly produced virions that preferentially
infect activated CD4+ T cells; (4) 4-1BB-mediated signals to HIV-1
infected CD4+ T cells may bind to apoptosis, resulting in premature
death of infected cells.
[0136] The results demonstrate that 4-1BB expression was increased
in HIV-1 infected PBMC after in vitro activation correlating to
immune activation and disease progress. Ligation of 4-1BB in HIV-1
infected CD4+ T cells enhanced virus replication in vitro, as
mediated through an NF-.kappa.B pathway. If the 4-1BB
co-stimulatory pathway is disturbed or purposely, interfered with
at early stages of the infection it is likely that a lower virus
load will result. This in turn will serve to prevent the subsequent
loss of CD4+ T cells, and maintain immune competency. The increased
4-1BB expression on CD8+ T is correlated to the degree of
immunodeficiency in HIV-1 infection.
EXAMPLE 5
Role of 4-1BB in Human CD4+ T Cell Adhesion
[0137] The human 4-1BB protein is a co-stimulatory molecule for T
cells. By studying the role of 4ABB in T cell adhesion with human
primary T cells and two T cell lines, CEM and Jurkat, it was
discovered that 4-1BB co-stimulation induced T cell adhesion
dramatically anti-4-1BB signaling along with PMA/ionomycin
stimulation caused CEM cells, which express a high level of 4-1BB
uncharacteristic cell adhesion. In contrast, Jurkat cells which do
not express a detectable level of 4-1BB demonstrated nearly no
response to anti-4-1BB in cell adhesion to fibronectin (FN). When
Jurkat cells were transfected to produce: 4-1BB; they Acquired the
ability to adhere in response to FN by anti-4-1BB stimulations. An
absolute co-requirement for anti-CD3 stimulation in addition to
4-1BB signaling to cell adhesion in the Jurkat transfectants
suggests that adhesion caused by anti-4-1BB is mediated through
activation of adhesion signaling rather than a direct interaction
between 4-1BB and FN or anti-4-1BB. It is in this way that the
4-1BB co-stimulatory signal amplifies T cell activation, by
intermediating CD28 co-stimulation with adhesive responses.
Antibodies and Reagents for Adhesion Studies
[0138] Monoclonal anti-4-1BB, BK4, also called 4B4-1 (mouse IgG)
was used to stimulate and immunostain 4-1BB on T cells. The
anti-4-1BB was conjugated with biotin for flow cytometric analysis.
Anti-CD28, mAb 9.3 (mouse IgG2a) was a kind gift from Dr. C. H.
June (Naval Medical Research Institute, Bethesda, Md.). Monoclonal
anti-CD3 (OKT3) was purchased from Ortho Diagnostic (Westwood,
Mass.). Secondary cross-linking goat anti-mouse IgG (H+ L) was
purchased from Zymed (South San Francisco, Calif.). Blocking
anti-integrin b.sub.1, purchased from Immunotech (Westbrook, NEE).
4-1BB-Fc, a fusion protein consisting of the extracellular portion
of human 4-1BB coupled with the Fc region of human IgG.sub.1 was
obtained from Immunex (Seattle, Wash.) An isotype control mouse
IgG.sub.1 (MOPC-21) conjugated with biotin was purchased from
PharMingen (San Diego, Calif.). Human fibronectin was obtained from
Dr. Fred Pavalko (Indiana University). A premixed cocktail of
monoclonal antibodies and complement to isolate T cells was
purchased from One Lambda (Canoga Park, Calif.).
Flow-Cytometry
[0139] Selected T-cells were stimulated and re-stimulated with
anti-CD3 and anti-CD28 for 3 days and used for proliferation and
adhesion assay in response to different co-stimulatory mAbs. CEM, a
human leukemic T cell line, was stimulated with PMA (10 ng/ml) and
ionomycin (IgM) for 24 hr before adhesion assay. Jurkat human
leukemic T cell line was transfected with pcDNA3 with entire 4-1BB
cDNA and selected for neomycin resistant clones by limit dilution.
4-1BB expression levels on the cell surface of the transfectants
were determined by flow cytometry.
[0140] T cells (2.times.10.sup.5 cells) were suspended in a 200 ml
of 2 .mu.g/ml anti-4-1BB conjugated with biotin in staining
solution, PBS containing 1% BSA and incubated at 4.degree. C. for
30 minutes. The cells were subsequently washed three times,
re-suspended in 200 ml of phycoerythrin (PE) conjugated
streptavidin, 1 .mu.g/ml, and incubated for 30 minutes. After wash,
the samples were fixed with 1% paraformaldehyde prior to flow
cytometric analysis on the FACSan (Becton Dickinson, Mountainview,
Calif.). Biotin-conjugated mouse IgG, (MOPC-21, PharMingen, San
Diego, Calif.) was used for isotype control for anti-4-1BB
conjugated with biotin. Gates were set on live cells based on
forward versus side scatter profiles. 10,000 events were collected
for each sample.
