U.S. patent application number 09/794258 was filed with the patent office on 2001-12-27 for method to regulate cd40 signaling.
This patent application is currently assigned to National Jewish Medical and Research Center. Invention is credited to Gelfand, Erwin W., Johnson, Gary L..
Application Number | 20010055753 09/794258 |
Document ID | / |
Family ID | 21734211 |
Filed Date | 2001-12-27 |
United States Patent
Application |
20010055753 |
Kind Code |
A1 |
Gelfand, Erwin W. ; et
al. |
December 27, 2001 |
Method to regulate CD40 signaling
Abstract
The present invention relates to methods useful for identifying
compounds capable of specifically controlling CD40 regulation of
JNK or p38 activity useful for inhibiting immunoglobulin heavy
chain class switching, cytokine production and activation of cells
involved in an inflammatory response. The present invention also
includes kits to perform such assays and methods to control disease
related to such responses.
Inventors: |
Gelfand, Erwin W.;
(Englewood, CO) ; Johnson, Gary L.; (Boulder,
CO) |
Correspondence
Address: |
Angela Dallas-Pedretti
SHERIDAN ROSS P.C.
1560 Broadway, Suite 1200
Denver
CO
80202-5141
US
|
Assignee: |
National Jewish Medical and
Research Center
|
Family ID: |
21734211 |
Appl. No.: |
09/794258 |
Filed: |
February 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09794258 |
Feb 27, 2001 |
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09361436 |
Jul 26, 1999 |
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09361436 |
Jul 26, 1999 |
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08769747 |
Dec 19, 1996 |
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6132978 |
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60008877 |
Dec 19, 1995 |
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Current U.S.
Class: |
435/4 ; 435/7.21;
435/7.32 |
Current CPC
Class: |
G01N 2500/10 20130101;
C12Q 1/48 20130101; G01N 2333/70578 20130101; G01N 33/5052
20130101; G01N 2333/912 20130101 |
Class at
Publication: |
435/4 ; 435/7.21;
435/7.32 |
International
Class: |
C12Q 001/00; G01N
033/567; G01N 033/554; G01N 033/569 |
Goverment Interests
[0002] This invention was made in part with government support
under DK-37871 and GM-30324, both awarded by the National
Institutes of Health. The government has certain rights to this
invention.
Claims
What is claimed is:
1. A method to identify a compound that controls CD40 regulation of
JNK activity in a cell, comprising: (a) contacting a cell with a
putative regulatory compound, wherein the cell includes a CD40
protein and a Jun kinase protein; and (b) assessing the ability of
said putative regulatory compound to regulate the activity of said
Jun kinase.
2. The method of claim 1, wherein said assessment step comprises
determining the ability of said JUN kinase protein to regulate the
activity of a protein selected from the group consisting of c-Jun,
ATF-2, Ets-1 and other Ets family members activity in said
cell.
3. The method of claim 1, wherein said method further comprises
stimulating said cell, prior to said assessing step, with a ligand
of CD40.
4. The method of claim 1, wherein said method further comprises
stimulating said cell, prior to said assessment step, with a
stimulatory compound selected from the group consisting of soluble
gp39, membrane-bound gp39 and an antibody that binds specifically
to CD40.
5. The method of claim 1, wherein said cell is selected from the
group consisting of a mammalian, an invertebrate, a plant, an
insect, a fungal, a yeast and a bacterial cell.
6. The method of claim 1, wherein said cell is selected from the
group consisting of a mammalian, an amphibian and a yeast cell.
7. The method of claim 1, wherein said cell is selected from the
group consisting of a primate, a mouse and a rat cell.
8. The method of claim 1, wherein said cell is selected from the
group consisting of Pre-B cells, B lymphocytes, cancer cells,
fibroblasts, Langerhans cells, epithelial cells monocytes and
dendritic cells.
9. The method of claim 1, wherein said putative regulatory compound
is selected from the group consisting of a protein-based compound,
a carbohydrate-based compound, a lipid-based compound, a nucleic
acid-based compound, a natural organic compound, a synthetically
derived organic compound, an anti-idiotypic antibody and/or
catalytic antibody, or fragments thereof.
10. The method of claim 1, wherein said putative regulatory
compound is selected from the group consisting of a small organic
molecule, a peptide and a polypeptide.
11. A method to identify a compound that controls CD40 regulation
of p38 activity in a cell, comprising: (a) contacting a cell with a
putative regulatory compound, wherein the cell includes CD40
protein and p38 protein; and (b) assessing the ability of said
putative regulatory compound to regulate the activity of said p38
protein.
12. The method of claim 11, wherein said assessment step comprises
determining the ability of said p38 protein to regulate the
activity of a protein selected from the group consisting of ATF-2,
Ets-1 and other Ets family members activity in said cell.
13. The method of claim 11, wherein said method further comprises
stimulating said cell, prior to said assessing step, with a ligand
of CD40.
14. The method of claim 11, wherein said method further comprises
stimulating said cell, prior to said assessment step, with a
stimulatory compound selected from the group consisting of soluble
gp39, membrane-bound gp39 and an antibody that binds specifically
to CD40.
15. The method of claim 11, wherein said cell is selected from the
group consisting of Pre-B cells, B lymphocytes, cancer cells,
fibroblasts, Langerhans cells, epithelial cells monocytes and
dendritic cells.
16. The method of claim 11, wherein said putative regulatory
compound is selected from the group consisting of a small organic
molecule, a peptide and a polypeptide.
17. A regulatory compound identified by said compound's ability to
regulate a biological function selected from the group consisting
of immunoglobulin heavy chain class switching, cytokine production
and inflammatory cell activation, said compound being capable of
penetrating the plasma membrane of a cell and of inhibiting the
ability of CD40 protein to regulate JNK protein activity in said
cell.
18. The compound of claim 17, wherein said compound is capable of
regulating the activity of a protein selected from the group
consisting of CD40 and Jun kinase protein.
19. The compound of claim 17, wherein said regulation involves a
step selected from the group consisting of sequestering JNK protein
in an inactive complex, regulating the ligand binding activity of
CD40, regulating MEKK activity, regulating the phosphorylation of
JNK protein, regulating the interaction between JNK and JNKK,
regulating the ability of JNK to activate c-Jun, ATF-2, and Ets-1
and other Ets family members, regulating the, expression of
endogenous and/or heterologous nucleic acid molecules encoding CD40
or JNK protein, and combinations thereof.
20. A regulatory compound capable of penetrating the plasma
membrane of a cell and of inhibiting the ability of CD40 protein to
regulate p38 protein activity in said cell, said compound able to
regulate a biological function selected from the group consisting
of immunoglobulin heavy chain class switching, cytokine production
and inflammatory cell activation.
21. The compound of claim 20, wherein said compound is capable of
regulating the activity of a protein selected from the group
consisting of CD40 and p38 protein.
22. The compound of claim 20, wherein said regulation involves a
step selected from the group consisting of sequestering p38 protein
in an inactive complex, regulating the ligand binding activity of
CD40, regulating MEKK activity, regulating the phosphorylation of
p38 protein, regulating the interaction between p38 and MEK,
regulating the ability of p38 to activate ATF-2, and Ets-1 and
other Ets family members, regulating the expression of endogenous
and/or heterologous nucleic acid molecules encoding CD40 or p38
protein, and combinations thereof.
23. A method to inhibit immunoglobulin heavy chain class switching,
comprising inhibiting the activity of a protein selected from the
group consisting of Jun kinase protein and p38 protein.
24. The method of claim 23, wherein said activity being inhibited
is selected from the group consisting of sequestering JNK or p38
protein in an inactive complex, regulating the ligand binding
activity of CD40, regulating MEKK activity, regulating the
phosphorylation of JNK or p38 protein, regulating the interaction
between JNK and JNKK, regulating the ability of JNK to activate
c-Jun, ATF-2, and Ets-1 and other Ets family members, regulating
the interaction between p38 and MEK, regulating the ability of p38
to activate ATF-2, and Ets-1 and other Ets family members,
regulating the expression of endogenous or heterologous nucleic
acid molecules encoding a CD40, JNK or p38 protein, and
combinations thereof.
25. The method of claim 24, wherein said activity is inhibited
using a compound selected from the group consisting of a Jun kinase
activation site mimetope, a Jun kinase target site mimetope, a Jun
kinase kinase domain mimetope, a mutated Jun kinase protein, a Jun
kinase pseudosubstrate, a p38 activation site mimetope, a p38
target site mimetope, a p38 kinase domain mimetope, a mutated p38
protein, a p38 pseudosubstrate, a mutated CD40 protein, a CD40
antagonist, an antisense oligonucleotide, a ribozyme and an
expression plasmid encoding CD40 or Jun kinase.
26. A method to inhibit cytokine production by a cell having CD40,
comprising inhibiting the activity of a protein selected from the
group consisting of Jun kinase protein and p38 protein.
27. The method of claim 26, wherein said activity is inhibited
using a compound selected from the group consisting of a Jun kinase
activation site mimetope, a Jun kinase target site mimetope, a Jun
kinase kinase domain mimetope, a mutated Jun kinase protein, a Jun
kinase pseudosubstrate, a p38 activation site mimetope, a p38
target site mimetope, a p38 kinase domain mimetope, a mutated p38
protein, a p38 pseudosubstrate, a mutated CD40 protein, a CD40
antagonist, an antisense oligonucleotide, a ribozyme and an
expression plasmid encoding CD40 or Jun kinase.
