U.S. patent application number 10/079068 was filed with the patent office on 2002-06-27 for methods and compositions for enhancing the immunostimulatory effect of interleukin-12.
Invention is credited to Koblish, Holly, Lee, William M. F., Trinchieri, Giorgio.
Application Number | 20020081277 10/079068 |
Document ID | / |
Family ID | 26798528 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081277 |
Kind Code |
A1 |
Trinchieri, Giorgio ; et
al. |
June 27, 2002 |
Methods and compositions for enhancing the immunostimulatory effect
of interleukin-12
Abstract
Methods for enhancing the therapeutic and adjuvant use of IL-12
by reducing unwanted transient immunosuppression caused by IL-12 or
by high doses thereof involve co-administering IL-12 with an
effective amount of an agent that inhibits or neutralizes nitric
oxide (NO) in vivo. Enhanced vaccine therapy involves
co-administering the IL-12 adjuvant, a selected vaccine antigen and
the NO inhibiting/neutralizing agent. Additionally, the toxicity of
IL-12 treatment may be reduced by co-administering IL-12 with an
effective amount of the NO inhibiting or neutralizing agent. A
therapeutic composition characterized by reduced toxicity in
mammals contains IL-12, preferably a low dose thereof, and an NO
inhibiting or neutralizing agent in a pharmaceutically acceptable
carrier. A vaccine composition contains an effective adjuvanting
amount of IL-12, an effective amount of an NO inhibiting or
neutralizing agent, and an effective protective amount of a vaccine
antigen in a pharmaceutically acceptable carrier.
Inventors: |
Trinchieri, Giorgio;
(Charly, FR) ; Lee, William M. F.; (Wynnewood,
PA) ; Koblish, Holly; (Yardley, PA) |
Correspondence
Address: |
HOWSON AND HOWSON
ONE SPRING HOUSE CORPORATION CENTER
BOX 457
321 NORRISTOWN ROAD
SPRING HOUSE
PA
19477
US
|
Family ID: |
26798528 |
Appl. No.: |
10/079068 |
Filed: |
February 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10079068 |
Feb 20, 2002 |
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09395038 |
Sep 13, 1999 |
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6375944 |
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60101698 |
Sep 25, 1998 |
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Current U.S.
Class: |
424/85.2 ;
514/398; 514/509; 514/565; 514/634 |
Current CPC
Class: |
Y02A 50/41 20180101;
A61K 39/39 20130101; Y02A 50/412 20180101; Y02A 50/423 20180101;
A61K 2039/55511 20130101; Y02A 50/30 20180101; A61K 38/208
20130101; Y02A 50/383 20180101; Y02A 50/388 20180101; A61K
2039/55538 20130101; Y02A 50/466 20180101; A61K 38/208 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
514/398; 514/509; 514/565; 514/634 |
International
Class: |
A61K 038/20; A61K
031/198; A61K 031/21; A61K 031/155 |
Goverment Interests
[0002] This invention has been supported by grants from the
National Institutes of Health, Grant Nos. CA20833, AM34412,
CA10805, CA32898, CA65805, AI42334-01, and CA77851 and from the
Department of the Army, Grant No. DAMD17-94-J-4027. The United
States government has an interest in this invention.
Claims
What is claimed is:
1. A method for reducing the immunosuppressive effects of IL-12
treatment comprising: co-administering with said IL-12, an
effective amount of a nitric oxide inhibiting and/or neutralizing
agent.
2. The method according to claim 1 wherein said co-administration
comprises simultaneously administering said agent with said
IL-12.
3. The method according to claim 1 wherein said co-administration
comprises sequentially administering said agent, and said
IL-12.
4. The method according to claim 3 wherein said co-administration
comprises administering said IL-12 before said agent.
5. The method according to claim 1 wherein said agent is an
inhibitor of nitric oxide generation is an inhibitor of nitric
oxide synthase.
6. The method according to claim 5 wherein said agent is specific
for inducible nitric oxide synthase.
7. The method according to claim 5 wherein said inhibitor is
selected from the group consisting of L-N.sup.G monomethyl arginine
(L-NMMA), L-N.sup.G nitroarginine (L-NORAG), L-N.sup.G
nitroarginine methylester (L-NAME), L-N.sup.G nitroarginine
p-nitroanilide (L-NAPNA), L-N.sup.G aminoarginine (L-NAA),
L-N.sup.G cyclopropylarginine, L-N.sup.G allylarginine, asymmetric
L-N.sup.GN.sup.G dimethylarginine (L-ADMA),
L-N.sup..omega.iminoethyl ornithine (L-NIO), 7-nitro indazole
(7-NI), 2,7 dinitro indazole, 3-bromo 7-nitro indazole,
aminoguanidine, N,N'-diaminoguanidine, dimethylguanidine,
diphenyleneiodonium, iodoniumdiphenyl, di-2-thienyliodonium,
chlorpromazine, trifluoperazine, pimozide, clozapine,
calmidazolium, 2,4 diamino-6-hydroxypyrimidine, methotrexate,
N-acetyl-5-hydroxytryptamine, miconazole, ketoconazole,
clotrimazole, imidazole, 1-, 2- and 4-phenylimidazole, methylene
blue, NO, carbon monoxide, ebselen, phencyclidine, and
antineoplastic agents (doxorubicin, aclarubicin).
8. The method according to claim 7 wherein said agent is
L-NAME.
9. The method according to claim 7 wherein said agent is
L-NMMA.
10. The method according to claim 1 wherein said agent is a nitric
oxide scavenger.
11. The method according to claim 10 wherein said scavenger is
selected from the group consisting of N-acetyl cysteine,
pyrrolidine dithiocarbamate, and hemoglobin.
12. A method for reducing the toxicity of IL-12 treatment
comprising: co-administering with an effective dose of said IL-12,
an effective amount of a nitric oxide inhibiting and reducing
agent.
13. The method according to claim 12 wherein said co-administration
comprises simultaneously administering said agent with said
IL-12.
14. The method according to claim 12 wherein said co-administration
comprises sequentially administering said agent, and said
IL-12.
15. The method according to claim 12 wherein said co-administration
comprises administering said IL-12 before said agent.
16. The method according to claim 12 wherein said effective amount
of IL-12 is a low dose thereof.
17. The method according to claim 12 wherein said agent is an
inhibitor of nitric oxide synthase.
18. The method according to claim 17 wherein said agent is specific
for inducible nitric oxide synthase.
19. The method according to claim 17 wherein said inhibitor is
selected from the group consisting of L-N.sup.G monomethyl arginine
(L-NMMA), L-N.sup.G nitroarginine (L-NORAG), L-N.sup.G
nitroarginine methylester (L-NAME), L-N.sup.G nitroarginine
p-nitroanilide (L-NAPNA), L-N.sup.G aminoarginine (L-NAA),
L-N.sup.G cyclopropylarginine, L-N.sup.G allylarginine, asymmetric
L-N.sup.GN.sup.G dimethylarginine (L-ADMA),
L-N.sup..omega.iminoethyl ornithine (L-NIO), 7-nitro indazole
(7-NI), 2,7 dinitro indazole, 3-bromo 7-nitro indazole,
aminoguanidine, N,N'-diaminoguanidine, dimethylguanidine,
diphenyleneiodonium, iodoniumdiphenyl, di-2-thienyliodonium,
chlorpromazine, trifluoperazine, pimozide, clozapine,
calmidazolium, 2,4 diamino-6-hydroxypyrimidine, methotrexate,
N-acetyl-5-hydroxytryptamine, miconazole, ketoconazole,
clotrimazole, imidazole, 1-, 2- and 4-phenylimidazole, methylene
blue, NO, carbon monoxide, ebselen, phencyclidine, and
antineoplastic agents (doxorubicin, aclarubicin).
20. The method according to claim 19 wherein said agent is
L-NAME.
21. The method according to claim 19 wherein said agent is
L-NMMA.
22. The method according to claim 12 wherein said agent is a nitric
oxide scavenger.
23. The method according to claim 22 wherein said scavenger is
selected from the group consisting of N-acetyl cysteine,
pyrrolidine dithiocarbamate, and hemoglobin.
24. A therapeutic composition comprising IL-12, characterized by
reduced toxicity in mammals, said composition comprising an
effective dose of said IL-12 and an effective amount of a nitric
acid inhibiting and/or neutralizing agent in a pharmaceutically
acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
No. 09/395,038, filed Sep. 13, 1999, which claims the benefit of
U.S. Provisional Patent Application No. 60/101,698, filed Sep. 25,
1998, now abandoned.
BACKGROUND OF THE INVENTION
[0003] The present invention relates generally to pharmaceutical
compositions and methods of use thereof which involve using IL-12
as a therapeutic agent or an adjuvant, particularly in vaccines
against cancer; and more particularly, relates to methods and
compositions for enhancing IL-12 activity.