[0141] Immobilization of antibodies and FN to microtiter plates
96-well flat bottom polystyrene plates (Costar, Cambridge, Mass.)
were coated overnight at 40.degree. C. with anti-4-1BB, anti-CD28,
at 10 .mu.g/ml in PBS or otherwise at the concentrations indicated
in the text. In some cases, the antibody solution was included with
FN or BSA as a control at 0.1 .mu.g/ml each. The plates were then
rinsed to remove non-adherent proteins and cells were immediately
added to the plates after final wash. To block anti-4-1BB, the T
cells were labeled with [-5'Cr] sodium chromate, 200 .mu.g/ml at
37.degree. C. for 1 hr, and transferred to 96-well plate
(5.times.10.sup.1 cells/well) coated with anti-CD28, anti-4-1BB or
both anti-CD28 and anti-4-1BB.
[0142] For adhesion assays for primary and Jurkat cells, the plates
coated with FN in addition to the antibodies were used. After cells
were incubated at 37.degree. C. in a carbon monoxide-incubator for
the indicated time periods, unbound cells were removed by washing
the plates with prewarmed culture medium three times and the cells
remained bound to plates were lysed in 1% SDS. The lysates were
counted for radioactivity and the percentage of bound cells were
calculated as a ratio bound to total cpm added to the wells. The
adhesion assay for CEM cells were undertaken in the plates where FN
was omitted in antibody coating. The adhesion assay for the Jurkat
4-1BB transfectants were performed in the presence or absence of
soluble anti-CD3 at 1 .mu.g/ml.
Assays for Proliferation
[0143] The primary T cells which were repeatedly stimulated with
anti-CD3 and anti-CD28 as described above were subjected to further
activation by soluble anti-CD3, 1 with secondary anti-mouse IgG, 5
.mu.g/ml in the 96-well plates previously coated with
co-stimulatory anti-CD28 or anti-4-1BB or with with anti-CD28 at 10
.mu.g/ml plus additional anti-4-1BB at indicated concentrations. In
some cases, the cells were co-stimulated with immobilized
co-stimulatory antibodies including sub-optimal 0.1 .mu.g/ml FN.
After 3 days, proliferation rates were measured by 6-hr
pulse-labeling with [.sup.3]H thymidine, 1.0/well.
[0144] 4-1BB signal induced uncharacteristic-cell adhesion of
PMA/ionomycin-stimulated CEM cells to culture plate. Although both
CD28 and 4-1BB are able to co-stimulate T cells similarly for
proliferation and IL-2 production, one obvious difference of the
two molecules is their expression modes. 4-1BB expression is highly
regulated as compared to CD28 which expresses constitutively in the
most of T cells. The 4-1BB molecule from most of the CD4+ human
lymphoma was not detected. The T cell lines which were tested
included Jurkat, CEM, Molt-4, and HUT-78. However, CEM cells were
exceptional in readily inducing 4-1BB upon PMA/ionomycin
stimulation to an extremely high levels compared to those induced
by activated primary T cells. CEM cells became blastic and
aggregated after PMA/ionomycin stimulation, but remained
non-adherent to culture plate.
[0145] During the experiments it became clear that the stimulated
CEM cells responded vigorously to plate-bound anti-4-1BB completely
spreading to culture plate. The responses were so intense that no
extraneous stimulating adhesion receptor ligands were necessary.
More than 80% of the PMA/ionomycin-stimulated CEM cells were firmly
attached to culture plates in response to anti-4-1BB within 1 hr.
On the contrary, the CEM cells which were not previously stimulated
and, therefore, produced no detectable 4-1BB did not respond to
anti-4-1BB at all. Since CD28 is a primary co-stimulatory molecule,
CEM cell response to anti-CD28 was tested to determine whether this
induction of CEM adhesion was unique to 4-1BB co-stimulation. In
contrast to anti-4-1BB, anti-CD28 did not induce cell adhesion for
both stimulated and un-stimulated CEM cells.
[0146] The distinctive co-stimulatory outcomes from 4-1BB and CD28
in CEM cell adhesion indicate that the two co-stimulatory molecules
deliver non-overlapping signals. Several anti-CD4 mAbs whose
antigens are abundant on CEM cell surface but observed no such
responses as seen with anti-4-1BB were tested. The 4-1BB signal
altered morphology of CEM cells. It was also determined that
noticeable morphological changes in CEM cells immediately following
firm attachment to culture plates in response to anti-4-1BB. The
CEM cells remained in dispersed form during culture and usually
became clustered after PMA/ionomycin stimulation. There were
dramatic changes in cell shape when the stimulated cells were
exposed to immobilized anti-4-1BB. Anti-4-1BB signal following
PMA/ionomycin stimulation completely altered the round cells to
elongated fibroblastic shapes with sharp spikes. This observation
suggest that 4-1BB signal may induce cytoskeletal rearrangement
which can allow the cells to adapt to cell adhesion to culture,
plate. A similar morphological change with anti-CD28 was not
observed.