28. A method to treat an animal with a disease selected from the
group consisting of a disease involving an allergic response and a
disease involving an autoimmune disease, said method comprising
administering to an animal an effective amount of a therapeutic
composition comprising a compound that controls CD40 regulation of
the activity of a protein selected from the group consisting of Jun
kinase and p38 protein.
29. The method of claim 28, wherein said disease involving an
allergic response is selected from the group consisting of asthma,
allergic rhinitis, allergic hypersensitivity, atopic dermatitis and
acute bronchopulmonary aspergillosis.
30. The method of claim 28, wherein said disease involving an
autoimmune disease is selected from the group consisting of
rheumatoid arthritis and systemic lupus erythematosus.
31. A kit to identify compounds that controls CD40 regulation of
JNK activity in a cell, said kit comprising: (a) a cell comprising
a CD40 protein and a Jun kinase protein; and (b) a means for
detecting regulation of said Jun kinase protein.
32. The kit of claim 32, wherein said cell is selected from the
group consisting of Pre-B cells, B lymphocytes, cancer cells,
fibroblasts, Langerhans cells, epithelial cells monocytes and
dendritic cells.
33. A kit to identify compounds that controls CD40 regulation of
p38 activity in a cell, said kit comprising: (a) a cell comprising
CD40 protein and p38 protein; and (b) a means for detecting
regulation of said p38 protein.
34. The kit of claim 33, wherein said cell is selected from the
group consisting of Pre-B cells, B lymphocytes, cancer cells,
fibroblasts, Langerhans cells, epithelial cells monocytes and
dendritic cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from U.S.
Provisional Application Serail No. 60/008,877, filed Dec. 19,
1995.
FIELD OF THE INVENTION
[0003] The present invention relates to a process for identifying
compounds that control CD40 regulation of Jun kinase and p38 MAP
kinase activity. The present invention also relates to a method to
treat allergies and autoimmunity by regulating the activity of Jun
kinase and p38 MAP kinase in a cell.
BACKGROUND OF THE INVENTION
[0004] The B lymphocyte surface antigen receptor membrane
immunoglobulin has important functions in the binding and
internalization of antigen, as well as in transducing signals
through the plasma membrane which lead to cell activation,
differentiation and apoptosis. Crosslinking of the receptor
stimulates the Ras/Raf-1/MEK cascade with activation of
p42.sup.erk2 MAPK and p90.sup.rsk. A second important B cell
surface antigen receptor is CD40. CD40 is a 45-50 kD transmembrane
glycoprotein expressed on all mature B cells. CD40 is a member of
the TNF receptor family and has homology to the receptors for nerve
growth factor, TNF-.alpha., Fas and CD30. The ligand for CD40
(CD40L, gp39) is expressed on activated T lymphocytes and
activation through CD40 plays an important role in T cell-dependent
immunoglobulin isotype switching. The signal transduction pathways
through CD40 are not well delineated.
[0005] Certain biological functions of a B lymphocyte (B cell) are
tightly regulated by signal transduction pathways within B cells.
Signal transduction pathways maintain the balanced steady state
functioning of a cell. Disease states can arise when the steady
state function of a cell becomes harmful to an animal. For example,
allergic reactions occur due to undesired production of IgE
antibodies specific for an antigen. In addition, autoimmunity can
occur due to an animal mounting an undesired immune response
against a self-antigen. Signal transduction pathways in a cell can
be responsible for regulating cellular biological functions. As
such, regulation of signal transduction pathways can assist in the
regulation of undesired cellular biological functions.
[0006] Despite a long-felt need to understand and discover methods
for regulating cells involved in various disease states, the
complexity of signal transduction pathways has precluded the
development of products and processes for regulating cellular
function by manipulating signal transduction pathways in a cell. As
such, there remains a need for products and processes that permit
the implementation of predictable controls of signal transduction
in cells, thus enabling the treatment of various diseases that are
caused by undesired cellular function.
SUMMARY OF THE INVENTION
[0007] The present invention provides a solution to the complex
problem of identifying regulatory compounds which can be used to
regulate cellular function, including CD40 regulation of Jun kinase
(JNK) and p38 MAP kinase (p38) activity. Despite the complexity of
signal transduction networks in cells, the present invention
provides for an efficient method for regulating JNK or p38 activity
and identifying compounds capable of specifically regulating JNK or
p38 activity.
[0008] The present invention is particularly advantageous because
it provides for a method to identify compounds that can regulate
the production of IgE by an animal without substantially
interfering with the production of IgG and IgA by an animal. As
such, unlike traditional immunosuppressive reagents which suppress
an animal's immune response indiscriminately, a compound identified
by a method of the present invention enables an animal to mount an
immune response against foreign pathogens by producing IgG and IgA
antibodies. Thus, those of skill in the art will immediately
recognize the advantages arising from this invention which include
the identification and uses of compounds which are useful for the
treatment of allergic and autoimmune diseases but not disruptive to
an animal's overall immune response.
[0009] One embodiment of the present invention includes a method to
identify a compound that controls CD40 regulation of Jun kinase
(JNK) activity in a cell, comprising: (1) contacting a cell with a
putative regulatory compound, wherein the cell includes a CD40
protein and a Jun kinase protein; and (2) assessing the ability of
the putative regulatory compound to regulate the activity of the
Jun kinase. Another embodiment of the present invention includes a
method to identify a compound that controls CD40 regulation of p38
activity in a cell, comprising: (1) contacting a cell with a
putative regulatory compound, wherein the cell includes CD40
protein and p38 protein; and (2) assessing the ability of the
putative regulatory compound to regulate the activity of the p38
protein. In particular, these methods of the present invention
include a step of stimulating the cell, prior to the assessing
step, with a ligand of CD40.
[0010] Also included in the present invention is a regulatory
compound identified by said compound's ability to regulate a
biological function selected from the group consisting of
immunoglobulin heavy chain class switching, cytokine production and
inflammatory cell activation, the compound being capable of
penetrating the plasma membrane of a cell and of inhibiting the
ability of CD40 protein to regulate JNK protein or p38 activity in
the cell.
[0011] The present invention includes a method to inhibit
immunoglobulin heavy chain class switching, comprising inhibiting
the activity of a protein selected from the group consisting of Jun
kinase protein and p38 protein. The present invention also includes
a method to inhibit cytokine production by a cell having CD40,
comprising inhibiting the activity of a protein selected from the
group consisting of Jun kinase protein and p38 protein.
[0012] One aspect of the present invention includes a method to
treat an animal with a disease selected from the group consisting
of a disease involving an allergic response and an autoimmune
disease, said method comprising administering to an animal an
effective amount of a therapeutic composition comprising a compound
that controls CD40 regulation of the activity of a protein selected
from the group consisting of Jun kinase and p38 protein.
[0013] Another aspect of the present invention includes a kit to
identify compound that controls CD40 regulation of JNK or p38
activity in a cell, the kit comprising: (1) a cell comprising a
CD40 protein, and a Jun kinase or a p38 protein; and (2) a means
for detecting regulation of the Jun kinase or p38 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 illustrates the activation of ERK protein following
treatment with anti-IgM antibody but not after treatment with
anti-CD40 antibody.
[0015] FIG. 2 illustrates the time course of JNK activation after
treatment of Ramos cells with 1 .mu.g/ml anti-CD40 antibody.
[0016] FIG. 3 illustrates the dose response of anti-CD40 antibody
activated JNK in Ramos cells treated using various concentrations
of anti-CD40 antibody for 15 min.
[0017] FIG. 4 illustrates the dose response of JNK activity to
anti-CD40 antibody stimulation in tonsillar B cells treated for 15
min.
[0018] FIG. 5 illustrates activation of JNK using soluble gp39, in
which dilutions of culture supernatants containing soluble gp39
were added to Ramos cells for 15 min.
[0019] FIG. 6 illustrates the absence of stimulation of JNK
activity in Ramos cells by anti-IgM antibody at different time
periods.
[0020] FIG. 7 illustrates the absence of stimulation of JNK
activity in tonsillar B cells at different time periods.
[0021] FIG. 8 illustrates the activation of Ras following treatment
of cells with anti-IgM antibody but not after treatment with
anti-CD40 antibody.
[0022] FIG. 9 illustrates the activation of MEKK protein following
treatment with anti-CD40 antibody.
[0023] FIG. 10 illustrates the activation of JNK in Ramos cells by
anti-IgM or anti-CD40 antibody alone, or after preincubation with
anti-CD40 antibody followed by incubation with anti-IgM
antibody.
[0024] FIG. 11 illustrates activation of p38 in the presence or
absence of anti-CD40 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention relates to a method for identifying
compounds that regulate CD40 regulation of JNK or p38 activity and
products identified using such method. As used herein, the phrase
"signal transduction pathway" refers to at least one biochemical
reaction, but more commonly a series of biochemical reactions,
which result from interaction of a cell with a stimulatory
compound. The interaction of a stimulatory compound with a cell
generates a "signal" that is transmitted through a signal
transduction pathway, ultimately resulting in JNK or p38
activation. Compounds inhibitory to signal transduction pathways
(antagonists) are also useful and can be identified by the methods
of the present invention.
[0026] A signal transduction pathway of the present invention can
include a variety of signal transduction molecules that play a role
in the transmission of a signal from one portion of a cell to
another portion of a cell. As used herein, the term "molecule"
refers to a protein, a lipid, a nucleic acid or an ion, and at
times is used interchangeably with such terms. In particular, a
signal transduction molecule refers to a protein, a lipid, a
nucleotide, or an ion involved in a signal transduction pathway.
