U.S. patent application number 14/065171 was filed with the patent office on 2014-05-29 for use of il-23 antagonists for treatment of infection.
This patent application is currently assigned to MERCK SHARP & DOHME CORP.. The applicant listed for this patent is MERCK SHARP & DOHME CORP.. Invention is credited to Alissa A. Chackerian, Robert A. Kastelein, Manfred Kopf, Luigina Romani.
Application Number | 20140147442 14/065171 |
Document ID | / |
Family ID | 40130357 |
Filed Date | 2014-05-29 |
United States Patent
Application |
20140147442 |
Kind Code |
A1 |
Kopf; Manfred ; et
al. |
May 29, 2014 |
USE OF IL-23 ANTAGONISTS FOR TREATMENT OF INFECTION
Abstract
Methods and compositions comprising antagonists of IL-23 are
provided for the treatment of infections, such as chronic
bacterial, viral and fungal infections.
Inventors: |
Kopf; Manfred; (Zurich,
CH) ; Romani; Luigina; (Perugia, IT) ;
Kastelein; Robert A.; (Portola Valley, CA) ;
Chackerian; Alissa A.; (Sunnyvale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MERCK SHARP & DOHME CORP. |
Rahway |
NJ |
US |
|
|
Assignee: |
MERCK SHARP & DOHME
CORP.
Rahway
NJ
|
Family ID: |
40130357 |
Appl. No.: |
14/065171 |
Filed: |
October 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12525003 |
Apr 30, 2010 |
8586035 |
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PCT/US08/01717 |
Feb 8, 2008 |
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14065171 |
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60889475 |
Feb 12, 2007 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/172.1; 514/44A |
Current CPC
Class: |
A61P 31/14 20180101;
A61P 37/02 20180101; C07K 16/244 20130101; A61K 2039/505 20130101;
A61P 31/20 20180101; A61P 31/04 20180101; A61P 31/12 20180101; A61P
37/04 20180101; A61K 31/7088 20130101; A61P 31/00 20180101; A61K
31/713 20130101; A61P 31/18 20180101; A61P 31/10 20180101; C07K
16/2866 20130101 |
Class at
Publication: |
424/135.1 ;
424/172.1; 424/133.1; 514/44.A |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 31/7088 20060101 A61K031/7088; A61K 31/713
20060101 A61K031/713 |
Claims
1. A method of treating a subject having a chronic infection
selected from the group consisting of a viral infection, a fungal
infection and a bacterial infection, comprising administering an
effective amount of an antagonist of IL-23.
2. (canceled)
3. The method of claim 1 wherein the chronic infection is a fungal
infection.
4. The method of claim 3, wherein the fungal infection is selected
from the group consisting of candidiasis, aspergillosis,
cryptococcosis and onychomycosis.
5-9. (canceled)
10. The method of claim 1 wherein the chronic infection is a
mycobacterial infection.
11. (canceled)
12. The method of claim 10 wherein the mycobacterial infection is
TB.
13-15. (canceled)
16. The method of claim 1 wherein the chronic infection is a viral
infection caused by a virus selected from the group consisting of
HIV, HPV, HBV and HCV.
17-20. (canceled)
21. A method of enhancing a Th1 immune response in a subject having
a chronic infection comprising administering an antagonist of
IL-23.
22. The method of claim 21 wherein the enhanced Th1 immune response
comprises a 2-fold or greater increase in the percentage of
CD4.sup.+ T cells expressing IFN-.gamma. compared with the
percentage of CD4.sup.+ T cells expressing IFN-.gamma. prior to
administering said antagonist of IL-23.
23. The method of claim 21 wherein the enhanced Th1 immune response
comprises a 2-fold or greater decrease in the percentage of
CD4.sup.+ T cells expressing IL-17 compared with the percentage of
CD4.sup.+ T cells expressing IL-17 prior to administering said
antagonist of IL-23.
24. (canceled)
25. The method of claim 1 further comprising administering at least
one of an antagonist of IL-17A, IL-6 or TGF-.beta..
26-28. (canceled)
29. The method of claim 1, wherein the antagonist of IL-23 is a
binding compound that binds to IL-23p19.
30. The method of claim 1, wherein the antagonist of IL-23 is a
binding compound that binds to IL-23R.
31. The method of claim 29 wherein the binding compound is an
antibody or antigen binding fragment thereof.
32. (canceled)
33. The method of claim 31 wherein the binding compound is an
antibody fragment selected from the group consisting of Fab, Fab',
Fab'-SH, Fv, scFv, F(ab').sub.2, a single chain antibody, and a
diabody.
34. The method of claim 31 wherein the antibody is a humanized or
fully human antibody or antigen binding fragment thereof.
35-38. (canceled)
39. The method of claim 1 wherein the antagonist of IL-23 is an
siRNA or an antisense nucleic acid.
40-41. (canceled)
42. The method of claim 1 wherein the subject having the infection
is immune compromised.
43-50. (canceled)
51. The method of claim 30 wherein the binding compound is an
antibody or antigen binding fragment thereof.
52. The method of claim 51 wherein the binding compound is an
antibody fragment selected from the group consisting of Fab, Fab',
Fab'-SH, Fv, scFv, F(ab').sub.2, a single chain antibody, and a
diabody.
53. The method of claim 51 wherein the antibody is a humanized or
fully human antibody or antigen binding fragment thereof.
Description
[0001] This application claims benefit of U.S. Provisional Patent
Application No. 60/889,475, filed Feb. 12, 2007, the disclosure of
which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to treatment of
infections. Specifically, the invention relates to administration
of antagonist of IL-23, e.g. antibodies, to subjects exhibiting
infections, such as chronic bacterial, fungal or viral
infections.
BACKGROUND OF THE INVENTION
[0003] A number of pathogens cause chronic infections. Various
viruses, fungi and bacteria, for example, can cause persistent
infections that fail to resolve.
[0004] As the number of severe fungal infections continues to rise,
the need for methods and compositions for their treatment is more
urgent. Major fungal pathogens include Candida albicans,
Aspergillus fumigatus and Cryptococcus neoformans, with estimated
annual incidence for invasive mycoses involving these pathogens of
72-228 (for Candida species), 12-34 (for Aspergillus species) and
30-66 (for C. neoformans) infections per million population in the
United States. Pfaller et al. (2006) Clin. Infect. Dis. 43:S3-14.
The rise in fungal infections is primarily due to the increasing
number of immuno-compromised patients as a result of medical
advances (transplantation and chemotherapy), and as a result of the
increasing population of AIDS patients. More than 80% of fungal
infections in immunocompromised patients are caused by Candida
species. Cryptococcosis is the second most prevalent fungal
infection in AIDS patients following candidiasis. Aspergillosis is
responsible for at least 30% of the infections in cancer and organ
transplant patients and has a high mortality rate.
[0005] Although fluconazole has been an effective drug against
fungal pathogens for a number of years, resistance is increasing.
Alternatives such as amphotericin B have serious drawbacks,
including such side effects as fever, kidney damage, anemia, low
blood pressure, headache, nausea, vomiting and phlebitis.
[0006] Bacterial infections remain an important issue despite the
prevalence of antibiotics, in light of an increased population of
immune compromised individuals and a widespread development of
antibiotic resistant bacterial strains. Immune compromised
individuals include the elderly, transplant recipients,
chemotherapy patients, and individuals with acquired immune
deficiency syndrome (AIDS). Nearly two million patients in the
United States get an infection in the hospital each year, and 70%
of the bacteria responsible for those infections are resistant to
at least one antibiotic. NIAID Fact Sheet, "The Problem of
Antimicrobial Resistance," April 2006. In recent years, about
90,000 people in the United States die from infections, up from
13,300 in 1992. Although most bacterial infections remain
susceptible to a prolonged course of therapy of at least one
antibiotic (e.g. continuous intravenous administration of
vancomycin), there is no guarantee that this will remain true with
future pathogenic bacteria. Methicillin resistant Stapholococcus
aureus (MRSA) is a prime example of a multiple-antibiotic-resistant
microbe that represents a significant public health challenge. In
2002, the Centers for Disease Control (CDC) reported the first case
of a S. aureus infection that was completely resistant to
vancomycin (dubbed VRSA) in a patient in Michigan. Persistent
bacterial pathogens also include Salmonella spp., Brucella spp. and
Chlamydia spp.
[0007] Mycobacteria are a diverse and widely distributed group of
aerobic, nonsportulating, nonmotile bacilli that have a high
cell-wall lipid content and a slow growth rate. Members of the
genus Mycobacterium vary in virulence, e.g., from harmless to
species with significant pathogenicity, for example, M.
tuberculosis, the causative agent in tuberculosis (TB). TB is the
second leading infectious cause of death in the world. It is
estimated that about two billion people, or one third of the
world's population, are infected with M. tuberculosis. Eight
million new cases and nearly three million deaths occur annually.
TB is directly responsible for 7% of all deaths world wide, and the
global epidemic is likely to worsen as a result of the spread of
drug-resistant organisms and the ongoing HIV epidemic. See, e.g.,
Dale and Federman (eds.) (2002) WebMD Scientific American Medicine,
WebMD Professional Publishing, New York, N.Y.
[0008] Most current methods to treat TB involve the use of broad
spectrum anti-infective agents such as isoniazid, rifampin,
pyrazinamide, ethambutol, streptomycin, ciprofloxacin, and
ofloxacin. Such agents, however, can cause toxicities in various
organs, and with the growth of several antibiotic resistant strains
of TB, are losing efficaciousness. Reducing the mycobacterial
burden in the lungs of tuberculosis patients with the use of a
variety of non-antibiotic agents can prevent disease formation,
transmission, and death.
[0009] Chronic viral infections also represent a significant threat
to public health. Failure to completely eradicate viral infections
such as hepatitis C virus (HCV) or human immunodeficiency virus
(HIV) can lead to subsequent reactivations and complications such
as liver cancer or acquired immune deficiency syndrome (AIDS),
respectively. Robertson & Hasenkrug (2006) Springer Semin.
Immun. 28:51. In addition, human papillomavirus (HPV) genotypes 16,
18, 31, 33, 45, and 56 account for more than 95% of cases of
cervical cancer. Berzofsky et al. (2004) J. Clin. Invest. 114:450.
It is estimated that chronic infections arise in virtually 100% of
cases of HIV infection, 55-85% of cases of HCV infection, and over
30% of cases of HPV. Berzofsky et al. (2004).
[0010] The need exists for improved methods and compositions for
treatment and/or prevention of bacterial, viral and fungal
infections. Such methods and compositions are preferably less toxic
and/or more efficacious that existing treatment methods and
compositions.
SUMMARY OF THE INVENTION
[0011] The present invention meets these needs and more by
providing compositions, medicaments and methods of using
antagonists of IL-23 to combat bacterial, viral and fungal
infections.
[0012] In one aspect the invention relates to methods of treatment
of a subject having an infection, suspected of having an infection,
or at risk of acquiring an infection, involving administration of
an antagonist of IL-23. In one embodiment the antagonist is a
binding compound, such as an antibody or binding fragment thereof,
that binds to IL-23 or the p19 subunit thereof. In some embodiments
the binding of the antibody blocks binding of IL-23 or its p19
subunit to the IL-23 receptor or the IL-23R subunit thereof. In
another embodiment the antagonist of IL-23 binds to IL-23 receptor
or the IL-23R subunit thereof. In some embodiments the antagonist
that binds to IL-23 receptor, or the IL-23R subunit thereof, and
blocks binding to IL-23 or the p19 subunit thereof. In another
aspect the invention relates to compositions for use in said
methods of treatment.
[0013] In some embodiments the infectious disorder comprises an
infectious disease, such as a bacterial, mycobacterial, viral or
fungal infection. In one embodiment the infectious disorder is a
mycobacterial infection caused by M. bovis, M. leprae, or M.
tuberculosis. In one embodiment the infectious disorder is TB. In
another embodiment the infectious disorder is a fungal infection
selected from the group consisting of onychomycosis, candidiasis,
aspergillosis, cryptococcosis. In yet another embodiment the
infectious disorder is a fungal infection caused by C. albicans
(e.g. chronic mucocutaneous candidiasis, thrush), C. neoformans or
A. fumigatus. In a further embodiment the infectious disorder is a
viral infection, e.g. a viral infection caused by human
immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C
virus (HCV) or human papillomavirus (HPV).
[0014] In other embodiments, the infectious disorder is a chronic
infection. In various embodiments the chronic infection has
persisted, despite at least one previous attempt to resolve the
infection, for 1, 2, 4, 6, 9, 12, 18, 24, 36 or 48 months or
longer. In various embodiments the previous attempt to resolve the
chronic infection involves treatment with antimicrobial agents,
antibiotics, antiviral agents, or antifungal agents.
[0015] In one embodiment the invention relates to combination
therapy combining administration of an antagonist of IL-23 with at
least one other therapeutic modality, such as another therapeutic
agent. In various embodiments, the other therapeutic agent is an
IL-17A antagonist, an IL-17F antagonist, an IL-12 agonist
(including IL-12), a TGF-.beta. antagonist, or an IL-6 antagonist.
In another embodiment the other therapeutic agent is one or more
antifungal agent selected from the group consisting of
posaconazole, fluconazole, voriconazole, itraconazole,
ketoconazole, liarozole, irtemazol, clotrimazole, miconazole,
econazole, butoconazole, oxiconazole, sulconazole, tioconazole, and
terconazole, substituted thiazoles, thiadiazole, oxadiazole,
caspofungin, amphotericin B, nystatin, pimaricin, flucytosine
(5-fluorocytosine), naftifine, terbinafine, butenafine,
thiocarbonate tolnaftate, griseofulvin, amiodarone, ciclopirox,
sulbentine, amorolfine, clioquinol, gentian violet, potassium
iodide, sodium thiosulfate, carbol-fuchsin solution, and the
echinocandins (e.g. caspofungin acetate, micafungin and
anidulafungin).
[0016] In another embodiment the other therapeutic agent is one or
more antimicrobial agent selected from the group consisting of
isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin,
ciprofloxacin, vancomycin or ofloxacin.
[0017] In another embodiment the other therapeutic agent is one or
more antiviral agent selected from the group consisting of
abacavir, acyclovir, amantadine, amprenavir, delavirdine,
didanosine, efavirenz, famciclovir, indinavir, an interferon alfa
(IFN-.alpha.), ribavirin, lamivudine, nelfinavir, nevirapine,
oseltamivir, penciclovir, ribavirin, ritonavir, saquinavir,
stavudine, valacyclovir, zalcitabine, zanamivir, zidovudine
(azidodeoxythymidine, AZT).
[0018] In one aspect the invention relates to methods of enhancing
a type 1 (Th1) immune response in a subject having an infection or
suspected of having an infection. In various embodiments,
enhancement of the Th1 response is reflected by an increase in the
percentage of CD4.sup.+ T cells expressing IFN-.gamma., a decrease
in the percentage of CD4.sup.+ T cells expressing IL-17A, or both,
when compared to the percentage of T cells prior to treatment with
an antagonist of IL-23. In various embodiments the increase or
decrease is 1.5-, 2-, 3-, 5-, 10-, 20-, 50-fold or more. In another
aspect the invention relates to compositions for use in said
methods of enhancing a Th1 response.
[0019] In various embodiments the other therapeutic agent is
administered before, and/or concurrently with, and/or after
administration of the antagonist of IL-23. In one embodiment, an
antagonist of IL-17A is administered before and/or concurrently
with the antagonist of IL-23. In another embodiment, an
antimicrobial, antifungal or antiviral agent is administered
concurrently with the antagonist of IL-23.
[0020] In another aspect the invention relates to methods of
treatment of a subject having an infection, suspected of having an
infection, or at risk of acquiring an infection, involving
administration of antagonists of IL-17A and/or IL-17F, such as
antagonistic antibodies to the cytokines themselves or to their
respective receptors or receptor subunits.
[0021] In other embodiments the antagonist of IL-23 comprises a
polynucleotide. In various embodiments the polynucleotide is an
antisense polynucleotide (e.g. antisense RNA) or a small
interfering RNA (siRNA). In one embodiment the polynucleotide
antagonist of IL-23 is delivered in gene therapy vector, such as an
adenovirus, lentivirus, retrovirus or adenoassociated virus vector.
In another embodiment the polynucleotide antagonist of IL-23 is
delivered as a therapeutic agent.
[0022] In yet another embodiment the antagonist of IL-23 comprises
a soluble receptor polypeptide. In one embodiment the antagonist of
IL-23 is a soluble fragment derived from the extracellular domain
of IL-23R.
[0023] In various embodiments the antagonist of IL-23 is an
antibody or antigen binding fragment thereof. In various embodiment
the antibody or antigen binding fragment thereof comprises a
polyclonal antibody, a monoclonal antibody, a humanized antibody, a
fully human antibody; an antibody fragment (e.g. Fab, Fab',
Fab'-SH, Fv, scFv, F(ab').sub.2, and a diabody). In other
embodiments the antagonist comprises a peptide mimetic of an
antibody. In still further embodiments the antibody or antigen
binding fragment thereof is detectably labeled. In one embodiment,
the antagonist of IL-23 is an antibody, or antigen binding fragment
thereof, that exhibits reduced complement activation,
antibody-dependent cellular cytotoxicity (ADCC), or both. In one
embodiment the IL-23 antagonist antibody or fragment thereof with
reduced effector function is an anti-IL-23 receptor (e.g.
anti-IL-23R) antibody or fragment. In various embodiments the
antibody with reduced effector function is an antibody fragment
(e.g. Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2), an IgG4, or has
altered glycosylation.
[0024] In one embodiment, the invention relates to treatment of an
infection, e.g. a chronic fungal, bacterial or viral infection, by
administering an effective amount of a bispecific antibody that
binds specifically to any two proteins selected from the group
consisting of IL-23p19, IL-23R, IL-17A, IL-17F, IL-17RA, IL-17RC,
IL-6 and TGF-.beta.. In one embodiment the proteins are human
proteins.
[0025] In one embodiment the antagonist of IL-23 is specific for
IL-23 (or its receptor) and does not antagonize IL-12 (or its
receptor). In various embodiments antagonism is measured by an in
vitro binding assay (e.g. an ELISA) or by a bioassay (e.g. BaF3
cell proliferation or promotion of Th17 cell production). In
various embodiments the ratio of the IC50 for inhibition of binding
of IL-12 to its receptor to the IC50 for inhibition of binding of
IL-23 to its receptor (IC50.sub.IL-12/IC50.sub.IL-23) is 1.5, 2, 3,
4, 5, 7, 10, 15, 20, 50, 100 or more.
[0026] In one embodiment the methods and compositions of the
present invention are used to treat TB, and the success of the
treatment is measured by a reduction in bacterial burden. In
various embodiments the mycobacterial burden is measured by a
tuberculin test, a Mantoux test, or presence of mycobacterial DNA
or RNA in a clinical sample.
