U.S. patent application number 10/354767 was filed with the patent office on 2003-10-02 for maturation of dendritic cells with immune response modifying compounds.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Ahonen, Cory L., Tomai, Mark A., Vasilakos, John P..
Application Number | 20030186440 10/354767 |
Document ID | / |
Family ID | 22939138 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186440 |
Kind Code |
A1 |
Tomai, Mark A. ; et
al. |
October 2, 2003 |
Maturation of dendritic cells with immune response modifying
compounds
Abstract
A method of inducing the maturation of dendritic cells by
stimulating immature dendritic cells with an imidazoquinoline type
immune response modifying compound. Dendritic cells that have been
matured in this manner display increased antigen presenting ability
and may be used as immunotherapeutic agents.
Inventors: |
Tomai, Mark A.; (Oakdale,
MN) ; Vasilakos, John P.; (Woodbury, MN) ;
Ahonen, Cory L.; (Hanover, NH) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
22939138 |
Appl. No.: |
10/354767 |
Filed: |
January 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10354767 |
Jan 30, 2003 |
|
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09248439 |
Feb 11, 1999 |
|
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6558951 |
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Current U.S.
Class: |
435/377 ;
424/93.7; 435/372; 514/292 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 2039/5154 20130101; Y02A 50/30 20180101; C12N 5/0639 20130101;
Y02A 50/41 20180101; C12N 2501/999 20130101 |
Class at
Publication: |
435/377 ;
435/372; 424/93.7; 514/292 |
International
Class: |
A61K 045/00; A61K
031/4745; C12N 005/08 |
Claims
What is claimed is:
1. A method of preparing a cellular adjuvant for the treatment of a
disease comprising: (a) maturing dendritic cells in vitro by
treating the dendritic cells with an imidazoquinoline type immune
response modifying compound and (b) exposing the mature dendritic
cells to an antigen associated with said disease.
2. The method of claim 1 wherein the disease is a neoplastic
disease and the antigen is derived from neoplastic cells.
3. The method of claim 1 wherein the disease is caused by an
infectious agent and the antigen is derived from the infectious
agent.
4. The method of claim 1 wherein the antigen is recombinantly
derived.
5. A method of treating a disease comprising administering a
therapeutically effective dose of the cellular adjuvant of claim 1
to a mammal in need of such treatment.
6. A method of treating a disease comprising administering a
therapeutically effective dose of dendritic cells that have been
matured by stimulation with an imidazoquinoline type IRM to mammal
in need of such treatment.
7. The method of claim 6 wherein the disease is a neoplastic
disease.
8. The method of claim 6 wherein the disease is a Th2 mediated
disease.
9. A cellular adjuvant prepared by the method of claim 1.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/248,439, filed Feb. 11, 1999.
SUMMARY OF THE INVENTION
[0002] The invention relates to the use of synthetic immune
response modifiers to induce the maturation of dendritic cells in
vitro. The invention additionally relates to methods of maturing
dendritic cells, to methods of enhancing the antigen presenting
ability of dendritic cells, and of enhancing T-cell stimulation
using synthetic immune response modifiers. The invention further
relates to cellular adjuvants prepared with the dendritic cells
that have been matured according to the method of the
invention.
BACKGROUND OF THE INVENTION
[0003] Dendritic cells are known to play an important role in the
immune system, both for their potent antigen presenting ability and
their ability to initiate T-cell mediated immune responses. Indeed,
dendritic cells ("DC") activate T-cells more efficiently than any
other known antigen presenting cell, and may be required for the
initial activation of naive T-cells in vitro and in vivo. These
cells are generally present in the body at locations that are
routinely exposed to foreign antigens, such as the skin, lung, gut,
blood, and lymphoid tissues. In general, DC are broadly classified
as immature or mature. Immature DC endocytose and process antigen
efficiently, but express low levels of costimulatory molecules. In
contrast, mature DC display increased levels of costimulatory
molecules CD40, CD80 and CD86, as well as HLA-DR. In addition,
mature DC express CD83 and secrete increased amounts of various
cytokines and chemokines that aid T-cell activation.
[0004] In addition to naive T-cell activation, DC can influence the
balance of the Th1/Th2 immune response. Several reports have
indicated that DC preferentially activate Th1 responses, with the
major determining factor being IL-12 secretion from the activated
DC. Macatonia et al., J. Immunol. 154:5071 (1995). Hilkens et al.,
Blood 90:1920 (1997). Other reports have shown that DC can induce
the generation of either Th1 or Th2 clones. Roth, et al., Scand. J.
Immunol. 43:646 (1996). The evidence indicates that multiple
factors influence the ability of DC to initiate a Th1 or Th2
response, including the DC to T-cell ratio, the DC tissue of
origin, the amount of antigen used to prime the DC, the expression
of costimulatory molecules and the antigen injection route.
[0005] The pivotal role played by DC in antigen presentation and
T-cell activation has resulted in considerable interest in the use
of DC in immunotherapy. This is particularly evident in the areas
of vaccinology and cancer immunotherapy. Although much effort has
been devoted to the development of successful vaccines using
recombinant DNA, successful clinical use of DNA vaccines has not
been achieved. Recent evidence indicates that effective
immunization with DNA vaccines requires recombinant protein
expression from DC. Further, enhanced immunity in animal models has
been achieved utilizing DNA vaccines that encode for cytokines or
that contain CpG oligonucleotide sequences that upregulate DC
maturation. Recently, autologous DC obtained from cancer patients
have been used for cancer immunotherapy. See, e.g., WO98/23728.
Accordingly, efficient ex vivo methods for generating DC are
prerequisite for successful immunotherapy.
[0006] In general, the process of ex vivo DC generation consists of
obtaining DC precursor cells and then differentiating the cells in
vitro into DC before introduction back into the patient. However,
the DC must be terminally differentiated, or they will
de-differentiate into monocytes/macrophages and lose much of their
immunopotentiating ability. Ex vivo DC maturation has been
successfully accomplished with monocyte conditioned medium;
recombinant cytokines such as TNF-.alpha., IL-1 and IL-6; bacterial
products such as LPS, bacterial DNA and cross-linking CD40; and
transfection with genes that encode cytokines or costimulatory
molecules. While these methods are capable of producing mature DC,
there are disadvantages to using recombinant molecules and cellular
supernatants for maturing DC. These include inconsistent quality
and yield from lot to lot of these reagents and the introduction of
exogenous proteins into patients, which may be toxic or result in
autoimmunity. Such reagents can also be expensive to produce,
making the cost of immunotherapy prohibitively expensive. There is
a need for a method of maturing DC in vitro that is reliable and
efficient, without the drawbacks of the currently known
methods.
SUMMARY OF THE INVENTION
[0007] We have found that certain immune response modifier (IRM)
compounds can induce the maturation of DC in vitro. These compounds
are small molecules that can be readily produced at a consistent,
high level of purity and potency. By using these compounds one can
efficiently and consistently mature DC, which can then be used as
immunotherapeutic agents. The IRM compounds useful in the method of
the invention are generally of the imidazoquinoline type; that is,
they have a structure that contains the imidazoquinoline ring
system or a similar ring system, such as imidazopyridine or
imidazonaphthyridine.
[0008] Accordingly, the invention provides a method of in vitro
maturation of dendritic cells comprising treating said dendritic
cells with an imidazoquinoline type immune response modifying
compound, as well as a population of dendritic cells produced by
this method.
[0009] The invention further provides a method of enhancing the
antigen presenting ability of dendritic cells comprising treating
said dendritic cells with an imidazoquinoline type immune response
modifying compound.
[0010] In addition, the invention provides a method of preparing a
cellular adjuvant for the treatment of a disease comprising the
steps of maturing dendritic cells in vitro by treating the
dendritic cells with an imidazoquinoline type immune response
modifying compound and exposing the mature dendritic cells to an
antigen associated with said disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a graphical depiction of the ability of the IRM
compound 4-amino-2-ethoxymethyl-<, <-dimethyl-1H-imidazo
[4,5-c]quinolin-1-ethanol (R-848) to enhance cell surface
expression of CD83 and CD86.
[0012] FIG. 2 shows the ability of R-848 to enhance the cell
surface expression of co-stimulatory molecules on MO-DC.
[0013] FIG. 3 shows the maturation of DC as measured by cell
surface expression of various markers after 6 hours of stimulation
with 2 .mu.g/ml R-848.
[0014] FIG. 4 depicts the results of treating MO-DC with R-848 on
T-cell proliferation and T-cell cytokine production as seen by a
primary MLR.
[0015] FIG. 5 shows the response of R-848 treated MO-DC to tetanus
toxoid.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The IRM Compounds
[0017] Compounds useful in the methods of the invention include
imidazoquinoline type IRM compounds. In general, the term
"imidazoquinoline type IRM compounds" refers to compounds
containing an imidazoquinoline ring system or a similar ring system
that have the ability to modify the immune response. Preferred
imidazoquinoline type IRM compounds-contain one or more of the
following ring systems: imidazoquinoline; imidazopyridine; 6,7
fused cycloalkylimidazopyridine; 1,2-bridged imidazoquinoline;
imidazonaphthyridine; and imidazotetrahydronaphthyridine.