[0147] The adhesion and morphological changes caused by anti-4-1BB,
however, did not affect cell proliferation rate. 4-1BB
co-stimulatory signal induced Jurkat cell adhesion to FN. Although
CEM cells provided a good model for eliciting 4-1BB roles in T cell
activation, there were obstacles for interpreting the results
mainly because of PMA/ionomycin stimulation to produce 4-1BB during
4-1BB stimulation. The simultaneous multiple signals made difficult
to discern the 4-1BB action responsible for the final outcomes. To
circumvent the problem, stable T cell transfectants that
constitutively expressed 4-1BB without prior stimulation to induce
4-1BB expression were developed. Jurkat cells did not express
detectable level of 4-1BB. Jurkat cells were transfected to produce
4-1BB with 4-1BB cDNA inserted in expression plasmid pcDNA3. After
G418 selection, three transfectant clones producing different
levels of 4-1BB, 17-2,8-1 and 2-7 from lowest to highest in order,
were obtained. The 4-1BB expressions of the parental and each
Jurkat transfectant measured by flow cytometry are shown in. The
4-1BB expression level of the highest 4-1BB producer, transfectant,
2-7 was still about 10 times lower than those seen in CEM.
Therefore, parental Jurkat and these transfectants for
4-1BB-mediated cell adhesion to plate-bound a sub-optimal FN (0.1
.mu.g/ml) which primarily supports integrin-mediated adhesion in T
cells were used.
[0148] The cell adhesion assay was performed using the plates
coated with isotype control b.sub.1, anti-CD28, anti-4-1BB or both
anti-CD28 and anti-4-1BB in addition to FN and measured the cells
attached to the plate after 1-5-minutes in culture at 37.degree. C.
with or without soluble anti-CD3. The 4-1BB transfectants but not
parental Jurkat cells promptly responded to anti-4-1BB only in the
presence anti-CD3. The data from the Jurkat 4-1BB transfectants
clearly indicated that cell adhesion in response to anti-4-1BB
occurred in a 4-1BB expression level-dependent manner. The highest
response was seen in tranfectants 2-7 but lowest in transfectant
17-2. Under the same conditions, the control Ig and anti-CD28 mAbs.
At saturation concentration did not induce such cell adhesion for
the all the cells tested.
[0149] To examine whether 4-1BB and CD28 signals interact each
other, the level of cell adhesion in the presence of both
anti-4-1BB and anti-CD28 was determined. The two co-stimulatory
signals resulted in synergistically higher cell adhesion.
Therefore, 4-1BB appears to require CD28 signalling for maximal
adhesion response. The anti-4-1BB-mediated adhesive responses
detected in the presence of anti-CD3 were totally abolished when
anti-CD3 was absent. The critical requirement of anti-CD3 in
4-1BB-mediated cell adhesion indicates that this cell adhesion was
not caused simply by an interaction between 4-1BB and anti-4-1BB or
FN but rather intermediate CD3 signaling to cell adhesion pathways.
Taken together, 4-1BB and CD28 are both co-stimulate T cells but
the 4-1BB roles are different form CD28 in inducing T cell adhesion
to FN. The cell adhesion through anti-4-1BB signal did not change
proliferation rate in Jurkat cells as seen in CEM. 4-1BB expression
progressively increased by repeated CD3 activation. The link with
previous studies and human peripheral T cells was strengthened
through a series of flow cytometry experiments with 4-1BB
expression patterns during anti-CD3 activation.
[0150] The expression levels of 4-1BB are detected only in about 2%
of the T cells freshly prepared from healthy individuals. The
levels were increased through repeated in vitro anti-CD3 and
anti-CD28 activation. T cells stimulated with anti-CD3 and
anti-CD28 for 3 to 4 days exhibited about 20% cells in 4-1BB
positive but the levels were gradually declined following the peak.
The numbers of activated cells in IL-2 without anti-CD3 and
anti-CD28 was expanded. The 4-1BB expression levels were
progressively decreased during the culture in IL-2. However, a
dramatic increase of 4-1BB both in the cell number in 4-1BB
positive and the expression levels were obtained upon
re-stimulation with anti-CD3 and anti-CD28 resulting in greater
than 50% of the cells positive with 4-1BB. The repeated-cycles of
stimulation progressively increased 4-1BB expression levels even
higher when additional anti-4-1BB signal is added to anti-CD3 and
anti-CD28 stimulation. Theses observations suggest that the
4-1BB-producing cell population may be in proliferative advantage
due to additional 4-1BB co-stimulatory input during repeated
stimulation.
[0151] A large portion of T cells still registered expression of
4-1BB even after prolonged activation with respect to 4-1BB
expression. 4-1BB signaling induced primary T cell adhesion mainly
through integrins. The human primary T cells activated by two 3-day
consecutive cycles of stimulation with anti-CD3 and anti-CD28 were
prepared to increase 4-1BB expression on the cells. The cells were
maintained for 7 days in IL-2 prior to re-stimulation. Following
each stimulation step, a determination was made as to whether the
activated primary T cells could be induced for cell adhesion to
sub-optimal FN by 4-1BB signal as observed in CEM and Jurkat cells.