Signal transduction molecules of the present invention include, for
example, cell surface receptors and intracellular signal
transduction molecules. As used herein, the phrase "cell surface
receptor" includes molecules and complexes of molecules capable of
receiving a signal and the transmission of such a signal across the
plasma membrane of a cell. The phrase "intracellular signal
transduction molecule," as used herein, includes those molecules or
complexes of molecules involved in transmitting a signal from the
plasma membrane of a cell through the cytoplasm of the cell, and in
some instances, into the cell's nucleus. The phrase "stimulatory
compound", as used herein, includes ligands capable of binding to
cell surface receptors to initiate a signal transduction pathway,
as well as intracellular initiator molecules capable of initiating
a signal transduction pathway from inside a cell. One aspect of the
present invention includes a cell-based assay to identify
compounds, referred to herein as "putative regulatory compounds",
which are capable of regulating CD40 regulation of JNK or p38
activity. As used herein, the term "putative" refers to compounds
having an unknown or previously unappreciated regulatory activity
in a particular process. As such, the term "identify" is intended
to include all compounds, the usefulness of which as a regulatory
compound of JNK activity is determined by a method of the present
invention.
[0027] One embodiment of the present invention relates to a method
to identify a compound that controls CD40 regulation of JNK
activity in a cell, comprising: (1) contacting a cell with a
putative regulatory compound, wherein the cell includes a CD40
protein and a Jun kinase (JNK) protein; and (2) assessing the
ability of the putative regulatory compound to regulate the
activity of the Jun kinase. The assessment step preferably involves
determining the phosphorylation of JNK upon ligation of the CD40
using antibodies specific for CD40 and/or CD40 ligand (gp39;
described in Noelle et al., Proc. Natl. Acad. Sci. USA
89:6550-6554, 1992). JNK regulates the activity of the
transcription factor JUN which is involved in controlling the
growth and differentiation of different cell types, such as B
cells, T cells, neural cells or fibroblasts. JNK is a member of the
Ras signal transduction pathway, which includes molecules such as
MEKK protein Jun, ATF and Myc protein.
[0028] Another embodiment of the present invention relates to a
method to identify a compound that controls CD40 regulation of p38
activity in a cell, comprising: (1) contacting a cell with a
putative regulatory compound, wherein the cell includes a CD40
protein and a p38 protein; and (2) assessing the ability of the
putative regulatory compound to regulate the activity of the
p38.
[0029] The present method can further comprise assessing the
ability of a putative regulatory compound to inhibit:
immunoglobulin heavy chain class switching in a cell; cytokine
production by a cell; or activation of inflammatory cells (i.e.,
cells involved in an inflammatory response). Methods for
determining immunoglobulin heavy chain class switching are to those
of skill in the art. For example, Southern blots can be performed
using DNA probes specific for genes encoding different classes of
immunoglobulin heavy chains to look for rearrangement of the DNA
encoding the different classes. Alternatively, immunoassays can be
performed on proteins produced by the treated cell using antibodies
specific for different classes of immunoglobulin heavy chains.
Methods for determining cytokine production are known to those of
skill in the art. For example, cell responsiveness assays using
cells capable of responding to a cytokine can be used to test the
disruption of cytokine production by a putative regulatory
compound. In addition, immunoassays using antibodies specific for a
cytokine can be used to test the disruption of cytokine production
by a putative regulatory compound. Methods for determining
inhibition of inflammatory cell activation are known to those of
skill in the art by testing the ability of an inflammatory cell to
perform a desired biological function in the presence or absence of
a putative regulatory protein.
[0030] Suitable cells for use with the present invention include
any cell that has CD40, and JNK or p38 protein. Such cells can
include normal cells or transformed cells (i.e., with a
heterologous nucleic acid molecule) that express CD40, and JNK
and/or p38 in a native physiological context (e.g., Pre-B cells, B
lymphocytes, cancer cells, fibroblasts, Langerhans cells,
epithelial cells monocytes and dendritic cells). Alternatively,
cells for use with the present invention can include spontaneously
occurring variants of normal cells, or genetically engineered
cells, that have altered signal transduction activity, such as
enhanced responses to particular ligands. Signal transduction
variants of normal cells can be identified using methods known to
those in the art. For example, variants can be selected using
fluorescence activated cell sorting (FACS) based on the level of
calcium mobilization by a cell in response to a ligand. Genetically
engineered cells can include recombinant cells of the present
invention (described in detail below) that have been transformed
with, for example, a recombinant molecule encoding a signal
transduction molecule of the present invention.
[0031] Cells for use with the present invention include mammalian,
invertebrate, plant, insect, fungal, yeast and bacterial cells.
Preferred cells include mammalian, amphibian and yeast cells.
Preferred mammalian cells include primate, non-human primate, mouse
and rat, with human cells being preferred.
[0032] In one embodiment, a cell suitable for use in the present
invention has a functional CD40 on the surface of the cell. A
functional CD40 can comprise a full-length or a portion of a CD40
that is capable of transmitting a signal across the plasma membrane
of a cell, upon ligation with an anti-CD40 antibody or a CD40
ligand, in such a manner that immunoglobulin heavy chain class
switching results. Preferably, a cell of the present invention
expresses a CD40 derived from a human, mouse or rat, with human
cells being preferred.
[0033] In another embodiment, a cell suitable for use in the
present invention has one or more intracellular signal transduction
molecules capable of transmitting a signal through the cytoplasm of
the cell, resulting in JNK and/or p38 activation. An intracellular
signal transduction molecule as described herein can be produced in
a cell by expression of a naturally occurring gene and/or by
expression of a heterologous nucleic acid molecule transformed into
the cell.
[0034] A preferred cell of the present invention has, amongst other
signal transduction molecules, MEKK protein, Jun, ATF Myc protein,
p38, phosphotidylinositol-3 kinase (PI-3 kinase), Jun kinase kinase
(JNKK), Elk-1 and other Ets family members, phospholipase C .gamma.
(PLC.gamma.) and intracellular calcium.
[0035] In a preferred embodiment, a cell for use with the present
invention includes the human Burkitt's lymphoma cell line,
Ramos.
[0036] Signal transduction molecules referred to herein include the
natural full-length protein, or can be a functionally equivalent
protein in which amino acids have been deleted (e.g., a truncated
version of the protein), inserted, inverted, substituted and/or
derivatized (e.g., phosphorylated, acetylated, glycosylated,
carboxymethylated, myristoylated, prenylated or palmitoylated amino
acids) such that the modified protein has a biological activity
and/or function substantially similar to that of the natural
protein. Modifications can be accomplished by techniques known in
the art, including, but not limited to, direct modifications to the
protein or modifications to the gene encoding the protein. Such
modifications to the gene encoding the protein can include using,
for example, classic or recombinant DNA techniques to effect random
or targeted mutagenesis (see, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press, 1989, which is incorporated herein by reference in its
entirety).
[0037] Functionally equivalent proteins can be selected using
assays established to measure the biological activity of the
protein. For example, a functionally equivalent cell surface
receptor would have a similar ability to bind a particular ligand
as would the corresponding natural cell surface receptor protein.
As a further example, a functionally equivalent intracellular
signal transduction protein would have a similar ability to
associate with and regulate the activity of another intracellular
molecule as would the corresponding natural intracellular signal
transduction protein.
[0038] In certain embodiments, a cell of the present invention is
transformed with at least one heterologous nucleic acid molecule. A
nucleic acid molecule as described herein can be DNA, RNA, or
hybrids or derivatives of either DNA or RNA. Nucleic acid molecules
as referred to herein can include regulatory regions that control
expression of the nucleic acid molecule (e.g., transcription or
translation control regions), full-length or partial coding
regions, and combinations thereof. It is to be understood that any
portion of a nucleic acid molecule can be produced by: (1)
isolating the molecule from its natural milieu; (2) using
recombinant DNA technology (e.g., PCR amplification, cloning); or
(3) using chemical synthesis methods. A gene includes all nucleic
acid sequences related to a natural cell surface receptor gene such
as regulatory regions that control production of a cell surface
receptor encoded by that gene (such as, but not limited to,
transcription, translation or post-translation control regions) as
well as the coding region itself.
[0039] A nucleic acid molecule can include functional equivalents
of natural nucleic acid molecules encoding a protein. Functional
equivalents of natural nucleic acid molecules can include, but are
not limited to, natural allelic variants and modified nucleic acid
molecules in which nucleotides have been inserted, deleted,
substituted, and/or inverted in such a manner that such
modifications do not substantially interfere with the nucleic acid
molecule's ability to encode a molecule of the present invention.
Preferred functional equivalents include sequences capable of
hybridizing under stringent conditions (i.e., sequences having at
least about 70% identity), to at least a portion of a signal
transduction protein encoding nucleic acid molecule according to
conditions described in Sambrook et al., ibid.
[0040] As guidance in determining what particular modifications can
be made to any particular nucleic acid molecule, one of skill in
the art should consider several factors that, without the need for
undue experimentation, permit a skilled artisan to appreciate
workable embodiments of the present invention. For example, such
factors include modifications to nucleic acid molecules done in a
manner so as to maintain particular functional regions of the
encoded proteins including, a ligand binding site, a target binding
site, a kinase catalytic domain, etc. Functional tests for these
various characteristics (e.g., ligand binding studies and signal
transduction assays such as kinase assays, and other assays
described in detail herein and those known by those in the art)
allows one of skill in the art to determine what modifications to
nucleic acid sequences would be appropriate and which would
not.