[0027] In some embodiments of the present invention the subject
having an infection has been previously treated for the infection
with other methods or compositions. In one embodiment, the previous
treatment was not effective in eliminating infection. In another
embodiment the subject having an infection, suspected of having an
infection, or at risk of acquiring an infection, is
immunocompromised, e.g. as a result of AIDS, transplant or
chemotherapy.
[0028] The invention further encompasses use of antagonists of
IL-23 in the manufacture of a medicament for the treatment of one
or more infectious diseases selected from the group consisting of a
fungal infection, a persistent fungal infection, candidiasis,
chronic mucocutaneous candidiasis (CMC), aspergillosis,
cryptococcosis, a viral infection, a persistent viral infection,
HIV infection, HBV infection, HCV infection, a persistent bacterial
infection, mycobacterial infection, M. tuberculosis infection, M.
bovis infection, and M. leprae infection. In some embodiments, the
medicament may comprise one or more additional therapeutic agents.
In other embodiments the medicament of the present invention may be
used in conjunction with one or more other therapeutic agents.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A-1E show the results of experiments on the role of
IL-23/IL-17-dependent pathway in susceptibility to candidiasis.
Mice were injected intragastrically with 10.sup.8 virulent Candida.
Results are pooled from 3 experiments (6 mice per group per
experiment).
[0030] FIG. 1A shows percent (%) survival over time for
p19.sup.-/-, p35.sup.-/-, p40.sup.-/- and C57BL/6(WT) mice.
[0031] FIG. 1B shows fungal growth (CFU) in the stomach three and
ten days after the infection. Results were statistically different
(p<0.05, indicated by *) for p19.sup.-/-, p35.sup.-/- or
p40.sup.-/- mice when compared with C57BL/6 mice at both three and
ten day timepoints.
[0032] FIG. 1C shows p35 and p19 mRNA expression (one day after the
infection) and IL-12.beta.2R and IL-23R mRNA expression (three days
after the infection) in MLN. mRNA expression was measured by
real-time RT-PCR.
[0033] FIG. 1D shows the frequencies of IFN-.gamma.-, IL-17- or
IL-4-producing MLN CD4+ cells a week after the infection. The
frequency of cytokine-producing cells was measured by ELISPOT
assay, and values are the mean number of cytokine-producing cells
(.+-.SE) per 10.sup.5 cells.
[0034] FIG. 1E shows the levels of inflammatory cytokines (IL-17,
IL-23, IFN-.gamma., IL-12) in the stomach homogenates three days
after the infection. Cytokines were measured by ELISA (pg/ml).
[0035] In FIGS. 1C-1E, differences were statistically significant
(p<0.05) when comparing infected (+) to uninfected (-) mice (*),
and when comparing p19.sup.-/- or p35.sup.-/- mice to C57BL/6 mice
(**), as indicated in the figures.
[0036] FIGS. 2A and 2B show the results of experiments on the role
of IL-23/IL-17-dependent pathway in susceptibility to
aspergillosis. Mice were infected intranasally with
2.times.10.sup.7 Aspergillus resting conidia. Results shown in
FIGS. 2A and 2B are pooled from four experiments (six
animals/group).
[0037] FIG. 2A shows fungal growth (chitin content, expressed as
.mu.g glucosamine/organ) in the lung three days after the
infection. Differences were statistically significant (p<0.05)
when comparing p19.sup.-/-, p35.sup.-/- or p40.sup.-/- mice to
C57BL/6 mice (*).
[0038] FIG. 2B shows p35/p19 mRNA expression (one day after the
infection) and IL-12.beta.2R/IL-23R mRNA expression (three days
after the infection) in TLN. Messenger RNA expression was measured
by RT-PCR. Differences were statistically significant (P<0.05)
when comparing infected (+) to uninfected (-) mice (*), and when
comparing p19.sup.-/- or p35.sup.-/- mice to C57BL/6 mice (**), as
indicated in the figure.
[0039] FIGS. 3A-3C show the results of experiments on the
importance of the IL-23/IL-17-dependent pathway in susceptibility
to fungal infections. Mice were infected as in FIGS. 1 and 2, and
treated with 200 .mu.g of p19- or IL-17-neutralizing antibodies 5 h
after the infection, or with 1 mg TGF-.beta. neutralizing antibody
5 and 24 h after the infection.
[0040] FIG. 3A shows fungal growth in the stomach or lung of mice
with candidiasis (C. albicans) or aspergillosis (A. fumigatus)
three days after the infection. Differences were statistically
significant (p<0.05) when comparing treated (+) to untreated (-)
mice (*), as indicated in the figure.
[0041] FIG. 3B shows the frequencies of IFN-.gamma.- or
IL-17-producing CD4+ cells from MLN or TLN from mice with
candidiasis or aspergillosis, respectively, as determined by
ELISPOT assay. Values are the mean number of cytokine-producing
cells (.+-.SE) per 10.sup.5 cells. FIG. 3B further shows actual
IL-17 production (one week after the infection) in culture
supernatants of antigen-stimulated unfractionated MLN or TLN.
Differences were statistically significant (p<0.05) when
comparing infected to uninfected (Ct) mice (*), and when comparing
treated (+) to untreated (-) mice (**), as indicated in the
figure.
[0042] FIG. 3C shows fungal growth in the stomach of mice with
candidiasis treated with p19 neutralizing antibodies as above,
three days after the infection. Differences were statistically
significant (p<0.05) when comparing treated (+) to untreated (-)
mice (*), and when comparing IL-4.sup.-/-, IFN-.gamma..sup.-/-,
p35.sup.-/- or IFN-.gamma..sup.-/-/p35.sup.-/- mice to BALB/c mice
(**), as indicated in the figure.
[0043] FIGS. 4A-4D show the results of experiments on IL-23 and
IL-12 production in DC subsets in response to fungi. Bone marrow DC
obtained in the presence of GM-CSF+IL-4 (GM-DC) or FLT3-L (FL-DC)
were stimulated with fungi and assessed for cytokine
expression.
[0044] FIG. 4A shows real time RT-PCR analysis of cytokine mRNA
expression, and FIG. 4B shows cytokine expression as measured by
ELISA (pg/ml). Zymosan, LPS (10 .mu.g/ml) or CpG-ODN 2006 (0.06
.mu.M) were used as positive controls. DC were exposed to yeasts at
10:1 ratio. Differences were statistically significant (p<0.05)
when comparing exposed to unexposed ("None") DC (*), as indicated
in the figure.
[0045] FIG. 4C shows IL-12 and IL-23 production in splenic CD11c+DC
from p19.sup.-/- or p35.sup.-/- mice. Mice were stimulated with
fungi before the measurement of cytokines in culture
supernatants.
[0046] FIG. 4D shows IL-12 and IL-23 production in splenic CD11c+DC
from C57BL/6 mice exposed to fungi for 12 h in the presence (+) or
absence (-) of IL-12 or IL-23 (10 ng/ml), or in the presence of
neutralizing anti-IL-12 or anti-IL-23 antibodies (10 .mu.g/ml), as
indicated in the figure.
[0047] FIGS. 5A-5C show the results of experiments on IL-23
production by inflammatory DC in response to fungi, and
specifically whether such production is TLR- and T
cell-dependent.
[0048] FIG. 5A shows IL-23 production (pg/ml) in splenic CD11c+DC
from different types of mice exposed to fungi 12 h earlier. Pooled
results from four experiments are shown. Differences were
statistically significant (p<0.05) when comparing exposed to
unexposed ("None") DC (*), as indicated in the figure.
[0049] FIG. 5B shows expression of cytokines in various cell
cultures and co-cultures. Splenic CD4+T cells from C57BL/6 (WT) or
p35.sup.-/- mice were cultured in the presence of the corresponding
splenic DC either unpulsed (groups 2 and 5) or pulsed with Candida
yeasts (Ag) (groups 3 and 6). Cytokines (IL-12, IL-23, IFN-.gamma.,
IL-17) were measured by ELISA five days post-pulse. Groups 1 and 4
are C57BL/6 or p35.sup.-/- DC stimulated with fungi and no T cells.
Groups 7 and 8 are p35.sup.-/- or C57BL/6 CD4+ T cells cultivated
with C57BL/6 or p35.sup.-/- DC, respectively, in the presence of
the fungus. Differences are statistically significant (p<0.05,
indicated by *) when groups 3 and 7 are compared to group 1 for
IFN-.gamma. production, and when groups 6 and 8 are compared to
group 4 for IL-23 and IL-17 production, as indicated in the
figure.
[0050] FIG. 5C shows data similar to those shown in FIG. 5B, except
that some of the samples include anti-IL-23 or anti-TGF-.beta.
antibodies. Splenic CD4+T cells from C57BL/6 (WT) (groups 1-3) or
p35.sup.-/- (groups 4-6) mice were cultured in the presence of the
corresponding splenic DC. Cultures were pulsed with Candida yeasts
(Ag) for 5 days in the presence of 10 .mu.g/ml of IL-23 or
TGF-.beta. neutralizing antibodies, and cytokines (IFN-.gamma.,
IL-17) were quantified in culture supernatants by ELISA.
Differences are statistically significant (p<0.05, indicated by
*) when groups 2 and 3 are compared to group 1 for IFN-.gamma. and
IL-17 production, and when group 5 is compared to group 4 for IL-17
production, as indicated in the figure.
[0051] FIGS. 6A-6E show the results of experiments on the ability
of IL-23 and IL-17 to impair antifungal effector functions and
subvert the anti-inflammatory program of PMN.
[0052] FIG. 6A shows fungicidal activity in PMN from C57BL/6 (WT),
p19.sup.-/- or p35.sup.-/- mice after incubation with unopsonized
yeasts (30 min) or conidia (60 min) at an effector to fungal cell
ratio of 5:1, at 37.degree. C. Results are plotted as the
percentage of colony forming units inhibition (mean.+-.SE). Results
reflect pooled data from three experiments. Differences were
statistically significant (p<0.05) when comparing p19.sup.-/- or
p35.sup.-/- PMN to C57BL/6 (WT) PMN (*), as indicated in the
figure.
[0053] FIG. 6B shows fungicidal activity of PMN from C57BL/6 (WT)
mice exposed to IL-23 or IL-17 at the indicated concentrations.
Differences were statistically significant (p<0.05) when
comparing cytokine-exposed PMN to unexposed PMN (*).
[0054] FIGS. 6C and 6D shows fungicidal activity of PMN from
C57BL/6 (WT) mice exposed to various combinations of IFN-.gamma.
(50 ng/ml), IL-23 (100 ng/ml) and IL-17 (100 ng/ml) for 60 min.
Fungicidal activity was measured against Candida yeasts or
Aspergillus conidia (FIG. 6C). MMP9/MPO production was also
measured (FIG. 6D). Production of gelatinase and myeloperoxidase
was assessed by gelatin zymography and Western blot analysis was
performed on culture supernatants. Gels show bands corresponding to
the active 92 kDa MMP9 and the 60 kDa MPO. Differences were
statistically significant (p<0.05) when comparing
cytokine-exposed PMN to unexposed PMN (*), and when comparing
(IFN-.gamma.+IL-23)- or (IFN-.gamma.+IL-17)-exposed PMN to
IFN-.gamma.-exposed PMN (**), as indicated in the figure.
[0055] FIG. 6E shows bands on a Western blot. PMN were exposed in
vitro to various combinations of IFN-.gamma., IL-23 and IL-17 for
12 h. IDO protein expression was then determined by Western
Blotting. IDO-expressing MC.sub.24 transfectants and
mock-transfected MC.sub.22 cells served as positive and negative
controls, respectively. .beta.-tubulin serves as a loading
control.
DETAILED DESCRIPTION
[0056] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise. Unless otherwise indicated, exemplary embodiments
provided herein are not to be considered to limit the scope of the
invention. Such exemplary embodiments may be preceded by such
phrases as "e.g.," "for example," "in one embodiment" or other such
non-limiting language, or their exemplary nature may be apparent
from the context (e.g. the "Examples"). Unless indicated otherwise,
terms such a "does not inhibit" are intended to be relative rather
than absolute. For example, an agent that inhibits IL-23 but "does
not" inhibit IL-12 refers to an agent that is less effective at
inhibiting IL-12 than IL-23 when the agent is present at a given
concentration in comparable assays for the two cytokines.
[0057] All references cited herein are incorporated by reference in
their entireties to the same extent as if each individual
publication, database entry, patent application, or patent, was
specifically and individually incorporated by reference.
I. Definitions
[0058] "Activation," "stimulation," and "treatment," as it applies
to cells or to receptors, may have the same meaning, e.g.,
activation, stimulation, or treatment of a cell or receptor with a
ligand, unless indicated otherwise by the context or explicitly.
"Ligand" encompasses natural and synthetic ligands, e.g.,
cytokines, cytokine variants, analogues, muteins, and binding
compositions derived from antibodies. "Ligand" also encompasses
small molecules, e.g., peptide mimetics of cytokines and peptide
mimetics of antibodies. "Activation" can refer to cell activation
as regulated by internal mechanisms as well as by external or
environmental factors. "Response," e.g., of a cell, tissue, organ,
or organism, encompasses a change in biochemical or physiological
behavior, e.g., concentration, density, adhesion, or migration
within a biological compartment, rate of gene expression, or state
of differentiation, where the change is correlated with activation,
stimulation, or treatment, or with internal mechanisms such as
genetic programming.
[0059] "Activity" of a molecule may describe or refer to the
binding of the molecule to a ligand or to a receptor, to catalytic
activity; to the ability to stimulate gene expression or cell
signaling, differentiation, or maturation; to antigenic activity,
to the modulation of activities of other molecules, and the like.
"Activity" of a molecule may also refer to activity in modulating
or maintaining cell-to-cell interactions, e.g., adhesion, or
activity in maintaining a structure of a cell, e.g., cell membranes
or cytoskeleton. "Activity" can also mean specific activity, e.g.,
[catalytic activity]/[mg protein], or [immunological activity]/[mg
protein], concentration in a biological compartment, or the like.
"Proliferative activity" encompasses an activity that promotes,
that is necessary for, or that is specifically associated with,
e.g., normal cell division, as well as cancer, tumors, dysplasia,
cell transformation, metastasis, and angiogenesis.
[0060] "Administration" and "treatment," as it applies to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid. "Administration"
and "treatment" can refer, e.g., to therapeutic, pharmacokinetic,
diagnostic, research, and experimental methods. Treatment of a cell
encompasses contact of a reagent to the cell, as well as contact of
a reagent to a fluid, where the fluid is in contact with the cell.
"Administration" and "treatment" also means in vitro and ex vivo
treatments, e.g., of a cell, by a reagent, diagnostic, binding
composition, or by another cell. "Treatment," as it applies to a
human, veterinary, or research subject, refers to therapeutic
treatment, prophylactic or preventative measures, to research and
diagnostic applications. "Treatment" as it applies to a human,
veterinary, or research subject, or cell, tissue, or organ,
encompasses contact of IL-23 or IL-23R antagonist to a human or
animal subject, a cell, tissue, physiological compartment, or
physiological fluid. "Treatment of a cell" also encompasses
situations where the IL-23 or IL-23R antagonist contacts IL-23R
complex (IL-23R/IL-12Rbetal heterodimer), e.g., in the fluid phase
or colloidal phase, but also situations where the antagonist does
not contact the cell or the receptor.
[0061] "Binding composition" refers to a molecule, small molecule,
macromolecule, antibody, a fragment or analogue thereof, or soluble
receptor, capable of binding to a target. "Binding composition"
also may refer to a complex of molecules, e.g., a non-covalent
complex, to an ionized molecule, and to a covalently or
non-covalently modified molecule, e.g., modified by
phosphorylation, acylation, cross-linking, cyclization, or limited
cleavage, which is capable of binding to a target. "Binding
composition" may also refer to a molecule in combination with a
stabilizer, excipient, salt, buffer, solvent, or additive, capable
of binding to a target. "Binding" may be defined as an association
of the binding composition with a target where the association
results in reduction in the normal Brownian motion of the binding
composition, in cases where the binding composition can be
dissolved or suspended in solution.
[0062] The binding compounds of the invention may comprise
bispecific antibodies. As used herein, the term "bispecific
antibody" refers to an antibody, typically a monoclonal antibody,
having binding specificities for at least two different antigenic
epitopes. In one embodiment, the epitopes are from the same
antigen. In another embodiment, the epitopes are from two different
antigens. Methods for making bispecific antibodies are known in the
art. For example, bispecific antibodies can be produced
recombinantly using the co-expression of two immunoglobulin heavy
chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature
305: 537-39. Alternatively, bispecific antibodies can be prepared
using chemical linkage. See, e.g., Brennan, et al. (1985) Science
229: 81. Bispecific antibodies include bispecific antibody
fragments. See, e.g., Holliger, et al. (1993) Proc. Natl. Acad.
Sci. U.S.A. 90: 6444-48, Gruber, et al., J. Immunol. 152: 5368
(1994).
[0063] A "classical TH1-type T cell" is a T cell that expresses
interferon-gamma (IFN.gamma.) to an extent greater than expression
of each of IL-4, IL-5, or IL-13, while a "classical TH2-type T
cell" is a T cell that expresses IL-4, IL-5, or IL-13, each to an
extent greater than expression of IFN.gamma.. "Extent" is typically
4-fold or more, more typically 8-fold or more, and most typically
16-fold or more than for a classical TH2-type cell.
[0064] "Memory T cells" as defined herein are a subset of
long-lived T cells with prior exposure to a given antigen. Memory T
cells can be present in an organism for years, allowing a rapid
response to subsequent challenges by the same antigen. The
phenotype for mouse memory T cells is defined as
CD4+.sup.highCD45RB.sup.low. The phenotype of human memory T cells
is defined as CD45RA.sup.neg/low CD45R0.sup.high. IL-23 treatment
of these memory T cells results in proliferation and expression of
IL-17. Unless otherwise indicated "IL-17," as used herein, refers
to IL-17A. See, e.g., Moseley et al. (2003) Cytokine & Growth
Factor Rev. 14:155.
[0065] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids that encode identical or essentially identical amino
acid sequences or, where the nucleic acid does not encode an amino
acid sequence, to essentially identical nucleic acid sequences.
Because of the degeneracy of the genetic code, a large number of
functionally identical nucleic acids may encode any given
protein.