[0004] Interleukin 12 (IL-12) is an immunoregulatory cytokine with
potent antitumor, antiparasitic, antiviral and antimicrobial
effects [M. J. Brunda, Res. Imm., 146:622 (1995); G. Trinchieri,
Annu. Rev. Immunol., 13:251 (1995)]. Many of its activities are
attributable to its ability to induce Th1 CD4+T cell
differentiation, CD8+T cell cytotoxicity and natural killer (NK)
cell activation. IL-12 is critical to the development of
cell-mediated immunity (CMI), being a potent inducer of gamma
interferon (IFN.gamma.) from T and NK cells. IL-12 is also
proinflammatory through its ability to induce production of
IFN.gamma., tumor necrosis factor alpha (TNF.alpha.),
granulocyte-macrophage colony stimulating factor (GM-CSF) and other
cytokines by T and NK cells. IL-12 is required for the development
of Th1 responses, is necessary for delayed type hypersensitivity
(DTH) responses, and is an enhancer of NK cell cytotoxicity [G.
Trinchieri, Blood, 84: 4008 (1994); G. Muller et al., J. Immunol.,
155: 4661 (1995)]. Monocytes are the principal producers of IL-12
in peripheral blood mononuclear cells (PBMC), and
monocyte/macrophages are thought to be the principal producing
cells in vivo [Trinchieri, cited above; A. D'Andrea et al., J. Exp.
Med., 176: 1387 (1992); R. T. Gazzinelli et al, Proc. Natl. Acad.
Sci. U.S.A. 90: 6115 (1993)].
[0005] IFN.gamma. is a particularly important mediator of IL-12
effects. Among other actions, IFN.gamma. activates macrophages and
induces the production of nitric oxide. IFN.gamma. also acts on
many other types of cells, including tumor cells, and its ability
to upregulate MHC expression, slow cell proliferation, and inhibit
angiogenesis may contribute to IL-12's antitumor effects [Voest, E.
E. et al, J. Natl. Cancer Inst., 87:5813 (1995); Sgadari, C. et al,
Blood, 87:3877 (1996); and Coughlin, C. M. et al, J. Clin. Invest.
101:1441 (1998)].
[0006] Therapeutic effects of IL-12 administered systemically have
been reported [e.g., F. P. Heinzel et al, J. Exp. Med., 177:1505
(1993) among others]. IL-12 has also been found to be an effective
adjuvant for a variety of vaccine antigens [U.S. Pat. No.
5,723,127]. However, despite its desirable therapeutic effects, the
therapeutic use of recombinant IL-12 (rIL-12) can be accompanied by
severe toxicities. Dose and schedule dependent toxicities have been
seen during clinical trials [Atkins, M. B. et al., Clin. Can. Res.,
3:409 (1997); Cohen, J., Science, 270:908 (1995)] and in mice
[Coughlin, C. M. et al., Cancer. Res., 57:2460 (1997)].
Administration of rmIL-12 during LCMV infection in mice has been
associated with adverse immunological effects manifested by higher
viral loads, decreased anti-viral CTL activity, and poorer outcome.
TNF.alpha.is implicated in rmIL-12 suppressive effects during LCMV
infection [Orange, J. S. et al., J. Immunol., 152:1253 (1994)].
[0007] Further, while studying the effects of rmIL-12 in A/J mice
during vaccination of genetically modified irradiated, SCK tumor
cells, the present inventors observed that IL-12 transiently
suppressed cellular immune responses in mice. High doses of the
cytokine transiently suppressed tumor protection in vivo and
proliferative responses of splenocytes to T cell mitogens in vitro
[Kurzawa, H. et al., Cancer Res., 58:491 (1998)]. These effects of
high-dose rmIL-12 were generalized, affecting responses to
allogeneic vaccination and splenocyte mitogenic responses in naive
mice of many strains, and appeared to result from impairment of
immune effector function rather than failure to induce
immunity.
[0008] Therapeutic applications of IL-12 may benefit from reduction
or elimination of its transient immunosuppressive side effects.
Approaches to reducing or eliminating IL-12 immunosuppression
include using fewer doses of the cytokine. While this approach may
be beneficial [Noguchi, Y. E. et al, Proc. Natl. Acad. Sci.. USA,
92:2219 (1995)], finding the "proper" regime of IL-12
administration is likely to be quite involved and the results
idiosyncratic. Inhibiting IFN.gamma. action is another alternative
for avoiding IL-12 immunosuppression, but is an impractical
approach which is severely compromised by the fact that IFN.gamma.
may be the primary mediator of IL-12 therapeutic effects.
[0009] Thus, there remains a need in the art for methods and
compositions which can eliminate the immunosuppressive effect of
IL-12, particularly in situations where an enhanced and rapid
adjuvant effect is desirable, and in situations where lower doses
of IL-12 are desired for therapy.
SUMMARY OF THE INVENTION
[0010] In one aspect, the invention provides a method for enhancing
the adjuvant effect of IL-12 comprising co-administering with IL-12
and a vaccine antigen, an effective amount of an agent that
inhibits or reduces the generation of, or that breaks down,
absorbs, metabolizes or eliminates, nitric oxide in vivo (hereafter
referred to as "nitric oxide inhibiting and/or neutralizing
agent"). The vaccine antigen may be a mammalian tumor cell antigen
or an antigen from a pathogenic microorganism.
[0011] In another aspect, the invention provides a method for
reducing the immunosuppressive effects of IL-12 treatment
comprising co-administering with IL-12, an effective amount of the
nitric oxide inhibiting and/or neutralizing agent described
above.
[0012] In another aspect, the invention provides a method for
reducing the toxicity of IL-12 treatment comprising
co-administering with an effective dose of IL-12, an effective
amount of a nitric oxide inhibiting and/or neutralizing agent. The
effective dose of IL-12 may be a low dose thereof
[0013] Still another aspect of the invention is a therapeutic
composition comprising IL-12, characterized by reduced toxicity in
mammals, which comprises IL-12, preferably a low dose thereof, and
an effective amount of a nitric oxide inhibiting and/or
neutralizing agent in a pharmaceutically acceptable carrier.
[0014] In yet a further aspect, the invention provides an adjuvant
composition suitable for use with a vaccine antigen comprising an
effective adjuvanting amount of IL-12 and an effective amount of a
nitric oxide inhibiting and/or neutralizing agent in a
pharmaceutically acceptable carrier.
[0015] In still a further aspect, the invention provides a vaccine
composition comprising an effective adjuvanting amount of IL-12, an
effective amount of a nitric oxide inhibiting and/or neutralizing
agent and an effective protective amount of a vaccine antigen in a
pharmaceutically acceptable carrier.
[0016] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A is a bar graph which reports mean footpad swelling
(+standard error or S.E.) for C57BL/6 mice genetically deficient
for IFN.gamma. and wild-type control mice which are (a)
unvaccinated (open bars; 2 mice); (b) vaccinated with HKB cells and
given phosphate buffered saline (PBS; black bars; 4 mice); (c)
vaccinated with HKB cells and given recombinant murine
Interleukin-12 (rmIL-12) on days 0-4 and 7-11 (gray bars; 4 mice).
All mice were challenged with irradiated SCK cells in the right
foot, and with PBS control injection in the left foot on day
12.
[0018] FIG. 1B is a bar graph which reports mean footpad swelling
(+S.E.) for SV129.times.C57BL/6 mice deficient for both p55 and p75
TNF receptors and control mice which are (a) vaccinated with HKB
cells and given IL-12 (gray bars) or (b) vaccinated with HKB cells
and given PBS (black bars) on days 0-4 and 7-11. Delayed type
hypersensitivity (DTH) assessment was performed on day 12.
[0019] FIG. 2 is a graph illustrating percentage (+S.E.) of
stimulation measured as tritiated thymidine incorporation for
splenocytes from rmIL-12-treated, and PBS-treated, C57BL/6 mice
which were allowed to adhere to 96 well plates for 90 minutes,
overlaid with nonadherent cells and stimulated with either 2.5
.mu.g/ml ConconavalinA (Con A; black bars), 100 U/ml IL-12 (hatched
bars), or 10.sup.5 mitomycin C treated A/J (H-2.sup.a) splenocytes
(stippled bars). Cultures of adherent and nonadherent cells from
spleens of PBS-treated mice are reported. Coculture data are from
triplicate determinations and are significantly different from
control mixtures (adherent and nonadherent cells from PBS treated
mice, p<0.05) where indicated (*).
[0020] FIG. 3A is a bar graph illustrating percentage (+S.E.) of
stimulation measured as tritiated thymidine incorporation for
cocultures established from splenic adherent and nonadherent cells
of PBS-treated mice or from the adherent cells of rmIL-12-treated
mice and nonadherent cells of PBS-treated mice. Antibodies XMG6 (to
IFN.gamma.), XT22 (to TNF.alpha.), AE5 (to IL-10) and C17.8 (to
IL-12) were added to cocultures containing adherent cells from
spleen of rmIL-12-treated mice. Data from Con A-(black bars) and
IL-12-(hatched bars) stimulated cultures are from triplicate
determinations and are significantly different from control
cocultures (p<0.05) where indicated (*).