Particularly preferred IRM compounds contain an
imidazoquinoline-4-amine ring system. Compounds useful in the
methods of the invention will also typically have the ability to
induce production of one or more of the cytokines TNF-, IL-1, IL-6
and IL-12 when administered to a host or applied in vitro to
dendritic cells or monocyte/macrophages.
[0018] Immune response modifier compounds useful in the method of
the invention include compounds defined by Formulas I-IX(b) below.
Preferred 1H-imidazo [4,5-c]quinolin-4-amines are defined by
Formulas I-V: 1
[0019] wherein
[0020] R.sub.11 is selected from the group consisting of alkyl of
one to ten carbon atoms, hydroxyalkyl of one to six carbon atoms,
acyloxyalkyl wherein the acyloxy moiety is alkanoyloxy of two to
four carbon atoms or benzoyloxy, and the alkyl moiety contains one
to six carbon atoms, benzyl, (phenyl)ethyl and phenyl, said benzyl,
(phenyl)ethyl or phenyl substituent being optionally substituted on
the benzene ring by one or two moieties independently selected from
the group consisting of alkyl of one to four carbon atoms, alkoxy
of one to four carbon atoms and halogen, with the proviso that if
said benzene ring is substituted by two of said moieties, then said
moieties together contain no more than six carbon atoms;
[0021] R.sub.21 is selected from the group consisting of hydrogen,
alkyl of one to eight carbon atoms, benzyl, (phenyl)ethyl and
phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being
optionally substituted on the benzene ring by one or two moieties
independently selected from the group consisting of alkyl of one to
four carbon atoms, alkoxy of one to four carbon atoms and halogen,
with the proviso that when the benzene ring is substituted by two
of said moieties, then the moieties together contain no more than
six carbon atoms; and
[0022] each R.sub.1 is independently selected from the group
consisting of alkoxy of one to four carbon atoms, halogen, and
alkyl of one to four carbon atoms, and n is an integer from 0 to 2,
with the proviso that if n is 2, then said R.sub.1 groups together
contain no more than six carbon atoms; 2
[0023] wherein
[0024] R.sub.12 is selected from the group consisting of straight
chain or branched chain alkenyl containing two to ten carbon atoms
and substituted straight chain or branched chain alkenyl containing
two to ten carbon atoms, wherein the substituent is selected from
the group consisting of straight chain or branched chain alkyl
containing one to four carbon atoms and cycloalkyl containing three
to six carbon atoms; and cycloalkyl containing three to six carbon
atoms substituted by straight chain or branched chain alkyl
containing one to four carbon atoms; and
[0025] R.sub.22 is selected from the group consisting of hydrogen,
straight chain or branched chain alkyl containing one to eight
carbon atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl,
(phenyl)ethyl or phenyl substituent being optionally substituted on
the benzene ring by one or two moieties independently selected from
the group consisting of straight chain or branched chain alkyl
containing one to four carbon atoms, straight chain or branched
chain alkoxy containing one to four carbon atoms, and halogen, with
the proviso that when the benzene ring is substituted by two such
moieties, then the moieties together contain no more than six
carbon atoms; and
[0026] each R.sub.2 is independently selected from the group
consisting of straight chain or branched chain alkoxy containing
one to four carbon atoms, halogen, and straight chain or branched
chain alkyl containing one to four carbon atoms, and n is an
integer from zero to 2, with the proviso that if n is 2, then said
R.sub.2 groups together contain no more than six carbon atoms;
3
[0027] wherein
[0028] R.sub.23 is selected from the group consisting of hydrogen,
straight chain or branched chain alkyl of one to eight carbon
atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl, (phenyl)ethyl
or phenyl substituent being optionally substituted on the benzene
ring by one or two moieties independently selected from the group
consisting of straight chain or branched chain alkyl of one to four
carbon atoms, straight chain or branched chain alkoxy of one to
four carbon atoms, and halogen, with the proviso that when the
benzene ring is substituted by two such moieties, then the moieties
together contain no more than six carbon atoms; and
[0029] each R.sub.3 is independently selected from the group
consisting of straight chain or branched chain alkoxy of one to
four carbon atoms, halogen, and straight chain or branched chain
alkyl of one to four carbon atoms, and n is an integer from zero to
2, with the proviso that if n is 2, then said R.sub.3 groups
together contain no more than six carbon atoms; 4
[0030] wherein
[0031] R.sub.14 is --CHR.sub.xR.sub.y wherein R.sub.y is hydrogen
or a carbon-carbon bond, with the proviso that when R.sub.y is
hydrogen R.sub.x is alkoxy of one to four carbon atoms,
hydroxyalkoxy of one to four carbon atoms, 1-alkynyl of two to ten
carbon atoms, tetrahydropyranyl, alkoxyalkyl wherein the alkoxy
moiety contains one to four carbon atoms and the alkyl moiety
contains one to four carbon atoms, 2-, 3-, or 4-pyridyl, and with
the further proviso that when R.sub.y is a carbon-carbon bond
R.sub.y and R.sub.x together form a tetrahydrofuranyl group
optionally substituted with one or more substituents independently
selected from the group consisting of hydroxy and hydroxyalkyl of
one to four carbon atoms;
[0032] R.sub.24 is selected from the group consisting of hydrogen,
alkyl of one to four carbon atoms, phenyl, and substituted phenyl
wherein the substituent is selected from the group consisting of
alkyl of one to four carbon atoms, alkoxy of one to four carbon
atoms, and halogen; and
[0033] R.sub.4 is selected from the group consisting of hydrogen,
straight chain or branched chain alkoxy containing one to four
carbon atoms, halogen, and straight chain or branched chain alkyl
containing one to four carbon atoms, and n is an integer from 0 to
2, with the proviso that if n is 2 then said R.sub.4 groups
together contain no more than six carbon atoms; 5
[0034] wherein
[0035] R.sub.15 is selected from the group consisting of: hydrogen;
straight chain or branched chain alkyl containing one to ten carbon
atoms and substituted straight chain or branched chain alkyl
containing one to ten carbon atoms, wherein the substituent is
selected from the group consisting of cycloalkyl containing three
to six carbon atoms and cycloalkyl containing three to six carbon
atoms substituted by straight chain or branched chain alkyl
containing one to four carbon atoms; straight chain or branched
chain alkenyl containing two to ten carbon atoms and substituted
straight chain or branched chain alkenyl containing two to ten
carbon atoms, wherein the substituent is selected from the group
consisting of cycloalkyl containing three to six carbon atoms and
cycloalkyl containing three to six carbon atoms substituted by
straight chain or branched chain alkyl containing one to four
carbon atoms; hydroxyalkyl of one to six carbon atoms; alkoxyalkyl
wherein the alkoxy moiety contains one to four carbon atoms and the
alkyl moiety contains one to six carbon atoms; acyloxyalkyl wherein
the acyloxy moiety is alkanoyloxy of two to four carbon atoms or
benzoyloxy, and the alkyl moiety contains one to six carbon atoms;
benzyl; (phenyl)ethyl; and phenyl; said benzyl, (phenyl)ethyl or
phenyl substituent being optionally substituted on the benzene ring
by one or two moieties independently selected from the group
consisting of alkyl of one to four carbon atoms, alkoxy of one to
four carbon atoms, and halogen, with the proviso that when said
benzene ring is substituted by two of said moieties, then the
moieties together contain no more than six carbon atoms;
[0036] R.sub.25 is 6
[0037] wherein
[0038] R.sub.S and R.sub.T are independently selected from the
group consisting of hydrogen, alkyl of one to four carbon atoms,
phenyl, and substituted phenyl wherein the substituent is selected
from the group consisting of alkyl of one to four carbon atoms,
alkoxy of one to four carbon atoms, and halogen;
[0039] X is selected from the group consisting of alkoxy containing
one to four carbon atoms, alkoxyalkyl wherein the alkoxy moiety
contains one to four carbon atoms and the alkyl moiety contains one
to four carbon atoms, hydroxyalkyl of one to four carbon atoms,
haloalkyl of one to four carbon atoms, alkylamido wherein the alkyl
group contains one to four carbon atoms, amino, substituted amino
wherein the substituent is alkyl or hydroxyalkyl of one to four
carbon atoms, azido, chloro, hydroxy, 1-morpholino, 1-pyrrolidino,
alkylthio of one to four carbon atoms; and
[0040] R.sub.5 is selected from the group consisting of hydrogen,
straight chain or branched chain alkoxy containing one to four
carbon atoms, halogen, and straight chain or branched chain alkyl
containing one to four carbon atoms, and n is an integer from 0 to
2, with the proviso that if n is 2, then said R.sub.5 groups
together contain no more than six carbon atoms.