[0066] As to amino acid sequences, one of skill will recognize that
an change in a nucleic acid, peptide, polypeptide, or protein
sequence that substitutes an amino acid or a small percentage of
amino acids in the encoded sequence for a conserved amino acid is a
"conservatively modified variant." Conservative substitution tables
providing functionally similar amino acids are well known in the
art. An example of a conservative substitution is the exchange of
an amino acid in one of the following groups for another amino acid
of the same group (U.S. Pat. No. 5,767,063 issued to Lee, et al.;
Kyte and Doolittle (1982) J. Mol. Biol. 157: 105-132):
(1) Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, or Met;
[0067] (2) Neutral hydrophilic: Cys, Ser, Thr;
(3) Acidic: Asp, Glu;
(4) Basic: Asn, Gln, H is, Lys, Arg;
[0068] (5) Residues that influence chain orientation: Gly, Pro;
(6) Aromatic: Trp, Tyr, Phe;
[0069] (7) Small amino acids: Gly, Ala, Ser.
[0070] "Effective amount" encompasses an amount sufficient to
ameliorate or prevent a symptom or sign of the medical condition.
Effective amount also means an amount sufficient to allow or
facilitate diagnosis. An effective amount for a particular patient
or veterinary subject may vary depending on factors such as the
condition being treated, the overall health of the patient, the
method route and dose of administration and the severity of side
effects. See, e.g., U.S. Pat. No. 5,888,530. An effective amount
can be the maximal dose or dosing protocol that avoids significant
side effects or toxic effects. The effect will result in an
improvement of a diagnostic measure or parameter by at least 5%,
usually by at least 10%, more usually at least 20%, most usually at
least 30%, preferably at least 40%, more preferably at least 50%,
most preferably at least 60%, ideally at least 70%, more ideally at
least 80%, and most ideally at least 90%, where 100% is defined as
the diagnostic parameter shown by a normal subject. See, e.g.,
Maynard, et al. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK.
[0071] "Exogenous" refers to substances that are produced outside
an organism, cell, or human body, depending on the context.
"Endogenous" refers to substances that are produced within a cell,
organism, or human body, depending on the context.
[0072] "Infectious disease" refers to microbial, e.g., bacterial,
viral, and/or fungal, infections of an organism, organ, tissue, or
cell.
[0073] An "IL-17-producing cell" means a T cell that is not a
classical TH1-type T cell or classical TH2-type T cell.
"IL-17-producing cell" also means a T cell that expresses a gene or
polypeptide (e.g., mitogen responsive P-protein; chemokine ligand
2; interleukin-17 (IL-17); transcription factor ROR-.gamma.T
related; suppressor of cytokine signaling 3. etc.), where
expression with treatment by an IL-23 agonist is greater than
treatment with an IL-12 agonist, where "greater than" is defined as
follows. Expression with an IL-23 agonist is ordinarily at least
5-fold greater, typically at least 10-fold greater, more typically
at least 15-fold greater, most typically at least 20-fold greater,
preferably at least 25-fold greater, and most preferably at least
30-fold greater, than with IL-12 treatment. Expression can be
measured, e.g., with treatment of a population of substantially
pure IL-17 producing cells.
[0074] Moreover, "IL-17-producing cell" includes a progenitor or
precursor cell that is committed, in a pathway of cell development
or cell differentiation, to differentiating into an IL-17-producing
cell, as defined above. A progenitor or precursor cell to the IL-17
producing cell can be found in a draining lymph node (DLN).
Additionally, "IL-17-producing cell" encompasses an IL-17-producing
cell, as defined above, that has been, e.g., activated, e.g., by a
phorbol ester, ionophore, and/or carcinogen, further
differentiated, stored, frozen, desiccated, inactivated, partially
degraded, e.g., by apoptosis, proteolysis, or lipid oxidation, or
modified, e.g., by recombinant technology.
[0075] "Inhibitors" and "antagonists" refer to inhibitory molecules
for the inhibition of, e.g., a ligand, receptor, cofactor, a gene,
cell, tissue, or organ. A modulator of a gene, a receptor, a
ligand, or a cell, is a molecule that alters an activity of the
gene, receptor, ligand, or cell, where activity can be activated,
inhibited, or altered in its regulatory properties. The modulator
may act alone, or it may use a cofactor, e.g., a protein, metal
ion, or small molecule Inhibitors are compounds that decrease,
block, prevent, delay activation, inactivate, desensitize, or down
regulate, e.g., a gene, protein, ligand, receptor, or cell.
Activators are compounds that increase, activate, facilitate,
enhance activation, sensitize, or up regulate, e.g., a gene,
protein, ligand, receptor, or cell. An inhibitor may also be
defined as a composition that reduces, blocks, or inactivates a
constitutive activity. An "antagonist" is a compound that opposes
the actions of an agonist. An antagonist prevents, reduces,
inhibits, or neutralizes the activity of an agonist. An antagonist
can also prevent, inhibit, or reduce constitutive activity of a
target, e.g., a target receptor, even where there is no identified
agonist.
[0076] An antagonist of IL-23, for example, includes any agent that
disrupts the biological activity of IL-23, such as amplification
and survival of Th17 cells as described in greater detail infra.
Antagonists of IL-23 receptor and IL-23R are subsets of antagonists
of IL-23 because they serve to block the activity of IL-23 by
blocking IL-23 signaling.
[0077] To examine the extent of inhibition, for example, samples or
assays comprising a given protein, gene, cell, or organism, are
treated with a potential activator or inhibitor and are compared to
control samples without the inhibitor. Control samples, i.e., not
treated with antagonist, are assigned a relative activity value of
100% Inhibition is achieved when the activity value relative to the
control is about 90% or less, typically 85% or less, more typically
80% or less, most typically 75% or less, generally 70% or less,
more generally 65% or less, most generally 60% or less, typically
55% or less, usually 50% or less, more usually 45% or less, most
usually 40% or less, preferably 35% or less, more preferably 30% or
less, still more preferably 25% or less, and most preferably less
than 25%. Activation is achieved when the activity value relative
to the control is about 110%, generally at least 120%, more
generally at least 140%, more generally at least 160%, often at
least 180%, more often at least 2-fold, most often at least
2.5-fold, usually at least 5-fold, more usually at least 10-fold,
preferably at least 20-fold, more preferably at least 40-fold, and
most preferably over 40-fold higher.
[0078] Endpoints in activation or inhibition can be monitored as
follows. Activation, inhibition, and response to treatment, e.g.,
of a cell, physiological fluid, tissue, organ, and animal or human
subject, can be monitored by an endpoint. The endpoint may comprise
a predetermined quantity or percentage of, e.g., an indicium of
reduced bacterial burden, oncogenicity, or cell degranulation or
secretion, such as the release of a cytokine, toxic oxygen, or a
protease. The endpoint may comprise, e.g., a predetermined quantity
of ion flux or transport; cell migration; cell adhesion; cell
proliferation; potential for metastasis; cell differentiation; and
change in phenotype, e.g., change in expression of gene relating to
inflammation, apoptosis, transformation, cell cycle, or metastasis.
See, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158; Hood and
Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme et al. (2003)
Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.
Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.
Genomics Hum. Genet. 3:101-128; Bauer et al. (2001) Glia
36:235-243; Stanimirovic and Satoh (2000) Brain Pathol.
10:113-126.
[0079] An endpoint of inhibition is generally 75% of the control or
less, preferably 50% of the control or less, more preferably 25% of
the control or less, and most preferably 10% of the control or
less. Generally, an endpoint of activation is at least 150% the
control, preferably at least two times the control, more preferably
at least four times the control, and most preferably at least 10
times the control.
[0080] "Knockout" (KO) refers to the partial or complete reduction
of expression of at least a portion of a polypeptide encoded by a
gene, e.g., encoding a subunit of IL-23 or IL-23 receptor, where
the gene is endogenous to a single cell, selected cells, or all of
the cells of a mammal. KO also encompasses embodiments where
biological function is reduced, but where expression is not
necessarily reduced, e.g., a polypeptide that contains an inserted
inactivating peptide. Disruptions in a coding sequence or a
regulatory sequence are encompassed by the knockout technique. The
cell or mammal may be a "heterozygous knockout", where one allele
of the endogenous gene has been disrupted. Alternatively, the cell
or mammal may be a "homozygous knockout" where both alleles of the
endogenous gene have been disrupted. "Homozygous knockout" is not
intended to limit the disruption of both alleles to identical
techniques or to identical outcomes at the genome.
[0081] A composition that is "labeled" is detectable, either
directly or indirectly, by spectroscopic, photochemical,
biochemical, immunochemical, isotopic, or chemical methods. For
example, useful labels include .sup.32P, .sup.33P, .sup.35S,
.sup.14C, .sup.3H, .sup.125I, stable isotopes, fluorescent dyes,
electron-dense reagents, substrates, epitope tags, or enzymes,
e.g., as used in enzyme-linked immunoassays, or fluorettes. See,
e.g., Rozinov and Nolan (1998) Chem. Biol. 5:713-728.
[0082] "Ligand" refers, e.g., to a small molecule, peptide,
polypeptide, and membrane associated or membrane-bound molecule, or
complex thereof, that can act as an agonist or antagonist of a
receptor. "Ligand" also encompasses an agent that is not an agonist
or antagonist, but that can bind to the receptor. Moreover,
"ligand" includes a membrane-bound ligand that has been changed,
e.g., by chemical or recombinant methods, to a soluble version of
the membrane-bound ligand. By convention, where a ligand is
membrane-bound on a first cell, the receptor usually occurs on a
second cell. The second cell may have the same or a different
identity as the first cell. A ligand or receptor may be entirely
intracellular, that is, it may reside in the cytosol, nucleus, or
some other intracellular compartment. The ligand or receptor may
change its location, e.g., from an intracellular compartment to the
outer face of the plasma membrane. The complex of a ligand and
receptor is termed a "ligand receptor complex." Where a ligand and
receptor are involved in a signaling pathway, the ligand occurs at
an upstream position and the receptor occurs at a downstream
position of the signaling pathway.
[0083] A "marker" relates to the phenotype of a cell, tissue,
organ, animal, e.g., of an IL-17 producing cell. Markers are used
to detect cells, e.g., during cell purification, quantitation,
migration, activation, maturation, or development, and may be used
for both in vitro and in vivo studies. An activation marker is a
marker that is associated with cell activation.
[0084] "Purified cell" encompasses, e.g., one or more "IL-17
producing cells" that is substantially free of other types of
cells, e.g., contamination by other types of T cells. Purity can be
assessed by use of a volume that is defined by geometric
coordinates or by a compartment comprising, e.g., a flask, tube, or
vial. A "purified IL-17 producing cell" can be defined by, e.g., a
compartment where the "IL-17 producing cells" normally constitute
at least 20% of all the cells, more normally at least 30% of all
the cells, most normally at least 40% of all the cells, generally
at least 50% of all the cells, more generally at least 60% of all
the cells, most generally at least 70% of all the cells, preferably
at least 80% of all the cells, more preferably at least 90% of all
the cells; and most preferably at least 95% of all the cells.
[0085] "Small molecule" is defined as a molecule with a molecular
weight that is less than 10 kD, typically less than 2 kD, and
preferably less than 1 kD. Small molecules include, but are not
limited to, inorganic molecules, organic molecules, organic
molecules containing an inorganic component, molecules comprising a
radioactive atom, synthetic molecules, peptide mimetics, and
antibody mimetics. As a therapeutic, a small molecule may be more
permeable to cells, less susceptible to degradation, and less apt
to elicit an immune response than large molecules. Small molecules,
such as peptide mimetics of antibodies and cytokines, as well as
small molecule toxins are known in the art. See, e.g., Casset et
al. (2003) Biochem. Biophys. Res. Commun. 307:198-205; Muyldermans
(2001) J. Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol.
18:1251-1256; Apostolopoulos et al. (2002) Curr. Med. Chem.
9:411-420; Monfardini et al. (2002) Curr. Pharm. Des. 8:2185-2199;
Domingues et al. (1999) Nat. Struct. Biol. 6:652-656; Sato and Sone
(2003) Biochem. J. 371:603-608; U.S. Pat. No. 6,326,482.
[0086] "Specifically" or "selectively" binds, when referring to a
ligand/receptor, antibody/antigen, or other binding pair, indicates
a binding reaction which is determinative of the presence of the
protein in a heterogeneous population of proteins and other
biologics. Thus, under designated conditions, a specified ligand
binds to a particular receptor and does not bind in a significant
amount to other proteins present in the sample. The antibody, or
binding composition derived from the antigen-binding site of an
antibody, of the contemplated method binds to its antigen, or a
variant or mutein thereof, with an affinity that is at least two
fold greater, preferably at least ten times greater, more
preferably at least 20-times greater, and most preferably at least
100-times greater than the affinity with any other antibody, or
binding composition derived thereof. In a preferred embodiment the
antibody will have an affinity for the desired target that is
greater than about 10.sup.9 liters/mol, as determined, e.g., by
Scatchard analysis. Munsen et al. (1980) Analyt. Biochem.
107:220-239.
[0087] An antibody that "specifically binds" to IL-23 or IL-23
receptor does not bind to proteins that do not comprise the
IL-23-derived sequences, i.e. "specificity" as used herein relates
to IL-23 specificity, and not any other sequences that may be
present in the protein in question. For example, as used herein, an
antibody that "specifically binds" to IL-23 will typically bind to
FLAG-hIL-23, which is a fusion protein comprising IL-23 and a
FLAG.RTM. peptide tag, but it does not bind to the FLAG.RTM.
peptide tag alone or when it is fused to a protein other than
IL-23. Depending on the context, specificity for IL-23 may also
refer to the ability to bind to IL-23 (or its receptor) but not to
other proteins, such as IL-12 (or its receptor).
II. General
[0088] Although IL-23 and IL-12 are both heterodimeric cytokines
sharing a common subunit and a common receptor subunit, recent
results have suggested that their roles in inflammation and host
defense are more antagonistic than overlapping. Interleukin-23
(IL-23) is a heterodimeric cytokine comprised of two subunits,
i.e., p19 and p40. The p19 subunit is structurally related to IL-6,
granulocyte-colony stimulating factor (G-CSF) and the p35 subunit
of IL-12. The p40 subunit is part of the cytokine IL-12, which is
composed of p35 and p40. Heterodimeric IL-12 is often referred to
as IL-12p70. IL-23 mediates signaling by binding to a heterodimeric
receptor, comprised of IL-23R and IL-12R.beta.1. The IL-12R.beta.1
subunit is shared by the IL-12 receptor, which is composed of
IL-12R.beta.1 and IL-12R.beta.2. IL-23 and IL-23 receptor, and
their respective IL-23-specific subunits, are disclosed at WO
99/05280, WO 01/18051, WO 00/73451, and WO 01/85790.
[0089] A number of early studies on IL-12 involved a genetic
deficiency in p40 (p40 knockout mouse; p40KO mouse), but it was
subsequently realized with the discovery of IL-23 that such mice
were deficient in both IL-12 and IL-23. Oppmann et al. (2000)
Immunity 13:715-725; Wiekowski et al. (2001) J. Immunol.
166:7563-7570; Parham et al. (2002) J. Immunol. 168:5699-708;
Frucht (2002) Sci STKE 2002, E1-E3; Elkins et al. (2002) Infection
Immunity 70:1936-1948. These results changed the interpretation of
many of the early observations that were originally thought to
relate to IL-12 and the Th1 response.
[0090] Recent studies have suggested a greater diversification of
the CD4+ T-cell effector repertoire than that encompassed by the
Th1/Th2 paradigm. Th17 cells are now thought to be a separate
lineage of effector Th cells contributing to immune pathogenesis
previously attributed to the Th1 lineage. Although the pathways
leading to Th17 differentiation are still unclear (Dong (2006) Nat
Rev Immunol 6:329), IL-23 is a critical cytokine for the generation
and maintenance of this lineage (Trinchieri et al. (2003) Immunity
19:641). While both IL-12 and IL-23 can induce IFN-.gamma.
expression in CD4+T-cells, IL-23 alone facilitates production of
the proinflammatory cytokine IL-17 by Th cells.
[0091] Despite many similarities, there is increasing evidence that
IL-12 and IL-23 drive divergent immunological pathways. Th cells
primed for IL-17 (Th17 cells) are responsible for various
organ-related autoimmune diseases (Harrington et al. (2006) Curr.
Opin. Immunol. 18:349), including experimental autoimmune
encephalomyelitis (EAE) (Langrish et al. (2005) J. Exp. Med.
201:233), arthritis (Murphy et al. (2003) J. Exp. Med. 198:1951),
colitis (Yen et al. (2006) J. Clin. Invest. 116:1310) and
autoimmune myocarditis (Sonderegger et al. (2006) Eur. J. Immunol.
36:2844). Moreover, although less clear, the production of high
levels IL-23/IL-17, more than IL-12/IFN-.gamma., better correlates
with disease severity and immunopathology in diverse infections.
Hunter (2005) Nat. Rev. Immunol. 5:521; Rutitzky (2005) J. Immunol.
175:3920. Other studies have suggested that IL-12 and IL-23 have
distinct roles in host defense against Klebsiella pneumoniae
(Happel et al. (2005) J. Exp. Med. 202:761) and Citrobacter
rodentium (Mangan et al. (2006) Nature 441:231). These studies
suggest that IL-12 and IL-23 have distinct roles in promoting
antimicrobial immune responses and diseases in vivo.
[0092] The different roles of IL-12 and IL-23 in inflammation and
host defense have important implications for chronic infections,
such as chronic fungal infection. Although inflammation is required
for prompt control of fungal infections, resolution of inflammation
is essential for maintaining the balance between protection and
immunopathology in infections and associated diseases. Han and
Ulevitch (2005) Nat. Immunol. 6:1198. Prolonged inflammation is a
hallmark of a wide range of chronic diseases and autoimmunity. Han
& Ulevitch (2005). For Candida, failure to resolve inflammation
is associated with defective fungal clearance. This unresolved
Candida infection results in chronic mucocutaneous candidiasis
(CMC). CMC is associated with autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy, a condition of
dysfunctional T cell activity. Ryan et al. (2005) J. Allergy Clin.
Immunol. 116:1158. CMC also encompasses a variety of clinical
disorders of unknown immunopathogenesis. Lilic (2002) Curr. Opin.
Infect. Dis. 15:143. For Aspergillus, persistent inflammation with
intractable infection is common in non-neutropenic patients after
allogeneic hematopoietic stem cell transplantation (Ortega et al.
(2006) Bone Marrow Transplant 37:499) as well as in allergic fungal
diseases (Schubert (2006) Clin. Rev. Allergy Immunol. 30:205). For
the last two decades the immunopathogenesis of fungal infections
and associated inflammatory diseases has been explained primarily
in terms of Th1/Th2 balance as affected by a combination of
different types of regulatory T cells (T reg). Romani (2004) Nat.
Rev. Immunol. 4:1; Romani and Puccetti (2006) Trends Microbiol.
14:183.