[0021] FIG. 3B is a bar graph of the same experiment as described
for FIG. 3A, except that N.sup.G-monomethyl-L-arginine (L-NMMA), a
nitric oxide synthase inhibitor, and N.sup.G-monomethyl-D-arginine
(D-NMMA), the inactive isoform, were added to the coculture
containing adherent cells from rmIL-12-treated mice. Data from Con
A-(black bars) and IL-12-(hatched bars) stimulated cultures are
from triplicate determinations and are significantly different from
control cocultures (p<0.05) where indicated (*).
[0022] FIG. 4A is a bar graph illustrating mean footpad swelling
(+S.E.) from 3 mice/treatment group for mice rendered genetically
deficient in inducible nitric oxide synthase (iNOS), i.e. iNOS-/-
mice, and wild-type C57BL/6 mice vaccinated with irradiated HKB
cells and given rmIL-12 (gray bars) or PBS (black bars) injections.
Footpad injections for DTH assessment were performed on day 12, and
swelling measured 24 hours later.
[0023] FIG. 4B is a bar graph illustrating the percentage of
thymidine incorporation for mitogenic stimulation (Con C: black
bars; IL-12: hatched bars) and allogeneic stimulation (stippled
bars) of splenocytes, performed as described in FIGS. 2A, 2B, 3A
and 3B above.
[0024] FIG. 5 is a graph indicating the percentage of mice with
tumors for female A/J mice vaccinated with SCK.GM cells and given
either PBS (solid gray lines), or rmIL-12 (solid black lines), or
rmIL-12+N.sup..omega.-nit- ro-L-arginine methyl ester (L-NAME)
(hatched black lines) or rmIL-12 and N.sup..omega.-nitro-D-arginine
methyl ester (D-NAME) (double dashed black lines) on days 0-4 and
7-11. Mice were challenged fourteen days after vaccination with SCK
cells in the opposite flank and tumorigenesis scored daily. The (*)
designates statistical differences at p<0.05 for rmIL-12 and
L-NAME treated mice vs. rmIL-12 and D-NAME treated mice and vs.
rmIL-12 treated mice. Data are compiled from two separate
experiments that produced consistent results (15-17 mice per group
total).
[0025] FIG. 6 is a graph reporting percentage of mice with tumors
for female A/J mice (8 mice per group) vaccinated with SCK cells
and treated with either PBS (solid gray lines), rmIL-12 (solid
black lines), rmIL-12+L-NAME (hatched black lines) or
rmIL-12+D-NAME (double dashed black lines) on days 0-4 and 7-11.
Mice were challenged fourteen days after vaccination with SCK cells
in the opposite flank. Tumorigenesis was scored daily.
[0026] FIG. 7 is a graph reporting percentage of mice with tumors
for female A/J mice injected with SCK cells and given either PBS
(solid gray lines), rmIL-12 (solid black lines), rmIL-12+L-NAME
(hatched black lines) or rmIL-12 and D-NAME (double dashed black
lines) on days 0-4 and 7-11. Tumorigenesis was scored daily. Data
are compiled from two separate experiments that produced consistent
results (11-12 mice per group total).
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention provides a solution to the unfilled
need in the art for methods and compositions which enhance the
therapeutic and adjuvant use of IL-12. The inventors provide
herewith both methods and compositions for reducing and/or
eliminating the suppression of cellular immune response caused by
administration of recombinant interleukin 12 (rIL-12), particularly
where IL-12 is employed as a vaccine adjuvant in mammalian
patients, preferably humans. Moreover, the present invention also
provides methods and compositions for enhancing the biological
activity of IL-12, in therapeutic treatment of mammalian patients
based on overcoming the cause of the unwanted suppression which has
previously led to the use of more toxic doses of IL-12 in clinical
trials of disease treatment.
I. Mechanism of the Invention
[0028] As discovered by the inventors in the course of the work
discussed in the examples below, recombinant IL-12, when
administered to mammals undergoing alloimmunization, has been found
to transiently, but profoundly, suppress in vivo and in vitro
allogeneic responses and in vitro splenocyte mitogenic responses.
In vivo and in vitro evidence indicates that neutralizing
anti-IFN.gamma. antibodies, but not anti-TFN.alpha., nor antibodies
to other cytokines, can prevent rIL-12-induced suppression (see
Examples 1 and 2 below). The importance of the role of IFN.gamma.
in this immune suppression is demonstrated by splenocyte
fractionation studies in IFN.gamma..sup.-/- and
IFN.gamma.R1.sup.-/- mice which are not immunosuppressed by rIL-12,
which reveal that adherent cells from rIL-12 treated mice suppress
the mitogenic response of normal nonadherent cells to Conconavalin
A (ConA) and IL-12.
[0029] The inventors have determined that the mechanism of rIL-12
immune suppression results from inhibition of T cell proliferation
by nitric oxide (NO) generated by macrophages activated by the
IFN.gamma. produced in response to rIL-12. Experiments using
splenocyte fractionation, an inhibitor of NO generation, and mice
genetically deficient in inducible nitric oxide synthase (iNOS-/-
mice) revealed that adherent cells of the spleen, through
stimulated production of NO by nitric oxide synthase (NOS) are
responsible for suppressing T cell mitogenesis in vitro and
cellular immune responses in vivo, Having thus identified the
mechanism, the inventors found that addition of an inhibitor of NOS
restored mitogenic responses, and iNOS.sup.-/- mice were not
immunosuppressed by IL-12. These results demonstrate that
suppression of T cell responses were due to NO produced by
macrophages activated by high levels of IFN.gamma. induced by
rIL-12.
[0030] When inhibitors of nitric oxide generation were given with
rIL-12 during vaccination of animals with irradiated tumor cells,
immunosuppression was averted and the ability of rIL-12 to enhance
induction of protective antitumor immunity was revealed,
demonstrating that rIL-12 is an effective vaccine adjuvant whose
efficacy may be initially masked by its transient immunosuppressive
effect.
[0031] Without being bound by theory, the inventors believe that
events leading to immune suppression by rIL-12 administration
(particularly high dosage IL-12 administration) are initiated by
its induction of IFN.gamma. production by host lymphocytes. Levels
of IFN.gamma. production high enough to activate macrophages and
induce NOS activity generate high levels of NO which impairs the
proliferation of T cells in response to mitogens. That adherent
cells rather than T cells are primarily responsible for the
pathogenesis of rIL-12 immune suppression is supported by the fact
that T cells from the spleens of rIL-12-treated mice are normally
mitogenic when cocultured with adherent cells from normal mice or
from rIL-12-treated iNOS.sup.-/- mice. Impairment is transient
presumably because T cell proliferative responses recover as
IFN.gamma. production and consequent macrophage activation wanes
following completion of rIL-12 therapy.
[0032] The identification of NO as a mediator of rIL-12-induced
immunosuppression is consistent with its known activities. NO, a
key component of host defense mechanisms against invading
pathogens, is produced by NOS in macrophages activated by
IFN.gamma. and other cytokines [MacMicking, J. et al, Annu. Rev.
Immunol. 15:323 (1997)]. Impaired splenocyte mitogenesis during
Salmonella typhimurium, Trypanasoma cruzi, Toxoplasma gondii and
Listeria monocytogenes infections is due to NO production
associated with high levels of IFN.gamma., endogenous IL-12 and
other proinflammatory cytokines [Hunter, C. A. et al, Immunol., 84:
16 (1995); Gregory, S. H. et al, J. Immunol., 150:2901 (1993);
Schwacha, M. G. et al, Infection and Immunity. 65:4897 (1997); and
Fernandez-Gomez, R. et al, J. Immnunol., 160:3471 (1998)]. When
splenocytes from these mice are fractionated, the adherent
population containing macrophages and producing nitric oxide
appears to suppress splenic T cell mitogenic responses, and this
suppression is reversed by inhibitors of NOS [Candolfi, E. et al,
Infection and Immunity, 62:1995 (1994)]. Thus, diverse processes
that induce NO production of macrophages [Hunter, cited above;
Gregory, cited above; and Fecho, K. et al, J. Immunol., 152:5845
(1994)] can impair T cell proliferative responses.
[0033] Based on the inventors' discovery, to this list of processes
that induce NO production can now be added rIL-12 (including, for
example, high doses of IL-12) which, through induction of high
levels of IFN.gamma. and in the absence of additional inflammatory
stimuli, sufficiently activates macrophages to induce
immunosuppression.