[0041] Preferred 6,7 fused cycloalkylimidazopyridine-4-amine IRM
compounds are defined by Formula VI below: 7
[0042] wherein m is 1, 2, or 3;
[0043] R.sub.16 is selected from the group consisting of hydrogen;
cycloalkyl of three, four, or five carbon atoms; straight chain or
branched chain alkyl containing one to ten carbon atoms and
substituted straight chain or branched chain alkyl containing one
to ten carbon atoms, wherein the substituent is selected from the
group consisting of cycloalkyl containing three to six carbon atoms
and cycloalkyl containing three to six carbon atoms substituted by
straight chain or branched chain alkyl containing one to four
carbon atoms; fluoro- or chloroalkyl containing from one to ten
carbon atoms and one or more fluorine or chlorine atoms; straight
chain or branched chain alkenyl containing two to ten carbon atoms
and substituted straight chain or branched chain alkenyl containing
two to ten carbon atoms, wherein the substituent is selected from
the group consisting of cycloalkyl containing three to six carbon
atoms and cycloalkyl containing three to six carbon atoms
substituted by straight chain or branched chain alkyl containing
one to four carbon atoms; hydroxyalkyl of one to six carbon atoms;
alkoxyalkyl wherein the alkoxy moiety contains one to four carbon
atoms and the alkyl moiety contains one to six carbon atoms;
acyloxyalkyl wherein the acyloxy moiety is alkanoyloxy of two to
four carbon atoms or benzoyloxy, and the alkyl moiety contains one
to six carbon atoms, with the proviso that any such alkyl,
substituted alkyl, alkenyl, substituted alkenyl, hydroxyalkyl,
alkoxyalkyl, or acyloxyalkyl group does not have a fully carbon
substituted carbon atom bonded directly to the nitrogen atom;
benzyl; (phenyl)ethyl; and phenyl; said benzyl, (phenyl)ethyl or
phenyl substituent being optionally substituted on the benzene ring
by one or two moieties independently selected from the group
consisting of alkyl of one to four carbon atoms, alkoxy of one to
four carbon atoms, and halogen, with the proviso that when said
benzene ring is substituted by two of said moieties, then the
moieties together contain no more than six carbon atoms;
[0044] and --CHR.sub.xR.sub.y
[0045] wherein
[0046] R.sub.y is hydrogen or a carbon-carbon bond, with the
proviso that when R.sub.y is hydrogen R.sub.x is alkoxy of one to
four carbon atoms, hydroxyalkoxy of one to four carbon atoms,
1-alkynyl of two to ten carbon atoms, tetrahydropyranyl,
alkoxyalkyl wherein the alkoxy moiety contains one to four carbon
atoms and the alkyl moiety contains one to four carbon atoms, 2-,
3-, or 4-pyridyl, and with the further proviso that when R.sub.y is
a carbon-carbon bond R.sub.y and R.sub.x together form a
tetrahydrofuranyl group optionally substituted with one or more
substituents independently selected from the group consisting of
hydroxy and hydroxyalkyl of one to four carbon atoms,
[0047] R.sub.26 is selected from the group consisting of hydrogen,
straight chain or branched chain alkyl containing one to eight
carbon atoms, straight chain or branched chain hydroxyalkyl
containing one to six carbon atoms, morpholinoalkyl wherein the
alkyl moiety contains 1 to 4 carbon atoms, benzyl, (phenyl)ethyl
and phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being
optionally substituted on the benzene ring by a moiety selected
from the group consisting of methyl, methoxy, and halogen; and
[0048] --C(R.sub.S)(R.sub.T)(X) wherein R.sub.S and R.sub.T are
independently selected from the group consisting of hydrogen, alkyl
of one to four carbon atoms, phenyl, and substituted phenyl wherein
the substituent is selected from the group consisting of alkyl of
one to four carbon atoms, alkoxy of one to four carbon atoms, and
halogen;
[0049] X is selected from the group consisting of alkoxy containing
one to four carbon atoms, alkoxyalkyl wherein the alkoxy moiety
contains one to four carbon atoms and the alkyl moiety contains one
to four carbon atoms, haloalkyl of one to four carbon atoms,
alkylamido wherein the alkyl group contains one to four carbon
atoms, amino, substituted amino wherein the substituent is alkyl or
hydroxyalkyl of one to four carbon atoms, azido, alkylthio of one
to four carbon atoms, halogen, hydroxy, morpholino, and
morpholinoalkyl wherein the alkyl moiety contains one to four
carbon atoms, and
[0050] R.sub.6 is selected from the group consisting of hydrogen,
fluoro, chloro, straight chain or branched chain alkyl containing
one to four carbon atoms, and straight chain or branched chain
fluoro- or chloroalkyl containing one to four carbon atoms and at
least one fluorine or chlorine atom.
[0051] Preferred imidazopyridine-4-amine IRM compounds are defined
by Formula VII below: 8
[0052] wherein
[0053] R.sub.17 is selected from the group consisting of hydrogen;
--CH.sub.2R.sub.W wherein R.sub.W is selected from the group
consisting of straight chain, branched chain, or cyclic alkyl
containing one to ten carbon atoms, straight chain or branched
chain alkenyl containing two to ten carbon atoms, straight chain or
branched chain hydroxyalkyl containing one to six carbon atoms,
alkoxyalkyl wherein the alkoxy moiety contains one to four carbon
atoms and the alkyl moiety contains one to six carbon atoms, and
phenylethyl; and --CH.dbd.CR.sub.ZR.sub.Z wherein each R.sub.Z is
independently straight chain, branched chain, or cyclic alkyl of
one to six carbon atoms;
[0054] R.sub.27 is selected from the group consisting of hydrogen,
straight chain or branched chain alkyl containing one to eight
carbon atoms, straight chain or branched chain hydroxyalkyl
containing one to six carbon atoms, alkoxyalkyl wherein the alkoxy
moiety contains one to four carbon atoms and the alkyl moiety
contains one to six carbon atoms, benzyl, (phenyl)ethyl and phenyl,
the benzyl, (phenyl)ethyl or phenyl substituent being optionally
substituted on the benzene ring by a moiety selected from the group
consisting of methyl, methoxy, and halogen; and morpholinoalkyl
wherein the alkyl moiety contains one to four carbon atoms; and
[0055] R.sub.67 and R.sub.77 are independently selected from the
group consisting of hydrogen and alkyl of one to five carbon atoms,
with the proviso that R.sub.67 and R.sub.77 taken together contain
no more than six carbon atoms, and with the further proviso that
when R.sub.77 is hydrogen then R.sub.67 is other than hydrogen and
R.sub.27 is other than hydrogen or morpholinoalkyl, and with the
further proviso that when R.sub.67 is hydrogen then R.sub.77 and
R.sub.27 are other than hydrogen.
[0056] Preferred 1,2-bridged imidazoquinoline-4-amine IRM compounds
are defined by Formula VIII below: 9
[0057] wherein
[0058] Z is selected from the group consisting of:
[0059] --(CH.sub.2).sub.p-- wherein p is 1 to 4;
[0060] --(CH.sub.2).sub.a--C(R.sub.DR.sub.E)(CH.sub.2).sub.b--,
wherein a and b are integers and a+b is 0 to 3, R.sub.D is hydrogen
or alkyl of one to four carbon atoms, and R.sub.E is selected from
the group consisting of alkyl of one to four carbon atoms, hydroxy,
--OR.sub.F wherein R.sub.F is alkyl of one to four carbon atoms,
and --NR.sub.GR'.sub.G wherein R.sub.G and R'.sub.G are
independently hydrogen or alkyl of one to four carbon atoms;
and
[0061] --(CH.sub.2).sub.a--(Y)--(CH.sub.2).sub.b-- wherein a and b
are integers and a+b is 0 to 3, and Y is O, S, or --NR.sub.j--
wherein R.sub.j is hydrogen or alkyl of one to four carbon
atoms;
[0062] and wherein q is 0 or 1 and R.sub.8 is selected from the
group consisting of alkyl of one to four carbon atoms, alkoxy of
one to four carbon atoms, and halogen.