The percentages of the cells attached to the plates coated with
anti-CD28 or anti-CD28 and anti-4-1BB in addition to sub-optimal FN
at 0.1 .mu.g/ml in the presence of soluble anti-CD3 after 1 hr was
determined. While anti-CD3 alone or anti-CD3 and anti-4-1BB induced
negligible adhesion to FN, anti-CD28 co-stimulation resulted in
about 16% bound to the sub-optimal FN-coated plate. However, a
dramatic increase in cell adhesion was seen when the activated T
cells were co-stimulated by both anti-4-1BB and anti-CD28 resulting
in 50% of the cells firmly attached to the plates after 1 hr.
Considering that only about 50% of the stimulated T cells used in
the assay expressed 4-1BB, the cell adhesion observed may have been
the maximum levels achievable.
[0152] The cells further activated by repeated re-stimulation
gradually lost the responsiveness to signal anti-CD28 alone
significantly but not to the combined signals from both anti-CD28
and anti-4-1BB. Integrins, mainly (X401 (CD49d/CD29) and
a.sub.5b.sub.1 (CD49e/CD29) are primary adhesion molecules
responsible to transmit the FN interaction. To determine whether
4-1BB-induced cell adhesion to FN was mediated by the integrins,
the cells were incubated with blocking mAb to integrin b.sub.1
before adhesion assays. The adhesion induced by anti-4-1BB was
effectively inhibited by more than 50% as a result of the
pretreatment with anti-integrin. The results suggest that 4-1BB may
activate integrins to promote FN interaction rather than involve in
direct association with FN. Murine 4-1BB has been known to have
strong affinity to FN but the direct interaction between 4-1BB and
FN does not seem to be a major force in this cell adhesion. The FN
augmented synergistic 4-1BB effects on CD28 co-stimulation. Because
4-1BB and CD28 cooperated for promoting T cell adhesion to FN, a
determination was made as to whether cell adhesive response affect
the synergy between 4-1BB and CD28 signals for proliferative
responses.
[0153] It was hypothesized that if 4-1BB worked cooperatively with
CD28 was mediated by cell adhesion, the 4-1BB effect should be
farther amplified by FN. In fact, it has been shown that FN itself
can co-stimulate T cells reducing antigen threshold for T cell
activation. To test the possibility, the effects of FN in
proliferative responses of the activated primary T cells
co-stimulated by anti-CD28, anti-4-1BB, or both antibodies were
determined. To maximize the anti-4-1BB effects, a sub-optimal
anti-CD28 concentration at 0.5 .mu.g/ml was selected because it
leads only marginal proliferation by anti-CD3. Under the
conditions, a determination was made with regard to the anti-4-1BB
effects on anti-CD28-mediated proliferation with or without FN.
Anti-4-1BB alone co-stimulate highly purified human T cells with
anti-CD3 modestly. However, anti-4-1BB allowed the sub-optimal
anti-CD28 to lead high proliferative response indicating that there
was a definite cooperation between CD28 and 4-1BB in
co-stimulation. Next addressed was whether this 4-1BB cooperation
with CD28 can be amplified by FN. The results of the experiments
clearly demonstrate that the synergistic cooperation between CD28
and 4-1BB was greatly enhanced by FN. The sub-optimal FN
concentration used in the experiment effected little for CD28 or
4-1BB co-stimulation. The cells under sub-optimal CD28 and FN
influences were able to fully respond to anti-CD3 activation by the
presence of 4-1BB signal. This indicates that the primary role of
4-1BB is to sensitize T cells to respond to antigenic activation by
integrating TCR co-stimulation and cell adhesion.
[0154] The anti-4-1BB effects on amplifying T cell proliferative
response were totally abolished when 4-1BB-Fc, a competitive
blocking reagent to 4-1BB was included in the culture indicating
the specificity of the anti-4-1BB. 4-1BB signaling reduced
threshold of CD3 signal required for CD28 co-stimulation.
co-stimulatory signal can reduce antigen threshold. The
strengthened co-stimulatory input may reduce the threshold of
antigen stimulation. To determine whether synergistic 4-1BB effects
on CD28 co-stimulation could further reduce the threshold of CD3
signal, a titration of the anti-CD3 concentrations necessary for
activating T cells as measured by proliferation, was measured.
[0155] Activated primary T cells were proliferated by varying
anti-CD3 concentrations, 0.01, 0.1 and 1 mg/ml under the different
co-stimulatory conditions by anti-CD28, anti-4-1BB or both
anti-CD28 and anti-4-1BB. The 4-1BB synergistic effects on CD28
co-stimulation allowed high proliferation with 0.1 mg/ml of
anti-CD3, which would lead to relatively low proliferative response
if the cells were co-stimulated with either anti-CD28 or anti-4-1BB
alone. Such a high proliferative response was achieved by more than
10 fold higher anti-CD3 (1 mg/ml) when the cells-were co-stimulated
by anti-CD28 alone. Therefore, effective anti-CD3 concentration
could be reduced by fold by 4-1BB engagement to CD28
co-stimulation. The anti-4-1BB co-stimulation alone, however,
resulted in modest proliferation. The results demonstrate that the
4-1BB signal gave rise a pronounced impact in reducing threshold of
anti-CD3 by cooperating with CD28.