II. Methods of the Invention
[0034] Therefore according to the present invention the therapeutic
and vaccine adjuvant use of IL-12 can be enhanced by eliminating or
reducing its transient immunosuppressive effects, which normally
occur due to administration of recombinant IL-12. This elimination
or reduction of the unwanted immunosuppressive effects of IL-12 can
be accomplished by co-administering with IL-12 an effective amount
of an agent which can retard or prevent nitric oxide synthesis in
vivo by macrophages in response to IFN.gamma. stimulation.
Alternatively, this elimination or reduction of the unwanted
immunosuppressive effects of IL-12 can be accomplished by
co-administering with IL-12 an effective amount of an agent which
can break down, absorb, metabolize, eliminate or "neutralize" the
immunosuppressive activity of NO in vivo. For ease of reference,
these agents are referred to collectively as "NO inhibiting and/or
neutralizing agents".
[0035] For example, the adjuvant effect of IL-12 when administered
with a selected vaccine antigen, e.g., a mammalian tumor cell
antigen or an antigen from a pathogenic microorganism (see e.g.,
U.S. Pat. No. 5,723,127), may be enhanced by co-administering to a
mammalian patient the IL-12, the vaccine antigen, and an effective
amount of an NO inhibiting and neutralizing agent. Such
co-administration may include simultaneously administering the NO
inhibiting and/or neutralizing agent with IL-12 and the antigen.
Alternatively, co-administration may involve sequentially
administering the NO inhibiting and neutralizing agent, the IL-12
and the antigen in any desired order. For example, it is desirable
to administer IL-12 before the vaccine composition, e.g., about one
or more days before the vaccine. As another example, the NO
inhibiting and/or neutralizing agents can be administered after the
IL-12 is administered but, preferably before the gamma interferon
is induced, e.g., within 2 to 24 hours thereafter. More preferably,
to enhance the IL-12 adjuvant effect, the NO inhibiting and/or
neutralizing agent is administered within about one to about two
hours after the IL-12 and preferably with the vaccine antigen
administration. Other orders of administration may readily be
selected by one of skill in the art.
[0036] In still another embodiment of the present invention, a
method for reducing the immunosuppressive effects of IL-12 when the
IL-12 is used therapeutically comprises co-administering with
IL-12, an effective amount of an NO inhibiting and neutralizing
agent of this invention. Such co-administration may include
simultaneously administering the inhibitor with IL-12.
Alternatively, co-administration may involve sequentially
administering the NO inhibiting and neutralizing agent and the
IL-12 in any desired order. Preferably, to enhance the IL-12
therapeutic effect, the NO inhibiting and/or neutralizing agent is
administered within about 2 hours of the IL-12. This method is
useful with the currently employed "high doses" of IL-12 which have
been used in clinical trials of therapeutic treatment with IL-12.
Still another desirable embodiment of this aspect of the invention
enables one to reduce the toxicity of IL-12 treatment by
co-administering an effective amount of an NO inhibiting and/or
neutralizing agent with a "low dose" of IL-12. The
co-administration of the NO inhibiting and/or neutralizing agent
with a dose of IL-12 which may currently be considered suboptimal
to accomplish the desired biological effects of IL-12 treatment
(i.e., a dose comprises between about 10 to about 200 ng IL-12 per
kg body weight, and preferably between about 50 to about 100 ng/kg)
is anticipated to enhance the IL-12 effect. The method of the
present invention enables the use of the low doses of IL-12 in
therapy, which will reduce the toxic side effects noted with the
currently employed high doses (about 100-1000 ng/kg).
[0037] To perform the above defined methods of this invention, the
following components are needed, i.e., IL-12, the NO inhibiting
and/or neutralizing agent, and optionally, the vaccine antigen.
A. Interleukin 12
[0038] Interleukin-12 (IL-12), originally called natural killer
cell stimulatory factor, is a heterodimeric cytokine described, for
example, in M. Kobayashi et al, J Exp. Med, 1709:827 (1989) and in
U.S. Pat. No. 5,457,038, and in the related published International
Patent Application WO90/05147 and European patent application No.
441,900. The expression and isolation of IL-12 protein in
recombinant host cells, the DNA and amino acid sequences of the 30
kd and 40 kd subunits of the heterodimeric human IL-12 are provided
in the above recited documents, incorporated herein by reference.
Research quantities of recombinant human and murine IL-12 are also
available from Genetics Institute, Inc., Cambridge, Mass.
[0039] Fragments of IL-12 which share the same biological activity
of the full-length protein as well as the DNA sequences which
encode IL-12 or fragments thereof may also be employed as the IL-12
of the compositions. Such biologically active fragments may be
obtained by conventional recombinant engineering methods of
fragmenting a protein. Any fragment may be readily assessed for
IL-12 biological activity by testing in an assay which measures the
induction of interferon-.gamma. secretion by human lymphocytes [M.
Kobayashi et al, J. Exp. Med., 1709:827 (1989)]. It should be
understood by one of skill in the art, that such identification of
suitable biologically active fragments of IL-12 for use in the
composition of this invention involves only a minor amount of
routine experimentation.
[0040] For use in the methods and compositions of this invention,
IL-12 may be employed as biologically active heterodimeric protein
or peptide fragments. Where it is used throughout the examples, the
term IL-12 refers to the heterodimeric protein unless smaller
fragments thereof are specifically identified.
[0041] Based on clinical trials and other experiments, the
therapeutic dosages of IL-12 that are now in use, and which have
been reported to cause toxic side effects range from about 100 to
500 ng or more IL-12 protein/kg patient body weight. "Low doses" of
IL-12 protein which have previously shown minimal therapeutic
benefit, but which may be used according to this invention range
from between about 10 to 100 ng IL-12 protein per kg patient body
weight.
[0042] When used as an adjuvant for a selected vaccine composition
containing an antigen, the dosage amounts will depend upon the
cancer or pathogen for which the vaccine is designed, the nature of
the antigen, the dosage amounts of the antigen as well as the
species and physical and medical conditions (e.g., general healthy,
weight, etc.) of the vaccinate. As one example, an effective
adjuvanting dosage or amount of IL-12 protein is desirably between
about 0.1 .mu.g to about 0.1 mg of IL-12 protein per about 25 .mu.g
of antigen. In view of this teaching, one of skill in the art will
know that the adjuvanting amount of IL-12 for any particular
vaccine will be readily defined by balancing the efficacy and
toxicity of the IL-12 and antigen combination with the IL-12
enhancing effect of the NO inhibiting and/or neutralizing agent.
One of skill in the art of vaccine composition is expected to be
able to readily determine suitable amounts of IL-12 to adjuvant
particular vaccines.
[0043] Therapeutic administration of IL-12 protein or peptide may
take any route of administration and such routes as e.g.,
subcutaneous, intraperitoneal, oral, intramuscular, intravascular,
intranasal, etc., may be used for therapeutic or vaccine
administration. When it is administered as an adjuvant with a
vaccine composition, IL-12 is administered by the same route as the
vaccinal antigen.
[0044] Still another mode of delivering IL-12 to the mammalian
patient as an adjuvant or as a therapeutic is in the form of DNA.
Nucleic acid delivery compositions and methods are known to those
of skill in the art and may be employed rather than administration
of the IL-12 protein, as desired. IL-12 may be employed in the
methods of this invention or in the compositions described herein
as DNA sequences, either administered as naked DNA, or associated
with a pharmaceutically acceptable carrier and provide for in vivo
expression of the IL-12 protein or peptide. So-called `naked DNA`
may be used to express the IL-12 protein or peptide fragment in
vivo in a patient. See, e.g., J. Cohen, Science, 259:1691-1692
(Mar. 19, 1993); E. Fynan et al, Proc. Natl. Acad. Sci., 90:
11478-11482 (December 1993); J. A. Wolff et al, Biotechniques,
11:474-485 (1991) which describe similar uses of `naked DNA`, all
incorporated by reference herein. For example, IL-12 DNA for use as
an adjuvant may be incorporated, or transduced, into a pathogenic
microorganism itself, if the whole pathogen itself is to be
employed as the vaccinal antigen. Alternatively, IL-12 DNA may be
administered therapeutically or as part of the vaccine composition
e.g., by injection.
[0045] Alternatively, IL-12 DNA may be administered as part of a
vector or as a cassette containing the IL-12 DNA sequences
operatively linked to a promoter sequence. See, e.g., International
Patent Application PCT WO94/01139, published Jan. 20, 1994.
Briefly, the DNA encoding the IL-12 protein or desired fragment
thereof may be inserted into a nucleic acid cassette. This cassette
may be engineered to contain, in addition to the IL-12 sequence to
be expressed, other optional flanking sequences which enable its
insertion into a vector. This cassette may then be inserted into an
appropriate DNA vector downstream of a promoter, an MRNA a leader
sequence, an initiation site and other regulatory sequences capable
of directing the replication and expression of that sequence in
vivo. This vector permits infection of vaccinate's cells and
expression of the IL-12 in vivo.