[0093] Although inflammation is an essential component of the
protective response to fungi, its dysregulation may significantly
worsen fungal diseases. As disclosed herein, IL-23 and IL-17
negatively regulate IL-12/Th1-mediated immune resistance to fungi
and play an inflammatory role previously attributed to uncontrolled
Th1 cell responses. IL-23 is known to promote the survival of Th17
cells (which produce IL-17 and cause inflammation) and antagonize
the IL-12-mediated Th1 response (which involves production of
interferon-.gamma. (IFN-.gamma.)). Langrish et al. (2004) Immunol.
Rev. 202:96. As demonstrated herein, IL-23 counter-regulation of
IL-12 production and Th1 responses results in uncontrolled
inflammation and growth of Candida albicans and Aspergillus
fumigatus, two major human fungal pathogens. Both IL-23 and IL-17
subvert the inflammatory program and anti-fungal activity of
neutrophils, resulting in severe tissue inflammatory pathology
associated with infection. In summary, IL-23-driven inflammation
promotes infection and impairs antifungal immune resistance. See
also Zelante et al. (2007) Eur. J. Immunol. 37:2695, and related
commentary at Cooper (2007) Eur. J. Immunol. 37:2680. Modulation of
the inflammatory response by antagonism of IL-23 represents a
represents a promising strategy to stimulate protective immune
responses to fungi.
[0094] The present invention provides compositions and methods for
the treatment of infection, such as chronic infections, by blocking
the activity of IL-23 and/or IL-17 to reduce the effects of Th17
cells and allow a robust Th1 response to emerge and eliminate the
infected cells or organisms. In the optimal case the result is a
sterile cure in which the infection is fully resolved (i.e.
treatment may be discontinued without recurrence of the
infection).
[0095] The same reasoning regarding the role of Th17 cells in
maintaining a counter-productive inflammatory state applies in the
case of chronic viral and bacterial infection, such as tuberculosis
(TB). Cytokines are the soluble mediators of immune cells. The
following cytokines have been detected in pleural or
bronchoalveolar lavage (BAL) fluids of patients infected with TB:
IL-113, TNF.alpha., IFN.gamma., TGF.beta., and IL-12. See, e.g.,
Crystal, et al. (eds.) (1997) The Lung Scientific Foundations,
Lippincott-Raven, New York, N.Y., pp. 2381-2394. IFN-.gamma. and
TNF.alpha. have been shown to play important roles in the control
of mycobacterial infections. See, e.g., Cooper et al. (1993) J.
Exp. Med. 178:2243; Flynn et al. (1993) J. Exp. Med. 178:2249;
Kindler et al. (1989) Cell 56:731; Cheuse et al. (1994) Am. J.
Pathol. 145:1105. To the extent that IL-23 promotes the survival of
Th17 cells, which reduces IL-12 driven IFN-.gamma. production,
antagonism of IL-23 activity may be expected to enhance resolution
of bacterial infection.
[0096] The literature suggest that treatment with antagonists of
IL-23 may be safer than treatment with antagonists of IL-12, e.g.
in the treatment of autoimmune disorders or chronic infection.
Chackerian et al. describe experiments in which elimination of
IL-23 activity, either through antibody neutralization or genetic
elimination in p19.sup.-/- knockout (KO) mice, did not compromise
immunity to mycobacterial (BCG) infection. Chackerian et al. (2006)
J. Exp. Med. 74:6092. The course of infection in IL-23p19 KO mice
was indistinguishable from that in wildtype mice, and the numbers
of bacterial colony forming units in anti-IL-23p19-treated mice did
not differ from the number in isotype-control-treated mice. In
contrast, IL-12 deficient KO mice failed to control the growth of
BCG, and antibody blocking of IL-12 correlated with significantly
higher numbers of CFU in the spleen, livers and lungs as compared
to isotype-control-treated mice. These results suggest that IL-23
does not play a significant role in host defense against
mycobacteria in the presence of IL-12, and therefore that selective
inhibition of IL-23 may be safer than treatments that involve IL-12
neutralization (either with or without concurrent IL-23
neutralization). The results presented herein extend these results
to suggest that antagonists of IL-23 are not only safer, in that
they don't compromise host defense, but they may in fact be
beneficial in helping to resolve chronic infections caused by
dysregulation of IL-23/IL-17 inflammation.
[0097] The experiments in Chackerian et al. (2006) were not
designed to address the issue of whether blocking IL-23 would
enhance clearance of pre-existing chronic mycobacterial infections.
Control mice (WT mice, untreated or treated with isotype control
antibody only) were able to effectively clear the infections,
rather than developing a chronic infection. In both the KO mice and
the antibody-blocking experiments in Chackerian et al. (2006),
IL-23 activity was eliminated prior to infection with intravenous
BCG, rather than after infection. The experiments described herein
involved fungal, rather than mycobacterial, infections. In
addition, the experiments described at Examples 4 and 5 herein
include experiments using intragastric and intranasal
administration of fungal pathogens, rather than intravenous
administration. Direct delivery of these fungal pathogens to lung
and stomach provides a more physiologically relevant disease model
than intravenous delivery. The tissues infected with fungal
pathogens in the experiments described herein have been suggested
as the tissues in which the Th17 response may have its most
important physiological role, i.e. the mucosal barrier of the lung
and gut. Cua and Kastelein (2006) Nature Immunol. 7:557. In
addition, anti-IL-23p19 and anti-IL-17 antibodies were administered
five hours after infection in the experiments disclosed herein,
rather than prior to infection.
III. Experimental Results in Fungal Infections
[0098] Prolonged inflammation is a hallmark of a wide range of
chronic diseases and autoimmunity. Han & Ulevitch (2005).
Before the discovery of IL-23 and its recently documented role in
autoimmune inflammation (Cua et al. (2003) Nature 421:744; Langrish
et al. (2005) J. Exp. Med. 201:233), IL-12, by initiating and
maintaining Th1 responses, was thought to be responsible for
overreacting immune and autoimmune disorders. This was also true of
fungal infections and diseases where immunoregulation proved to be
essential in fine-tuning inflammation and uncontrolled Th1/Th2
antifungal reactivity. Ryan et al. (2005); Romani (2004); Romani
& Puccetti (2006).
[0099] The results of the present study show that the IL-23/IL-17
axis, and not an uncontrolled Th1 response, is associated with
defective pathogen clearance, failure to resolve inflammation and
to initiate protective immune responses to Candida and Aspergillus.
Thus, the new findings may serve to accommodate the paradoxical
association of chronic inflammatory responses with intractable
forms of fungal infections where fungal persistence occurs in the
face of an ongoing inflammation.
[0100] Both IL-23 and IL-17 impaired the antifungal effector
activities of PMN even in the presence of IFN-.gamma., a finding
suggesting that the Th17 effector pathway prevails over the Th1
pathway. In addition, both cytokines activated the inflammatory
program of PMN by counteracting the IFN-.gamma.-dependent
activation of indoleamine 2,3-dioxygenase (IDO), known to limit the
inflammatory status of PMN against fungi (Bozza et al. (2005) J.
Immunol. 174:2910), as well as by inducing the release of MMP9 and
MPO which likely accounts for the high inflammatory pathology and
tissue destruction associated with Th17 cell activation.
[0101] The action on IDO is of interest. IDO is expressed in C.
albicans and is involved in tryptophan auxotrophy-dependent
inhibition of fungal germination. Bozza et al. (2005). Similar to
IDO blockade, and as opposed to IFN-.gamma. (Kalo-Klein et al.
(1990) Infect. Immun. 58:260), IL-17 promoted fungal germination
(data not shown), a finding suggesting an action on fungal IDO, an
enzyme that is highly responsive to signals from the mammalian host
immune system. Mellor and Munn (2004) Nat. Rev. Immunol. 4:762.
Therefore, the function of the Th17 pathway may go beyond its
ability to promote inflammation and subvert antimicrobial immunity,
as already described for other infections (McKenzie et al. (2006)
Trends Immunol. 27:17), to include an action on fungal morphology
and virulence. This may translate in concomitant IL-4+Th2 cell
activation, known to be strictly dependent on high levels fungal
growth (Mencacci et al. (1996) Infect. Immun. 64:4907) and further
preventing Th1 functioning.
[0102] As already described for other infections (Cruz et al.
(2006) J. Immunol. 177:1416; Park et al. (2005) Nat. Immunol.
6:1133), the Th1 or Th17 pathways were reciprocally regulated in
both fungal infections. This finding suggests that the occurrence
of either pathway in response to fungi is under strict
environmental control. Regulation may occur at different stages.
One obvious level of regulation is represented by IFN-.gamma. which
is known to regulate the induction of Th17 cells. Cruz et al.
(2006); Park et al. (2005). The IL-23/IL-17 axis was indeed
heightened in condition of IFN-.gamma. deficiency in both
infections, and the number of IFN-.gamma.-producing cells increased
upon IL-17 neutralization. These data are in line with the notion
that IFN-.gamma. is required for IL-12 responsiveness in mice with
candidiasis. Cenci et al. (1998) J. Immunol. 161:3543.
[0103] More important, the production of IL-12 was higher in
p19.sup.-/- DC and production of IL-23 higher in p35.sup.-/- DC,
and both cytokines were cross-regulated in WT DC. These findings
suggest that these cytokines are reciprocally regulated at the
level of DC production. Becker et al. (2006) J. Immunol. 177:2760.
However, because inflammatory DC more than tolerogenic DC appear to
produce IL-23 in response to fungi, this implies that the Th1/Th17
balance also depends on the reciprocal regulation between DC
subsets at different body sites.
[0104] The finding that IL-23 is produced in response to fungi in
condition of high-threat inflammation, that is by inflammatory DC
in response to high yeast number through the TLR-/MyD88 pathway,
has important implications. Not only does it point to IL-23 as an
important molecular link between the inflammatory processes and
fungal virulence, but it also establishes a scenario whereby a
vicious circle may be at work. Because p19.sup.-/- mice produce
less IL-17 and TGF-.beta. showed a non-essential role in Th17
activation and/or maintenance against fungi, it is conceivable that
IL-23 acts as a proximal mediator of IL-17. In this scenario, the
uncontrolled fungal growth may perpetuate the activation of
pathogenic Th17 cells implicating concomitant activation of
nonprotective Th2 cells.
[0105] One interesting observation in this study was that although
microbial stimuli may be a major inducer of IL-23 secretion,
adaptive immune processes may also modulate its production. In
support of this we have provided evidence that IL-23 secretion by
DC was dramatically increased in the presence of T cells, a finding
suggesting that activated T cells may provide a positive feedback
loop for further induction of IL-23.
[0106] The above considerations may help to accommodate fungi,
either commensals or ubiquitous, within the immune homeostasis and
its dysregulation. If the ability to subvert the inflammatory
program through the activation of the IL-23/IL-17 axis may
eventually lead to immune dysregulation, their ability to activate
T reg cells, integral and essential components of protective
immunity to either Candida or Aspergillus (Romani & Puccetti
(2006)), may represent a mechanism whereby dysregulated immunity is
prevented. In this regard, a functional antagonism between Th17 and
T reg cells has been described (Bettelli & Kuchroo (2005) J.
Exp. Med. 201:169), including the inhibitory role of IL-10 in the
development of IL-17-producing cells in vivo. Kullberg et al.
(2006) J. Exp. Med. 203:2485. It is possible therefore that a
reciprocal pathway for the generation of Th17 and T reg cells may
also take place in fungal infections. We have found no evidence of
CD4+CD25+ T reg cell activation in p35.sup.-/- mice after
infection, a finding suggesting that Th17 and T reg cells are
mutually exclusive. CD4+CD25+ T reg cells were instead observed in
p19.sup.-/- mice, despite a significant decrease of IL-10
production, which is consistent with the ability of IL-23 to
up-regulate IL-10 production by T cells. Vanden Eijnden et al.
(2005) Eur. J. Immunol. 35:469-475.
[0107] Another important observation of the present study is that
the IL-23/IL-17-dependent pathway may provide some antifungal
resistance in condition of IFN-.gamma. deficiency, through a
p35-dependent pathway. That IL-23 may serve a protective role in
condition of IL-12 deficiency has already been reported in chronic
cryptococcosis (Kleinschek et al. (2006) J. Immunol. 176:1098),
mycobacterial infection (Khader et al. (2005) J. Immunol. 175:788)
and acute pulmonary Klebsiella pneumoniae infection (Happel et al.
(2005) Infect. Immun. 73:5782), where the protection correlated
with an ability of IL-23 to activate antigen-specific
IFN-.gamma.-producing CD4+T cells, independently of IL-12p70, and
to recruit PMN mediating pathogen clearance. Happel et al. (2005)
J. Exp. Med. 202:761. As a matter of fact, in experimental
Helicobacter hepaticus-induced colitis, IL-23 has clearly been
shown to drive both IFN-.gamma.- and IL-17-producing cells.
Kullberg et al. (2006). Our results seem to suggest a further level
of cross-regulation between the Th1 and the Th17 pathways in
infections that implicates a p35-dependent pathway in the action of
IL-23. Ultimately, the ability of IL-23 to process initial
inflammatory danger signals before the onset of the appropriate
immune effector functions dominated by the IL-12-dependent axis
(McKenzie et al. (2006)) is consistent with antagonist as well as
collaborative activities between this pair of cytokines.
[0108] Collectively, the data presented in this study demonstrate a
previously undefined role for the IL-23-dependent Th17 lineage in
fungal infections that has important implications for mechanisms of
host defense, immune homeostasis and immunity to fungi. Moreover,
they show a molecular connection between the failure to resolve
inflammation and lack of antifungal immune resistance. The current
results suggest strategies for immune therapy of fungal infections
that attempt to limit inflammation to stimulate an effective immune
response.
IV. IL-23 Antagonists
[0109] Antagonists of IL-23 include any substance or method capable
of inhibiting one or more biological activities of IL-23. Such
activities include binding to the IL-23 (comprising p19 and p40
subunits), IL-23 receptor (comprising IL-23R and IL-12R.beta.1
subunits) and promotion and maintenance of Th17 cells. Antagonists
may comprise, e.g., small molecules, antibodies or antibody
fragments, peptide mimetics, aptamers (e.g. as disclosed in U.S.
Patent Application Publication No. 2006-0193821), soluble receptor
derived from on the extracellular region of a subunit of the IL-23
receptor, and nucleic acid based antagonists.
[0110] Nucleic acid-based antagonists of IL-23 include antisense
nucleic acids and siRNA directed to the IL-23p19 gene or the IL-23R
gene. For general siRNA methodology, see WO 2006/06060598. See also
Arenz and Schepers (2003) Naturwissenschaften 90:345; Sazani and
Kole (2003) J. Clin. Invest. 112:481; Pirollo et al. (2003)
Pharmacol. Therapeutics 99:55; Wang et al. (2003) Antisense Nucl.
Acid Drug Devel. 13:169. Antisense and siRNA molecules can be
designed based on the known sequences of human IL-23p19 and IL-23R
mRNA. mRNA and amino acid sequences for human IL-23p19 are found at
GenBank Accession Nos. NM.sub.--016584 and NP.sub.--057668,
respectively. cDNA and amino acid sequences for human IL-23R are
found at GenBank Accession Nos. AF461422 and AAM44229,
respectively. The invention also provides compositions for RNA
interference.
[0111] Methods of producing and using siRNA are disclosed, e.g., at
U.S. Pat. No. 6,506,559 (WO 99/32619); U.S. Pat. No. 6,673,611 (WO
99/054459); U.S. Pat. No. 7,078,196 (WO 01/75164); U.S. Pat. No.
7,071,311 and PCT publications WO 03/70914; WO 03/70918; WO
03/70966; WO 03/74654; WO 04/14312; WO 04/13280; WO 04/13355; WO
04/58940; WO 04/93788; WO 05/19453; WO 05/44981; WO 03/78097. U.S.
patents are listed with related PCT publications. Exemplary methods
of using siRNA in gene silencing and therapeutic treatment are
disclosed at PCT publications WO 02/096927 (VEGF and VEGF
receptor); WO 03/70742 (telomerase); WO 03/70886 (protein tyrosine
phosphatase type IVA (Prl3)); WO 03/70888 (Chk1); WO 03/70895 and
WO 05/03350 (Alzheimer's disease); WO 03/70983 (protein kinase C
alpha); WO 03/72590 (Map kinases); WO 03/72705 (cyclin D); WO
05/45034 (Parkinson's disease). Exemplary experiments relating to
therapeutic uses of siRNA have also been disclosed at Zender et al.
(2003) Proc. Nat'l. Acad. Sci. (USA) 100:7797; Paddison et al.
(2002) Proc. Nat'l. Acad. Sci. (USA) 99:1443; and Sah (2006) Life
Sci. 79:1773. siRNA molecules are also being used in clinical
trials, e.g., of chronic myeloid leukemia (CML) (ClinicalTrials.gov
Identifier: NCT00257647) and age-related macular degeneration (AMD)
(ClinicalTrials.gov Identifier: NCT00363714).
[0112] Although the term "siRNA" is used herein to refer to
molecules used to induce gene silencing via the RNA interference
pathway (Fire et al. (1998) Nature 391:806), such siRNA molecules
need not be strictly polyribonucleotides, and may instead contain
one or more modifications to the nucleic acid to improve its
properties as a therapeutic agent. Such agents are occasionally
referred to as "siNA" for short interfering nucleic acids. Although
such changes may formally move the molecule outside the definition
of a "ribo"nucleotide, such molecules are nonetheless referred to
as "siRNA" molecules herein. Other variants of nucleic acids used
to induce gene silencing via the RNA interference pathway include
short hairpin RNAs ("shRNA"), for example as disclosed in U.S. Pat.
Application Publication No. 20060115453. Nucleic acid-based
inhibitors may be delivered, e.g., by transformation with a
recombinant vector such as a plasmid or a virus (e.g. as naked
DNA), or by gene therapy with any of known gene therapy vector
(e.g. adeno-associated virus (AAV), adenovirus, a retrovirus or a
lentivirus). Nucleic acids may be delivered by transformation,
electroporation, biolistic bombardment or other methods known in
the art.
[0113] Antibody antagonists of IL-23 for use in the compositions
and methods of the present invention include antibodies to IL-23
and antibodies to IL-23 receptor. Exemplary antagonist antibodies
to IL-23 include the anti-human IL-23p19 antibodies, and fragments
thereof, as disclosed in commonly-assigned U.S. Provisional Patent
Application Nos. 60/891,409 and 60/891,413 (both filed 23 Feb.
2007), in U.S. Patent Application Publication Nos. 2007-0009526 and
2007-0048315, and in International Patent Publication Nos. WO
2007/076524, WO 2007/024846 and WO 2007/147019. Antibody
antagonists to IL-23 also include antibodies that bind to the
IL-12p40 subunit when that subunit is bound to IL-23p19, but not
when it is bound to IL-12p35. See, e.g., U.S. Patent Application
Publication No. 2005-0137385 and U.S. Pat. No. 7,252,971. Exemplary
antagonist antibodies to IL-23 include anti-human IL-23 receptor
antibodies, e.g. anti-IL-23R antibodies, and fragments thereof.