[0063] Preferred imidazonaphthyridine-4-amine and
imidazotetrahydronaphthy- ridine-4-amine IRM compounds are defined
by Formulas IX(a) and IX(b) below: 10
[0064] wherein
[0065] A is .dbd.N--CR.dbd.CR--CR.dbd.; .dbd.CR--N.dbd.CR--CR.dbd.;
.dbd.CR--CR.dbd.N--CR.dbd.; or .dbd.CR--CR.dbd.CR--N.dbd.;
[0066] R.sub.19 is selected from the group consisting of:
[0067] -hydrogen;
[0068] --C.sub.1-20 alkyl or C.sub.2-20 alkenyl that is
unsubstituted or substituted by one or more substituents selected
from the group consisting of:
[0069] -aryl;
[0070] -heteroaryl;
[0071] -heterocyclyl;
[0072] --O--C.sub.1-20alkyl,
[0073] --O--(C.sub.1-20alkyl).sub.0-1-aryl;
[0074] --O--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0075] --O--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0076] --C.sub.1-20alkoxycarbonyl;
[0077] --S(O).sub.0-2--C.sub.1-20alkyl;
[0078] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-aryl;
[0079] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0080] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0081] --N(R.sub.39).sub.2;
[0082] --N.sub.3;
[0083] oxo;
[0084] -halogen;
[0085] --NO.sub.2;
[0086] --OH; and
[0087] --SH; and
[0088] --C.sub.1-20alkyl-NR.sub.39-Q-X--R.sub.49 or
--C.sub.2-20alkenyl-NR.sub.39-Q-X--R.sub.49 wherein Q is --CO-- or
--SO.sub.2--; X is a bond, --O-- or --NR.sub.39-- and R.sub.49 is
aryl; heteroaryl; heterocyclyl; or --C.sub.1-20 alkyl or C.sub.2-20
alkenyl that is unsubstituted or substituted by one or more
substituents selected from the group consisting of:
[0089] -aryl;
[0090] -heteroaryl;
[0091] -heterocyclyl;
[0092] --O--C.sub.1-20alkyl,
[0093] --O--(C.sub.1-20alkyl).sub.0-1-aryl;
[0094] --O--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0095] --O--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0096] --C.sub.1-20alkoxycarbonyl;
[0097] --S(O).sub.0-2--C.sub.1-20alkyl;
[0098] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-aryl;
[0099] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0100] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0101] --N(R.sub.39).sub.2;
[0102] --NR.sub.39--CO--O--C.sub.1-20alkyl;
[0103] --N.sub.3;
[0104] oxo;
[0105] -halogen;
[0106] --NO.sub.2;
[0107] --OH; and
[0108] --SH; or R.sub.49 is 11
[0109] wherein Y is --N-- or --CR--;
[0110] R.sub.29 is selected from the group consisting of:
[0111] -hydrogen;
[0112] --C.sub.1-10alkyl;
[0113] --C.sub.2-10alkenyl;
[0114] -aryl;
[0115] --C.sub.1-10alkyl-O--C.sub.1-10-alkyl;
[0116] --C.sub.1-10alkyl-O--C.sub.2-10alkenyl; and
[0117] --C.sub.1-10alkyl or C.sub.2-10alkenyl substituted by one or
more substituents selected from the group consisting of:
[0118] --OH;
[0119] -halogen;
[0120] --N(R.sub.39).sub.2;
[0121] --CO--N(R.sub.39).sub.2;
[0122] --CO--C.sub.1-10alkyl;
[0123] --N.sub.3;
[0124] -aryl;
[0125] -heteroaryl;
[0126] -heterocyclyl;
[0127] --CO-aryl; and
[0128] --CO-heteroaryl;
[0129] each R.sub.39 is independently selected from the group
consisting of hydrogen and C.sub.1-10 alkyl; and
[0130] each R is independently selected from the group consisting
of hydrogen, C.sub.1-10alkyl, C.sub.1-10alkoxy, halogen and
trifluoromethyl, 12
[0131] wherein
[0132] B is --NR--C(R).sub.2--C(R).sub.2--C(R).sub.2--;
--C(R).sub.2--NR--C(R).sub.2--C(R).sub.2--;
[0133] --C(R).sub.2--C(R).sub.2--NR--C(R).sub.2-- or
--C(R).sub.2--C(R).sub.2--C(R).sub.2--NR--;
[0134] R.sub.19 is selected from the group consisting of:
[0135] -hydrogen;
[0136] --C.sub.1-20alkyl or C.sub.2-20alkenyl that is unsubstituted
or substituted by one or more substituents selected from the group
consisting of:
[0137] -aryl;
[0138] -heteroaryl;
[0139] -heterocyclyl;
[0140] --O--C.sub.1-20alkyl;
[0141] --O--(C.sub.1-20alkyl).sub.0-1-aryl;
[0142] --O--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0143] --O--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0144] --C.sub.1-20alkoxycarbonyl;
[0145] --S(O).sub.0-2--C.sub.1-20alkyl;
[0146] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-aryl;
[0147] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0148] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0149] --N(R.sub.39).sub.2;
[0150] --N.sub.3;
[0151] oxo;
[0152] -halogen;
[0153] --NO.sub.2;
[0154] --OH; and
[0155] --SH; and
[0156] --C.sub.1-20alkyl-NR.sub.39-Q-X--R.sub.49 or
--C.sub.2-20alkenyl-NR.sub.39-Q-X--R.sub.49 wherein Q is --CO-- or
--SO.sub.2--; X is a bond, --O-- or --NR.sub.39-- and R.sub.49 is
aryl; heteroaryl; heterocyclyl; or --C.sub.1-20alkyl or C.sub.2-20
alkenyl that is unsubstituted or substituted by one or more
substituents selected from the group consisting of:
[0157] -aryl;
[0158] -heteroaryl;
[0159] -heterocyclyl;
[0160] --O--C.sub.1-20alkyl,
[0161] --O--(C.sub.1-20alkyl).sub.0-1-aryl;
[0162] --O--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0163] --O--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0164] --C.sub.1-20alkoxycarbonyl;
[0165] --S(O).sub.0-2--C.sub.1-20alkyl;
[0166] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-aryl;
[0167] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heteroaryl;
[0168] --S(O).sub.0-2--(C.sub.1-20alkyl).sub.0-1-heterocyclyl;
[0169] --N(R.sub.39).sub.2;
[0170] --NR.sub.39--CO--O--C.sub.1-20alkyl;
[0171] --N.sub.3;
[0172] oxo;
[0173] -halogen;
[0174] --NO.sub.2;
[0175] --OH; and
[0176] --SH; or R.sub.49 is 13
[0177] wherein Y is --N-- or --CR--;
[0178] R.sub.29 is selected from the group consisting of:
[0179] -hydrogen;
[0180] --C.sub.1-10alkyl;
[0181] --C.sub.2-10alkenyl;
[0182] -aryl
[0183] --C.sub.1-10alkyl --O--C.sub.1-10-alkyl;
[0184] --C.sub.1-10alkyl-O--C.sub.2-10alkenyl; and
[0185] --C.sub.1-10alkyl or C.sub.2-10 alkenyl substituted by one
or more substituents selected from the group consisting of:
[0186] --OH;
[0187] -halogen;
[0188] --N(R.sub.39).sub.2;
[0189] --CO--N(R.sub.36).sub.2;
[0190] --CO--C.sub.1-10alkyl;
[0191] --N.sub.3;
[0192] -aryl;
[0193] -heteroaryl;
[0194] -heterocyclyl;
[0195] --CO-aryl; and
[0196] --CO-heteroaryl;
[0197] each R.sub.39 is independently selected from the group
consisting of hydrogen and C.sub.1-10 alkyl; and
[0198] each R is independently selected from the group consisting
of hydrogen, C.sub.1-10alkyl, C.sub.1-10alkoxy, halogen and
trifluoromethyl.
[0199] The substituents R.sub.11-R.sub.19 above are generally
designated "1-substituents", as they are located at the 1-position
of the various ring systems. Preferred 1-substituents include alkyl
containing one to six carbon atoms and hydroxyalkyl containing one
to six carbon atoms. More preferably the 1-substituent is
2-methylpropyl or 2-hydroxy-2-methylpropyl.
[0200] The substituents R.sub.21-R.sub.29 above are generally
designated "2-substituents", due to their placement at the
2-position of the various ring systems. Preferred 2-substituents
include hydrogen, alkyl of one to six carbon atoms, alkoxyalkyl
wherein the alkoxy moiety contains one to four carbon atoms and the
alkyl moiety contains one to four carbon atoms, and hydroxyalkyl of
one to four carbon atoms. More preferably the 2-substituent is
hydrogen, methyl, butyl, hydroxymethyl, ethoxymethyl or
methoxyethyl.
[0201] In instances where n can be zero, one, or two, n is
preferably zero or one.
[0202] As used herein, the terms "alkyl", "alkenyl", and the prefix
"-alk" are inclusive of both straight chain and branched chain
groups and of cyclic groups, i.e. cycloalkyl and cycloalkenyl.
These cyclic groups can be monocyclic or polycyclic and preferably
have from 3 to 10 ring carbon atoms. Exemplary cyclic groups
include cyclopropyl, cyclopentyl, cyclohexyl and adamantyl. Alkyl
and alkenyl groups contain from 1 to 10 (or 2 to 10) carbon atoms
unless otherwise specified.
[0203] The term "aryl" as used herein includes carbocyclic aromatic
rings or ring systems. Examples of aryl groups include phenyl,
naphthyl, biphenyl, fluorenyl and indenyl. The term "heteroaryl"
includes aromatic rings or ring systems that contain at least one
ring hetero atom (e.g. O, S, N). Suitable heteroaryl groups include
furyl, thienyl, pyridyl, quinolinyl, tetrazolyl, imidazolyl, and so
on.
[0204] "Heterocyclyl" includes non-aromatic rings or ring systems
that contain at least one ring hetero atom (e.g. O, S, N).
Exemplary heterocyclic groups include pyrrolidinyl,
tetrahydrofuranyl, morpholinyl, thiazolidinyl, imidazolidinyl and
the like.