[0046] Numerous types of appropriate vectors are known in the art
for protein expression and may be designed by standard molecular
biology techniques. Such vectors are selected from among
conventional vector types including insects, e.g., baculovirus
expression, or yeast, fungal, bacterial or viral expression
systems. Methods for obtaining such vectors are well-known. See,
Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition,
Cold Spring Harbor Laboratory, New York (1989); Miller et al,
Genetic Engineering, 8:277-298 (Plenum Press 1986) and references
cited therein. Recombinant viral vectors, such as retroviruses or
adenoviruses, are preferred for integrating the exogenous DNA into
the chromosome ofthe cell.
[0047] Also where desired, the regulatory sequences in such a
vector which control and direct expression of the IL-12 gene
product in the transfected cell include an inducible promoter.
Inducible promoters are those which "turn on" expression of the
gene when in the presence of an inducing agent. Examples of
suitable inducible promoters include, without limitation, the sheep
metallothionine (MT) promoter, the mouse mammary tumor virus
(MMTV), the tet promoter, etc. The inducing agents may be a
glucocorticoid such as dexamethasone, for, e.g., the MMTV promoter,
or a metal, e.g., zinc, for the MT promoter; or an antibiotic, such
as tetracycline for tet promoter. Still other inducible promoters
may be selected by one of skill in the art, such as those
identified in International patent application WO95/13392,
published May 18, 1995, and incorporated by reference herein. The
identity of the inducible promoter is not a limitation of this
invention.
[0048] When IL-12 nucleic acid sequences are employed as the
therapeutic agent or adjuvant either as `naked DNA` operatively
linked to a selected promoter sequence or transduced into a strain
of the pathogenic microorganism, rather than the protein itself,
the amounts of DNA to be delivered and the routes of delivery may
parallel the IL-12 protein amounts for adjuvant or therapeutic
delivery described above and may also be determined readily by one
of skill in the art.
[0049] The IL-12 useful in the form of protein, peptide or nucleic
acids and fragments thereof may be produced by now-conventional
synthetic or recombinant methods. See, e.g., conventional texts
such as Sambrook et al, "Molecular Cloning. A Laboratory Manual",
2.sup.nd edition, Cold Spring Harbor Laboratory, Cold Spring, N.Y.
(1989). Alternatively, IL-12 may be purchased from pharmaceutical
companies.
B. Agents that Interfere with NO Generation and/or Neutralize its
Activity in vivo
[0050] Another component useful in the methods and compositions of
this invention are agents that either inhibit NO generation by
macrophages, or agents that neutralize NO activity in vivo. Among
such agents useful in the methods and compositions of the present
invention are inhibitors of the enzyme nitric oxide synthase (NOS).
Agents which inhibit the enzyme NOS include the following:
N.sup.G-monomethyl-L-arginine (L-NMMA) (Sigma) and
N.sup..omega.-nitro-L-arginine methyl ester (L-NAME) (Sigma). The
ideal inhibitor would be one effective selectively for inducible
NOS, and not for other constitutive NOS enzymes. See, for example,
those compounds listed in Table 3 of J. E. Ogden and P. K. Moore,
Trends Biotechnol., 13:70-78 (1995), including, without limitation,
L-N.sup.G monomethyl arginine (L-NMMA), L-N.sup.G nitroarginine
(L-NORAG), L-N.sup.G nitroarginine methylester (L-NAME), L-N.sup.G
nitroarginine p-nitroanilide (L-NAPNA), L-N.sup.G aminoarginine
(L-NAA), L-N.sup.G cyclopropylarginine, L-N.sup.G allylarginine,
asymmetric L-N.sup.GN.sup.G dimethylarginine L-ADMA),
L-N.sup..omega.iminoethyl ornithine (L-NIO), 7-nitro indazole
(7-NI), 2,7 dinitro indazole, 3-bromo 7-nitro indazole,
aminoguanidine, N,N'-diarninoguanidine, dimethylguanidine,
diphenyleneiodonium, iodoniumdiphenyl, di-2-thienyliodonium,
chlorpromazine, trifluoperazine, pimozide, clozapine,
calmidazolium, 2,4 diamino-6-hydroxypyrimidine, methotrexate,
N-acetyl-5-hydroxytryptamine, miconazole, ketoconazole,
clotrimazole, imidazole, 1-, 2- and 4-phenylimidazole, methylene
blue, NO, carbon monoxide, ebselen, phencyclidine, and
antineoplastic agents (doxorubicin, aclarubicin). One of skill in
the art provided with this specification and with knowledge
conventional in the art is expected to readily select the
appropriate agent for use in the various aspects of this
invention.
[0051] Agents which breakdown, absorb, eliminate, metabolize or
scavenge (i.e., neutralize) NO may also be employed in the methods
and compositions of this invention. Such agents include
dithiocarbamates, such as diethyldithiocarbamate,
pyrrolidinedithiocarbamate, N-methyl-D-glucamine dithiocarbamate
[A. M. Komarov et al, Biochim. Biophys Acta, 1361(3):229-234
(1997)] and hemoglobin [M. Ikeda et al, J. Am. Soc. Nephrol.,
7(10):2213-2218 (1996)]. Some reactive oxygen species scavengers
may also be similarly useful [A. K. Hughes et al, Kidney Int.,
49(1):181-189 (1996)].
[0052] These NO inhibiting and/or neutralizing agents may be safely
administered to a mammalian patient in dosages of from about 0.1
mg/kg to about 50 mg/kg. Other safe dosages may be selected from a
review of the art on any particular agent. They may be administered
simultaneously with the IL-12, before the IL-12 or after the IL-12
when used to enhance therapeutic IL-12 effects. The NO inhibiting
and/or neutralizing agents may also be administered in any order,
i.e., with, before or after, the IL-12 and the vaccine antigen,
where IL-12 is being employed as a vaccine adjuvant. If IL-12 is
administered as DNA, the inhibitor may be needed at a later
time.
[0053] The NO inhibiting and/or neutralizing agents identified
above may be obtained commercially, for example, from Sigma
Corporation or other companies. Alternatively the peptide or
protein NO inhibiting and/or neutralizing agents or these agents in
nucleic acid form may be prepared synthetically or recombinantly,
as described above for the IL-12.
C. The Vaccine Antigen
[0054] Where the IL-12 is administered as an adjuvant, the vaccine
antigen may be a cancer antigen, such as a mammalian tumor cell
surface antigen, or a cancer cell transfected with, and capable of
expressing, a selected antigen, e.g., B7.
[0055] Alternatively, the vaccine antigen may be obtained from
pathogenic microorganisms (e.g., bacteria, protozoa, helminths,
viruses and parasites) which are the causative agents of diseases
such as HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus,
poliovirus, influenza virus, meningitis virus, measles virus, mumps
virus, rubella, pertussis, encephalitis virus, papilloma virus,
yellow fever virus, respiratory syncytial virus, parvovirus,
chikungunya virus, hemorrhagic fever viruses, Klebsiella, and
Herpes viruses, particularly, varicella, cytomegalovirus and
Epstein-Barr virus, leprosy and tuberculosis, leishmaniasis and
malaria or schistosomiasis. This list is not inclusive and one of
skill in the art would understand that one could expand this list
to include an antigen from any pathogenic organism which may be
useful in a vaccine.
[0056] Such vaccine antigens may be prepared as is known to one of
skill in the art of vaccine preparation by several means, for
example, synthetically or recombinantly, as described above for the
IL-12, depending on the identity of the antigen, or the antigens
may be naturally isolated from the pathogen.
[0057] Such antigens may be administered as whole killed organisms,
or as heat- or chemically- inactivated organisms or portions
thereof, or produced as DNA as discussed above in detail for
IL-12.
III. Compositions of the Invention
[0058] Based on the above disclosures, the present invention also
encompasses several forms of pharmaceutical compositions containing
the IL-12 and NO inhibiting and/or neutralizing agents, and
optionally the vaccine antigen.
[0059] Thus, as one embodiment the invention provides a therapeutic
composition comprising a therapeutically effective amount of IL-12
and an effective amount of an NO inhibiting and/or neutralizing
agent in a pharmaceutically acceptable carrier. As discussed above,
such a therapeutically effective dosage of IL-12 in this
composition may include the higher dosages of current clinical
trials. More preferably, the composition uses lower doses of IL-12
and is characterized by a lower toxicity for mammalian, preferably
human, patients. Such a composition may optionally contain other
pharmaceutical ingredients which are known to one of skill in the
pharmaceutical art to provide timed delivery, or provide coatings,
stabilizers, preservatives, etc, to the active ingredients.