Exemplary antagonist antibodies to IL-23R are disclosed in
commonly-assigned U.S. Provisional Patent Application No.
60/892,104 (filed 28 Feb. 2007) and 60/945,183 (filed 20 Jun.
2007). Antagonists of IL-23 also include bispecific antibodies.
[0114] Regions of increased antigenicity can be used for antibody
generation. Regions of increased antigenicity of human p19 occur,
e.g., at amino acids 16-28; 57-87; 110-114; 136-154; and 182-186 of
GenBank AAQ89442 (gi:37183284). Regions of increased antigenicity
of human IL-23R occur, e.g., at amino acids 22-33; 57-63; 68-74;
101-112; 117-133; 164-177; 244-264; 294-302; 315-326; 347-354;
444-473; 510-530; and 554-558 of GenBank AAM44229 (gi: 21239252).
Analysis was by a Parker plot using Vector NTI.RTM. Suite
(Informax, Inc., Bethesda, Md.). The present invention also
provides an IL-23 antagonist that is a soluble receptor, i.e.,
comprising an extracellular region of IL-23R, e.g., amino acids
1-353 of GenBankAAM44229, or a fragment thereof, where the
extracellular region or fragment thereof specifically binds to
IL-23. Mouse IL-23R is GenBank NP.sub.--653131 (gi:21362353).
Muteins and variants are contemplated, e.g., pegylation or
mutagenesis to remove or replace deamidating Asn residues.
[0115] Additional potential methods of antagonizing the activity of
IL-23 for use in the methods and compositions of the present
invention include administering filamentous hemagglutinin (FHA) (WO
2006/109195) and vaccinating to generate an immune response against
IL-23 (WO 2005/058349).
[0116] In one embodiment, an antagonist of an IL-17 producing
(Th17) cell encompasses a reagent that specifically modulates the
activity of a Th17 cell, e.g., without substantial influence on the
activity of, e.g., a naive T cell, Th1-type T cell, TH2-type T
cell, epithelial cell, and/or endothelial cell. The reagent can
modulate expression or activity of, e.g., a transcription factor
(e.g. ROR.gamma.t) or adhesion protein, of the IL-17 producing
cell. In addition, an antagonist of IL-23, TGF-.beta., or IL-6 may
decrease the creation and survival of Th17 cells, and an antagonist
of IL-17 may decrease the inflammatory effects (e.g. neutrophil
recruitment) of such cells.
[0117] Monoclonal, polyclonal, and humanized antibodies can be
prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He et al.
(1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem.
274:27371; Baca et al. (1997) J. Biol. Chem. 272:10678; Chothia et
al. (1989) Nature 342:877; Foote and Winter (1992) J. Mol. Biol.
224:487; U.S. Pat. No. 6,329,511). Fully human antibodies may also
be prepared, in which the entirety of the antibody sequence is
derived from human germline sequences. Such fully human antibodies
may be prepared from transgenic animals engineered to express human
immunoglobulin genes, or by methods such as phage display and the
like. See, e.g., Lonberg (2005) Nature Biotechnol. 23:1117; Vaughan
et al. (1998) Nature Biotechnol. 16:535. Antibody fragments include
Fab, Fab', Fab'-SH, Fv, single-chain Fv (scFv), F(ab').sub.2, and a
diabodies. Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL
ANTIBODIES, vol. 113, Rosenburg and Moore eds., Springer-Verlag,
New York, pp. 269-315; Holliger and Hudson (2005) Nature
Biotechnol. 23:1126-1136.
[0118] The antibodies of the present invention also include
antibodies with modified (or blocked) Fc regions to provide altered
effector functions. See, e.g., U.S. Pat. No. 5,624,821; WO
2003/086310; WO 2005/120571; WO 2006/0057702; Presta (2006) Adv.
Drug Delivery Rev. 58:640-656. Such modification can be used to
enhance or suppress various reactions of the immune system, with
possible beneficial effects in diagnosis and therapy. Alterations
of the Fc region include amino acid changes (substitutions,
deletions and insertions), glycosylation or deglycosylation, and
adding multiple Fc. Changes to the Fc can also alter the half-life
of antibodies in therapeutic antibodies, and a longer half-life
would result in less frequent dosing, with the concomitant
increased convenience and decreased use of material. Altered
effector functions may be achieved by introducing specific
mutations in the Fc portion of IgG1, e.g. by altering Asn297, e.g.
to Ala or Gln (N297A or N297Q). See Presta (2005) J. Allergy Clin.
Immunol. 116:731 at 734-35. Effector functions may also be changed
by selecting different constant domains. For example, if a
particular intended use of an antibody (or fragment) of the present
invention were to call for altered effector functions, a heavy
chain constant domain other than IgG1 may be used. Although IgG1
antibodies provide for long half-life and for effector functions,
such as complement activation and antibody-dependent cellular
cytotoxicity (ADCC), such activities may not be desirable for all
uses of the antibody. In such instances an IgG4 constant domain,
for example, may be used. Altered effector functions are of
particular relevance in the case of antibodies to IL-23 receptor
(e.g. to IL-23R), since in one embodiment (not the only embodiment)
the goal is not to induce killing of cells expressing IL-23
receptor, but instead merely to block IL-23 signaling in such
cells. In this embodiment, the goal is to shift Th cells from the
Th17 lineage toward the Th1 lineage, in which case cell killing
would be unproductive.
[0119] Purification of antigen is not necessary for the generation
of antibodies. Immunization can be performed by DNA vector
immunization, see, e.g., Wang et al. (1997) Virology 228:278.
Alternatively, animals can be immunized with cells bearing the
antigen of interest. Splenocytes can then be isolated from the
immunized animals, and the splenocytes can fused with a myeloma
cell line to produce a hybridoma (Meyaard et al. (1997) Immunity
7:283; Wright et al. (2000) Immunity 13:233; Preston et al. (1997)
Eur. J. Immunol. 27:1911). Resultant hybridomas can be screened for
production of the desired antibody by functional assays or
biological assays, that is, assays not dependent on possession of
the purified antigen. Immunization with cells may prove superior
for antibody generation than immunization with purified antigen
(Kaithamana et al. (1999) J. Immunol. 163:5157).
[0120] Antibody to antigen and ligand to receptor binding
properties can be measured, e.g., by surface plasmon resonance
(Karlsson et al. (1991) J. Immunol. Methods 145:229; Neri et al.
(1997) Nat. Biotechnol. 15:1271; Jonsson et al. (1991)
Biotechniques 11:620) or by competition ELISA (Friguet et al.
(1985) J. Immunol. Methods 77:305; Hubble (1997) Immunol. Today
18:305). Antibodies can be used for affinity purification to
isolate the antibody's target antigen and associated bound
proteins. See, e.g., Wilchek et al. (1984) Meth. Enzymol.
104:3.
[0121] Antibodies will usually bind with at least a K.sub.D of
about 10.sup.-6 M, typically at least 10.sup.-7 M, more typically
at least 10.sup.-8 M, preferably at least about 10.sup.-9 M, and
more preferably at least 10.sup.-10 M, and most preferably at least
10.sup.-11 M. See, e.g., Presta et al. (2001) Thromb. Haemost.
85:379; Yang et al. (2001) Crit. Rev. Oncol. Hematol. 38:17;
Carnahan et al. (2003) Clin. Cancer Res. (Suppl.) 9:3982s.
[0122] Soluble receptors comprising the extracellular domain of
IL-23R are useful in the compositions and methods of the present
invention. Soluble receptors can be prepared and used according to
standard methods. See, e.g., Jones et al. (2002) Biochim. Biophys.
Acta 1592:251; Prudhomme et al. (2001) Expert Opinion Biol. Ther.
1:359; Fernandez-Botran (1999) Crit. Rev. Clin. Lab Sci.
36:165-224.
[0123] In one embodiment the compositions and methods of the
present invention require antagonism of IL-23 and not antagonism of
IL-12. There are currently several potential therapeutic agents
under development that target the IL-12p40 subunit of both IL-12
and IL-23 that would block the activity of both IL-23 and IL-12.
Such agents would not be suitable for use in this embodiment of the
compositions and methods of the present invention since they would
inhibit the robust IL-12-mediated Th1 response that the invention
is intended to promote. Although it is in principle possible to
develop an agent that binds to IL-12p40 only in the context of
IL-23 but not in the context of IL-12 (see U.S. Patent Application
Publication No. 2005-0137385 and U.S. Pat. No. 7,252,971), it is
likely that the majority of agents targeting IL-12p40 will inhibit
IL-12 and thus not be suitable for the present invention. The same
argument applies with the shared receptor subunit of IL-23 and
IL-12, IL-12R.beta.1. Although it is in principle possible to
develop an agent that binds to IL-12R.beta.1 only in the context of
IL-23 receptor but not in the context of IL-12 receptor, it is
likely that the majority of agents targeting IL-12R.beta.1 will
inhibit IL-12 receptor and thus not be suitable for the present
invention. In contrast, agents that bind to and antagonize subunits
specific to IL-23 or its receptor, i.e. p19 and IL-23R,
respectively, are likely to be specific inhibitors of IL-23 rather
than IL-12, and thus more suitable for use in the compositions and
methods of the present invention.
[0124] Whether a potential therapeutic agent specifically inhibits
IL-23 rather than IL-12 may be determined by any method known in
the art. For example a potential IL-23-specific antagonist may be
tested for its ability to block the binding of IL-23 to its
receptor, or IL-12 to its receptor. Such blocking assays may be
performed in solution (e.g. by fluorescence-activated cell sorting)
or on a solid support (e.g. by enzyme-linked immunosorbent
assay--ELISA). IL-23 and IL-12 receptor blocking can also be
measured in a bioassay, such as a Ba/F3 cell proliferation assay.
See e.g. Ho et al. (1995) Mol. Cell. Biol. (1995) 15:5043. In such
binding assays, the potency and specificity of a potential IL-23
antagonist may be expressed as an IC50, or the concentration of the
potential antagonist necessary to achieve a 50% reduction in IL-23
binding (or biological activity dependent on binding) under a given
set of assay conditions. A lower IC50 indicates a more effective
antagonist. The IL-23 specificity of a potential antagonist may be
expressed as the ratio of the IC50 for inhibition of binding of
IL-12 to its receptor to the IC50 for inhibition of binding of
IL-23 to its receptor (IC50.sub.IL-12/IC50.sub.IL-23). In various
embodiments a potential IL-23 specific antagonist is considered to
be IL-23 specific if this ratio (IC50.sub.IL-12/IC50.sub.IL-23) is
1.5, 2, 3, 4, 5, 7, 10, 15, 20, 50, 100 or more. In preferred
embodiments the levels of IL-23 and IL-12 used in inhibition assays
are adjusted to ensure that at least one, and preferably both of
the IL-23 and IL-12 assays, are performed in the linear dose
response concentration range.
[0125] IL-23 and IL-12 also have different biological functions
that may be used to determine specificity of antagonism. In
contrast to IL-12, IL-23 preferentially stimulates memory as
opposed to naive T cell populations in both human and mouse. IL-23
activates a number of intracellular cell-signaling molecules, e.g.,
Jak2, Tyk2, Stat1, Stat2, Stat3, and Stat4. IL-12 activates this
same group of molecules, but Stat4 response to IL-23 is relatively
weak, while Stat4 response to IL-12 is strong. Oppmann et al.
(2000); Parham et al. (2002) J. Immunol. 168:5699.
[0126] A potential IL-23-specific antagonist may also be tested for
its ability to inhibit the amplification and survival of Th1 and
Th17 cells by IL-12 and IL-23. An IL-23-specific antagonist will
preferentially inhibit the IL-23-mediated amplification and
survival of Th17 cells, but not the IL-12-mediated amplification
and survival of Th1 cells. Th17 cells characteristically secrete
IL-17 whereas Th1 cells characteristically secrete IFN-.gamma..
Data from an exemplary Th1/Th17 assay is found at FIG. 2 of
Langrish et al. (2005) J. Exp. Med. 201:233, which demonstrates
that IL-23 promotes amplification and survival of IL-17 producing
CD4.sup.+ T cells, whereas IL-12 promotes amplification and
survival of IFN-.gamma.-producing CD4.sup.+ T cells. In one
embodiment of the present invention, an agent is considered to be
an "IL-23-specific" antagonist (relative to IL-12) when it is able
to inhibit IL-23-mediated amplification and survival of Th17 cells,
while not inhibiting IL-12-mediated amplification and survival of
Th1 cells Inhibition of Th17/Th1 cell proliferation can be
expressed as an IC50, or the concentration of the agent necessary
to achieve a 50% reduction in the activity of IL-23 in promoting
the amplification and survival of a particular T cell subset
producing IL-17 or IFN-.gamma. under a given set of assay
conditions. An exemplary assay is provided at Example 13. The
potency of an IL-23 antagonist in a bioassay like the one described
in Example 13 may be expressed as the IC50.sub.IL-23, i.e. the
concentration of antagonist needed to reduce the activity of IL-23
to 50% of its uninhibited value. An analogous IC50.sub.IL-12 may be
determined for IL-12 and its activity in promoting production of
IFN-.gamma. producing cells. The IL-23-specificity of the
antagonist can then be expressed as the ratio
IC50.sub.IL-12/IC50.sub.IL-23. In various embodiments, the
IC50.sub.IL-12/IC50.sub.IL-23 ratio for a validated IL-23-specific
antagonist is 1.5, 2, 3, 4, 5, 7, 10, 15, 20, 50, 100 or more.
[0127] Production of IL-17A and IFN-.gamma. may be measured by
intracellular cytokine flow cytometry by fluorescence activated
cell sorting (FACS.RTM. analysis) with fluorescent reagents that
bind to the cytokines, essentially as described in Langrish et al.
(2005). It is important to define the threshold level of IL-17A or
IFN-.gamma. in a live CD4' T cell for that cell to be considered
"IL-17 producing" or "IFN-.gamma. producing." In one embodiment the
threshold level is defined as the level at which 5% of live CD4' T
cells are "IL-17 producing" or "IFN-.gamma. producing" in a control
sample of untreated cells. Exemplary untreated cells include
draining lymph node (DLN) cells isolated from SJL mice (The Jackson
Laboratories, Bar Harbor, Me., USA) immunized with proteolipid
protein (PLP) cultured in the presence of PLP.
V. Compositions and Methods
[0128] To prepare pharmaceutical or sterile compositions including
an antagonist of IL-23, the antagonist is admixed with a
pharmaceutically acceptable carrier or excipient, see, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984).
Formulations of therapeutic agents may be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous solutions
or suspensions (see, e.g., Hardman, et al. (2001) Goodman and
Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill,
New York, N.Y.; Gennaro (2000) Remington: The Science and Practice
of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;
Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral
Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms Tablets, Marcel Dekker, NY; Lieberman,
et al. (eds.) (1990) Pharmaceutical Dosage Forms Disperse Systems,
Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity
and Safety, Marcel Dekker, Inc., New York, N.Y.).
[0129] The route of administration is by, e.g., topical or
cutaneous application, injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intracerebrospinal, intralesional, or pulmonary
routes, or by sustained release systems or an implant. Injection of
gene transfer vectors into the central nervous system has been
described. See, e.g., Cua et al. (2001) J. Immunol. 166:602; Sidman
et al. (1983) Biopolymers 22:547; Langer et al. (1981) J. Biomed.
Mater. Res. 15:167; Langer (1982) Chem. Tech. 12:98; Epstein et al.
(1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al. (1980)
Proc. Natl. Acad. Sci. USA 77:4030; U.S. Pat. Nos. 6,350,466 and
6,316,024.
[0130] Selecting an administration regimen for a therapeutic agent
depends on several factors, including the serum or tissue turnover
rate of the agent, the level of symptoms, the immunogenicity of the
agent, and the accessibility of the target cells in the biological
matrix. Preferably, an administration regimen maximizes the amount
of therapeutic agent delivered to the patient consistent with an
acceptable level of side effects. Accordingly, the amount of agent
delivered depends in part on the particular entity and the severity
of the condition being treated. Guidance in selecting appropriate
doses of antibodies, cytokines, and small molecules are available.
See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific
Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal
Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.;
Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in
Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al.
(2003) New Engl. J. Med. 348:601; Milgrom et al. (1999) New Engl.
J. Med. 341:1966; Slamon et al. (2001) New Engl. J. Med. 344:783;
Beniaminovitz et al. (2000) New Engl. J. Med. 342:613; Ghosh et al.
(2003) New Engl. J. Med. 348:24; Lipsky et al. (2000) New Engl. J.
Med. 343:1594.
[0131] Antibodies, antibody fragments, and cytokines can be
provided by continuous infusion, or by doses at intervals of, e.g.,
one day, one week, or 1-7 times per week. Doses may be provided
intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscularly, intracerebrally, intraspinally, or by
inhalation. In various embodiments the mode of administration is
selected based on the primary locus of infection, e.g. the lung or
GI tract.
[0132] A preferred dose protocol is one involving the maximal dose
or dose frequency that avoids significant undesirable side effects.
A total weekly dose is generally at least about 0.05 .mu.g/kg, 0.2
.mu.g/kg, 0.5 .mu.g/kg, 1 .mu.g/kg, 10 .mu.g/kg, 100 .mu.g/kg, 0.2
mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, or 50 mg/kg. See,
e.g., Yang et al. (2003) New Engl. J. Med. 349:427; Herold et al.
(2002) New Engl. J. Med. 346:1692; Liu et al. (1999) J. Neurol.
Neurosurg. Psych. 67:451; Portielji et al. (2003) Cancer Immunol.
Immunother. 52:133. The desired dose of a small molecule
therapeutic, e.g., a peptide mimetic, natural product, or organic
chemical, is about the same as for an antibody or polypeptide, on a
moles/kg basis.
[0133] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side effects, see, e.g., Maynard
et al. (1996) A Handbook of SOPs for Good Clinical Practice,
Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and
Good Clinical Practice, Urch Publ., London, UK.
[0134] Typical veterinary, experimental, or research subjects
include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs,
horses, and humans.
[0135] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the infection or infection levels.
Preferably, a biologic to be used is derived from the same species
as the animal targeted for treatment, or is modified to mimic a
protein derived from the same species (e.g. humanized antibodies),
thereby minimizing a humoral response to the reagent.
[0136] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art. See,
e.g., Hardman et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill,
New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice:A Practical Approach,
Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., PA. An effective amount of
therapeutic will decrease the symptoms typically by at least 10%;
usually by at least 20%; preferably at least about 30%; more
preferably at least 40%, and most preferably by at least 50%.