[0205] The aryl, heteroaryl and heterocyclyl groups may be
unsubstituted or substituted by one or more substituents selected
from the group consisting of C.sub.1-20alkyl, hydroxy, halogen,
N(R.sub.10).sub.2 where each R.sub.10 is independently selected
from the group consisting of hydrogen, C.sub.1-10alkyl, NO.sub.2,
C.sub.1-20alkoxy, C.sub.1-20alkylthio, trihalomethyl,
C.sub.1-20acyl, arylcarbonyl, heteroarylcarbonyl,
(C.sub.1-10alkyl).sub.0-1-aryl,
(C.sub.1-10alkyl).sub.0-1-heteroaryl, nitrile,
C.sub.1-20alkoxycarbonyl, oxo, arylalkyl wherein the alkyl group
has from 1 to 10 carbon atoms, and heteroarylalkyl wherein the
alkyl group has from 1 to 10 carbon atoms.
[0206] The invention is inclusive of the compounds described herein
in any of their pharmaceutically acceptable forms, including salts,
isomers such as diastereomers and enantiomers, solvates,
polymorphs, and the like.
[0207] Of the foregoing IRM compounds, those having the
imidazoquinoline structure are preferred. In particular,
imidazoquinoline-4-amine compounds of formulas I and V are
preferred. The compounds
4-amino-2-ethoxymethyl-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinolin-
-1-ethanol and 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine
are especially preferred.
[0208] The IRM compounds useful in the methods of the invention can
be prepared using methods that are known in the art, as seen for
example in U.S. Pat. Nos. 4,689,338, 5,389,640, 5,268,376,
4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905,
5,395,937, 5,756,747, 4,988,815, 5,175,296, 5,741,908, 5,367,076,
5,693,811 and 5,525,612, and in copending U.S. patent application
Ser. No. 09/210,114 all of which are incorporated by reference
herein.
[0209] Maturation of Dendritic Cells
[0210] The IRM compounds described above have been found to induce
the maturation of DC ex vivo. In general, mature DC display
properties such as cytokine secretion, the expression of particular
cell surface markers, and an enhanced ability to stimulate
T-cells.
[0211] Dendritic cells that can be matured using the method of the
invention can be obtained from any source, which sources can be
readily determined by those of skill in the art. For example, the
immature DC can be obtained by isolating the DC from tissues such
as blood, spleen, bone marrow, skin (e.g., Langerhans cells) and
the like or by inducing the differentiation of monocytes or stem
cells using methods known in the art. A preferred method of
obtaining DC comprises the cytokine-induced differentiation of
human peripheral blood mononuclear cells. This method has been
described, for example by Romani et al., J. Immunol. Methods
196:137 (1996) and Bender et al., J. Immunol. Methods 196:121
(1996). A particularly preferred method comprises culturing CD14+
peripheral blood monocytes with GM-CSF and IL-4 using the method
described by Romani, supra.
[0212] The DC thus obtained will be in an immature state, generally
possessing a high capability for antigen capture and processing,
but relatively low T-cell stimulatory capacity. To acquire optimal
T-cell stimulating capacity, the DC must be in a stable, mature
state. Mature DC can be identified by a number of properties,
including their expression of the cell surface marker CD83 and by
the behavior displayed during the mixed lymphocyte reaction. In
this reaction mature DC will cause increased proliferation of naive
allogeneic T-cells and/or increased production of dendritic cell
cytokines. Preferably, the mature DC will induce at least a two-
fold increase in the proliferation of naive allogeneic T-cells
and/or will display at least a three- fold increase in the
production of dendritic cell cytokines, particularly IL-12 and
TNF-.alpha., as compared to DC that have been obtained from the
same source but have not been contacted with any exogenous stimuli
("immature DC"). While immature DC may display some of the
properties described above, they display them to a much lesser
extent than DC which have been matured by exposure to exogenous
stimuli such as an imidazoquinoline type IRM compound. The mature
DC should be stable and not revert to their immature state, as the
immature DC are much less potent stimulators of T-cell
activity.
[0213] The method of the invention comprises the maturation of DC
by stimulating the DC with an imidazoquinoline type IRM in an
amount and for a time sufficient to cause the DC to mature. It is
understood that the DC are incubated in a tissue culture medium
under conditions readily determinable to those of skill in the art.
The specific amount of IRM used and the time of exposure will vary
according to a number of factors that will be appreciated by those
of skill in the art, including the origin of the DC to be matured,
the potency and other characteristics of the IRM compound used, and
so on. However, it is currently preferred that the IRM be used at a
concentration of about 0.1 to about 10 .mu.g/ml, preferably about
0.5 to about 2.0 .mu.g/ml. The IRM compound is solubilized before
being added to the DC containing medium, preferably in water or a
physiological buffer. However, if necessary the compound can be
solubilized in a small amount of an organic solvent such as DMSO
and then diluted or added directly to the DC containing medium.
[0214] The DC are stimulated by the IRM compound for a sufficient
amount of time to allow the DC to become fully mature. This can be
determined by periodically withdrawing samples of the DC containing
medium and assaying for one of the above described properties, such
as secretion of dendritic cell cytokines. In general, the DC can be
said to be fully mature when the measured property has attained its
maximal level and is no longer increasing with time. Although the
time of exposure will vary according to factors understood by those
of skill in the art (including but not limited to the origin of the
DC, the concentration and potency of the IRM, and so on), in
general approximately 16 to 24 hours of stimulation are required
for the DC to become fully mature.
[0215] Dendritic cells that have been matured by exposure to one or
more imidazoquinoline type IRMs express CD83 and display enhanced
expression of CD80, CD86 and CD40. In addition, IRM matured DC
secrete a number of cytokines, particularly pro-inflammatory
cytokines such as TNF-.alpha., IFN-.alpha., IL-6, IL-1, IL-12
p40.
[0216] Use of IRM Matured Dendritic Cells
[0217] Dendritic cells that have been matured by exposure to
imidazoquinoline type IRMs have enhanced antigen presenting ability
as compared to immature DC and can be used in a variety of ways to
enhance the immune response of a subject. For example, the mature
DC can be injected directly into a patient. In this case, the DC
are preferably monocyte derived DC wherein the monocytes have been
obtained from the same patient.
[0218] The DC can also be used in a number of immunotherapies.
Examples of such therapies include ex vivo cell transplantation
therapies for treating disorders of the immune system, such as
AIDS; the ex vivo expansion of T-cells, particularly antigen
specific T-cells which can then be used to treat disorders
characterized by deterioration of the immune system; the generation
of monoclonal antibodies that recognize DC-specific markers; the
preparation of antigen activated DC according to methods known in
the art; and development of vaccines and vaccine adjuvants.
[0219] Preferred uses of DC that have been matured by exposure to
one or more imidazoquinoline type IRMs include those that make use
of antigen activated DC and/or DC modified antigens. The antigen
activated DC, or cellular adjuvants, of the invention are generally
prepared by exposing DC matured according to the method of the
invention to an antigen. The antigen may be protein, carbohydrate
or nucleic acid in nature and may be derived from any suitable
source, including neoplastic cells (e.g., tumor cells) and
infectious agents (e.g., bacterium, virus, yeast, parasite).
Alternatively, the antigen can be derived by recombinant means.
[0220] The cellular adjuvant of the invention can be used in the
treatment of diseases. For example, cellular adjuvants prepared by
exposing the mature DC to tumor derived antigens can be
administered to a patient, thereby provoking an anti-tumor immune
response in the patient. Similarly, infectious diseases can be
treated by administering to the patient cellular adjuvants prepared
by exposing the DC to antigens derived from the infectious
agent.
[0221] Dendritic cells that have been matured by the method of the
invention produce cytokines such as IL-12 and IFN-< that favor
the generation of Th1 immune responses. The ability to bias the
immune response towards the Th1, as opposed to the Th2, response,
can provide a means for treatment of Th2 mediated diseases.
Examples of such diseases include asthma; allergic rhinitis;
systemic lupus erythematosis; eczema; atopic dermatitis Ommen's
syndrome (hyperseosinophilia syndrome); certain parasitic
infections such as cutaneous and systemic leishmaniais, toxoplasma
infection and trypanosome infection; certain fungal infections, for
example candidiasis and histoplasmosis; and certain intracellular
bacterial infections such as leprosy and tuberculosis.
[0222] Experimental
[0223] Materials and Methods
[0224] Culture Medium. Complete RPMI (cRPMI) medium was used
throughout this study. cRPMI consists of RPMI 1640 with 25 mM HEPES
(Life Technologies, Gaithersburg, Md.) supplemented with 10% heat
inactivated FCS (Hyclone, Logan, Utah), 1 mM sodium pyruvate, 0.1
mM non-essential amino acids, 1 mM L-glutamine and 50 .mu.g/ml
gentamicin sulphate (Life Technologies).