[0060] In still another embodiment, the invention provides an
adjuvant composition suitable for use with a vaccine antigen, which
comprises an effective adjuvanting amount of IL-12 and an effective
amount of an NO inhibiting and/or neutralizing agent in a
pharmaceutically acceptable carrier. Yet another composition of the
invention is a vaccine composition comprising an effective
adjuvanting amount of IL-12, an effective amount of an NO
inhibiting and/or neutralizing agent, and an effective protective
amount of a vaccine antigen in a pharmaceutically acceptable
carrier.
[0061] These compositions can contain each component as a peptide
or protein or chemical pharmaceutical compounds, in dosages as
described above in Part II.
[0062] Alternatively, these compositions may contain the components
as nucleic acids, as described above. The IL-12 and NO inhibiting
and/or neutralizing agent, and optionally the vaccine antigen, as
DNA, may be incorporated, or transduced, into one or multiple DNA
molecules, i.e., plasmid vectors, of which many types are known, or
into one or more viral vectors, preferably poxvirus or adenovirus
vectors, for delivery of the IL-12, NO inhibiting and/or
neutralizing agent, and optional vaccine antigen DNA into the
patient. When incorporated into another DNA molecule, the DNA
sequence encoding the IL-12 and/or NO inhibiting and/or
neutralizing agent, and/or vaccine antigen is operatively linked
with regulatory sequences which direct the expression of the
encoded protein or fragment in vivo. Briefly, a cassette may be
engineered to contain, in addition to the IL-12 and/or NO
inhibiting and/or neutralizing agent and/or vaccine antigen
sequence to be expressed, other flanking sequences which enable
insertion into a vector. This cassette may then be inserted into an
appropriate DNA vector downstream of a promoter, an MRNA leader
sequence, an initiation site and other regulatory sequences capable
of directing the replication and expression of the desired
component sequence(s) in a host cell.
[0063] When administered as naked DNA or as part of plasmid or
viral vectors, the sequences encoding IL-12, the NO inhibiting
and/or neutralizing agent and the optional vaccine antigen may be
present on separate DNA molecules which are admixed for
administration, or may be assembled as part of a single
polycistronic molecule, under the control of the same or different
regulatory sequences.
[0064] For either the protein compositions or DNA compositions
described above, suitable pharmaceutically acceptable carriers can
facilitate administration of proteins, DNA or chemical compounds
but are physiologically inert and/or nonharmful. Carriers may be
selected by one of skill in the art. Exemplary carriers include
sterile saline, lactose, sucrose, calcium phosphate, gelatin,
dextrin, agar, pectin, peanut oil, olive oil, sesame oil, and
water. Additionally, the carrier or diluent may include a time
delay material, such as glycerol monostearate or glycerol
distearate alone or with a wax. In addition, slow release polymer
formulations can be used. Optionally, this composition may also
contain conventional pharmaceutical ingredients, such as
preservatives, or chemical stabilizers.
[0065] Alternatively, or in addition to the compounds of the
invention, other agents useful in treating cancer, or useful in
treating any accompanying bacterial or viral infection, e.g.,
antivirals, or immunostimulatory agents and cytokine regulation
elements, or costimulatory molecules, such as B7, are expected to
be useful in the components of this invention. Such agents may
operate in concert with the therapeutic compositions of this
invention and may be delivered to the patient as DNA or protein, or
as a conventional pharmaceutical synthetic agent. The development
of therapeutic compositions containing these agents is within the
skill of one in the art in view of the teachings of this
invention.
[0066] The dosages of the components of these compositions are
discussed above with respect to the methods of administration.
[0067] The following examples illustrate various aspects of this
invention and do not limit the invention, the scope of which is
embodied in the appended claims. The animals identified below are
employed in the following examples: female C57BL/6 (H-2.sup.b)
mice, 5-8 weeks old [Harlan-Sprague-Dawley (Indianapolis, Ind.)];
IFN.gamma..sup.-/- and iNOS.sup.-/- C57BL/6 mice and wild-type
controls and female A/J (H-2.sup.a) mice, 5-8 weeks old [Jackson
Laboratories (Bar Harbor, Me.)]; IFN.gamma.R1.sup.-/- .times.SV129
mice and controls (stemmed from a breeding pair that was a gift
from Dr. Michel Aguet) [Huang, S. et al, Science, 259:1742 (1993)].
TNF.alpha. p55 and p75 receptor.sup.-/- C57BL/6.times.SV129 mice
and controls were provided by Dr. Phillip Scott and Michelle
Nashleanas (University of Pennsylvania, Philadelphia, Pa.) with
permission from Genentech (South San Francisco, Calif.) and Dr.
Horst Bluethmann and Roche (Basel, Switzerland) [Kalb, A. et al.,
J. Biol. Chem. 271:28097 (1996); Rothe, J. et al, Nature, 364:798
(1993)].
[0068] Where the examples below refer to HKB cells, such cells were
established from a spontaneous tumor that arose in an aged,
unmanipulated female A/J mouse and maintained in RPMI with 10%
fetal calf serum (FCS) and penicillin/streptomycin. They are MHC
class I+ and nontumorigenic in A/J mice when 10.sup.6 cells are
injected simultaneously (sc). SCK murine mammary carcinoma cells
and SCK.GM cells were described in Kurzawa, H., cited above.
EXAMPLE 1:
[0069] IFN.gamma. Mediates rIL-12 Induced Immunosuppression
[0070] To understand the mechanisms underlying this suppression
without the confounding influence of tumor burden or infection with
pathogens, the effects of rIL-12 on allogeneic immune responses
were observed. In vivo and in vitro evidence indicates that
alloimmunization is transiently but profoundly suppressed by
high-dose rIL-12. It seems to impair immune effector mechanisms,
because responses in mice with established immunity are also
suppressed. It does not appear to impair induction of immunity,
since rIL-12 given during tumor cell vaccination provides enhanced
protective antitumor immunity after the period of
immunosuppression. rIL-12 impairment of cellular immune responses
is consistently associated with and likely due to impaired T cell
mitogenic responses. The roles of IFN.gamma. and TNF.alpha. were
examined in rmIL-12 induced suppression of responses to
alloimmunization in the following experiments:
[0071] A. C57BL/6 (H-2.sup.b) mice were vaccinated with irradiated
allogeneic HKB (H-2.sup.a) cells suspended in PBS at 10.sup.7
trypan blue-excluding cells/ml. Cells were irradiated with 6000
rads from a .sup.137Cs source, and mice were vaccinated with
10.sup.6 cells s.c. (day 0). Mice were given rmIL-12 (Genetics
Institute, Andover, Mass.) by intraperitoneal injection (ip) with
500 ng/day on days 0-4 and 7-11 (10 injections). Control mice
received phosphate buffered saline (PBS) injections. The vaccinated
mice received 1 mg of neutralizing anti-IFN.gamma. (XMG6) and/or
anti-TNF.alpha. (XT22) monoclonal antibodies (mAbs) on days -1, 3
and 7. Mice were subsequently assayed for delayed type
hypersensitivity (DTH) and for mitogen and alloantigen stimulation
of splenocytes as described below.
[0072] The mitogen and alloantigen stimulation of splenocytes assay
was performed as follows: In vitro mitogenic stimulation of
splenocytes with 2.5 mg/ml Con A or 100 U/ml rmIL-12 was performed
as described [Kurzawa, cited above]. Proliferative responses to
allogeneic antigens (mixed lymphocyte reaction, MLR) was measured
when splenocytes from the mice were stimulated with 10.sup.5
mitomycin C-treated A/J splenocytes. Splenocyte fractionation was
performed by allowing 10.sup.5 splenocytes to adhere for 90 minutes
in 96 well plates, after which the nonadherent cells were removed
and cocultured with adherent cells from different wells for assay.
When added, antibodies (XMG6 for IFN.gamma., XT22 for TNF.alpha.,
AE5 for IL-10 and C17.8 for IL-12) were used at 10 mg/ml final
concentration. After 72 hours exposure to mitogen, cultures were
pulsed with 1 .mu.Ci.sup.3H-thymidine for 16 hours, cells were
harvested and .sup.3H incorporation was measured by scintillation
counting. Supernatants from cultures assayed for IFN.gamma. by
radioimmunoassay (RIA) (using antibodies AN18 and XMG6 [Wysocka, M.
et al. 1995 Eur. J Immunol. 25:672] were harvested 24 or 72 hours
after stimulation.
[0073] The DTH assessment was performed as follows: For assessment
of allogeneic DTH responses, mice were injected with 50 .mu.l PBS
containing 10.sup.6 irradiated SCK cells in the right footpad and
with 50 .mu.l PBS in the left footpad. Footpad thickness
measurements were taken just before injection and 24 hours later
using a Starrett pocket gauge (Athol, Mass.). Data are presented as
the difference in footpad swelling induced by SCK cells and by
PBS.