[0156] To verity 4-1BB effects on CD28 co-stimulation, the T cell
proliferative responses 0.1 mg/ml anti-CD3 under the co-stimulation
with increasing amounts of anti-4-1BB at the fixed anti-CD28
concentration at 10 mg/ml were measured. The results showed that
the synergistic 4-1BB effects on CD28 co-stimulation was
dose-dependent manner. Therefore, it was shown that 4-1BB signal
synergized CD28-mediated T cell activation by enhancing adhesive
response thereby being able to allow high proliferative responses
with the lower anti-CD3 concentration. This 4-1BB function may be
important for the T cells needed being continuously activated or
survived in limited co-stimulatory signals during chronic immune
reaction. In particular, 4-1BB role may be crucial in amplifying
cytotoxic T cell responses to eradicate weakly immunogenic tumor
cells which often down regulates T cell immune surveillance.
Promoted adhesive responses by 4-1BB may be a key
intermediating-pathways responsible for 4-1BB-mediated
amplification in CD8+ T cell cytotoxicity for tumor cells.
T Cell Integrin Activation
[0157] Up-regulation of integrin activity is induced by activation
of T cells within minutes, suggesting a qualitative, alteration in
function of the integrin receptor. The integrins co-stimulate T
cells activated by sub-mitogenic levels of anti-CD3 in the presence
of FN or other appropriate ECM proteins. Integrins a.sub.4b.sub.1
and a.sub.5b.sub.1 which bind to fibronectin (FN) or vesicular cell
adhesion molecule-1 (VCAM-1), characteristically play a predominant
role in mediating FN co-stimulated T cell proliferation and
intracellular Ca.sup.+2 signaling. FN, mediated adhesion of T cells
indicates activation and avidity of integrin.
[0158] The receptors which activate T cells include CD3/TCR complex
and CD2, CD7, CD28 and chemokines. Treatment of T cells with
phorbol ester, PMA or Ca+ ionophore, ionomycin also up-regulates
integrin activity, implicating both protein kinase C and
intracellular calcium with this regulatory events. Integrins are
associated intracellular cytoskeletal proteins and termini of actin
stress fiber bundles in cell attachment structures known as focal
adhesions. Stimulation of the integrins ultimately leads to
Rho-dependent focal adhesion formation that is accompanied by the
tyrosin phosphorylation of paxillin as well as changes in the
activity of the members of FAK, Src and Csk. Cross-linking of CD28,
a glycoprotein on T cells also results in increased adhesion to FN.
VCAM-1 and intercellular adhesion molecule-1 (ICAM-1).
CD28-mediated regulation 1:1 of b.sub.1-integrin-dependent adhesion
involves the association of phosphatidylinositol-3-kinase (PI 3-K).
CD28 has been well characterized as a co-stimulatory molecule for T
cell activation, regulating IL-2 gene expression.
[0159] The 4-1BB effects on cell adhesion absolutely required
anti-CD3 activation but not anti-CD28. Thus, 4.1BB function is
tightly controlled by antigenic activation. There was synergy
between CD28 and 4-1BB signals in Jurkat cell adhesion and thus the
4-1BB signal may function to complement CD28 co-stimulation for
downstream pathways facilitating the TCR signal to adapt to
inside-out adhesive responses.
[0160] Following the initial activation of primary T cells, a
series of adhesive responses to cell specific ligands or ECM also
co-stimulate T cell proliferation. Integrin activation requires TCR
signal to enhance avidity to their ligands. The expression of both
4-1BB and adhesion molecules expression may require prolonged TCR
activation, and 4-1BB may activate cell adhesion maintaining the
activated T cells to be at high activation stages which enhance
4-1BB expression by positive feedback amplifying loop especially in
weak antigen presentation. The expression of 4-1BB was heavily
dependent in vitro CD28 co-stimulation and therefore, correlation
of 4-1BB and CD28 co-stimulation in primary T cells is more complex
to interpret than those in Jurkat 4-1BB transfectants.
[0161] The cooperation of the two molecules shown by the essential
co-existence of 4-1BB and CD28 in further lowering the threshold of
anti-CD3 signal is primarily attributable to the activity of
adhesion molecules. Therefore, 4-1-BB has the effect of lowering
the threshold of antigen receptor signaling, thereby affecting
effector T cell function. TCR signaling intensities which is
dominantly influenced by co-stimulation, also effects the cytokines
such as IF and IL-2 production patterns. The 4-1BB-mediated
amplified cytotoxic T cell responses may well result from
up-regulated avidity of adhesive molecules on cytotoxic T cells or
the ability of the H4-1BB to reduce the antigenic threshold for
poorly immunogenic tumors.
Common Abbreviations
[0162] TABLE-US-00001 CTL cytolytic T lymphocyte HTL helper T
lymphocyte LGL large granular lymphocytes NK natural killer cells
ConA concanavalin A DTT dithiothreitol mAb monoclonal antibody.