[0225] Reagents. Peripheral blood derived CD14.sup.+ cells were
differentiated into DC using recombinant human GM-CSF and
recombinant human IL-4 at 800 U/ml and 25 ng/ml, respectively
(R&D Corporation, Minneapolis, Minn.), as described by Romani
and Bender, supra. Tetanus toxoid (Calbiochem, La Jolla, Calif.)
was solubilized in cRPMI and used at 10 .mu.g/ml. The compound
R-848 (S-28463), 4-amino-2-ethoxymethyl-.alp-
ha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol,
M.W.=314.4, was prepared by 3M Pharmaceuticals, St. Paul, Minn. For
cell culture studies, the HCl salt was dissolved in pyrogen-free,
sterile water and stored as a stock solution at 4.degree. C. for up
to 4 months. Endotoxin levels were below the detectable level [1
pg/mg] in the Limulus amebocyte assay. A stock solution of
bacterial LPS from Escherichia coli 055:B5 (Sigma Chemical, St.
Louis, Mo.) was dissolved at 1 mg/ml in pyrogen-free water and
stored at 4.degree. C. until use.
[0226] Generation of Monocyte-Derived Dendritic Cells (MO-DC). PBMC
were isolated with Histopaque HybriMax-1077 density gradient
(Sigma) from healthy volunteers after obtaining informed consent.
CD14.sup.+ cells were purified by positive selection using
CD14.sup.+ microbeads in conjunction with the MiniMACS system
(Miltenyi Biotech, Auborn, Calif.) by following the manufacturer's
instructions. Purity, as assessed by flow cytometry, was greater
than 90%. The CD14.sup.+ cells were cultured at 2-5.times.10.sup.6
cells per 3 ml cRPMI in 6-well plates (Costar, Cambridge, Mass.)
with 800 U/ml GM-CSF and 25 ng/ml IL-4 as previously described by
Romani and Bender, supra. Fresh medium containing GM-CSF and IL-4
was added every three days. MO-DC were routinely used between days
7 and 8 of culture. As a control, depleted lymphocytes were
cultured in the same fashion.
[0227] In Vitro MO-DC Stimulation. MO-DC were stimulated with 0.1
to 8 .mu.g/ml R-848 (1 .mu.g/ml=3.2 FM) or 1 .mu.g/ml LPS for 1-96
hours. Cells were subsequently analyzed by flow cytometry for the
expression of various cell surface markers, and the cell culture
supernatants were analyzed for various cytokines and chemokines by
ELISA.
[0228] Cell Surface and Intracellular Flow Cytometry. Evaluation of
cell surface marker expression was performed by flow cytometric
analysis using the following monoclonal antibodies: FITC-conjugated
CD1a, clone NA1/34 HLK (Accurate Chemical, Westbury, N.Y.);
PE-conjugated CD14, clone M.phi.P9, PE-conjugated CD80, clone
L307.4, PE- and FITC-conjugated HLA-DR, clone L243, PE- and
FITC-conjugated .gamma.1/.gamma.2a isotype control, clones X40 and
X39 (all from Becton Dickinson, Mountain View, Calif.);
PE-conjugated CD40, clone EA-5 (Biosource International, Camarillo,
Calif.); PE-conjugated CD83, clone HB15a, PE- and FITC-conjugated
.gamma.1/.gamma.1 isotype control, clone 679.1Mc7 (Immunotech,
Marseille, France), PE-conjugated CD86, clone 2331(Pharmingen, San
Diego, Calif.). Cells (5.times.10.sup.5) were incubated for 15
minutes incubation at 4.degree. C. with purified IgD (Becton
Dickinson) to block non-specific binding, and then the cells were
stained for 30 minutes with the antibodies at 4.degree. C. in PBS
containing 10% FCS and 0.1% sodium azide. After washing in PBS, the
cells were analyzed using a FACScan flow cytometer and Cell Quest
software (Becton Dickinson).
[0229] Allogeneic Lymphocyte Activation. T-cells were isolated
using T-Cell Purification Columns according to manufacturer's
specifications (R&D Systems, Minneapolis, Minn.). Allogeneic
MO-DC stimulator cells were pulsed for various times with medium
alone, R-848, or LPS for 1, 6 or 24 hrs and then washed and treated
with 50 .mu.g/ml mitomycin C (Sigma) for 20 minutes at 37.degree.
C. Dendritic cells were subsequently washed, resuspended in cRPMI
and added at various concentrations (1-32.times.10.sup.3 per well)
to purified responder T-cells (1.times.10.sup.5 per well) in
96-well flat-bottomed microtiter plates (BD Labware) in a total
volume of 200 .mu.l. Triplicate cultures were maintained at
37.degree. C. for 96 hours after which time cell proliferation was
assessed by incorporation of [.sup.3H]-thymidine ([.sup.3H]-TdR)
(Amersham, Arlington Heights, Ill.). Each well received 1 .mu.Ci
[.sup.3H]TdR and was harvested 18 hours later. Results are
presented as mean CPM.+-.SEM of triplicate wells. Supernatants were
collected from the same cultures prior to pulsing with [.sup.3H]TdR
and analyzed for IFN-.gamma., IL-5 and IL-2.
[0230] Autologous T Cell Activation. Autologous T cells and
R-848-treated MO-DC were prepared as described for allogeneic T
cell stimulation. MO-DC were cultured with R-848 [2 .mu.g/ml] and
tetanus toxoid [10 .mu.g/ml] for 24 hours. The MO-DC were washed
and cultured at graded doses with PBMC-derived CD3.sup.+ T cells
for 7 days. Cell proliferation and analysis were determined as
described. Supernatants were also collected from the same cultures
prior to pulsing with [.sup.3H]TdR and analyzed for WN-.gamma. and
IL-5.
[0231] Cytokine Analysis. Cytokine levels were measured by ELISA.
Human TNF-.alpha., IL-12 (p40/p70), IFN.gamma., IL-4 and IL-2 kits
were purchased from Genzyme (Cambridge, Mass.). Human IL-6 kits
were obtained from Biosource International (Camarillo, Calif.).
Human IL-5, IL-8, MIP-1.alpha., MCP-1 and RANTES were purchased
from R&D)Systems. All ELISA were run according to
manufacturer's specifications. WN levels were measured by bioassay
(40). IFN-.alpha. and IFN-.beta. specific antibodies were used to
determine which type I IFN was present in the MO-DC supernatants.
Results for all ELISAs are presented in pg/ml, whereas WN results
are presented in U/ml.
[0232] Statistical Analysis. Data were analyzed using a paired
Student's t-test, and the results were considered statistically
significant if p.ltoreq.0.05.
[0233] To assess the maturation potential of R-848 on DC, MO-DC
were treated with R-848 [0.1-8 .mu.g/ml] or LPS [1 .mu.g/ml] for 24
hours, and cell surface CD83 and CD86 expression were analyzed by
flow cytometry on the DC (gated) population as defined by the
forward scatter/side scatter characteristics (FIG. 1A). The results
in FIG. 1B demonstrate that R-848 enhances the expression of CD83
and CD86 on MO-DC as compared to unstimulated (vehicle) cells.
There was no increase in either CD83 or CD86 cell surface
expression with 0.1 .mu.g/ml R848. Enhanced CD86 expression is
evident with 0.4, 2 and 8 .mu.g/ml R-848. Enhanced cell surface
expression of CD83 is seen at 2 and 8 .mu.g/ml R-848. Both CD83 and
CD86 cell surface expression are also enhanced with LPS, which has
been shown to enhance the expression of these molecules on DC. FIG.
1C represents the quantitative CD83 and CD86 cell surface
expression in mean fluorescence intensity (MFI) of R-848 treated
MO-DC. R-848 induces an increase of both CD83 and CD86 expression
in a dose dependent manner, with CD86 expression increasing between
0.1-0.4 .mu.g/ml R-848. CD83 expression is significantly increased
between 0.4-2 .mu.g/ml R-848. Maximal increases in both CD83 and
CD86 expression are generated with 2 .mu.g/ml R-848, which
corresponds to an average increase of approximately 3- to 4-fold
for both CD80 and CD86. Comparatively, maximal CD83 and CD86 cell
surface expression induced with R-848 was equivalent to that
induced by LPS. Both the relative cell number and MFI data
correlate indicating an increased number of cells expressing these
antigens in response to R-848.
[0234] In addition to CD83 and CD86, other cell surface molecules
indicative of DC maturation were also examined by flow cytometry.
MO-DC were cultured with 2 .mu.g/ml R-848 for 24 hours, which gave
maximal CD83 and CD86 expression as shown in FIG. 1. The cells were
stained for cell surface expression of CD1a, CD80, CD83, CD86, CD40
and HLA-DR. FIG. 2A demonstrates that R-848 also enhances the
expression of CD80 and CD40, in addition to CD83 and CD86, as
compared to vehicle controls. FIGS. 2B and 2C represent the
quantitative differences in cell surface molecule expression.