[0074] The results of these experiments were as follows:
Recombinant murine (rm)IL-12 suppressed in vivo DTH and in vitro
mitogenic and alloproliferative responses. XMG6 (anti-IFN.gamma.)
completely restored DTH responses, XT22 (anti-TNF.alpha.) only
partially restored responses, and XMG6+XT22 restored responses no
better than XMG6 alone (data not shown). Thus, IFN.gamma. is
crucial for rmIL-12 suppression of immune responses and the role of
TNF.alpha. is less certain.
[0075] B. The role of these cytokines was examined more
definitively by testing the effect of rmIL-12 on alloimmunization
in mice genetically deficient for these cytokines or their
receptors.
[0076] C57BL/6 mice genetically deficient for IFN.gamma.
(IFN.gamma..sup.-/-) and wild-type C57BL/6 mice were vaccinated
with 10.sup.6 irradiated HKB cells and given 500 ng rmIL-12 or
phosphate buffered saline (PBS) on days 0-4 and 7-11 or left
unvaccinated. They were challenged with 10.sup.6 irradiated SCK
cells in the right foot, with PBS control injection in the left
foot on day 12. The DTH responses were measured as described above
in Part A.
[0077] The results of this experiment, illustrated in FIG. 1A,
demonstrate that a course of rmIL-12 suppressed DTH responses to
background levels in wild-type mice but had no suppressive effect
in the IFN.gamma..sup.-/- mice. In a companion experiment,
C57BL/6.times.SV129 mice deficient for the IFN.gamma. receptor and
vaccinated with HKB cells displayed similar results: rmIL-12
suppressed DTH responses in control but not IFN.gamma.R1.sup.-/-
(data not shown). From these results, IFN.gamma. was shown to be
crucial for rmIL-12 induced immunosuppression.
[0078] C. To examine the role of TNF.alpha., SV129.times.C57BL/6
mice deficient for both p55 and p75 TNF receptors (TNFR.sup.-/-
mice) and control mice were vaccinated with 10.sup.6 irradiated HKB
cells and given 500 ng IL-12 or PBS on days 0-4 and 7-11. DTH
assessment was performed on day 12, as described above in Part
A.
[0079] The results are illustrated in FIG. 1B HKB-vaccinated
TNFR.sup.-/- mice treated with rmIL-12 had depressed DTH responses
like wild-type C57BL/6.times.SV129 mice, indicating that TNF.alpha.
responses were dispensable for rmIL-12 immune suppression. However,
DTH responses without rmIL-12 were lower in the TNFR.sup.-/- mice,
suggesting that TNF.alpha. responses might be necessary for maximal
responses.
[0080] D. In earlier studies of the inventor, suppression of in
vitro splenocyte mitogenic responses correlated well with
suppression of in vivo immune responses. This correlation held up
in studies of IFN.gamma..sup.-/- and TNFR.sup.-/- mice: Con A,
IL-12 and allogeneic stimulation of splenocytes from rmIL-12
treated IFN.gamma..sup.-/- mice, as described above in Part A
resulted in normal proliferative responses, while responses of
splenocytes from rmIL-12-treated TNFR.sup.-/- mice were suppressed
(data not shown).
EXAMPLE 2:
[0081] Adherent Cells Mediate IL-12-induced Suppression of
Splenocyte Mitogenesis
[0082] The following experiments identified the cell population
responsible for the suppressed splenocyte mitogenic responses.
[0083] A. Splenocytes from rmIL-12- and PBS-treated C57BL/6 mice
were fractioned by adherence to 96 well plastic plates for 90
minutes. The cells were reconstituted in various combinations of
adherent and nonadherent cells by overlaying nonadherent cells on
the adherent cells. These cocultures were then stimulated with
either 2.5 .mu.g/ml Con A, 100 U/ml IL-12, or 10.sup.5 mitomycin C
treated A/J (H-2A) splenocytes, as described in the
mitogen/alloantigen assay of Example 1.
[0084] The data reported in FIG. 2 are from one of five experiments
that produced similar results and are shown as the percentage
(+S.E.) of stimulation elicited in cultures of adherent and
nonadherent cells from spleens of PBS-treated mice, with the
exception of the data indicated on the graph by an asterisk. The
latter data are from triplicate determinations and are
significantly different from control mixtures (adherent and
nonadherent cells from PBS treated mice, p<0.05). As expected,
cultures of adherent and nonadherent splenocytes from rmIL-12
treated mice had suppressed mitogenic responses compared to
cultures of adherent and nonadherent cells from PBS-treated mice.
Nonadherent cells from rmIL-12 treated mice cocultured with
adherent cells from PBS-treated mice had normal mitogenic
responses, indicating that mitogenesis of T cells from rmIL-12
treated mice is not intrinsically or irreversibly defective.
[0085] When nonadherent cells from control mice were mixed with
adherent cells from rmIL-12 treated mice, proliferative responses
were severely impaired, whether the stimulus was Con A, IL-12 or
alloantigen. These results indicate that T cells from rmIL-12
treated mice can respond to mitogens and generate an
antigen-specific mitogenic response in the presence of adherent
cells from normal mice and that adherent cells are largely
responsible for the defect following rmIL-12 therapy.
[0086] IFN.gamma. was readily detected by radioimmunoassay (RIA) in
cocultures of adherent cells from rmIL-12-treated mice and
nonadherent cells from PBS-treated mice at both 24 hours and 72
hours after stimulation with Con A, IL-12 or alloantigen (data not
shown).
[0087] B. Cocultures were established from splenic adherent and
nonadherent cells of PBS-treated mice or from the adherent cells of
rmIL-12-treated mice and nonadherent cells of PBS-treated mice.
Antibodies XMG6 (to IFN.gamma.), XT22 (to TNF.alpha.), AE5 (to
IL-10) and C17.8 (to IL-12) were added to a final concentration of
10 mg/ml in cocultures containing adherent cells from spleen of
rmIL-12-treated mice, also according to the assay described in
Example 1.
[0088] The results of this experiment, reported in FIG. 3A, from
Con A- and IL-12-stimulated cultures are from triplicate
determinations and are significantly different from control
cocultures (p<0.05) where indicated (*). This data demonstrates
that the addition of anti-IFN.gamma. antibody to these cocultures
restored mitogenic responses, while addition of antibodies to
IL-12, IL-10 or TNF.alpha. had little effect. These antibodies did
not suppress mitogenic responses in cocultures containing adherent
and nonadherent splenocytes from PBS-treated mice (data not shown),
indicating that they had no intrinsic suppressive effects that
shrouded any beneficial effects of cytokine neutralization. These
data support the results of in vivo experiments showing a critical
role for IFN.gamma. in rmIL-12 immune suppression. Adherent cells
are important for rmIL-12 suppression of in vitro mitogenic and
immunological responses and IFN.gamma. is necessary for this
effect.
EXAMPLE 3:
[0089] Adherent Cell-derived Nitric Oxide Inhibits Proliferative
and Immune Responses
[0090] The following experiments demonstrate that IFN.alpha.
induced nitric oxide (NO) from activated macrophages mediates
rmIL-12 induced immunosuppression.
[0091] Cocultures were established from splenic adherent and
nonadherent cells of PBS-treated mice or from the adherent cells of
rmIL-12-treated mice and nonadherent cells of PBS-treated mice.
Antibodies XMG6 (to IFN.gamma.), XT22 (to TNF.alpha.), AE5 (to
IL-10) and C17.8 (to IL-12) were added to a final concentration of
10 mg/ml in cocultures containing adherent cells from spleen of
rmIL-12-treated mice. An inhibitor of inducible nitric oxide
synthase (iNOS), L-NMMA (Sigma), was added to cocultures of
adherent cells from rmIL-12 treated mice and nonadherent cells from
control mice at a final concentration of 500 mM. Alternatively,
D-NMMA (Sigma), a noninhibitory isoform of L-NMMA, was added to
other cocultures as a control.
[0092] NO production was measured as nitrite concentration in
stimulated cell supernatants by the Greiss assay [Green, L. C. et
al, Anal. Biochem, 126:131 (1982)]. Supernatant (100 .mu.l) was
added to 96 well plates; 100 .mu.l of a 1:1 mixture of 1%
sulfanilamide dihydrochloride in 2.5% H.sub.3PO.sub.4 and 0.1%
naphthylethylenediamine dihydrochloride in 2.5% H.sub.3PO.sub.4 was
then added to samples. Plates were incubated at room temperature
for 10 minutes and A540 was determined using a microplate reader
with reference to sodium nitrite standard curves.
S-nitroso-N-acetyl-penillamine (SNAP, Sigma), an NO donor, was used
as an acellular source of NO and was added to splenocytes from
MB-vaccinated C57BL/6 mice.
[0093] The results of this experiment are reported in FIG. 3B, in
which data from Con A- and IL-12-stimulated cultures are from
triplicate determinations and are shown to be significantly
different from control cocultures (p<0.05) where indicated (*).