4-1BB protein expressed on activated T cells rh 4-1BB recombinant
human 4-1BB 4-1BB/L ligand to 4-1BB found on activatedmacropage and
mature B cells 4-1BB/AP fusion protein between 4-1BB and alkaline
phosphatase. SDS sodium dodecysulfate SSC 150 mM sodium chloride/15
mM sodium citrate, pH 7.0 TPA 12-0-tetradecanoylphorbol-13-acetate
Th helper T lymphocytes IL-2 interleukin 2 IL-3 interleukin 3 rIL-2
recombinant IL-2 CSF-GM granulocyte/macrophage colony-stimulating
factors cRNA complementary RNA ss single-stranded ds
double-stranded TCR T-cell antigen receptor PTA phorbol
12-tetradecanoate 13-acetate r recombinant mu murine hu human BFU-E
burst forming unit-erythroid, an erythroid progenitor cell CFU-GEMM
colony forming unit-granulocyte erythroid macrophage megakaryocyte,
a multipotential progenitor cell CFU-GM colony forming
unit-granulocyte macrophage, a granulocyte-macrophage progenitor
cell CFU-S colony forming unit-Spleen, a multipotential stem cell
H-ferritin the heavy chain subunit form of ferritin MGF mast cell
growth factor, a c-kit ligand CSF colony stimulating factors G
granulocyte M macrophage Epo erythropoietin IL interleukin LD low
density NALDT non-adherent low density T-lymphocyte depleted PMSF
phenylmethylsulfonyl fluoride PBS phosphate buffered saline AcNPV
Autographa californica nuclear polyhedrosis virus SDS sodium
dodecyl sulfate LPS lipopolysaccharide
CITATIONS AND REFERENCES INCORPORATED HEREIN BY REFERENCE
[0163] 1. Smith, C. A., Davis, T., Anderson, D., Solam, L.,
Beckmann, M. P., Jerzy, R., Dower, S. K., Cosrnan, D., and Goodwin,
R. G. 1990. A receptor for tumor necrosis factor defines an unusual
family of cellular and viral proteins. Science 248:1019-1023.
[0164] 2. Ebina, Y., L. Ellis, K. Jaruagin, M. Edery, L. Graf, E.
Clauser, J. On, F. Marizrz, Y. W. Kan, J. D. Goldfine, R. A. Roth
and W. J. Rutter, 1985, The human insulin receptor cDNA: the
structural basis for hormone-activated transmembrane signalling,
Cell 40:747. [0165] 3. Vassali, R., R. Tedghi, B.
Listowska-Bernstein, A. Tartakoff and J. C. Jaton, 1979, Evidence
for hydrophobic region within heavy chains of mouse B lymphocyte
membrane-bound IgM, Proc. Natl. Acad. Sci. USA 76:5515. [0166] 4.
Haskins, K., R. Kubo, J. White, M. Pigeon, J. Kappler and P.
Marrack, 1983, The major histocompatability complex-restricted
antigen receptor on T cells I Isolation with monoclonal antibody,
J. Exp. Med. 157:1149. [0167] 5. Lesslayer, W. and H. Gmunder,
1986, Biochemical characterization of the 9.3 antigens of human
T-cells: stimultaneous expression of disulfide-bonded 90-Kiladalton
dimers and free subunits at the cell surface, Mol. Immunol. 23:271
[0168] 6. Van Lier, R., J. Borst, T. Vroom, H. Klein, P. Mourik, W.
Zeijlemaker and C. Melife, 1987, Tissue distribution and
biochemical and functional properties of Tp5.5 (CD27) a novel T
cell differentiation antigen, J. Immunol. 139:1589. [0169] 7.
Mallett, S., S. Fossum and A. Barclay, 1990, Characterization of
the MRC OX40 antigen of activated CD4 positive T lymphocytes-a
molecule related to nerve growth factor receptor, EMBO J. 9':1603.
[0170] 8. Banchereau, J., P. Paoli, A., Valle, E. Garcia and F.
Roussel, 1991, Long-term human B cell lines dependent on
interleukin-4 and antibody to CD40, Science 251:70. [0171] 9.
Moeller, D. L., M. K. Jenkins and R. H. Schwartz, 1989, Clonal
expansion versus functional colonal inactivation: a co-stimulatory
signalling pathway determines the outcome of T cell antigen
receptor occupancy, Ann. Rev. Immunol, 7:445. [0172] 10. June, D.
H., J. A. Ledbetter, P. S. Linsley and C. B. Thompson, 1989, Role
of CD28 receptor in T cell activation, Immunol. Today 11:211.
[0173] 11. Yang, L., B. Jones, A. Aruffo, K. M. Sullivan, P. S.
Linsley and C. A. Janeway, Jr., 1992, Heat stable antigen is a
co-stimulatory molecule for CD4 T cell growth, J. Exp. Med.
175:437. [0174] 12. Yamori, T., 1992, Molecular mechanisms for
generation of neural diversity and specificity: foles of
polypeptide factors in development of post-mitotic neurons,
Neuroscience Res. 12:545. [0175] 13. Liu, Y. J., D. E. Joshua; G.