Consistent with the increase in CD83 and CD86 expression, R-848
treatment also induces a 2-fold increase in CD80 and CD40
expression over the vehicle treated MO-DC. Although R-848-induces
an increase in cell surface HLA-DR expression (FIGS. 2A and 2C),
the increase is not quantitatively significant. Similarly, the
R-848-induced decrease in CD1a expression is not statistically
significant. These trends in HLA-DR and CD1a expression following
R-848 stimulation were seen in all experiments, and in some
experiments, the differences were statistical significant between
R-848 and vehicle treated cells. LPS used at 1 .mu.g/ml enhanced
cell surface expression of CD40, CD80, CD86 and CD83 to similar
levels induced by R-848 (data not shown). The results in FIGS. 1
and 2 demonstrate that R-848 induces MO-DC maturation as defined by
increased CD83, CD80, CD86 and CD40 expression. These DC maturation
markers were also examined after 48, 72 and 96 hour stimulation
with R-848, and maximal DC maturation marker expression was
obtained after 24 hours in culture with 2 .mu.g/ml R-848.
[0235] R-848 Induces the Secretion of Pro-inflammatory Cytokines
and Chemokines from Monocyte-Derived Dendritic Cells
[0236] DC maturation results in the production of various cytokines
and chemokines. In addition, numerous cytokines produced by mature
DC such as TNF-.alpha. and IL-12 can induce or enhance DC
maturation. Therefore, we tested if R-848 induces MO-DC cytokine
and chemokine secretion characteristic of DC maturation. MO-DC were
cultured with various concentrations of R-848 for 24 hours as in
FIGS. 1 and 2. The supernatants were analyzed for secreted
cytokines and chemokines by ELISA or by bioassay. The results in
Table I indicate that MO-DC treated with R-848 produce
significantly more TNF-.alpha., IL-6, IL-12, IL-8, MIP-1.alpha. and
WN-.alpha. as compared to the vehicle control. Although
statistically significant levels of all the tested cytokines are
obtained with 2 .mu.g/ml R-848, IL-6, IL-8 and IL-12 appear to be
induced with R-848 between 0.1-0.4 .mu.g/ml, but the levels are not
statistically different than those produced by the vehicle-treated
MO-DC. MCP-1 levels were increased with 0.1-8 .mu.g/ml R-848, but
not significantly different from the levels produced by the control
cells. Neutralizing IFN-.alpha. inhibited greater than 95% of the
bioactivity, indicating that the IFN induced by R-848 was
IFN-.alpha.. Similar to R-848, LPS significantly enhanced
TNF-.alpha., IL-6, IL-12, MIP-1.alpha. and IFN-.alpha. as compared
to the vehicle control group. The maximal cytokine and chemokine
levels induced by LPS are comparable to the maximal levels induced
by R-848.
[0237] The length of time MO-DC need to be in contact with R-848
for maturation to occur was determined by pulsing the cells with
R-848 for various periods of time. Culture supernatants were
analyzed for cytokine secretion after various treatment times with
R-848 or LPS. TNF-.alpha. and IL-12 secretion were used as markers
of DC maturation on the basis of the results in Table I and on
previous studies. First, MO-DC were cultured with 2 .mu.g/ml R-848
or 1 .mu.g/ml LPS for 1, 6 or 24 hours, and the supernatants were
then analyzed for cytokine secretion immediately post culture
(Table II, Groups I, II and V). The results in Table II demonstrate
that MO-DC produce minimal amounts of TNF-.alpha. and L-12 after
one hour stimulation with R-848. A significant increase in
TNF-.alpha. and IL-12 protein is detected in the supernatants
following 6 hour stimulation with R-848. R-848 treatment for 24
hours also induces a significant increase in TNF-.alpha. and IL-12
secretion. The LPS groups produced both TNF-.alpha. and IL-12 with
the same kinetics as the R-848-treated groups, except LPS induced
approximately 2-fold more TNF-.alpha. than was induced by R-848.
LPS treated MO-DC produced approximately 5-fold more IL-12 than
R-848 treated MO-DC.
[0238] The results in Table II indicate that MO-DC require greater
than one hour stimulation with either R-848 or LPS in order to
secrete significant levels of TNF-.alpha.. and IL-12. Maximal
TNF-.alpha. secretion is achieved between one and six hours
stimulation, and maximal L-12 secretion requires between six and
twenty four hours stimulation with either R-848 or LPS.
[0239] In addition to TNF-.alpha. and IL-12 production, cell
surface markers of DC maturation were also examined by flow
cytometry following R-848 treatment for various times in order to
determine the length of time MO-DC need to be in culture with R-848
for optimal maturation marker expression. MO-DC pulsed for one hour
with 2 .mu.g/ml R-848 or 1 .mu.g/ml LPS, and then stained for DC
maturation markers, did not show enhanced expression of CD83, CD80,
CD86, CD40 or HLA-DR. MO-DC pulsed for 6 hours with R-848 and
stained immediately for maturation markers show a significant
increase in CD83 but not CD80, CD86, CD40 or HLA-DR (FIGS. 3A and
3B). Although CD40, CD86 and HLA-DR expression are elevated in the
R-848 treated group following 6 hours in culture, the differences
are not statistically significant as compared to the medium
control. Similar to R-848 treated MO-DC, LPS treated MO-DC showed
enhanced CD83 expression, but no change in CD40, CD80, CD86 and
HLA-DR expression.
[0240] MO-DC were pulsed for 1 or 6 hours with 2 .mu.g/ml R-848 or
1 .mu.g/ml LPS, washed free of stimulus, and then re-cultured for
an additional 23 hours (1 hour pulse) or 18 hours (6 hour pulse)
before cell surface DC maturation marker determination. MO-DC
pulsed for one hour with 2 .mu.g/ml R-848 or 1 .mu.g/ml LPS did not
show enhanced expression of CD83, CD80, CD86, CD40 or HLA-DR after
24 hours in culture. MO-DC pulsed for 6 hours with R-848 show a
significant increase in CD83 and CD40 expression, but not CD80,
CD86 or HLA-DR after 24 hours in culture (FIGS. 3C and 3D). The
expression of CD86 and HLA-DR markers are elevated above, but not
statistically different, than the medium control group. Comparable
results were obtained with similarly cultured LPS-stimulated
MO-DC.
[0241] Allogeneic T cell Proliferation and T cell Cytokine
Secretion are Increased by R-848-treated Monocyte-Derived Dendritic
Cells
[0242] To determine if the functional features of DC were altered
by imidazoquinoline-treatment, R-848-stimulated MO-DC were tested
in a primary MLR. MO-DC were treated with 0.1-8 .mu.g/ml R-848 or 1
.mu.g/ml LPS. After 24 hours, the MO-DC were washed free of
stimulating agent and cultured with allogeneic CD3-enriched
peripheral blood T cells for 96 hours, whereby cell proliferation
was assessed by [.sup.3H]thymidine incorporation. The results in
FIG. 4A demonstrate that R-848-treated MO-DC were more efficacious
stimulators of allogeneic T cell proliferation than vehicle-treated
cells, and R-848-treated cells were as effective as LPS-stimulated
cells. A significant difference in T cell proliferation is seen
when MO-DC are treated with 2 or 8 .mu.g/ml R-848 as compared to
vehicle-treated MO-DC.
[0243] MLR supernatants were analyzed for T cell cytokines
following 96 hours of culture. R-848-treated MO-DC enhance IL-2,
IL-5 and IFN-.gamma., secretion from allogeneic T cells as compared
to the vehicle control group (FIGS. 4B-4D). Concordant with the MLR
proliferation results in FIG. 4A, a significant 2- to 3-fold
enhancement of IL-2, IL-5 and IFN-.gamma. production was induced by
cultures containing MO-DC treated with 2 and 8 .mu.g/ml R-848 as
compared to the untreated MO-DC cultures. T cell cytokines induced
by R-848-stimulated MO-DC were equivalent to cytokine levels
induced by LPS-stimulated MO-DC. IL-2, IL-5 and IFN-.gamma.
production require MO-DC cultured with T cells, because cultures
containing only MO-DC or only T cells did not produce detectable
levels of IL-2, IL-5 or IFN-.gamma.. Additionally, T cells cultured
in the presence of R-848, without added MO-DC, do not produce IL-2,
IL-5 or IFN-.gamma.. These data indicate that R-848 enhances DC
function equivalent to that induced by LPS. Although maximal
proliferation was induced by MO-DC that were pulsed for 24 hours
with R-848, MO-DC treated for 6 hours with R-848 also significantly
enhanced allogeneic T cell proliferation as compared to untreated
MO-DC. When MO-DC were treated for less than 6 hours with R-848,
allogeneic T cell proliferation was not significantly increased as
compared to the untreated MO-DC controls.
[0244] Autologous T cell Proliferation and T cell Cytokine
Secretion are Increased by R-848-treated Monocyte-Derived Dendritic
Cells
[0245] The effect of R-848 on MO-DC function was also tested in an
autologous (syngeneic) anamnestic response to tetanus toxoid. MO-DC
were treated with 2 .mu.g/ml R-848 and 10 .mu.g/ml tetanus toxoid
for 24 hours. The MO-DC were washed free of compound and antigen
and then cultured with syngeneic CD3-enriched peripheral blood T
cells for 7 days at which time proliferation was assessed by
[.sup.3H]thymidine incorporation. The results in FIGS. 5A and 5B
indicate that tetanus toxoid-treated MO-DC and untreated MO-DC
induced the same amount of syngeneic T cell proliferation. However,
R-848-treated MO-DC increased T cell proliferation by 2-to 3-fold
as compared to the MO-DC that were not treated with R-848. Cytokine
secretion was also analyzed from the autologous MO-DC/T cell
system. IFN-.gamma. secretion was only detected in the supernatants
that contained MO-DC treated with both R-848 and tetanus toxoid
(FIGS. 5C and 5D). MO-DC treated with both R-848 and tetanus toxoid
produced 4- to 11-fold more IFN-.gamma. than MO-DC cultured only
with the tetanus toxoid antigen. IL-5 was not detected in any of
the same culture supernatants containing IFN-.gamma.. The data in
FIG. 5 indicate that memory T cell IFN-.gamma. secretion, but not
proliferation, is enhanced by R-848-treated MO-DC.