[0041] Transformation of a heterologous nucleic acid molecule
(e.g., a heterologous cell surface receptor encoding a nucleic acid
molecule) into a cell suitable for use in the present invention can
be accomplished by any method by which a gene is inserted into a
cell. Transformation techniques include, but are not limited to,
transfection, retroviral infection, electroporation, lipofection,
bacterial transfer and spheroplast fusion. Nucleic acid molecules
transformed into cells suitable for use in the present invention
can either remain on extra-chromosomal vectors or can be integrated
into the cell genome.
[0042] Expression of a nucleic acid molecule of the present
invention in a cell can be accomplished using techniques known to
those skilled in the art. Briefly, the nucleic acid molecule is
inserted into an expression vector in such a manner that the
nucleic acid molecule is operatively joined to a transcription
control sequence in order to be capable of effecting either
constitutive or regulated expression of the gene when the gene is
transformed into a host cell. Construction of desired expression
vectors can be performed by methods known to those skilled in the
art and expression can be in eukaryotic or prokaryotic systems An
expression system can be constructed from control elements,
including transcription control sequences, translation control
sequences, origins of replication, and other regulatory sequences
that are compatible with a host cell, operatively linked to nucleic
acid sequences using methods known to those of skill in the art.
(see, for example, Sambrook et al., ibid. ).
[0043] In one embodiment, a cell suitable for use in the present
invention is transformed with a nucleic acid molecule encoding
CD40, JNK and/or p38, as described in detail herein. In another
embodiment of the present invention, a cell suitable for use in the
present invention is transformed with a nucleic acid molecules
encoding at least one type of intracellular signal transduction
protein of the present invention. Preferred intracellular signal
transduction protein encoding nucleic acid molecules include, but
are not limited to, nucleic acid molecules encoding JNK, p38, MEKK,
Jun, ATF, Myc protein, p38, PI-3 kinase, CDC42, Rho, Rac, JAK
family of kinases (e.g. JAK1, JAK2, JAK3), STAT family of kinases,
JNKK, Elk-1 and other Ets family members, PLC.gamma. and
intracellular calcium.
[0044] It is within the scope of the present invention that a cell
can be transformed with both a nucleic acid molecule encoding at
least one type of signal transduction molecule and a nucleic acid
molecule encoding at least one type of cell surface receptor.
[0045] In one embodiment, the method of the present invention
comprises contacting a cell with a putative regulatory compound.
According to the present invention, putative regulatory compounds
include compounds that are suspected of being capable of regulating
CD40, JNK and/or p38 activity. The term "activity" refers to any
stage of activation of a signal transduction molecule by, for
example, conformational change of a molecule which results in the
acquisition of catalytic activity by the molecule; the
phosphorylation of a molecule, thereby resulting in the acquisition
or loss of catalytic activity by the molecule; or the translocation
of a molecule from one region of a cell to another, thereby
enabling the molecule to bind another molecule. The term "regulate"
refers to controlling the activity of a molecule and/or biological
function, such as enhancing or diminishing such activity or
function.
[0046] Putative compounds as referred to herein include, for
example, compounds that are products of rational drug design,
natural products and compounds having partially defined signal
transduction regulatory properties. A putative compound can be a
protein-based compound, a carbohydrate-based compound, a
lipid-based compound, a nucleic acid-based compound, a natural
organic compound, a synthetically derived organic compound, an
anti-idiotypic antibody and/or catalytic antibody, or fragments
thereof. A putative regulatory compound can be obtained, for
example, from libraries of natural or synthetic compounds, in
particular from chemical or combinatorial libraries (i.e.,
libraries of compounds that differ in sequence or size but that
have the same building blocks; see for example, U.S. Pat. Nos.
5,010,175 and 5,266,684 of Rutter and Santi, which are incorporated
herein by reference in their entirety) or by rational drug
design.
[0047] In a rational drug design procedure, the three-dimensional
structure of a compound, such as a signal transduction molecule can
be analyzed by, for example, nuclear magnetic resonance (NMR) or
x-ray crystallography. This three-dimensional structure can then be
used to predict structures of potential compounds, such as putative
regulatory compounds by, for example, computer modelling. The
predicted compound structure can then be produced by, for example,
chemical synthesis, recombinant DNA technology, or by isolating a
mimetope from a natural source (e.g., plants, animals, bacteria and
fungi). Potential regulatory compounds can also be identified using
SELEX technology as described in, for example, PCT Publication Nos.
WO 91/19813; WO 92/02536 and WO 93/03172 (which are incorporated
herein by reference in their entirety).
[0048] In particular, a naturally-occurring intracellular signal
transduction molecule can be modified based on an analysis of its
structure and function to form a suitable regulatory compound. For
example, a compound capable of regulating the kinase domain of JNK
can comprise a compound having similar structure to a residues 1-79
of the amino terminus of JNK. Such a compound can comprise a
peptide, a polypeptide or a small organic molecule.
[0049] Putative regulatory compounds can also include molecules
designed to interfere with CD40. For example, mutant CD40 can be
created that interfere with the coupling of the receptor to
intracellular signal transduction proteins. Alternatively, mutant
CD40 can be created that interfere with the binding of CD40 ligand
to CD40. Putative regulatory compounds can include agonists and
antagonists of CD40. Such agonists and antagonists can be selected
based on the structure of a naturally-occurring ligand to CD40.
[0050] The conditions under which the cell of the present invention
is contacted with a putative regulatory compound, such as by
mixing, are conditions in which the cell can exhibit JNK and/or p38
activity if essentially no other regulatory compounds are present
that would interfere with such activity. Achieving such conditions
is within the skill in the art, and includes an effective medium in
which the cell can be cultured such that the cell can exhibit JNK
and/or p38 activity. For example, for a mammalian cell, effective
media are typically aqueous media comprising RPMI 1640 medium
containing 10% fetal calf serum.
[0051] Cells of the present invention can be cultured in a variety
of containers including, but not limited to, tissue culture flasks,
test tubes, microtiter dishes, and petri plates. Culturing is
carried out at a temperature, pH and carbon dioxide content
appropriate for the cell. Such culturing conditions are also within
the skill in the art. For example, for Ramos cells, culturing can
be carried out at 37.degree. C., in a 5% CO.sub.2 environment.
[0052] Acceptable protocols to contact a cell with a putative
regulatory compound in an effective manner include the number of
cells per container contacted, the concentration of putative
regulatory compound(s) administered to a cell, the incubation time
of the putative regulatory compound with the cell, the
concentration of ligand and/or intracellular initiator molecules
administered to a cell, and the incubation time of the ligand
and/or intracellular initiator molecule with the cell.
Determination of such protocols can be accomplished by those
skilled in the art based on variables such as the size of the
container, the volume of liquid in the container, the type of cell
being tested and the chemical composition of the putative
regulatory compound (i.e., size, charge etc.) being tested.
[0053] In one embodiment of the method of the present invention, a
suitable number of cells are added to a 96-well tissue culture dish
in culture medium. A preferred number of cells includes a number of
cells that enables one to detect a change in JNK activity using a
detection method of the present invention (described in detail
below). A more preferred number of cells includes between about 1
and 1.times.10.sup.6 cells per well of a 96-well tissue culture
dish. Following addition of the cells to the tissue culture dish,
the cells can be preincubated at 37.degree. C., 5% C.sub.2O for
between about 0 to about 24 hours.
[0054] A suitable amount of putative regulatory compound(s)
suspended in culture medium is added to the cells that is
sufficient to regulate the activity of a CD40, JNK and/or p38
protein in a cell such that the regulation is detectable using a
detection method of the present invention. A preferred amount of
putative regulatory compound(s) comprises between about 1 nM to
about 10 mM of putative regulatory compound(s) per well of a
96-well plate. The cells are allowed to incubate for a suitable
length of time to allow the putative regulatory compound to enter a
cell and interact with a signal transduction molecule. A preferred
incubation time is between about 1 minute to about 48 hours.
[0055] In another embodiment of the method of the present
invention, cells suitable for use in the present invention are
stimulated with a stimulatory molecules capable of binding to CD40
of the present invention to initiate a signal transduction pathway
and create a cellular response. Preferably, cells are stimulated
with a stimulatory molecule following contact of a putative
regulatory compound with a cell. Suitable stimulatory molecules can
include, for example, antibodies that bind specifically to the
extracellular domain of CD40 and CD40 ligand. Preferred stimulatory
molecules include, but are not limited to, anti-human CD40 antibody
G28-5, soluble gp39, membrane-bound gp39 (e.g. gp39 bound to the
plasma membrane of a cell or gp39 incorporated into a synthetic
lipid-based substrate such as a liposome or micelle) and mixtures
thereof. A suitable amount of stimulatory molecule to add to a cell
depends upon factors such as the type of ligand used (e.g.,
monomeric or multimeric; permeability, etc.) and the abundance of
the receptor on a cell. Preferably, between about 1.0 nM and about
1 mM of ligand is added to a cell.
[0056] The method of the present invention include determining if a
putative regulatory compound is capable of regulating JNK
activation. Such methods include assays described in detail in the
Examples section. The method of the present invention can further
include the step of performing a toxicity test to determine the
toxicity of a putative regulatory compound.