T. Williams, C. A. Smith, J. Gordon and I. C. M. MacLennan, 1989,
Mechanism of antigen-driven selection in germinal centres, Nature,
342:929. [0176] 14. Jabara, H. H., S. M. Fu, R. S. Geha, and D.
Vercelli, 1990, CD40 and IgE: synergism between anti-CD40
momoclonal antibody and interleukin 4 in the induction of IgE
synthesis by highly purified human B cells, J. Exp. Med. 172:1861.
[0177] 15. Defrance, R., B. Vanbervliet, F. Briere, I. Durnad, F.
Roussle and J. Banchereau, 1992, Interleukin 10 and transforming
growth factor b cooperate to induce anti-CD40 activated naive human
B cells to secrete immunoglobulin A, J. Exp. Med. 175:671. [0178]
16. Donahue, T., Cigan, A., Pahich, E. and Valavicius, B.,
Mutations at a Zn(II) finger motif in the yeast elF-2b gene alter
ribosomal start-site selection during the scanning process, Cell 54
(1988) 621-632. [0179] 17. Carthew, R. W and Rubin, G. M., seven in
absentia, a gene required for specification of R7 cell rate in the
Drosophila eye, Cell, 63 (1990) 561-577. [0180] 18. Driscoll, D. M.
and Williams, J. G., Two divergently transcribed genes of
Dictyostelium discoideum are cyclic AMP-inducible and coregulated
during development, Mol. and Cell. Biol. 7 (1987) 4482-4489. [0181]
19. Chalupny, N. J., Peach, R., Hollenbaugh, D., Ledbetter, J. A.,
Farr, A. G. and Aruffo, A., 1992, Proc. Natl. Acad. Sci. USA
89:10360-10364. [0182] 20. Noelle, R. J., and Snow, E. C., 1991,
The FASEB J. 5:2770-2776. [0183] 21. Noelle, R. and Snow, E., 1990,
Immunol. Today 11:361-368. [0184] 22. Zurawski, G., Benedik, M.,
Kamb, B. J., Abrams, J. S., Zurawaki, S. M. and Lee, F. D. (1986)
Science 232.772-775. [0185] 23. Kinachi, T. (1986) Nature 325,
70-73. [0186] 24. Gershenfeld, H. K. and Weissman, I. L. Science
(1986) 232; 854-858. [0187] 25. Biggin, M., Gison, T. and Hung, G.
(1983 Proc. Natl. Acad. Sci. USA 80, 3963-3965. [0188] 26. Hodgkin,
P. D., Yamashita, L. C., Coffman, R. L. and Kehry, M. R., 1990, J.
Immunol. 145:2025-2034. [0189] 27. Barlett, W. C., McCann, J.,
Shephaer, D. M., Roy, M. and Noelle, R. J., 1990, J. Immunol.
145:3956-3962. [0190] 28. Kwon, B. S., Kestler, D. P., Eshhar, Z.,
Oh, K., and Wakulchik, M. 1989 Expression characteristics of two
potential T cell mediator genes. Cell. Immunol. 121:414422. [0191]
29. Armitage, R., Fanslow, W., Strockbine, L., Sato, T., Clifford,
K., MacDuff, B., Anderson, D., Gimpel, S., Davis-Smith, T.,
Maliszewski, C., Clark, E., Smith, C., Grabstein, K., Cosman, D.
and Spriggs, M., 1991, Nature 357:80-82. [0192] 30. Kwon, B.,
Kestler, D., Lee, E., Wakulchik, M. and Young J. (1988) J. Exp.
Med. (1988). [0193] 31. Noelle, R. J., Roy, M., Shepherd, D. M.,
Stamenkovic, I., Ledbetter, J. A. and Aruffo, A., 1992, Proc. Natl.
Acad. Sci. USA 89:6550-6554. [0194] 32. Hollenbaugh, D., Grosmaier,
L. S., Kullas, C. D., Chalupny, N. J., Braesch-Andersen, S.,
Noelle, R. J., Stamenkovic, I., Ledbetter, J. A. and Aruffo, A.,
1992, EMBO 11:4314-4321. [0195] 33. Schall, T. J., M. Lewis, K. J.
Koller, A. Lee, G. C. Rice, G. H. W. Wong, T. Gatanaga, G. A.
Granger, R. Lentz, H. Raab, W. J. Kohr and D. V. Goeddel, 1990,
Molecular cloning and expression of a receptor for human tumor
necrosis factor, Cell 61:361. [0196] 34. Klein, R., Nanduri, V.,
Jiig, S., Lamballe, F., Tapley, P., Bryant, S., Cordon-Cardo, C.,
Jones, K. R., Reichardt, L. F. and Barbacid, M., 1991, Cell
66:395403. [0197] 35. Armitage, R. J., Sato, T. A., Macduff, B. M,
Clifford, K. N., Alpert, A. R, Smith, C. A. and Fanslow, W. C.,
1992, Eur. J. Immunol. 22:2071-2076. [0198] 36. Hintzen, R. Q.,
deJong, R., Hack, E. E., Chamuleau, M., de Vries, E. F. R., ten
Berge, I. J. M., Borst, J. and van Lier, R. A. W.; 1991, J.