DETAILED DESCRIPTION OF THE DRAWINGS
[0246] FIG. 1. The immune response modifier R-848 enhances cell
surface expression of CD83 and CD86 on monocyte-derived dendritic
cells (MO-DC). MO-DC were generated in vitro from CD14.sup.+ PBMC
as described in Materials and Methods. MO-DC (2.times.10.sup.5)
were stimulated with 0.1-8 .mu.g/ml R-848 [0.32-26 .mu.M] or 1
.mu.g/ml LPS for 24 hours. A, The cells were subsequently stained
for CD83 and CD86 cell surface expression, and the MO-DC gated
population was analyzed by flow cytometry. B, The results are
expressed as the relative cell number that stain positively within
the gated population. The solid lines indicate R-848 or LPS
treatment, and the dotted lines indicate medium (vehicle) control.
The results in A and B are representative of six independent
experiments from six different donors. C, The results are expressed
as the mean fluorescence intensity (MFI).+-.SEM of six independent
experiments from six different donors. *p.ltoreq.0.05
[0247] FIG. 2. R-848 enhances cell surface expression of
co-stimulatory molecules on MO-DC. MO-DC (2.times.10.sup.5) were
stimulated with 2 .mu.g/ml R-848 for 24 hours. The cells were
subsequently stained for cell surface expression of CD80, CD86,
CD40, HLA-DR, CD83 and CD1a. A, The results are expressed as the
relative cell number that stain positively within the MO-DC gated
population and are representative of three independent experiments
from three different donors. The solid lines indicate R-848
treatment, and the dotted lines indicate medium (vehicle) control.
B, C, The results are expressed as the MFI.+-.SEM of at least three
independent experiments from three different donors.
*p.ltoreq.0.05
[0248] FIG. 3. Maturation of monocyte-derived dendritic cells
requires between 1 and 6 hours stimulation with R-848. MO-DC
(2.times.10.sup.5) were stimulated with 2 .mu.g/ml R-848 for 6
hours. A, B, The cells were subsequently stained for cell surface
expression of CD80, CD86, CD40, HLA-DR, CD83 and CD1a. C, D, The
cells were extensively washed, re-cultured for an additional 18
hours, and then subsequently stained for cell surface expression of
CD80, CD86, CD40, HLA-DR, CD83 and CD1a. The results are expressed
as MFI.+-.SEM of three independent experiments from three different
donors. *p.ltoreq.0.05
[0249] FIG. 4. T cell proliferation and T cell cytokine production
are increased by R-848-treated MO-DC in a primary MLR. MO-DC
(2.times.10.sup.5) were stimulated with 0.1-8 .mu.g/ml R-848 or 1
.mu.g/ml LPS for 24 hours. The cells were extensively washed and
cultured at graded doses with 1.times.10.sup.5 CD3 enriched
allogeneic T cells in triplicate. A, Proliferation was assessed by
[.sup.3H]thymidine incorporation after 96 hours. The results are
expressed as mean CPM.+-.SEM of three independent experiments from
three different donors. Statistically significant differences
(p.ltoreq.0.05) were determined between R-848 [2 and 8 .mu.g/ml]
and LPS treated groups as compared to vehicle [0 .mu.g/ml] treated
group at 4-32.times.10.sup.3 MO-DC. B-D, IL-2, IL-5 and IFN-.gamma.
protein were assessed from the culture supernatants as described in
Materials and Methods. The results are expressed as mean
pg/ml.+-.SEM of three independent experiments from three different
donors. Statistically significant differences (p.ltoreq.0.05) were
determined between R-848 [2 and 8 .mu.g/ml] and LPS treated groups
as compared to vehicle [0 .mu.g/ml] treated group at
8-32.times.10.sup.3 MO-DC.
[0250] FIG. 5. Autologous T cell proliferation and T cell cytokine
secretion are increased by R-848-treated MO-DC in an anamnestic
response to tetanus toxoid. MO-DC (2.times.10.sup.5) were
stimulated with 2 .mu.g/ml R-848 and 10 .mu.g/ml tetanus toxoid for
24 hours. The cells were extensively washed and cultured at graded
doses with 1.times.10.sup.5 CD3 enriched syngeneic T cells in
triplicate for seven days. A, B, Proliferation was assessed by
[.sup.3H]thymidine incorporation after seven days. C, D,
IFN-.gamma. protein was assessed from the culture supernatants as
described in Materials and Methods. The results are expressed as
mean pg/ml.+-.SEM of three independent experiments from three
different donors. The values indicated above some of the data
points represent p-values.ltoreq.0.05.
1TABLE I R-848 stimulates MO-DC cytokine and chemokine
secretion.sup.a Treatment [.mu.g/ml] IFN-.alpha. TNF-.alpha. IL-6
IL-8 IL-12 MCP-1 MIP-1.alpha. 0 (vehicle) 3 .+-. 2 5 .+-. 2 3 .+-.
2 305 .+-. 77 27 .+-. 11 1742 .+-. 646 46 .+-. 46 0.1 R-848 8 .+-.
3 6 .+-. 3 20 .+-. 12 425 .+-. 132 70 .+-. 21 3603 .+-. 2158 57
.+-. 57 0.4 R-848 8 .+-. 3 14 .+-. 4 399 .+-. 208 5125 .+-. 2430
106 .+-. 27 4864 .+-. 2213 511 .+-. 314 2.0 R-848 27 .+-. 7* 1540
.+-. 371* 6729 .+-. 1888* 50092 .+-. 10385* 15984 .+-. 3860* 12941
.+-. 5802 15412 .+-. 5244* 8.0 R-848 41.+-. 12* 2208 .+-. 240* 9690
.+-. 1269* 66988 .+-. 11863* 19640 .+-. 3966* 16249 .+-. 7661 25956
.+-. 5782* 1.0 LPS 59 .+-. 12* 2246 .+-. 438* 10134 .+-. 1687*
64668 .+-. 12407* 15593 .+-. 2755* 11006 .+-. 4485 36243 .+-. 8676*
.sup.aMO-DC (2 .times. 10.sup.5) were cultured for 24 hours in
cRPMI containing graded doses of R-848 or LPS at 37.degree. C. with
5% CO.sub.2. Culture supernatants were collected and stored at
-70.degree. C. until analysis by ELISA or by bioassay. Data are
given as mean .+-. SEM of five independent experiments from five
different donors. All values are in pg/ml, except IFN-.alpha. which
is in U/ml. *p .ltoreq. 0.05, as compared to cytokine levels in
vehicle control
[0251]
2TABLE II TNF-.alpha. and IL-12 production from MO-DC requires
between 1 and 6 hours stimulation with R-848.sup.a Treatment time
(hr).sup.b Treatment.sup.c TNF-.alpha. IL-12 1 vehicle 1 .+-. 1 73
+ 37 R-848 32 .+-. 10* 65 + 18 LPS 51 + 8* 44 + 19 6 vehicle 3 + 3
92 + 99 R-848 1053 + 707* 4446 + 2438* LPS 2679 + 557* 6160 + 1109*
24 vehicle 3 + 4 107 + 32 R-848 335 + 201* 13153 + 5484* LPS 1675 +
665* 21167 + 1050* .sup.aMO-DC (2 .times. 10.sup.5) were cultured
for 24 hours in cRPMI containing graded doses of R-848 or LPS at
37.degree. C. with 5% CO.sub.2. Culture supematants were collected
and stored at -70.degree. C. until analysis by ELISA. Data are
given as mean pg/ml .+-. SEM of three independent experiments from
three different donors. .sup.bTreatment time (hr) is the length of
time MO-DC were in culture with R-848 or LPS. .sup.cMO-DC were
treated for the indicated times with 2 .mu.g/ml R-848, 1 .mu.g/ml
LPS or vehicle (PBS). *, p .ltoreq. 0.05, as compared to the
cytokine levels in the vehicle control.
[0252] The present invention has been described with reference to
several embodiments thereof. The foregoing detailed description and
examples have been provided for clarity of understanding only, and
no unnecessary limitations are to be understood therefrom. It will
be apparent to those skilled in the art that many changes can be
made to the described embodiments without departing from the spirit
and scope of the invention. Thus, the scope of the invention should
not be limited to the exact details of the methods, compositions
and structures described herein, but rather by the language of the
claims that follow.
* * * * *