[0137] The invention further provides use of antagonists of IL-23
or IL-23R, or both, in the manufacture of a medicament for the
treatment of an infectious disease, including but not limited to a
condition selected from the group consisting of a fungal infection,
a persistent fungal infection, candidiasis, CMC, aspergillosis,
cryptococcosis, a viral infection, a persistent viral infection,
HIV infection, HBV infection, HCV infection, a baceterial
infection, mycobacterial infection, an M. tuberculosis infection,
an M. bovis infection and an M. leprae infection. In some
embodiments, the medicament may comprise one or more additional
therapeutic agents. In other embodiments the medicament of the
present invention may be used in conjunction with one or more other
therapeutic agents.
VI. Anti-idiotypic Antibodies
[0138] The invention further provides anti-idiotypic antibodies
directed to therapeutic anti-IL-23 or anti-IL-23R antibodies of the
present invention. An anti-idiotypic antibody is an antibody that
recognizes unique determinants generally associated with the
antigen-binding region of another antibody. The anti-idiotypic
antibody can be prepared by immunizing an animal of the same
species and genetic type (e.g., mouse strain) as the source of the
original anti-IL-23 antibody with the anti-IL-23 antibody or a CDR
containing region thereof. The immunized animal then generates
antibodies to the idiotypic determinants of the immunizing antibody
to produce an anti-idiotypic antibody. The anti-idiotypic antibody
may also be used as an immunogen to induce an immune response in
yet another animal, producing a so-called "anti-anti-Id
antibody."
[0139] Anti-idiotypic antibodies may be used, for example, to
determine the level of a therapeutic anti-IL-23 (or anti-IL-23R)
antibody in a subject, e.g. in a bodily fluid (e.g. blood) of a
subject undergoing anti-IL-23 therapy. Determination of the level
of anti-IL-23 (or anti-IL-23R) antibody in a subject may be useful
in maintaining a desired level of anti-IL-23 antibody in a subject
since dosing may be modified in response to such determinations.
Dosing may be increased or decreased (in frequency and/or amount
per administration) to obtain a circulating level of anti-IL-23
antibody within a desired range of values. The desirable range may
be determined by medical practitioners by methods typical in the
art, and may depend on the therapeutic index for the anti-IL-23 (or
anti-IL-23R) antibody or fragment thereof.
[0140] An anti-idiotypic antibody may be supplied in a form
suitable for easy detection, including antibodies with polypeptide
tags (e.g. the FLAG.RTM. tag), or coupled to dyes, isotopes,
enzymes, and metals. See, e.g., Le Doussal et al. (1991) New Engl.
J. Med. 146:169; Gibellini et al. (1998) J. Immunol. 160:3891;
Hsing and Bishop (1999) New Engl. J. Med. 162:2804; Everts et al.
(2002) New Engl. J. Med. 168:883. Various assay formats exist, such
as radioimmunoassays (RIA), ELISA, and lab on a chip. U.S. Pat.
Nos. 6,176,962 and 6,517,234.
VII. Kits
[0141] This invention further provides antagonists of IL-23 in kits
for use in treatment of subjects (human or non-human) suffering
from infections, such as chronic bacterial, mycobacterial, viral
and fungal infections. In one embodiment, the kit comprises a
compartment for containing an antagonist of IL-23, the antagonist
of IL-23 itself (such as an antibody), and optionally instructions
for use, one or more additional therapeutic agent or agents, and
one or more medical devices for administration (e.g. a syringe or a
disposable injector such as the Redipen.TM. injector device). The
antagonist of IL-23 may be any of the agents described herein,
including but not limited to, anti-p19 antibodies or p19-binding
fragments thereof, anti-IL-23R antibodies or IL-23R-binding
fragments thereof, or soluble IL-23R fragments.
[0142] The one or more additional therapeutic agents include, but
are not limited to, non-steroidal anti-inflammatories (NSAIDS),
steroids, IL-12 or an agonist thereof, and antagonists of cytokines
such as IL-17A, IL-17F, TGF-13, IL-6, or their respective
receptors. Antagonists for cytokines include antibodies that bind
to the cytokine, its subunits, or its receptor. Although not all
antibodies that bind to cytokines or their receptors are
necessarily antagonists, such antagonist activity can readily be
assessed by techniques commonly known in the art, such as a
bioassay or receptor binding assay. Nucleic acid and amino acid
sequences for various (human) cytokines and receptors are known,
including IL-17A (NM.sub.--002190, NP.sub.--002181), IL-17F
(NM.sub.--052872, NP.sub.--443104); IL-17RA (NM.sub.--014339,
NP.sub.--055154); IL-17RC (transcript variants NM.sub.--153461,
NM.sub.--153460, NM.sub.--032732, and their respective
translations).
[0143] The invention further provides kits comprising
anti-idiotypic antibodies directed to therapeutic anti-IL-23 (or
anti-IL-23R) antibodies of the present invention. In one
embodiment, the kit comprises a compartment for containing the
anti-idiotypic antibody, the anti-idiotypic antibody itself, and
optionally instructions for use, one or more detection reagents,
one or more devices for detection of the anti-idiotypic antibody
(such as a microtiter plate), and one or more samples of the
anti-IL-23 antibodies to be detected (or other positive
control).
VIII. Uses
[0144] A prolonged asymptomatic preclinical period often occurs
prior to the development of tuberculosis. Thus, IL-23 and IL-23R
antagonist therapy can be commenced upon analysis of various
diagnostic markers of TB. Patients exhibiting a positive tuberculin
test or Mantoux test (see, e.g., Dale and Federman (2002)), as
compared to normal non-infected patients, can be given IL-23 or
IL-23R antagonist therapy to prevent the further growth of
mycobacteria, or to clear an existing non-pathological infection.
Patients with high levels of mycobacterium in biological samples,
e.g., BAL, may also benefit from IL-23 and IL-23R antagonist
therapy to prevent the further growth of mycobacteria and clear
bacterial burdens in the lungs. Similar treatment may be used for
patients having high mycobacterial DNA or RNA levels in clinical
samples or a positive niacin test in culture. Also envisioned is
the use of IL-23 and IL-23R antagonists in conjunction with
pathologically symptomatic TB infections to lessen or clear
bacterial burdens.
[0145] Bacterial infections that may be treated using the methods
and compositions of the present invention include, but are not
limited to, those caused by: Staphylococcus aureus, Staphylococcus
epidermidis; Streptococcus pneumoniae; Streptococcus agalactiae;
Streptococcus pyogenes; Enterococcus spp.; Bacillus anthracis;
Bacillus cereus; Bifidobacterium bifidum; Lactobacillus spp.;
Listeria monocytogenes; Nocardia spp.; Rhodococcus equi
(coccobacillus); Erysipelothrix rhusiopathiae Corynebacterium
diptheriae; Propionibacterium acnes; Actinomyces spp.; Clostridium
botulinum; Clostridium difficile; Clostridium perfringens;
Clostridium tetani; Mobiluncus spp., Peptostreptococcus spp.;
Neisseria gonorrhoeae; Neisseria meningitides; Moraxella
catarrhalis; Veillonella spp.; Actinobacillus
actinomycetemcomitans; Acinetobacter baumannii; Bordetella
pertussis; Brucella spp.; Campylobacter spp.; Capnocytophaga spp.;
Cardiobacterium hominis; Eikenella corrodens; Francisella
tularensis; Haemophilus ducreyi; Haemophilus influenzae;
Helicobacter pylori; Kingella kingae; Legionella pneumophila;
Pasteurella multocida; Klebsiella granulomatis; Citrobacter spp.,
Enterobacter spp.; Escherichia coli; Klebsiella pneumoniae; Proteus
spp.; Salmonella enteriditis; Salmonella typhi; Shigella spp.;
Serratia marcescens; Yersinia enterocolitica; Yersinia pestis;
Aeromonas spp.; Plesiomonas shigelloides; Vibrio cholerae; Vibrio
parahaemolyticus; Vibrio vulnificus; Acinetobacter spp.;
Flavobacterium spp.; Pseudomonas aeruginosa; Burkholderia cepacia;
Burkholderia pseudomallei; Xanthomonas maltophilia or
Stenotrophomonas maltophila; Bacteroides fragilis; Bacteroides
spp.; Prevotella spp.; Fusobacterium spp.; Spirillum minus;
Borrelia burgdorferi; Borrelia recurrentis; Bartonella henselae;
Chlamydia trachomatis; Chlamydophila pneumoniae; Chlamydophila
psittaci; Coxiella burnetii; Ehrlichia chaffeensis; Anaplasma
phagocytophilum; Legionella spp.; Leptospira spp.; Rickettsia
rickettsii; Orientia tsutsugamushi; Treponema pallidum.
[0146] Mycobacterial infections that may be treated using the
methods and compositions of the present invention include, but are
not limited to, those caused by: M abscessus, M. africanum, M.
asiaticum, Mycobacterium avium complex (MAC), M. avium
paratuberculosis, M. bovis, M. chelonae, M. fortuitum, M. gordonae,
M. haemophilum, M. intracellulare, M. kansasii, M. lentiflavum, M.
leprae, M liflandii, M. malmoense, M. marinum, M. microti, M.
phlei, M. pseudoshottsii, M. scrofulaceum, M. shottsii, M.
smegmatis, M. triplex, M. tuberculosis, M. ulcerans, M. uvium, and
M. xenopi.
[0147] The methods and compositions of the present invention may
also be used to treat fungal conditions, including but not limited
to, histoplasmosis, coccidioidomycosis, blastomycosis,
aspergillosis, penicilliosis, candidiasis and cryptococcosis. Risk
factors for mycoses include blood and marrow transplant,
solid-organ transplant, major surgery (especially gastrointestinal
surgery), AIDS, neoplastic disease, advanced age, immunosuppressive
therapy, and prematurity in infants.
[0148] Fungal pathogens causing infections (and clinical syndromes)
that may be treated using the methods and compositions of the
present invention include, but are not limited to, Candida albicans
(thrush, vaginal candidiasis, esophageal candidiasis), Cryptococcus
neoformans (meningitis), Histoplasma capsulatum (disseminated
infection with fever and weight loss), Coccidioides immitis
(diffuse and focal pulmonary disease), Blastomyces dermatitidis
(localized pulmonary disease and disseminated infection, including
meningitis), Aspergillus fumigatus (pulmonary disease with fever,
cough, and hemoptysis), and Penicillium marneffei (fever alone or
with pulmonary infiltrates, lymphadenopathy, or cutaneous lesions).
The methods and compositions of the present invention may also be
used to treat infections with Candida species C. glabrata, C.
parapsilosis, C. tropicalis, C. krusei, C. lusitaniae, C.
guilliermondii, and C. rugosa. The preceding fungal pathogens (and
clinical syndromes) are commonly associated with HIV infection.
[0149] The methods and compositions of the present invention may
also be used to treat infections with Candida species such as C.
glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae,
C. guilliermondii, and C. rugosa. The methods and compositions of
the present invention may also be used to treat infections with
Aspergillus species such as A. flavus, A. niger, A. ustus and A.
terreus. Additional fungal pathogens include Fusarium species (e.g.
F. moniliforme, F. solani, F. oxysporum) and Scedosporium species
(e.g. S. apiosperum, S. prolificans). Additional fungal diseases
include zygomycoses caused by species of Rhizopus (e.g. R. oryzae,
R. arrhizus), Rhizomucor, Absidia, Cunninghamella.
[0150] Antagonists of IL-23 and IL-23R may be used alone or in
conjunction with agents intended to enhance a Th1 response (e.g.
IL-12 or agonists thereof) or inhibit a Th17 response (e.g.
TGF-.beta. antagonists; IL-6 antagonists; IL-17A and/or IL-17F
antagonists), or both. Agonists and antagonists of the receptors
for these cytokines may also be used. Such agents may include
antibodies and antigen-binding fragments thereof, small molecules,
siRNA and antisense nucleic acids. Antagonists of IL-23 and IL-23R
may also be used in conjunction with anti-inflammatory agents, such
as corticosteroids, e.g. prednisone.
[0151] The IL-17 antagonist may inhibit the expression of IL-17A,
IL-17F, IL-17RA or IL-17RC or may inhibit IL-17 signaling by
directly or indirectly interacting with one or more of these
polypeptides to prevent a functional ligand-receptor interaction.
In some preferred embodiments, the IL-17 antagonist is an antibody
or antibody fragment that binds to and inhibits the activity of
either IL-17A, IL-17F, IL-17RA or IL-17RC. In one particularly
preferred embodiment, the IL-17 antagonist is a monoclonal antibody
that specifically binds to IL-17A. Exemplary antagonist antibodies
to IL-17A include the anti-human IL-17A antibodies, and fragments
thereof, disclosed in commonly-assigned U.S. patent application
Ser. No. 11/836,318 (filed 9 Aug. 2007), and in WO 2006/013107 and
WO 2006/054059. In another embodiment the IL-17 antagonist
comprises a bispecific antibody.
[0152] In one embodiment the IL-23 antagonist comprises a
bispecific antibody that binds to and inhibits the activity of
IL-23. Such bispecific antibodies may bind to IL-23p19 or IL-23R,
and may also bind to the IL-17A, IL-17F, IL-17RA, IL-17RC. In other
embodiments the IL-23 antagonist is a bispecific antibody that
binds to IL-23p19 and IL-17 and inhibits the activity of IL-23 and
IL-17. See, e.g., WO 2007/147019. Alternatively, IL-23 and IL-17
antagonist bispecific antibodies may bind to either IL-23 receptor
(e.g. IL-23R) or IL-17 receptor (IL-17RA or IL-17RC), respectively,
provided that they are antagonist antibodies. Bispecific antibodies
that antagonize both IL-17 and IL-23 activity can be produced by
any technique known in the art. For example, bispecific antibodies
can be produced recombinantly using the co-expression of two
immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein
et al. (1983) Nature 305:537-39. Alternatively, bispecific
antibodies can be prepared using chemical linkage. See, e.g.,
Brennan et al. (1985) Science 229:81. Bifunctional antibodies can
also be prepared by disulfide exchange, production of
hybrid-hybridomas (quadromas), by transcription and translation to
produce a single polypeptide chain embodying a bispecific antibody,
or transcription and translation to produce more than one
polypeptide chain that can associate covalently to produce a
bispecific antibody. The contemplated bispecific antibody can also
be made entirely by chemical synthesis. The bispecific antibody may
comprise two different variable regions, two different constant
regions, a variable region and a constant region, or other
variations.
[0153] Antagonists of IL-23 and IL-23R may be used alone or
co-administered with known antibacterials, such as isoniazid,
rifampin, pyrazinamide, ethambutol, streptomycin, ciprofloxacin,
and ofloxacin. Additional antibacterial agents include, but are not
limited to, alatrofloxacin, azithromycin, baclofen, benzathine
penicillin, cinoxacin, clarithromycin, clofazimine, cloxacillin,
demeclocycline, dirithromycin, doxycycline, erythromycin,
ethionamide, furazolidone, grepafloxacin, imipenem, levofloxacin,
lorefloxacin, moxifloxacin HCl, nalidixic acid, nitrofurantoin,
norfloxacin, ofloxacin, rifabutin, rifapentine, sparfloxacin,
spiramycin, sulphabenzamide, sulphadoxine, sulphamerazine,
ulphacetamide, sulphadiazine, sulphafurazole, sulphamethoxazole,
sulphapyridine, tetracycline, trimethoprim, trovafloxacin, and
vancomycin.
[0154] The methods and compositions of the present invention may be
used to treat persistent viral infections, including but not
limited to infections caused by HBV, HCV, HIV, human papillomavirus
(HPV). Such chronic infections represent a failure of the immune
response to eradicate the infection. Antagonists of IL-23 and
IL-23R may be used alone or in conjunction with other antiviral
agents, including but not limited to, abacavir, acyclovir,
amantadine, amprenavir, delavirdine, didanosine, efavirenz,
famciclovir, indinavir, an interferon alfa, ribavirin, lamivudine,
nelfinavir, nevirapine, oseltamivir, penciclovir, ribavirin,
ritonavir, saquinavir, stavudine, valacyclovir, zalcitabine,
zanamivir, zidovudine (azidodeoxythymidine, AZT). Preferred
interferon alfa agents include pegylated interferon alfa 2a and
pegylated interferon 2b. Exemplary forms of interferon alpha are
discussed in U.S. Pat. No. 6,923,966. The IL-23 antagonist may also
be used in combination with viral specific agents, such as HCV
protease or HCV polymerase inhibitors for chronic HCV infection,
and CCR5 antagonists for chronic HIV infection.
[0155] Antagonists of IL-23 and IL-23R may also be used in
conjunction with a therapeutic vaccine, e.g. gp120-depleted whole
killed virus for HIV infection, a recombinant E1 protein for HCV
infection, and viral E6 and E7 oncoproteins for HPV infection. See
Berzofsky et al. (2004). Such therapeutic vaccines include DNA
vaccines or viral vectors, optionally administered in a
heterologous priming and boosting regimen in which a DNA vaccine is
followed by a viral vector vaccine. Berzofsky et al. (2004).
[0156] Antagonists of IL-23 and IL-23R may be used alone or in
conjunction with other antifungal agents, including but not limited
to, posaconazole, fluconazole (U.S. Pat. No. 4,404,216),
voriconazole, itraconazole (U.S. Pat. No. 4,267,179), ketoconazole
(U.S. Pat. Nos. 4,144,346 and 4,223,036), liarozole, irtemazol,
clotrimazole, miconazole, econazole, butoconazole, oxiconazole,
sulconazole, tioconazole, and terconazole, substituted thiazoles,
thiadiazole, oxadiazole, caspofungin, amphotericin B, nystatin,
pimaricin, flucytosine (5-fluorocytosine), naftifine, terbinafine,
butenafine, thiocarbonate tolnaftate, griseofulvin, amiodarone,
ciclopirox, sulbentine, amorolfine, clioquinol, gentian violet,
potassium iodide, sodium thiosulfate, carbol-fuchsin solution, and
the echinocandins (e.g. caspofungin acetate, micafungin and
anidulafungin).
[0157] The IL-23 and IL-23R antagonists of the present invention
may be used in combination with standard antifungal agents at their
usual dosages when used as single agents, or at lower dosages if
there is any synergistic enhancement in efficacy when the drugs are
used together. Fluconazole may be administered, e.g., at 400-800
mg/day. Voriconazole may be administered at 4 mg/kg bid.
Itraconazole may be administered at 200-600 mg/day. Amphotericin B
desoxycholate (D-AmB) may be administered at 0.5-1 mg/kg/day.
General guidance as to the types of agents and treatment regimens
that may be combined with the compositions and methods of the
present invention may be found in practice guidelines published by
the Infectious Diseases Society of America (IDSA) at Pappas et al.
(2004) Clin. Infect. Dis. 38:161 (candidiasis) and Stevens et al.