Immunol., 147:29-35. [0199] 37. Mallett, S., and Barclay, A. N.
1991. A new super-family of cell surface proteins related to the
nerve growth factor receptor. Immunol. Today. 12:220-223. [0200]
38. Kwon, B. S., and Weissman, S. M. 1989. cDNA sequences of two
inducible T-cell genes. Proc. Natl. Acad. Sci. USA. 86:1963-1967.
[0201] 39. Johnson, D., Lanahan, A., Buck C. R., Sehgal, A.,
Morgan, C., Mercer, E., Bothwell, M., and Chao, M. 1986. Expression
and structure of the human NGF receptor. Cell 47:545-554.
[0202] 40. Stamenkovic, I., Clark, E., and Seed, B. 1989.
AB-lymphocyte activation molecule related to the nerve growth
factor receptor and induced by cytokines in carcinomas. EMBO. J.
8:1403-1408. [0203] 41. Pollok K E, Y-J Kim, Z Zhou, J Hurtado, K K
Kim, and B S Kwon. 1993. The inducible T cell antigen 4-1BB:
Analysis of expression and function. J Immunol 150:771. [0204] 42.
Antibody Lab Manual. 1988. Editors are E Harlow and D Lane. Cold
Spring Harbor Lab. [0205] 43. Pelchen-Matthews, A, J E Armes, G
Griffiths, and M Marsh. 1991. Differential endocytosis of CD4 in
lymphocytic and nonlymphocytic cells. J Exp Med. 173:575. [0206]
44. Biffen, M, D McMichael-Phillips, T Larson, A Venkitaraman, and
D Alexander. 1994. The CD45 tyrosine phosphatase regulates specific
pools of antigen receptor-associated P.sub.59 and CD4-associated
p56.sup.lck tyrosine kinases in human T-cells. EMBO J. 13:1920.
[0207] The foregoing description has been directed to particular
embodiments of the invention in accordance with the requirements of
the Patent Statutes for the purposes of illustration and
explanation. It will be apparent, however, to those skilled in this
art that many modifications and changes will be possible without
departure from the scope and spirit of the invention. It is
intended that the following claims be interpreted to embrace all
such modifications.
Sequence CWU 1
1
10 1 838 DNA Homo sapiens 1 aatcagcttt gctagtatca tacctgtcgc
agatttcatc atgggaaaca gctgttacaa 60 catagtagcc actctgttgc
tggtcctcaa ctttgagagg acaagatcat tgcaggatcc 120 ttgtagtaac
tgcccagctg gtacattctg tgataataac aggaatcaga tttgcagtcc 180
ctgtcctcca aatagtttct ccagcgcagg tggacaaagg acctgtgaca tatgcaggca
240 gtgtaaaggt gttttcagga ccaggaagga gtgttcctcc accagcaatg
cagagtgtga 300 ctgcactcca gggtttcact gcctgggggc aggatgcagc
atgtgtgaac aggattgtag 360 acaaggtcaa gaactgacaa aaaaaggttg
taaagactgt tgctttggga catttaacga 420 tcagaaacgt ggcatctgtc
gaccctggac aaactgttct ttggatggaa agtctgtgct 480 tgtgaatggg
acgaaggaga gggacgtggt ctgtggacca tctccagccg acctctctcc 540
gggagcatcc tctgtgaccc cgcctgcccc tgcgagagag ccaggacact ctccgcagat
600 catctccttc tttcttgcgc tgacgtcgac tgcgttgctc ttcctgctgt
tcttcctcac 660 gctccgtttc tctgttgtta aacggggcag aaagaaactc
ctgtatatat tcaaacaacc 720 atttatgaga ccagtacaaa ctactcaaga
ggaagatggc tgtagctgcc gatttccaga 780 agaagaagaa ggaggatgtg
aactgtgaaa tggaagtcaa tagggctgtt gggacttt 838 2 255 PRT Homo
sapiens 2 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu
Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys
Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn
Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala
Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly
Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala
Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly
Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110
Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115
120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly
Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val
Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser
Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser
Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala
Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235
240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245
250 255 3 20 DNA Homo sapiens 3 ttytgymgaa artayaaycc 20 4 20 DNA
Homo sapiens 4 ttytcstsca htggtggaca 20 5 20 DNA Homo sapiens 5
cccargswrc aggtyttrca 20 6 20 DNA Homo sapiens 6 ttytgrtcrt
traatgttcc 20 7 25 DNA Homo sapiens 7 aataagcttt gctagtatca tacct
25 8 30 DNA Homo sapiens 8 ttaagatctc tgcggagagt gtcctggctc 30 9 24
PRT Homo sapiens VARIANT (0)...(0) Xaa=Any amino acid 9 Cys Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa
His Xaa Xaa Xaa Cys Xaa Cys 20 10 18 PRT Homo sapiens 10 Phe Glu
Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala 1 5 10 15
Gly Thr
* * * * *