[0057] Another aspect of the present invention includes a kit to
identify compounds capable of regulating CD40 regulation of JNK or
p38 activity in a cell. Such a kit includes: (1) a cell comprising
CD40 protein, and JNK and/or p38 protein; and (2) a means for
detecting regulation of either the JNK or p38 protein. Such a means
for detecting the regulation of JNK protein include methods and
reagents known to those of skill in the art, for example, JNK
activity can be detected using, for example, activation assays
described in Example 2. Means for detecting the regulation of p38
protein also include methods and reagents known to those of skill
in the art. Suitable cells for use with a kit of the present
invention include cells described in detail herein. A preferred
cell for use with a kit includes, a human cell.
[0058] The present invention also includes the determination as to
whether a putative regulatory compound is capable of regulating a
biological response in a mammal. Such a method entails
administering a putative regulatory compound to an animal, such
compound being shown, using an assay of the present invention, to
regulate CD40, JNK and/or p38 activity in a cell. Such a
determination is useful for determining conditions under which a
putative regulatory compound can be administered to an animal as a
therapeutic composition. Thus, it is within the scope of the
present invention that those conditions stated herein for testing a
compound in an animal can be used when administering a therapeutic
composition of the present invention. In particular, a putative
regulatory compound can be administered to an animal to determine
if the compound is capable of regulating, for example, an
inflammatory response, a response to an infectious agent, an
autoimmune response, a metabolic response, a cardiovascular
response, an allergic response and/or an abnormal cellular growth
response in the animal. Acceptable protocols to administer putative
regulatory compounds to test the effectiveness of the compound
include individual dose size, number of doses, frequency of dose
administration, and mode of administration. Determination of such
protocols can be accomplished by those skilled in the art. A
suitable single dose is a dose that is capable of altering a
biological response in an animal when administered one or more
times over a suitable time period (e.g., from minutes to days or
weeks). Preferably, a dose comprises from about 1 nanogram of the
compound per kilogram of body weight (ng/kg) to about 1 gram of
compound per kilogram of body weight (gm/kg), more preferably 100
ng/kg to about 100 milligrams/kilogram (mg/kg), and even more
preferably from about 10 micrograms of compound per kilogram of
body weight to about 10 mg/kg. Modes of administration can include,
but are not limited to, aerosolized, subcutaneous, rectally,
intradermal, intravenous, nasal, oral, transdermal and
intramuscular routes. A putative regulatory compound can be
combined with other components such as a pharmaceutically
acceptable excipient and/or a carrier, prior to administration to
an animal. Examples of such excipients include water, saline,
Ringer's solution, dextrose solution, Hank's solution, and other
aqueous physiologically balanced salt solutions. Nonaqueous
vehicles, such as fixed oils, sesame oil, ethyl oleate, or
triglycerides may also be used. Other useful formulations include
suspensions containing viscosity enhancing agents, such as sodium
carboxymethylcellulose, sorbitol, or dextran. Excipients can also
contain minor amounts of additives, such as substances that enhance
isotonicity and chemical stability. Examples of buffers include
phosphate buffer, bicarbonate buffer and Tris buffer, while
examples of preservatives include thimerosal, m- or o-cresol,
formalin and benzyl alcohol. Standard formulations can either be
liquid injectables or solids which can be taken up in a suitable
liquid as a suspension or solution for injection. Carriers are
typically compounds that increase the half-life of a compound in
the treated animal. Suitable carriers include, but are not limited
to, polymeric controlled release vehicles, biodegradable implants,
liposomes, bacteria, viruses, oils, esters, and glycols. Preferred
controlled release formulations are capable of slowly releasing a
composition of the present invention into an animal. Suitable
controlled release vehicles include, but are not limited to,
biocompatible polymers, other polymeric matrices, capsules,
microcapsules, microparticles, bolus preparations, osmotic pumps,
diffusion devices, liposomes, lipospheres, and transdermal delivery
systems. Other controlled release vehicles of the present invention
include liquids that, upon administration to an animal, form a
solid or a gel in situ. Preferred controlled release vehicles are
biodegradable (i.e., bioerodible).
[0059] In another aspect of the present invention, the present
invention includes conducting a toxicity test on an animal to
determine the toxicity of a putative regulatory compound. Toxicity
tests for putative regulatory compounds can be performed, for
example, on animals after a putative regulatory compound has been
determined to have an effect at the cellular level on signal
transduction, such as the regulation of cellular inflammatory
responses. Such toxicity tests are within the skill of the art, and
generally involve testing the toxicity of a compound in vivo or in
vitro. A suitable method for testing the toxicity of a putative
regulatory compound in vivo can involve scientifically controlled
administration of the putative regulatory compound to a number of
animals and a period of observance in which the effects of the
compound on various aspects of the animal's biological functions
(e.g., occurrence of tissue damage, functioning of organs and
death) are noted. Suitable methods for testing the toxicity of a
putative regulatory compound in vitro can involve scientifically
controlled administration of the putative regulatory compound to a
cell and subsequent measurement of cell function, cytotoxicity, or
cell death. Cell function can be measured by any one of a wide
range of assays which will be apparent to one of skill in the art,
several of which are herein disclosed (e.g., tyrosine
phosphorylation, calcium mobilization and phosphoinositide assays).
Methods to measure cytotoxicity are well known in the art and
include measurement of the ability to reduce chromogenic substrates
such as the tetrazolium-based MTT or sulphorhodamine blue,
ATP-bioluminescence assays and fluorescence assays, for example
using the Fluorescent Green Protein, among many other readily
available assays (see, for example, Bellamy, Drugs 44 (5):690-708,
1992, which is incorporated herein by reference in its entirety).
Methods to measure cell death include, for example, Coomassie blue
staining, acridine orange staining, terminal deoxynucelotidyl
transferase (TDT) assays for measuring DNA fragmentation, neutral
red exclusion, and measuring changes in forward light scattering in
a flow cytometer.
[0060] Another aspect of the present invention includes a method to
regulate a cellular function selected from the group consisting of
immunoglobulin heavy chain class switching, cytokine production or
inflammatory cell activation, comprising regulating the activity of
a protein including CD40, JNK and/or p38. Regulation of activity of
such protein can be achieved by sequestering JNK and/or p38 protein
in an inactive complex, regulating the ligand binding activity of
CD40, regulating the phosphorylation of JNK and/or p38 protein,
regulating the interaction between JNK and JNKK, regulating the
ability of JNK to activate c-Jun, ATF-2, and Ets-1 and other Ets
family members, regulating the interaction between p38 and MEK,
regulating the ability of p38 to activate ATF-2, and Ets-1 and
other Ets family members, regulating the expression of endogenous
and/or heterologous nucleic acid molecules encoding a CD40, JNK
and/or p38 protein, and combinations thereof.
[0061] Suitable compounds for sequestering a JNK protein in an
inactive complex, include compounds that mimic the site at which
JNK protein interacts with JNKK, referred to herein as an
activation site JNK mimetope. Suitable compounds for sequestering a
p38 protein in an inactive complex, include compounds that mimic
the site at which p38 protein interacts with MEK, referred to
herein as an activation site p38 mimetope.
[0062] Suitable compounds for regulating the Interaction between
JNK and c-Jun, ATF-2, or Ets-1 or other Ets family members comprise
JNK target site mimetopes. Suitable compounds for regulating the
interaction between p38 and ATF-2, or Ets-1 or other Ets family
members comprise p38 target site mimetopes.
[0063] Suitable compounds for regulating the ligand binding
activity of CD40 include CD40 antagonists of extracellular ligands
to CD40.
[0064] Other suitable regulatory compounds of the present invention
include pseudosubstrates for a regulatory kinase domains of JNK or
p38, a JNK kinase domain mimetope, a p38 kinase domain mimetope and
a mutated CD40, JNK or p38 protein. Pseudosubstrates of a JNK
kinase domain include small organic molecules, peptides or
polypeptides that are phosphorylated by a JNK kinase domain in a
similar manner as a JNK substrate including c-Jun, ATF-2, or Ets-1
or other Ets family members. Similarly, pseudosubstrates of a p38
kinase domain include small organic molecules, peptides or
polypeptides that are phosphorylated by a p38 kinase domain in a
similar manner as a p38 substrate including ATF-2, or Ets-1 or
other Ets family members.
[0065] Suitable methods for regulating the expression of endogenous
and/or heterologous nucleic acid molecules encoding CD40, JNK
and/or p38 protein include methods known to those in the art. For
example, oligonucleotides for use in, for example, antisense-,
triplex formation-, ribozyme- and/or RNA drug-based technologies
can be used to reduce expression of endogenous nucleic acid
molecules encoding CD40, JNK and/or p38 protein. The present
invention, therefore, includes such oligonucleotides and methods to
interfere with the production of CD40, JNK and/or p38 protein by
use of one or more of such technologies. Appropriate expression
vectors can be developed by those skilled in the art based upon the
cell-type being transformed.
[0066] In accordance with the present invention, a "mimetope"
refers to any compound that is able to mimic the ability of a
regulatory reagent of the present invention. A mimetope can be a
peptide that has been modified to decrease its susceptibility to
degradation but that still retains regulatory activity. Other
examples of mimetopes include, but are not limited to,
protein-based compounds, carbohydrate-based compounds, lipid-based
compounds, nucleic acid-based compounds, natural organic compounds,
synthetically derived organic compounds, anti-idiotypic antibodies
and/or catalytic antibodies, or fragments thereof having desired
regulatory activity. A mimetope can be obtained by, for example,
screening libraries of natural and synthetic compounds for
compounds capable of altering the activity of CD40 or JNK, as
disclosed herein. A mimetope can also be obtained by, for example,
rational drug design. In a rational drug design procedure, the
three-dimensional structure of a compound of the present invention
can be analyzed by, for example, nuclear magnetic resonance (NMR)
or x-ray crystallography. The three-dimensional structure can then
be used to predict structures of potential mimetopes by, for
example, computer modelling. The predicted mimetope structures can
then be produced by, for example, chemical synthesis, recombinant
DNA technology, or by isolating a mimetope from a natural source
(e.g., plants, animals, bacteria and fungi).