The NO inhibitor L-NMMA reduced NO levels in the culture
supernatant by 58% and 94% in two independent measurements and
restored mitogenesis, when compared to addition of D-NMMA. If
secreted NO is responsible for suppression of mitogenesis, an
acellular source of NO should have a similar effect. Cultures with
NO levels as low as 2.6-3.5 mM from the addition of SNAP inhibited
mitogenic responses 83-98%. Together, these data indicate that
adherent splenocytes (probably macrophages) activated by rmIL-12
treatment to secrete NO are responsible for impaired T cell
mitogenic responses.
[0094] The ability of an iNOS inhibitor to reverse rmIL-12-induced
suppression of mitogenesis in vitro suggested that mice lacking
iNOS might be resistant to the immunosuppressive effects of
rmIL-12. iNOS-/- and wild-type C57BL/6 mice were vaccinated with
irradiated HKB cells and given a course of either rmIL-12 or PBS
injections, as described in the experiments above. Footpad
injections for DTH assessment were performed on day 12 as described
above in the DTH assay. Swelling 24 hours later is presented as the
mean (+S.E.) from 3 mice in each treatment group in FIG. 4A.
Mitogenic and allogeneic stimulation of splenocytes were also
performed as described in Example 1, and these results reported in
FIG. 4B.
[0095] From the data in FIG. 4A, iNOS.sup.-/- mice receiving
rmIL-12 had DTH responses that were at least as great as those of
PBS-treated iNOS.sup.-/- and wild-type mice that were substantially
higher than those of wild-type mice given rmIL-12. Although rmIL-12
induced splenomegaly in iNOS.sup.-/- mice like wild-type mice [Car,
B. D. et al. 1995 Am. J Path. 147:1693], their splenocytes had
proliferative responses like those of splenocytes from control mice
after in vitro stimulation with mitogens or alloantigens (FIG.
4B).
[0096] Together, these data show that macrophage-derived NO is
essential for rmIL-12-induced immunosuppression while
rmIL-12-induced splenomegaly and associated pathological changes
are not.
EXAMPLE 4:
[0097] IL-12 Vaccine Adjuvant Activity is more Apparent when NO
Generation is Inhibited
[0098] RmIL-12 does not suppress allogeneic responses in
iNOS.sup.-/- mice. Thus, whether iNOS inhibitors would prevent
immunosuppression in mice given rmIL-12 during tumor cell
vaccination was demonstrated as follows. Previously, the inventors
showed that vaccinating A/J mice with irradiated SCK tumor cells
engineered to secrete GM-CSF (SCK.GM cells) was highly protective,
but the administration of rmIL-12 abrogated protection two weeks
after vaccination (but had no deleterious effect four weeks after
vaccination) [Kurzawa et al, Cancer Res., 58: 491(1998)]. The
following experiments demonstrated that inhibition of iNOS function
reverses suppression of immunological protection.
[0099] A. Female A/J mice were vaccinated with 10.sup.6 irradiated
SCK.GM cells suspended in PBS at 10.sup.7 trypan blue-excluding
cells/ml. Cells were irradiated with 6000 rads from a .sup.137Cs
source, and mice were vaccinated with 10.sup.6 cells s.c. (day 0).
Mice were given either rmIL-12 (Genetics Institute, Andover, Mass.)
injected intraperitoneally (ip) 250 ng/day on days 0-4 and 7-11 (10
injections), or rmIL-12+L-NAME (an inhibitor of iNOS that acts
similarly to L-NMMA) injected at the same dosage and regimen, or
rmIL-12 and D-NAME (the inactive isoform) injected at the same
dosage and regimen, while control mice received PBS injections.
Vaccinated and naive A/J mice were challenged fourteen days after
vaccination with 2.5.times.10.sup.4 trypan blue-excluding SCK cells
in the opposite flank to assay for the presence of tumor immunity.
Tumorigenesis was scored daily.
[0100] The results are indicated in the graph of FIG. 5, in which
the difference in tumorigenesis between rmIL-12 treated mice given
L-NAME vs. either D-NAME or nothing is statistically significant at
p<0.05 (*). Data are compiled from two separate experiments that
produced consistent results (15-17 mice per group total). As
expected, SCK.GM vaccination protected the great majority of mice
from tumor cell challenge two weeks after vaccination, and rmIL-12
severely impaired this protection. L-NAME but not D-NAME prevented
this impairment (75% developed tumors). In mice not treated with
rmIL-12, L-NAME and D-NAME had no effect on SCK. GM-induced
protection (data not shown), showing that L-NAME acts by preventing
rmIL-12 suppression of SCK.GM vaccine efficacy.
[0101] RmIL-12 also impairs tumor protection in A/J mice with
established SCK immunity if it is given just prior to tumor cell
rechallenge [Kurzawa, cited above]. L-NAME but not D-NAME given
with rmIL-12 therapy prevented this impairment of immune rejection:
only 25% of rmIL-12 treated mice given L-NAME developed tumors,
whereas 75% of rmIL-12-treated mice given D-NAME developed tumors
(data not shown). Thus, L-NAME prevents rmIL-12 suppression of
established antitumor immune responses. In these studies, levels of
NO were not consistently measurable in mice given rmIL-12, so lower
levels in mice also given L-NAME could not be demonstrated.
[0102] B. Vaccination of A/J mice with irradiated wild-type SCK
cells protected only about 10% of mice from a tumor cell challenge,
i.e. SCK cells are intrinsically poorly immunogenic. Giving rmIL-12
with vaccination did not improve protection when mice were
challenged 14 days after vaccination, but did improve protection
when they were challenged at 28 days [Kurzawa, cited above]. Since
an iNOS inhibitor prevented transient immunosuppression by rmIL-12,
the following experiment was performed to determine whether its use
might reveal rmIL-12's effectiveness as a vaccine adjuvant at the
earlier time point.
[0103] Female A/J mice (8 mice per group) were vaccinated with
10.sup.6 irradiated SCK cells and received either PBS, rmIL-12,
rmIL-12+L-NAME or rmIL-12 and D-NAME on days 0-4 and 7-11. Mice
were challenged fourteen days after vaccination with
2.5.times.10.sup.4 SCK cells in the opposite flank. Tumorigenesis
was scored daily, as described above.
[0104] The results are demonstrated in FIG. 6. Only 38% of mice
given L-NAME with irradiated SCK cells and rmIL-12 developed tumors
when they were challenged on day 14, whereas 75% of mice given
D-NAME developed tumors. This indicated that rmIL-12 improves SCK
cell vaccine efficacy markedly and rapidly, but that the
improvement at day 14 was obscured by rmIL-12's immunosuppressive
effect. The level of protection with L-NAME at 14 days (62%) was
similar to the level of protection seen at 28 days in
SCK-vaccinated mice given rmIL-12 alone (75%) or rmIL-12 with
L-NAME (50%) of D-NAME (50%), indicating that use of L-NAME did not
impair long-term protection afforded by rmIL-12 and SCK
vaccination.
[0105] C. Use of an iNOS inhibitor to alleviate rmIL-12 immune
suppression would be problematic if it reduces the antitumor
efficacy of the cytokine--an important consideration since the
antitumor effects of rmIL-12 are diverse, and some of which are not
immunological [Voest, E. E. et al, J. Natl.Cancer Inst., 87:5813
(1995)]. Thus, the effect of L-NAME was tested on the antitumor
activity of rmIL-12 against SCK tumors.
[0106] Female A/J mice were injected with 2.5.times.10.sup.4 SCK
cells and either PBS, rmIL-12, rmIL-12+L-NAME or rmIL-12 and D-NAME
on days 0-4 and 7-11. Tumorigenesis was scored daily, as described
above.
[0107] The results of this experiment are reported in FIG. 7 and
are compiled from two separate experiments that produced consistent
results (11-12 mice per group total). Similar to what was seen in
the past [Coughlin, C. M. et al., Cancer Res. 55:4980 (1995)],
rmIL-12 injections started on the day of SCK cell injection delayed
tumor appearance by about 5 days (FIG. 7 shows medians of 9 days to
tumor appearance without rmIL-12 and 14 days to tumor appearance
with rmIL-12 ). Mice given L-NAME with their rmIL-12 developed
tumors after a median of 17 days which was 8 days later than in
untreated mice and 3 days later than in mice given rmIL-12 alone or
with D-NAME. L-NAME given without rmIL-12 had no effect on SCK
tumorigenesis (data not shown).
[0108] This experiment indicated that inhibition of iNOS function
potentiates rmIL-12 induced delay of SCK tumorigenesis.
[0109] All published documents are incorporated by reference
herein. Numerous modifications and variations of the present
invention are included in the above-identified specification and
are expected to be obvious to one of skill in the art. Such
modifications and alterations to the compositions and processes of
the present invention are believed to be encompassed in the scope
of the claims appended hereto.
* * * * *