(2000) Clin. Infect. Dis. 30:696 (aspergillosis). Practice
guidelines for the treatment of tuberculosis are found at
International Standards for Tuberculosis Care, published Mar. 22,
2006 and endorsed by the IDSA.
[0158] In some embodiments of the present invention the subject
having an infection, or suspected to have an infection, has been
previously treated for the infection using other methods or
compositions (i.e. not methods or compositions of the present
invention). The previous treatment may include treatment with any
of the antimicrobial agents, antibiotics, antifungal agents,
antiviral agents disclosed herein, or any other treatment method or
composition.
[0159] In some embodiments the subject will have a formal diagnosis
of infection, optionally with an identification of the etiological
agent, but in other embodiments the subject may not have a formal
diagnosis, or may have a partial diagnosis limiting but not fully
identifying the etiological agent. In other embodiments the subject
is only suspected of having an infection. In other embodiments the
subject is at risk of having or acquiring an infection, e.g. the
subject is undergoing immunosuppressive therapy, is at risk of
acquiring a fungal infection because of AIDS, etc. In some
embodiments the subject having an infection, or suspected to have
an infection, or at risk of having or acquiring an infection, is
immunocompromised, e.g. due to AIDS, chemotherapy, transplant, old
age.
[0160] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
EXAMPLE 1
General Methods
[0161] Standard methods in molecular biology are described
(Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;
Sambrook and Russell (2001) Molecular Cloning, 3.sup.rd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0162] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described. Coligan et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York. Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described. See, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies is described. Coligan et al. (2001) Current
Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1998).
Standard techniques for characterizing ligand/receptor interactions
are available. See, e.g., Coligan et al. (2001) Current Protcols in
Immunology, Vol. 4, John Wiley, Inc., New York.
[0163] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry,
2.sup.nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical
Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0164] Standard methods of histology of the immune system are
described. See, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams,
and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text
and Atlas, McGraw-Hill, New York, N.Y.
[0165] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available. See, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne, et al. (2000) Bioinformatics 16: 741-742; Menne et al.
(2000) Bioinformatics Applications Note 16:741; Wren et al. (2002)
Comput. Methods Programs Biomed. 68:177; von Heijne (1983) Eur. J.
Biochem. 133:17; von Heijne (1986) Nucleic Acids Res. 14:4683.
EXAMPLE 2
Fungal Infection Model
[0166] Mouse strains for the study of fungal infections were
obtained as follows. Female C57BL/6 and BALB/c mice, 8-10 wk old,
were purchased from Charles River (Calco, Italy). Homozygous
IL-12p35-, IL-23p19- or IL-12p40-deficient mice (hereafter referred
to as p35.sup.-/-, p19.sup.-/- and p40.sup.-/-, respectively),
TLR-2-, TLR-4-, MyD88- or TRIF-deficient mice (hereafter referred
to as TLR-2.sup.-/-, TLR-4.sup.-/-, MyD88.sup.-/- or TRIF.sup.-/-)
mice on C57BL/6 background were bred under specific pathogen-free
conditions at the Animal Facility of Perugia University, Perugia,
Italy. Breeding pairs of IFN-.gamma..sup.-/-/p35.sup.-/- mice, on
BALB/c background, were provided by Dr. M. Colombo (Istituto
Tumori, Milan, Italy). IFN-.gamma..sup.-/- and IL-4.sup.-/- mice,
on BALB/c background, were also bred at the Animal Facility of
Perugia University. Experiments were performed according to the
Italian Approved Animal Welfare Assurance A-3143-01.
[0167] Fungal infections and their treatments were studied as
follows. The origin and characteristics of the C. albicans strain
used in this study have already been described. Bacci et al. (2002)
J. Immunol. 168:2904. For gastrointestinal infection, 10.sup.8
Candida cells were injected intragastrically and quantification of
fungal growth was expressed as CFU per organ (mean.+-.SE) as
described. Bacci et al. (2002). For the intravenous infection, mice
received different amounts of the fungus in 0.5 ml, intravenously.
The strain of A. fumigatus and the culture conditions were as
described. Montagnoli et al. (2006) J. Immunol. 176:1712. Mice
received two doses of 2.times.10.sup.7 Aspergillus resting conidia
intranasally. Fungi were suspended in endotoxin-free solutions
(Detoxi-gel, Pierce, Rockford, Ill.) at <1.0 EU/ml, as
determined by the Limulus amebocyte lysate (LAL) method. Fungal
growth was quantified by the chitin assay, with results expressed
as micrograms of glucosamine/organ. For histology, tissues were
excised and immediately fixed in formalin, and sections (3-4 .mu.m)
of paraffin-embedded tissues were stained with periodic acid-Schiff
reagent and examined. Bacci et al. (2002); Montagnoli et al.
(2006). Infected animals were treated with 200 .mu.g of
p19-neutralizing Ab (Belladonna et al. (2006) Cytokine 34:161) or
IL-17A-neutralizing mAb (TC11-18H10, PharMingen, San Diego, Calif.)
administered i.p. five hours after infection. A total of 1 mg of
purified anti-TGF-.beta.1, -.beta.2, -.beta.3 mAb .quadrature.(2G7)
(Lucas et al. (1990) J. Immunol. 145:1415) was administered i.p. 5
and 24 h after the infection. Control mice were injected with PBS
because no differences were observed between PBS-treated and
isotype control-treated (each treatment) animals (n>6 for each
group).
[0168] Cells were purified as follows. Gr-1+ CD11b+
polymorphonuclear neutrophils (PMN, >98% pure on FACS analysis)
were isolated from the peritoneal cavity of mice by
magnetic-activating sorting using Ly-6G MicroBeads and MidiMacs
(Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+ T cells were
purified from the mesenteric lymph nodes (MLN), thoracic lymph
nodes (TLN) and spleens by magnetic-activated sorting using CD4
MicroBeads and MidiMacs (Miltenyi Biotech). DC were obtained from
bone marrow cells cultured in Iscove's modified medium in the
presence of 150 U/ml mouse rGM-CSF (Sigma-Aldrich, St. Louis, Mo.)
and 75 U/ml rIL-4 (R&D Systems, Minneapolis, Minn.) for 7 days
to obtain CD11b+ DC or 200 ng/ml FLT3-L (R&D Systems) for 9
days to obtain FL-DC. Romani et al. (2006) Blood 108:2265. Splenic
DC (>99% CD11c+ and <0.1% CD3+) consisting of 90-95% CD8-,
5-10% CD8+, and 1-5% B220+ cells) were purified by magnetic
activated sorting using CD11c MicroBeads and MidiMacs (Miltenyi
Biotech). Zymosan from Saccharomyces cerevisiae (10 .mu.g/ml,
Sigma-Aldrich), ultra-pure LPS from Salmonella minnesota Re 595 (10
.mu.g/ml, Labogen, Rho, Milan, Italy) and CpG oligonucleotides 2006
(CpGODN, 0.06 .mu.M) were used as described. Bellocchio et al.
(2004) J. Immunol. 173:7406.
[0169] DC cells were pulsed and cultured as follows. DC were
exposed to live unopsonized fungi, with and without 10 ng/ml
cytokines (from R&D Systems; Space Import-Export srl, Milan,
Italy; and BD Biosciences-PharMingen, San Diego, Calif.) or
neutralizing antibodies (10 .mu.g/ml), at a 1:1 cell:fungus ratio,
as described. Bacci et al. (2002); Montagnoli et al. (2006). Cells
were harvested for RT-PCR at 12 h of culture, and supernatants were
assessed for cytokine contents by ELISA. Splenic CD4+T cells
(10.sup.6/ml) were cultured in flat-bottomed 96-well plates in the
presence of 5.times.10.sup.5 Candida-pulsed splenic DC for 5 days,
with and without neutralizing antibodies (10 .mu.g/ml), before
cytokines quantification in culture supernatants. Unfractionated
MLN or TLN cells were cultured with inactivated fungi as described
(Montagnoli et al. (2006); Montagnoli et al. (2002) J. Immunol.
169:6298) before cytokine determination in culture supernatants 5
days later.
EXAMPLE 3
Antifungal Activity Assays
[0170] Assays of PMN phagocytosis of unopsonized Candida yeasts or
Aspergillus conidia, and fungicidal activity, were conducted as
described. Bellocchio et al. (2004). Results are expressed as the
percentage of CFU inhibition (mean.+-.SE). PMN were exposed to
varying concentrations of IL-17 or IL-23 or to 50 ng/ml
IFN-.gamma..+-.IL-23/IL-17 (100 ng/ml) for 12 h before western
blotting for IDO or for 60 min before the addition of fungi for an
additional 60 min for studies of fungicidal activity and MMP9/MPO
(mouse myeloperoxidase) determination. Gelatin zymography was
performed as described. Bellocchio et al. (2004). Gelatinolytic
activity of matrix metalloproteinase 9 (MMP9) was determined by
scanning the lysis band in the 72-kD area. For MPO determination,
samples were probed with rabbit polyclonal anti-human MPO Ab
(Calbiochem, San Diego, Calif.) and visualized using
electrochemiluminescence (ECL) (Amersham Pharmacia Biotech,
Piscataway, N.J.).
[0171] Indoleamine 2,3-dioxygenase (IDO) was detected by
immunoblotting with rabbit polyclonal IDO-specific antibody, as
described. Bozza et al. (2005). The positive control consisted of
IDO-expressing MC24 transfectants and the negative control was
mock-transfected MC22 cells.
[0172] Cytokines were quantified by real-time RT-PCR, ELISA and
ELISPOT assays, as follows. Real-time RT-PCR was performed using
the iCycler iQ.RTM. detection system (Bio-Rad, Hercules, Calif.)
and SYBR.RTM. Green chemistry (Finnzymes Oy, Espoo, Finland). Cells
were lysed and total RNA was extracted using RNeasy Mini Kit
(QIAGEN S.p.A., Milano, Italy) and was reverse transcribed with
Sensiscript Reverse Transcriptase (QIAGEN) according to the
manufacturer's directions. PCR primers were obtained from
Invitrogen (Carlsbad, Calif.). The PCR primers used were: [0173]
forward primer, 5'-CACCCTTGCCCTCCTAAACC (SEQ ID NO: 1), and [0174]
reverse primer, 5'-CAAGGCACAGGGTCATCATC (SEQ ID NO: 2), for mouse
IL-12p35; [0175] forward primer, 5'-CCAGCAGCTCTCTCGGAATC (SEQ ID
NO: 3), and [0176] reverse primer 5'-TCATATGTCCCGCTGGTGC (SEQ ID
NO: 4), for mouse IL-23p19; [0177] forward primer,
5'-CTTCTTAACAGCACGTCCTGG (SEQ ID NO: 5), and [0178] reverse primer
5'-GGTCTCAGATCTCGCAGGTCA (SEQ ID NO: 6), for IL-12R.beta.2; [0179]
forward primer, 5'-TGAAAGAGACCCTACATCCCTTGA (SEQ ID NO: 7), and
[0180] reverse primer 5'-CAGAAAATTGGAAGTTGGGATATGTT (SEQ ID NO: 8),
for IL-23R; [0181] forward primer, 5'-CGCAAAGACCTGTATGCCAAT (SEQ ID
NO: 9), and [0182] reverse primer, 5'-GGGCTGTGATCTCCTTCTGC (SEQ ID
NO: 10) for mouse .gamma.-actin.
[0183] PCR amplification of the housekeeping .gamma.-actin gene was
performed for each sample (triplicates) to control for sample
loading and allow normalization between samples as per the
manufacturer's instructions (Applied Biosystems, Foster City,
Calif.). Water controls were included to ensure specificity. The
thermal profile for SYBR.RTM. Green real time PCR was at 95.degree.
C. for 3 min, followed by 40 cycles of denaturation for 15 s at
95.degree. C. and an annealing/extension step of 1 min at
60.degree. C. Each data point was examined for integrity by
analysis of the amplification plot. The mRNA-normalized data were
expressed as relative cytokine mRNA in treated cells compared to
that of mock-infected cells. Cytokine content was assessed by
enzyme-linked immunosorbent assays (R&D Systems and, for IL-23,
eBioscience, Societa Italiana Chimici, Rome, Italy) on tissue
homogenates or supernatants of cultured cells. The detection limits
(pg/ml) of the assays were <16 for IL-12p70, <30 for IL-23,
<10 for IFN-.gamma., <3 for IL-10, <10 for IL-17 and
<4,6 for TGF-.beta.1. AID EliSpot assay kits (Amplimedical,
Buttigliera Alta, Turin, Italy) were used on purified MLN CD4+ T
cells co-cultured with Candida-pulsed DC for 3 days to enumerate
cytokine-producing cells.
[0184] Statistical analysis of the data was performed as follows.
The log-rank test was used for paired data analysis of the
Kaplan-Meier survival curves. Student's t-test or analysis of
variance (ANOVA) and Bonferroni's test were used to determine the
statistical significance of differences in organ clearance and in
vitro assays. Significance was defined asp <0.05. The data
reported are either from one representative experiment out of three
independent experiments or pooled from three to five experiments.
The in vivo groups consisted of 6-8 mice/group.
EXAMPLE 4
Role of IL-23/IL-17 in the Susceptibility to Candidiasis
[0185] To evaluate the contribution of the IL-23/IL-17 pathway to
C. albicans infection, we compared p19.sup.-/-, p35.sup.-/-,
p40.sup.-/- and C57BL/6 mice for susceptibility to gastrointestinal
infection in terms of survival, fungal growth, and tissue
pathology, as well as for parameters of inflammatory and adaptive
Th1/Th17 immunity. The results (FIGS. 1A-E) showed that resistance
to candidiasis was severely impaired in p35.sup.-/- mice, more than
50% of which succumbed to the infection (FIG. 1A) with an elevated
fungal growth in the stomach (FIG. 1B). In contrast, the ability to
restrict the fungal growth was greatly increased in p19.sup.-/-
mice as compared to C57BL/6 mice three and ten days after the
infection. Notably, p40.sup.-/- mice, deficient in both IL-12 and
IL-23, were less susceptible than p35.sup.-/- mice and more
susceptible than p19.sup.-/- mice to candidiasis, emphasizing the
differential roles of IL-12 for control of Candida. Mencacci et al.
(1998) J. Immunol. 161:6228. Similar results were observed after
intravenous infection of p35.sup.-/- and p19.sup.-/- mice, with a
mean survival time (MST) of 6.+-.2 versus 20.+-.3 days
(5.times.10.sup.5 fungal cell inoculum), and 4.+-.2 versus 15.+-.3
days (10.sup.6 fungal cell inoculum), respectively.
Histopathological examination of the stomach revealed the presence
of parakeratosis, acanthosis and limited inflammatory reaction in
C57BL/6, p19.sup.-/- or p40.sup.-/- mice, although p40.sup.-/-, and
in particular p19.sup.-/- mice, showed infiltrates of mononuclear
cells. In contrast, numerous fungal hyphae were present in the
keratinized layer in association with a massive infiltrate of PMN,
signs of epithelial necrosis and prominent acanthosis in the
stomach of p35.sup.-/- mice. These results suggest that the IL-23
and IL-12 pathways have divergent roles in candidiasis.
[0186] To correlate these findings with IL-12/Th1 and IL-23/Th17
immune responses, mice were assessed for p35, p19, IL-12R.beta.2
and IL-23R mRNA expression in MLN one or three days after the
infection (FIG. 1C), and frequencies of IFN-.gamma.-, IL-4- or
IL-17-producing CD4+ cells in MLN at day seven after infection
(FIG. 1D). We found increased levels p35 and IL-12R.beta.2, and
numbers of IFN-.gamma.+ cells, in p19.sup.-/- mice compared to
C57BL/6 mice demonstrating augmented IL-12/Th1 responses in the
absence of IL-23. In contrast, levels of p19 and IL-23R and number
of IL-17-producing cells were enhanced in mice lacking IL-12
(p35.sup.-/-). Expectedly, the number of IL-4-producing cells was
also considerably enhanced in p35.sup.-/- mice. These data
demonstrate a predominant Th1 response promoted by IL-12 and
limited by IL-23 in wild-type C57BL/6 mice. IL-12 suppresses IL-23
and IL-17 production, and vice versa--IL-23 inhibits IL-12 and
IFN-.gamma. .quadrature. production--indicating cross-regulation of
IL-23/Th17 and IL-12/Th1 pathways. These data suggest that an
elevated IL-23/Th17 response renders mice highly susceptible to
candidiasis.
EXAMPLE 5
Role of IL-23/IL-17 in the Susceptibility to Aspergillosis
[0187] To determine whether, similar to candidiasis, the activation
of the IL-23/IL-17 pathway correlates with susceptibility to
aspergillosis, p19.sup.-/-, p35.sup.-/-, p40.sup.-/- or C57BL/6
mice were assessed for susceptibility to pulmonary aspergillosis
and parameters of inflammatory and adaptive Th1/Th17 immunity. The
results (FIG. 2A) show that the fungal burden was reduced in
p35.sup.-/- mice, and to an even greater extent in p19.sup.-/-
mice, suggesting that IL-12, and especially IL-23, inhibit control
of (i.e. promote) Aspergillus infection. Histopathological
examination of the lung revealed the presence of a mild
inflammatory pathology in C57BL/6, p40.sup.-/- or p19.sup.-/- mice,
characterized by few infiltrates of inflammatory mononuclear cells
scattered in an otherwise intact lung parenchyma. Although the
number of infiltrating mononuclear cells was higher in p19.sup.-/-
mice, no signs of parenchyma destruction were observed. In
contrast, a massive infiltration of PMN (about 8-10 fold increase
of Gr1+CD11c-PMN) was present in the lungs of p35.sup.-/- mice
associated with signs of extensive interstitial pneumonia. Similar
to infection with Candida, expression of IL-12 and IL-23 and their
respective receptors were crossregulated, with upregulation of p35
and IL-12R.beta.2 in TLN of p19.sup.-/- mice, and upregulation of
p19 and IL-23R in TLN of p35.sup.-/- mice, compared to C57BL/6 mice
(FIG. 2B). In contrast, absence of both IL-12 and IL-23 in
p40.sup.-/- mice did not significantly alter expression of p35 and
p19 or their receptors IL-12R.beta.2 and IL-23R. Moreover, number
of IFN-.gamma.+ and IL-17+ producing CD4+ T cells was increased in
p19.sup.-/- and p35.sup.-/- mice, respectively, at day 7 after
infection (data not shown). In the lung, the levels of IL-12p70
were much higher in p19.sup.-/- (554.+-.44 pg/ml) than in C57BL/6
mice (68.+-.8 pg/ml), and IL-23 could be detected only in
p35.sup.-/- mice (79.+-.11). IL-17 was increased in p35.sup.-/-
mice (246.+-.17 pg/ml) compared to C57BL/6 mice (37.+-.7 pg/ml).