[0067] Another aspect of the present invention comprises
administering to an animal, a therapeutic composition capable of
regulating a biological function including immunoglobulin heavy
chain class switching, cytokine production or inflammatory cell
activation. A therapeutic composition of the present invention is
particularly useful for preventing or treating diseases involving
undesired immunoglobulin and/or cytokine production, or
inflammatory cell activation. In particular, a therapeutic
composition is useful for preventing or treating diseases involving
an allergic or autoimmune response. Preferably, a therapeutic
composition of the present invention is used to prevent or treat a
disease, including, but not limited to, allergic hypersensitivity,
asthma, rheumatoid arthritis, systemic lupus erythematosus (SLE),
allergic rhinitis, atopic dermatitis and acute bronchopulmonary
aspergillosis. A therapeutic composition is preferably administered
to a cell having CD40 and more preferably to cells including, but
not limited to, Pre-B cells, B lymphocytes, cancer cells,
fibroblasts, Langerhans cells, epithelial cells monocytes and
dendritic cells.
[0068] A variety of therapeutic compositions can be used to perform
the regulation method of the present invention. Such therapeutic
compositions include those compounds described in detail herein, in
particular, compounds identified using a method of the present
invention. A therapeutic composition of the present invention can
be formulated in an excipient that the animal to be treated can
tolerate. Examples of such excipients include those described in
detail above. In order to regulate heavy chain class switching in a
cell, a therapeutic composition of the present invention can be
administered in vivo (i.e., in an animal) or ex vivo (i.e., outside
of an animal, such as in tissue culture), in an effective manner
such that the composition is capable of regulating heavy chain
class switching.
[0069] An effective administration protocol (i.e., administering a
therapeutic composition in an effective manner) comprises suitable
dose parameters and modes of administration that result in
prevention or treatment of a disease. Effective dose parameters and
modes of administration can be determined using methods standard in
the art for a particular disease. Such methods include, for
example, determination of survival rates, side effects (i.e.,
toxicity) and progression or regression of disease. For example,
the effectiveness of dose parameters and modes of administration of
a therapeutic composition of the present invention can be
determined by assessing response rates. Such response rates refer
to the percentage of treated patients in a population of patients
that respond with either partial or complete remission.
[0070] In accordance with the present invention, a suitable single
dose size is a dose that is capable of preventing or treating an
animal with a disease when administered one or more times over a
suitable time period. Doses can vary depending upon the disease
being treated. For example, in the treatment of hypersensitivity, a
suitable single dose can be dependent upon the nature of the
immunogen causing the hypersensitivity.
[0071] It will be obvious to one of skill in the art that the
number of doses administered to an animal is dependent upon the
extent of the disease and the response of an individual patient to
the treatment. For example, in the case of allergic responses, the
immunogenicity of a compound may require more doses than a less
immunogenic compound. Thus, it is within the scope of the present
invention that a suitable number of doses, as well as the time
periods between administration, includes any number required to
cause treat a disease.
[0072] Therapeutic compositions can be administered directly to a
cell in vivo or ex vivo or systemically. Preferred methods of
systemic administration, include intravenous injection, aerosol,
oral and percutaneous (topical) delivery. Intravenous injections
can be performed using methods standard in the art. Aerosol
delivery can also be performed using methods standard in the art
(see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA
189:11277-11281, 1992, which is incorporated herein by reference in
its entirety). Oral delivery can be performed by complexing a
therapeutic composition of the present invention to a carrier
capable of withstanding degradation by digestive enzymes in the gut
of an animal. Examples of such carriers, include plastic capsules
or tablets, such as those known in the art. Topical delivery can be
performed by mixing a therapeutic composition of the present
invention with a lipophilic reagent (e.g., DMSO) that is capable of
passing into the skin.
[0073] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
Example 1
[0074] This example describes the activation of ERK by ligation of
surface IgM but not CD40.
[0075] The human Burkitt's lymphoma cell line Ramos was obtained
from the American Type Culture Collection (Rockville, Md.), and
cells were maintained in RPMI-1640 supplemented with 50 units/ml
penicillin-streptomycin, 2 mM glutamine, and 10% FCS. Exponentially
growing cells were used in all experiments.
[0076] A. Immunoblot Assays Using Ramos Cells
[0077] About 1.times.10.sup.6 Ramos cells were separately treated
with 10 .mu.g/ml anti-IgM F(ab')2 goat anti-human IgM antibody
(obtained from Zymed, San Francisco, Calif.) or 5 .mu.g/ml
anti-CD40 antibody (G28-5; obtained from Dr. E. A. Clark,
Washington University, Seattle, Wash.) for 0, 1, 5, 15, 30 or 60
min. Ramos cells were also treated with phorbol 12-myristate
13-acetate (PMA; obtained from Sigma, St Louis, Mo.). Immunoblot
analysis was performed on each of the samples using monoclonal
anti-ERK2 antibody in the following method. The treated cells were
lysed in 100 .mu.l of lysis buffer (25 mM Tris-HCl, pH 7.6, 50 mM
NaCl, 0.5% Na deoxycholate, 2% NP-40, 0.2% SDS, 1 mM PMSF, 50
.mu.g/ml aprotinin, 50 .mu.M leupeptin). Lysates were centrifuged
for 10 min at 14,000 rpm in an Eppendorf microcentrifuge, 90 .mu.l
of supernatants were mixed with 30 .mu.l of 4.times.Laemmli sample
buffer. Samples were boiled for 5 min. Twenty .mu.l of prepared
samples were electrophoresed through a 12% SDS-PAGE gel, and
proteins were transferred to nitrocellulose membranes. Membranes
were incubated in blocking buffer (25 mM Tris-HCl, pH 8.0, 125 mM
NaCl, 0.1% Tween 20, 2% BSA, 0.1% NaN.sub.3) at 4.degree. C.
overnight, then monoclonal mouse anti-ERK2 antibody (1:2000;
obtained from Upstate Biotechnology Incorporated, Lake Placid,
N.Y.) was added to the blocking buffer, and blots were incubated
for an additional 2 hours at room temperature. The blots were
washed three times in TBST (25 mM Tris-HCl, pH 8.0, 125 mM NaCl,
0.025% Tween 20) and incubated with AP-conjugated goat anti-mouse
Ig (1:10000 in TBST; obtained from Promega, Madison, Wis.) for 1
hour at room temperature. The blots were washed three times in TBST
and developed with the colorogenic substrates BCIP and NBT (Promega
protoblot AP system).
[0078] The results from the immunoblot indicated that the untreated
samples contained a single band (42 kD) reactive with anti-ERK2
antibody (lane 0'). In samples treated with PMA (100 ng/ml) or
anti-IgM for 20 min, a second band with immunoreactivity to
anti-ERK2 antibody appeared (lane PMA and anti-IgM lane 1'-60').
This lower mobility form represents the activated form due to
phosphorylation. The samples treated with anti-CD40 demonstrated
only a single band throughout the time course, thus indicating no
lower mobility shift in p42.sup.erk2. These data indicate that
anti-IgM antibody activates p42.sup.erk2 but anti-CD40 antibody
fails to activate p42.sup.erk2.
[0079] B. Immunoblot Assays Using Tonsillar Cells
[0080] The procedure of step A was repeated except using lysates
from freshly isolated tonsillar B cells (prepared from tonsils as
described in Takase et al., J. Cell Physiol. 162:246-255, 1995).
Following treatment with 5 .mu.g/ml anti-CD40 for the indicated
times also showed no shift in ERK2 mobility.
[0081] B. ERK Kinase Assay
[0082] Kinase activity was evaluated using EGFRF.sub.662-681
peptide as a substrate as described previously (Franklin et al., J.
Immunol. 153:4890-4898, 1994). Following stimulation, 10.sup.6
cells were lysed in 75 .mu.l of lysis buffer (70 mM
.beta.-glycerophosphate, pH 7.2, 100 mM Na.sub.3VO.sub.4, 2 mM
MgCl.sub.2, 1 mM EGTA, 0.5% Triton X-100, 5 .mu.g/ml leupeptin, 2
.mu.g/ml aprotinin and 1 mM DTT) and placed on ice for 15 min. Cell
lysates were centrifuged at 14,000 rpm for 10 min and 20 .mu.l of
supernatant were removed and mixed with 20 .mu.l of 2.times.kinase
buffer (50 mM .beta.-glycerophosphate, pH 7.2, 100 mM
Na.sub.3VO.sub.4, 20 mM MgCl.sub.2, 50 mg/ml IP-20, 1 mM EGTA, 400
.mu.M EGFR.sub.662-681 peptide, 200 .mu.M ATP and 0.225
mCi/ml[.gamma.-.sup.32P- ] ATP [ICN Biochemicals, Costa Mesa,
Calif.]). After 15 min at 30.degree. C., 10 .mu.l of 250 mM EDTA
was added, and 45 .mu.l of the reaction mixture was spotted onto
P-81 phosphocellulose paper (Whatman, Clifton, N.J.). The papers
were washed four times (5 min each) in 400 ml of 75 mM phosphoric
acid and then radioactivity bound to the filter paper was
determined by liquid scintillation counting. The assay system
contained both EGTA (1 mM) and IP-20 (25 mg/ml), reagents that
should effectively inhibit PKC, calcium/calmodulin, and
cAMP-dependent kinases.
[0083] The results (shown in FIG. 1) indicate the kinase activity
of samples treated with 100 ng/ml PMA for 20 min and 10 .mu.g/ml
anti-IgM for 5 min were about 30,000 counts per minute compared
with samples from unstimulated (control) cells (about 10,000 cpm)
or cells treated with 1 min (about 15,000 cpm), 5 min (about 17,000
cpm), or 10 .mu.g/ml anti-CD40 for 20 min (about 15,000 cpm). The
data represent incorporation of .sup.32p (.+-.SD) from separately
prepared duplicate samples from two independent experiments.
Statistically significant differences from untreated (0') samples
are represented by an asterisk (*) (p<0.05).
[0084] The results confirm the results obtained in the immunoblot
experiments. Activation of p42.sup.erk2 by anti-IgM was confirmed
by increases in EGFR.sub.662-681 peptide phosphorylation. In
contrast, the addition of anti-CD40 antibody at concentrations up
to 10 .mu.g/ml, failed to activate p42.sup.erk2 in Ramos cells.
Furthermore, we confirmed that anti-CD40 failed to activate
p42.sup.erk2 in freshly isolated tonsillar B cells.
Example 2
[0085] This example demonstrates that c-Jun kinase is activated by
anti-CD40 antibody and soluble gp39 but not by anti-IgM
antibody.
[0086] JNK activity was measured by solid-phase kinase assay using
GST-c-Jun (1-79) as a substrate following treatment with anti-IgM
antibody or anti-CD40 antibody (G28-5) in Ramos cells (three
independent experiments) and tonsillar B cells (two independent
experiments). GST-c-Jun (1-79) fusion protein was purified from
bacterial lysates using GSH-Sepharose beads (Pharmacia Biotech,
Uppsala, Sweden) at room temperature with gentle rocking using the
method described in Galcheva-Gargova et al. (Science 265:806-808,
1994). Following stimulation, 3.times.10.sup.6 cells were lysed in
lysis buffer (20 mM Tris-HCl, pH 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM
EGTA, 0.5% NP-40, 2 mM Na.sub.3VO.sub.4, 1 mM DTT, 1 mM PMSF, 20
.mu.g/ml aprotinin, 5 .mu.g/ml leupeptin). The lysates were mixed
with 10 .mu.l of GST-c-Jun (1-79) coupled to GSH-Sepharose beads.
The mixture was rotated at 4.degree. C. for 3 hr in a
microcentrifuge tube and pelleted by centrifugation at 14,000 rpm
for 5 min. The pelleted beads were washed 2 times in lysis buffer
and once in kinase buffer (20 mM Hepes, pH 7.5, 20 mM
.beta.-glycerophosphate, 10 mM MgCl.sub.2, 1 mM DTT, 50 mM
Na.sub.3VO.sub.4, 10 mM p-nitrophenyl phosphate), and then
resuspended in 40 .mu.l of kinase buffer containing 10 .mu.Ci of
[.gamma.-.sup.32p]ATP. After 20 min at 30.degree. C., the reaction
was terminated by adding 4.times.Laemmli sample buffer and boiling
for 3 min. Samples were resolved by 12% SDS-PAGE and subjected to
autoradiography. Phosphate incorporation was determined by
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The level
of .sup.32P incorporation (.+-.SD) into the substrate is
illustrated as the ratio of JNK activity to that of untreated
samples.
[0087] Results from a time course of JNK activation after treatment
of Ramos cell with 1 .mu.g/ml anti-CD40 antibody indicated a rapid
and marked increase in JNK activation within 1 min of treatment
with 1 .mu.g/ml anti-CD40 antibody (FIG. 2), reached peak levels
within 15 min and then began to decline by 30-60 min. Statistically
significant differences from untreated (040 ) samples are
represented by an asterisk (*) (p<0.05).
[0088] Results from a dose response study of anti-CD40
antibody-activated JNK in Ramos cells treated with various
concentrations of anti-CD40 antibody for 15 min. indicated that, in
the presence of 0.5 .mu.g/ml anti-CD40 antibody, levels of .sup.32P
incorporation were five-fold higher than control samples (FIG. 3).
JNK activity increased in a dose dependent fashion with peak levels
(about seven-fold) observed at a concentration of 2-5 .mu.g/ml
antibody. Statistically significant differences from untreated (0')
samples are represented by an asterisk (*) (p<0.05).
[0089] A dose-dependent response to anti-CD40 antibody was also
detected in tonsillar B cells (FIG. 4). Throughout the
dose-response curve lower levels of activation were observed in
tonsillar B cells relative to Ramos cells, but both cell types
clearly respond to CD40 ligation with a significant JNK
activation.
[0090] Results from the treatment of Ramos cells for 15 min with
dilutions of culture supernatants containing soluble gp39 (prepared
as described in Hollenbaugh et al., EMBO J. 11:4313-4321, 1992)
indicated that recombinant soluble gp39 also activated JNK in a
dose-dependent fashion in Ramos cells (FIG. 5) and tonsillar B
cells.
[0091] Results from blocking studies using dilutions of culture
supernatants containing soluble gp39 that were pre-incubated with
anti-gp39 antibody (2 .mu.g/ml; mAb39-1.106; described in Bajorath
et al., Biochemistry 34:1833-1844, 1995) for 5 min prior to
addition to Ramos cells indicated that anti-gp39 antibody prevented
activation of JNK by soluble gp39. JNK activation by UV irradiation
was unaffected by the presence of the antibody. The anti-gp39
antibody failed to block UV-irradiation-induced activation of
JNK.
[0092] Results from a time course cross-linking study using
anti-IgM antibody indicated that JNK activity was not increased
following surface IgM crosslinking even in the presence of 10
.mu.g/ml anti-IgM antibody in Ramos cells (FIG. 6) and tonsillar B
cells (FIG. 7).
[0093] Taken together, the results demonstrate that JNK is
activated by anti-CD40 antibody but not anti-IgM antibody,
indicating that anti-CD40 antibody activates JNK through a
different signaling pathway than that which mediates ERK activation
by anti-IgM antibody.
Example 3
[0094] This example demonstrates that anti-CD40 antibody activates
JNK through a Ras-independent pathway.
[0095] Metabolically labeled (.sup.32P)Ramos cells were untreated
(control) or treated with 10 .mu.g/ml anti-IgM or 5 .mu.g/ml
anti-CD40 for 1, 5 and 10 min, respectively. Ras was
immunoprecipitated using the Y13-259 anti-Ras antibody, and
radioactive GTP and GDP bound to Ras was measured as follows. Cells
(10.sup.7 cells) were labeled with .sup.32P-orthophosphate for 16
hr, and then stimulated. Ras was immunoprecipitated using the
Y13-259 anti-Ras antibody, and GTP was separated from GDP by thin
layer chromatography as described (Downward et al., Nature
346:719-723, 1990). The radiolabeled nucleotides were visualized by
autoradiography. Radioactivity was quantitated with a
PhosphorImager and the GTP/GTP+(1.5) GDP ratios were calculated.
The data were quantitated by PhosphorImager, and shown are the
GTP/GDP+(1.5) GDP ratios (in percent) for each condition. The
results represent three separate experiments. Statistically
significant differences from untreated (0') samples are represented
by an asterisk (*) (p<0.05).
[0096] The results, shown in FIG. 8, indicate that treatment with
anti-IgM antibody activated Ras. Anti-CD40 antibody treatment,
however, failed to activate Ras at concentrations that were
effective in JNK activation (described in Example 2). The results
demonstrate that the signals transduced following CD40 engagement
lead to JNK activation through a pathway that does not involve Ras
activation.
Example 4
[0097] This example demonstrates that Raf-1 does not participate in
the CD40-activated JNK pathway.
[0098] Ramos cells were untreated or treated with 100 ng/ml PMA, 10
.mu.g/ml anti-IgM or 5 .mu.g/ml anti-CD40 for 1, 2.5, 5, 10 or 20
min. Raf-1 was immunoprecipitated and a kinase assay was performed
using the following method. Cells (10.sup.7) were stimulated in
RPMI 1640 medium, and then lysed in RIPA (10 mM sodium phosphate,
pH 7.0, 150 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 1% Nonidet
P-40, 0.1% SDS, 1% aprotinin, 50 mM NaF, 200 mM Na.sub.3VO.sub.4,
0.1% 2-mercaptoethanol, 1 mM PMSF). The lysates were precleared by
protein A-Sepharose beads for 30 min at 4.degree. C. A purified
polyclonal anti-Raf-1 antibody was added to the lysates (1:100;
obtained from Santa Cruz Biotechnology, Santa Cruz, Calif.) and
incubated for 90 min at 4.degree. C. The immunocomplexes were
collected by protein A-Sepharose beads. The beads were then washed
3 times in RIPA and 3 times in a buffer containing 10 mM PIPES, pH
7.0, 100 mM NaCl, 2 .mu.g/ml aprotinin. A kinase mixture (40 .mu.l)
containing 10 mM PIPES, pH 7.0, 100 mM NaCl, 5 mM MnCl.sub.2, 2
.mu.g/ml aprotinin, 30 .mu.Ci of [.gamma.-.sup.32P]ATP and 100-200
ng of catalytically inactive MEK (KMMEK) was added to the beads.
KMMEK was expressed and purified as described (Gardner et al.,
Methods Enzymol. 238:258-270, 1994). The samples were incubated for
30 min at 30.degree. C. The kinase reaction was stopped by addition
of 4.times.Laemmli sample buffer and boiling for 3 min. The
proteins were resolved on 10% SDS-PAGE and transferred to a
nitrocellulose membrane. The membrane was probed using the same
anti-Raf-1 antibody and visualized as described above and subjected
to autoradiography.
[0099] The results indicate that the levels of KMMEK
phosphorylation following treatment with 5 .mu.g/ml anti-CD40
antibody were not different than control samples, whereas cells
treated with anti-IgM antibody resulted in increased KMMEK
phosphorylation. To verify similar loading of immunoprecipitated
Raf-1, an immunoblot was concomitantly performed using the same
antibody as was used for the immunoprecipitates. The Raf-1 mobility
shifts were consistent with the increased levels of kinase activity
measured using KMMEK. The magnitude of anti-IgM antibody activation
of Raf-1 was sufficient to activate ERK2 similarly as PMA. Raf-1,
which is an efficient activator of the ERK pathway, is not
measurably activated during JNK activation in response to CD40
ligation.
Example 5
[0100] This example demonstrates that anti-CD40 antibody activates
MEKK protein.
[0101] Ramos cells were treated with 2 .mu.g/ml anti-CD40 antibody
for 0, 0.5, 1, 2.5, 5 or 10 min. MEKK was immunoprecipitated and a
kinase assay was performed using the following methods. Following
stimulation, 5.times.10.sup.6 cells were lysed in 400 .mu.l of
extraction buffer (1% Triton X-100, 10 mM Tris-HCl [pH 7.4], 5 mM
EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serum albumin, aprotinin
[20 .mu.g/ml], 1 mM PMSF, and 2 mM Na.sub.3VO.sub.4). The lysates
were centrifuged for 10 min at 14,000 rpm and pellets were
discarded. The supernatants were incubated with the rabbit MEK
kinase (MEKK) antisera (1:100 dilution) raised against the MEKK
NH2-terminal fusion protein (described in Lange-Carter et al.,
Methods Enzymol. 255:290-301, 1995) for 2 hr at 4.degree. C. The
immune complexes were collected by protein A-Sepharose beads. The
beads were then washed twice in RIPA buffer and three times in a
buffer containing 10 mM PIPES, pH 7.0, 100 mM NaCl, 2 .mu.g/ml
aprotinin. A kinase mixture (40 .mu.l) containing 10 mM PIPES, pH
7.0, 100 mM NaCl, 5 mM MnCl.sub.2, 2 .mu.g/ml aprotinin, 30 .mu.Ci
[.gamma.-.sup.32P]ATP and 0.5 .mu.l of recombinant JNK activating
protein kinase (JNKK; described in Lin et al., Science 268:286-290,
1995) as a substrate was added to the beads. The samples were
incubated for 30 min at 30.degree. C. The kinase reaction was
stopped by addition of4.times.Laemmli sample buffer and boiling for
3 min. The proteins were resolved on 10% SDS-PAGE, transferred to a
nitrocellulose membrane and subjected to autoradiography. Phosphate
incorporation was quantitated by PhosphorImager.
[0102] The results of .sup.32P incorporation into JNKK are
illustrated in FIG. 9 as the ratio of MEKK activity of treated to
that of untreated samples. Statistically significant differences
from untreated (0') samples are represented by an asterisk (*)
(p<0.05). MEKK was activated rapidly, reaching maximal
stimulation by 30 sec after anti-CD40 antibody treatment, and then
decreased gradually with time. Immunoblots of the
immunoprecipitated MEKK with the anti-MEKK polyclonal antibody that
were used for immunoprecipitation revealed similar amounts of a 98
kD MEKK protein for each time point. These data indicate that an
MEKK is present in B-lymphoblastoid cells which regulates the JNK
pathway and is activated in response to CD40 ligation.
Example 6
[0103] This example demonstrates that anti-CD40 antibody rescues
apoptosis induced by anti-IgM antibody.
[0104] Ramos cells were untreated (control) or treated with 10
.mu.g/ml anti-IgM antibody or 2 .mu.g/ml anti-CD40 antibody;
co-stimulation of cells consisted of a 30 min preincubation with
anti-CD40 antibody followed by an incubation with anti-IgM
antibody. After 18 hr culture, DNA breaks derived from anti-IgM
induced apoptosis were evaluated using an in situ TdT assay using
the method as follows. For detection of DNA strand breaks in
individual cells, an in situ terminal deoxynucleotidyl transferase
(TdT) assay was employed based on the method of Gorczyca et al.
(Cancer Res. 53:3186-3192, 1993) with minor modifications. Cells
were treated for 18 hr as indicated. About 10.sup.6 cultured cells
were washed in PBS and suspended in 500 .mu.l of PBS. Paraform (4%,
170 .mu.l) was added and the mixture was stored on ice for 15 min.
Cells were then washed in cold PBS and fixed with 70% ethanol at
-20.degree. C. for an hour. Following washing in cold PBS, the
cells were resuspended in TdT reaction buffer (0.1 M potassium
cacodylate, pH 7.2, 2 mM CoCl.sub.2, 0.2 mM DTT, 20 U TdT, 2 nmol
fluorescein-dUTP, 10 mg/ml BSA). After 30 min at 37.degree. C.,
cells were washed once in 0.2% BSA/PBS and fluoresce staining was
evaluated on an EPICS Profile (Coulter, Hialeah, Fla.).
[0105] p42.sup.erk2 (5 min following treatment) and JNK activation
(15 min following treatment) were evaluated under identical
conditions as described in Example 1. The level of .sup.32P
incorporation (.+-.SD) from two independent experiments was
evaluated by PhosphorImager, and then plotted as the ratio of JNK
activation to that of untreated samples. Statistically significant
differences from untreated samples are represented by an asterisk
(*) (p<0.05).
[0106] The results indicate that DNA breaks were detected in 64.2%
of Ramos cells, 18 hr following treatment with 10 .mu.g/ml anti-IgM
antibody, whereas there was no shift in fluorescence intensity in
control (untreated) cells or in cells treated with 2 .mu.g/ml
anti-CD40 antibody. In the presence of 2 .mu.g/ml anti-CD40
antibody preincubated for 30 min prior to the addition of anti-IgM
antibody, however, DNA breaks induced by anti-IgM antibody were
reduced to 3.5% of the cells. Under identical conditions, an
immunoblot using anti-ERK2 monoclonal antibody indicated the
mobility shift in p42.sup.erk2 protein, 5 min following treatment
with 10 .mu.g/ml anti-IgM antibody in the presence of 2 .mu.g/ml
anti-CD40 antibody preincubated for 30 min. In addition, JNK
activity, measured by solid phase kinase assay using GST-c-Jun
fusion protein, was increased 15 min following treatment with
anti-CD40 antibody in the presence of anti-IgM antibody. Thus, CD40
ligation does not effect p42.sup.erk2 activation by sIgM and sIgM
ligation does not effect CD40 activation of JNK.
Example 7
[0107] This example describes the activation of p38 by CD40
ligation.
[0108] Neutrophils isolated by the plasma percoll method as
previously described (Haslett, C., Am. J. Pathol. 119, 101-110;
1985) were resuspended at about 25.times.10.sup.6 cells/ml in KRPD
containing 0.1% Human Heat Inactivated Platelet-Poor Plasma, 1 mM
PMSF, 10, .mu.g/ml leupeptin and 10 .mu.g/ml aprotinin. About
25.times.10.sup.6 PMN were preincubated for 30 minutes at
37.degree. C., then stimulated with 100 ng/ml LPS for varying time
intervals and reactions terminated by a 20 second centrifugation at
15,000 rpm. Cell pellets were lysed with 500 .mu.l cold RIPA (50 mM
Tris pH=7.2, 150 mM NaCl, 1.1% SDS, 0.1% sodium deoxycholate, 1%
Triton-X-100, 10 mM sodium pyrophosphate, 25 mM M glycerophosphate,
1 mM sodium orthovanadate and 2.1 .mu.g/ml aprotinin), and
centrifuged at 15,000 for 10 minutes at 4.degree. C. Triton soluble
lysates were initial precleared with Protein A Sepharose for 30
minutes at 4.degree. C., followed by Protein A Sepharose
immunoprecipitation with 5 .mu.l/sample polyclonal antibodies
specific for p38 and 15 .mu.l of bead suspension, for 120 minutes
at 4.degree. C. Beads were washed once in RIPA and twice in PAN (10
mM Pipes, 100 mM NaCl, pH=7.0, and 21 .mu.g/ml aprotinin). Beads
were resuspended 25 .mu.l kinase mix containing 20 mM Hepes,
pH=7.6, 200 mM MgCl.sub.2, 20 .mu.M ATP, 20 .mu.Ci
[.sup.32P].gamma.-ATP, 2 mM dTT, 100 .mu.M sodium orthovanadate, 25
mM B-glycerophosphate (pH=7.2) and a peptide comprising amino acids
1-110 of ATF-2. The samples were incubated for 15 minutes at
30.degree. C. with frequent mixing. Reactions were terminated with
2.times.Laemelli buffer and after boiling, proteins were separated
by SDS-PAGE, with quantification of activity by autoradiography and
phosphorimaging, and qualitative analysis of enzyme presence and
phosphorylation by Western Blotting.
[0109] The results are shown in FIG. 10 and indicate that p38 MAP
kinase is activated in neutrophils by lipopolysaccharide.
[0110] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims:
* * * * *