These data suggest that a heightened IL-23/IL-17-dependent
inflammatory response is also associated with susceptibility to
aspergillosis.
EXAMPLE 6
Role of TGF-.beta. in IL-23/IL-17-mediated Susceptibility to Fungal
Infection
[0188] To study the role of IL-17 in susceptibility to fungal
infections, we treated mice with an anti-IL-17 neutralizing
antibody shortly after fungal infection. Blockade of IL-17 greatly
increased resistance to both C. albicans and A. fumigatus, as
judged by a decreased fungal growth (FIG. 3A), tissue inflammation
and PMN infiltration (data not shown) in the relevant target
organs. Resistance was associated with an increased frequency of
IFN-.gamma.+Th1 cells and a decreased frequency of Th17 cells,
resulting in reduced amounts of IL-17 secreted by MLN cells (FIG.
3B). Similarly, neutralization of IL-23 by antibody increased
resistance to fungal infection and Th1 development and confirm our
data obtained in p19.sup.-/- mice (FIG. 3B). These results clearly
demonstrate that the IL-23/IL-17 pathway confers susceptibility to
fungal infection by inhibition of protective Th1 immunity.
[0189] Recent results suggest that TGF-.beta. together with IL-6
promotes Th17 development. Bettelli and Kuchroo (2005) J. Exp. Med.
201:169; Mangan et al. (2006) Nature 441:231; Veldhoen et al.
(2006) Immunity 24:179. We assessed the effect of TGF-.beta. on Th
cell development and fungal control in mice by treatment with
TGF-.beta.-neutralizing antibody. Notably, TGF-.beta. inhibition
did not affect development of IL-17 producing cells during both C.
albicans and A. fumigatus infection (FIG. 3B), and a slight but
significant reduction in fungal burden was observed only in mice
with Aspergillus but not with Candida (FIG. 3A) but in neither
infection was activation of CD4+Th17 cells affected by treatment.
Because TGF-.beta. neutralization effectively reduced TGF-.beta.
production in infections (from 46 to 24 pg/ml in the stomach and
from 36 to 15 pg/ml in the lung), we conclude that TGF-.beta. plays
a minor role in Th17-mediated susceptibility to fungal
infection.
EXAMPLE 7
Role of IL-23/IL-17 in Fungal Infection in the Absence of IL-12
[0190] The above data would suggest that one possible mechanism
through which the IL-23/IL-17 axis determines susceptibility to
fungal infections relies on the relative ability to restrain
protective Th1 responses. To formally prove it, blockade of IL-23
was done under conditions of either heightened (IL-4.sup.-/- mice)
or deficient (IFN-.gamma..sup.-/- mice) Th1 reactivity. Mice were
intragastrically infected with C. albicans and subjected to IL-23
blockade by means of neutralizing antibodies. Consistent with prior
publications (Romani et al. (1992) J. Exp. Med. 176:19; Cenci et
al. (1998) J. Immunol. 161:3543), the fungal load was lower in
IL-4.sup.-/- and higher in IFN-.gamma..sup.-/- mice compared to
BALB/c mice, demonstrating the importance of IFN-.gamma. for
control of infection. Similar to WT mice, blockade of IL-23 greatly
decreased the fungal burden in the stomach of IL-4.sup.-/- mice
(FIG. 3C) and concomitantly increased the IL-12p70/IFN-.gamma.
production in MLN (data not shown), suggesting that both the Th2
and IL-23/Th17 pathway additively antagonize protective antifungal
responses. Surprisingly, the elevated fungal burden in
IFN-.gamma..sup.-/- mice was further increased upon neutralization
of IL-23 (FIG. 3C), which resulted in decreased IL-23 production
(229 versus 21 pg/ml) IL-17 production (279 versus 95 pg/ml) in
anti-IL-23 treated mice. Thus, IL-23 can have a protective role in
fungal infection in the absence of IFN-.gamma.. However, IL-23 has
the opposite effect in the absence of IL-12p70, or in the absence
of both IL-12p70 and IFN-.gamma., .quadrature..quadrature. as
demonstrated by reduced fungal burden upon neutralization of IL-23
in p35.sup.-/- or doubly deficient IFN-.gamma..sup.-/-/p35.sup.-/-
mice (FIG. 3C). These data suggest that the protective role of
IL-23 in the absence of IFN-.gamma. is mediated by IL-12p70.
Notably, a moderate protective role of IL-23 in the absence of
IL-12p70 was also observed in tuberculosis, where IL-23 partially
replaced IL-12p70 in the induction of protective
IFN-.gamma.-producing CD4+ T cells. Khader et al. (2005).
EXAMPLE 8
Production of IL-23 and IL-12 in Dendritic Cells in Response to
Fungal Infection
[0191] It has already been shown that IL-23 is produced by human DC
in response to Aspergillus in vitro. Gafa et al. (2006) Infect.
Immun. 74:1480. We evaluated here whether IL-23 is produced by DC
in response to C. albicans and how it relates to the production of
IL-12 and IL-10, two cytokines essentially required for the
induction of protective tolerance to the fungus. Romani &
Puccetti (2006).
[0192] For this purpose, we generated bone marrow derived DCs in
the presence of either GM-CSF (GM-DC) or Flt3-L (FL-DC), which
share characteristics of myeloid DC and plasmacytoid DC,
respectively. Although FL-DC encompasses populations equivalent to
mixtures of freshly harvested splenic CD8+, CD8- and B220+LyC6+
plasmacytoid DC (Naik et al. (2005) J. Immunol. 174:6592), we have
recently demonstrated that the functional activity of FL-DC resides
in plasmacytoid DC or in the combination of CD8- and CD8+ DC.
Romani et al. (2006) Blood 108:2265. DC were stimulated in vitro
with yeasts or hyphae of the fungus and assessed for cytokine mRNA
expression and production. Zymosan and LPS were used as positive
controls of GM-DC and CpG-ODN as a positive control of FL-DC.
[0193] The results showed a dichotomy in the cytokine expression
and production by the two subsets of DC subsets in response to the
fungus. RT-PCR analysis revealed that p19 mRNA expression only
increased in GM-DC in response to yeasts more than hyphae; p35 mRNA
expression slightly increased in GM-DC in response to yeasts but,
similar to IL-10, greatly increased in FL-DC exposed to hyphae
(FIG. 4A). The measurement of actual cytokine production in culture
supernatants confirmed that IL-23 was produced by GM-DC in response
to yeasts, particularly at high fungus:DC ratios, as well as to
zymosan or LPS (FIG. 4B). The maximum level of IL-23 production was
observed at 12 h of incubation (FIG. 4B), and declined thereafter
(data not shown). Conversely, both IL-12p70 and IL-10 were mainly
produced by FL-DC stimulated with Candida hyphae, LPS or CpG-ODN
for 12 h (FIG. 4B) and continued to be elevated thereafter (data
not shown). Together, these data suggest that IL-23 is produced by
myeloid DC in response to the fungus, particularly in condition of
high level fungal growth and earlier than other directive
cytokines. The ability of distinct DC subsets to produce directive
cytokines in response to Candida may thus condition their
antifungal immunity in vivo. As a matter of fact, as already shown
for Aspergillus (Romani et al. (2006) Blood 108:2265),
Candida-pulsed FL-DC conferred protection and Candida-pulsed GM-DC
exacerbated the infection upon adoptive transfer into recipient
mice with candidiasis.
EXAMPLE 9
Cross-regulation of IL-23 and IL-12
[0194] To verify whether IL-12p70 and IL-23 production are
cross-regulated in response to the fungus, we measured IL-12p70 and
IL-23 secretion by splenic DC from p19.sup.-/-, p35.sup.-/- and
C57BL/6 control mice after exposure to either IL-12p70 or IL-23, or
the corresponding neutralizing antibodies. FIG. 4C shows that
IL-12p70 and IL-23 are indeed cross-regulated as the production of
IL-12p70 was higher in p19.sup.-/- DC and that of IL-23 higher in
p35.sup.-/- DC as compared to WT DC. Moreover, the exposure to
either IL-12p70 or IL-23 significantly decreased IL-23 or IL-12p70
secretion, respectively, by WT DC and the reverse was true in
condition of IL-12 or IL-23 neutralization (FIG. 4D). Because
RT-PCR revealed that unstimulated DC express both cytokine
receptors (data not shown), these data suggest the existence of a
paracrine loop by which IL-12p70 and IL-23 production by DC is
reciprocally regulated.
EXAMPLE 10
Role of TLR in IL-23 Production by Dendritic Cells
[0195] To define the possible TLR-dependency of IL-23 production in
response to fungi, we measured IL-23 production in response to
yeasts or conidia by GM-DC generated from TLR-2.sup.-/- or
TLR-4.sup.-/- mice as well as from MyD88.sup.-/- and TRIF.sup.-/-
mice. Akira and Takeda (2004) Nat. Rev. Immunol. 4:499. FIG. 5A
shows that both TLR2 and TLR4 are essential for IL-23 production by
signaling through MyD88, but not TRIF. Notably, IL-23 appeared to
be promoted even in the absence of the TRIF. Therefore, IL-23 is
produced by conventional DC in response to fungi through the
TLR/MyD88-dependent inflammatory pathway.
[0196] To define whether T cells may also regulate IL-23
production, we assessed levels of IL-23 produced in supernatants of
DC cultured with CD4+T cells. The results clearly showed that IL-23
production was-up-regulated in cultures of T cells stimulated with
Candida pulsed-DC from C57BL/6 and particularly p35.sup.-/- mice
(group 3 vs group 6, FIG. 5B), a finding suggesting that activated
T cells may provide a positive feedback loop for amplification of
IL-23 production. In addition, the results of criss-cross
experiments confirmed that IL-23-producing DC were necessary and
sufficient to activate IL-17-producing cells (groups 7 and 8).
Furthermore, neutralization of IL-23 by mAb added to co-cultures of
DC and T cells inhibited IL-17 production (group 4 versus group 5,
FIG. 5C), whereas TGF-.beta. .quadrature. neutralization affected
IFN-.gamma. (group 1 versus group 3) but not IL-17 production
(group 4 versus group 6).
EXAMPLE 11
Effect of IL-23 and IL-17 on Antifungal Effector Functions of
Polymorphonuclear Neutrophils
[0197] PMN are essential in the initiation and execution of the
acute inflammatory response to fungi. Romani (2004). The finding
that PMN were abundantly recruited to sites of infections, together
with early fungal growth in p35.sup.-/- mice, led us to hypothesize
that the IL-23/IL-17-dependent pathway could adversely affect the
anti-fungal effector functions of PMN. We evaluated therefore the
fungicidal activity of PMN from either p19.sup.-/- or p35.sup.-/-
mice, and from WT mice cultured with recombinant IL-23 or IL-17 in
the absence or presence of IFN-.gamma.. The killing activity was
significantly increased in p19.sup.-/- PMN and decreased in
p35.sup.-/- mice as compared to C57BL/6 PMN (FIG. 6A). Before
exposing PMN to these cytokines, we verified whether, similar to
IL-17R (Yao et al. (1995) Immunity 3:811-821), IL-23R was also
expressed on murine PMN. Quantitative RT-PCR revealed that
unstimulated PMN express IL-23R, whose expression was further
increased after stimulation with LPS (data not shown), a finding
suggesting that PMN are also responsive to IL-23.
[0198] Exposure to either cytokine impaired the killing activity of
WT PMN in a dose-dependent manner (FIG. 6B) in the absence and
presence of IFN-.gamma. (FIG. 6C). Therefore, IL-23 and IL-17
negatively regulated the antifungal effector functions of PMN,
which may account for the failure of p35.sup.-/- mice to
efficiently restrict fungal growth. Thus, although IL-17 is a
potent chemoattractant for PMN (Ye et al. (2001) J. Exp. Med.
194:519) such that decreased influx of peripheral PMN to infected
organs accounted for the high susceptibility of IL-17AR-deficient
mice to candidiasis (Huang et al. (2004) J. Infect. Dis. 190:624),
our results also point to a detrimental effect for IL-17 on PMN
function.
EXAMPLE 12
Effect of IL-23 and IL-17 on IDO-Dependent Anti-inflammatory
Program of Polymorphonuclear Neutrophils
[0199] We have already shown that IFN-.gamma.-mediated IDO
activation negatively regulates the inflammatory program of PMN
against Candida, such that IDO blockade resulted in the promotion
of an inflammatory state of PMN. Bozza et al. (2005). MMP-9 and MPO
are typical inflammatory markers that have been proposed to be
activated by IL-17. Kolls and Linden (2004) Immunity 21:467.
Therefore we evaluated the effects of both IL-23 and IL-17 on C.
albicans induced MMP-9, MPO and IFN-.gamma.-mediated IDO
production. Both IL-23 and, in particular, IL-17 increased MMP-9
and MPO considerably (FIG. 6D). IDO expression and inflammatory
response of WT PMN. In contrast, both cytokines completely
antagonized the induction of IDO by IFN-.gamma. (FIG. 6E).
Interestingly, the number of apoptotic PMN was significantly
decreased upon the exposure to both IL-23 and IL-17 (data not
shown), suggesting that these cytokines also enhance PMN viability.
This could be a further mechanism by which inflammation is
perpetuated by the Th17 pathway. Therefore, the ability to subvert
the inflammatory program of PMN along with the increased net
proteolytic load in inflamed tissues may account for the
inflammatory pathology associated with Th17 cell activation in
fungal infections.
EXAMPLE 13
Assay for IL-23-Specific Antagonists Based on IL-17 Production
[0200] In vitro studies using murine draining lymph node (DLN)
cells have demonstrated that eliminating IL-23 inhibits or
eliminates IL-17 producing cells, while adding IL-23 generates or
stimulates IL-17 secretion, as determined by fluorescence activated
cell sorting (FACS.RTM.) analysis. See WO 2004/071517, Langrish et
al. (2005) J. Exp. Med. 201:233. See also Aggarwal et al. (2003) J.
Biol. Chem. 278:1910. In these experiments, DLN cells were treated
with cytokine or antibodies for 5 days. Cells were isolated from
antigen-primed normal wild type mice, and cultured in the presence
of either rIL-12 or rIL-23. Analysis of the CD4' T cells in the DLN
cultures demonstrated that IL-12 promoted the development of
IFN-.gamma. producing cells, with loss of the IL-17 producing
population. In contrast, IL-23 promoted the development of IL-17
producing cells, with loss of the IFN-.gamma. producing population.
Anti-p19 antibodies reduced IL-17 production but did not affect
IFN-.gamma. levels, whereas anti-p35 antibodies did not change
IL-17 production. Taken together these results showed that IL-23
selectively promotes the development of IL-17 producing CD4' T
cells.
[0201] This difference in the biological activities of IL-23 and
IL-12 is used to assess the potency and specificity of potential
IL-23 antagonists, relative to IL-12, as follows.
[0202] The baseline data on IL-23 and IL-12 activity in the absence
of a potential IL-23-specific antagonist are obtained as follows.
Normal wild type SJL mice are immunized (s.c.) with proteolipid
peptide (PLP) emulsified in complete Freund's adjuvant, and with
(i.v.) pertussis toxin. Draining lymph nodes are removed at day 9
post-immunization, and mononuclear cells are either assessed for
intracellular IFN-.gamma. and IL-17 production right away (as
described below), or isolated and cultured in the presence of PLP
plus either rIL-12 or rIL-23 for 5 days. Cells are stimulated for 3
hours with PMA (50 ng/ml)/ionomycin (500 ng/ml) in the presence of
Golgi-plug for 4 h, then surface stained for CD4, permeabilized,
and intracellular stained for IFN-.gamma. and IL-17. Flow cytometry
plots are gated on alive CD4' T cells.
[0203] The effects of IL-23 and IL-12 are evaluated relative to the
control cells that were not cytokine treated. Typically, IL-23
treated cells will exhibit an increased percentage of IL-17
producing cells with no increase in IFN-.gamma. producing cells,
whereas IL-12 treated cells will exhibit an increased percentage of
IFN-.gamma. producing cells with no increase (or even a decrease)
in IL-12 producing cells.
[0204] The potency and specificity of a potential IL-23-specific
antagonist is determined by performing the same experiment in the
presence of the antagonist, or preferably at a series of
concentrations of antagonist. An IL-23 specific antagonist will
inhibit the activity of IL-23 (i.e. the antagonist will decrease
the percentage of IL-17 producing cells that would otherwise be
induced by IL-23), but not substantially reduce the activity of
IL-12. An agent that inhibits the activity of IL-12 or both IL-12
and IL-23 is not an IL-23-specific antagonist.
[0205] Optionally, a positive control may be included in which a
known anti-p19 antagonist antibody is used to specifically inhibit
the activity of IL-23.
EXAMPLE 14
Mycobacterial Infections
[0206] A method of demonstrating the efficacy of the compositions
and methods of the present invention in the treatment of
mycobacterial infections is provided. C57BL/6 mice are infected
with mycobacteria as follows. Theracys-BCG Live (Aventis Pasteur,
Inc., Swiftwater, Pa.), a freeze-dried preparation of the Connaught
strain of Bacille Calmette and Guerin and attenuated strain of M.
bovis, is reconstituted as recommended by the manufacturer. The
reconstituted bacteria are brought to a concentration of
approximately 6.times.10.sup.7 cfu/mL in 10% glycerol saline.
Aliquots are diluted to appropriate concentration in 0.02%
Tween-80/0.9% saline prior to injection into mice.
[0207] Six to eight week old female C57BL/6 mice are infected
intravenously via the lateral tail vein with approximately
3.5.times.10.sup.5 cfu of BCG. Mice are given 1 mg of the
appropriate monoclonal antibody (e.g. isotype control, anti
IL-23p19, or anti IL-23R) in 0.9% saline, administered
subcutaneously, one day prior to mycobacteria infection and again
1-2 weeks post mycobacteria infection. Mice are sacrificed at
appropriate time points after infection by CO.sub.2 narcosis.
[0208] The sacrificed BCG infected mice are analyzed as follows.
Blood is purged from the lungs by perfusing RPMI 1640 through the
right ventricle of the heart after the inferior vena cava is
severed. The left lung, the lower right liver lobe, and half the
spleen are aseptically removed. The tissues are homogenized in 0.9%
NaCl/0.02% Tween 80 with a Mini-Bead Beater-8 homogenizer (BioSpec
Products, Bartlesville, Okla.). Viable mycobacteria are quantitated
by plating 10-fold serial dilutions of organ homogenates onto 7H10
Middlebrook agar plates (Becton Dickinson, Sparks, Md.).
Colony-forming units (CFU) are manually counted after two weeks of
incubation at 37.degree. C. A statistically significant decrease in
bacterial burden (as measured by CFU) in animals treated with
anti-IL-23 antibodies (e.g. anti-IL-23p19 antibodies or anti-IL-23R
antibodies) as compared with control mice (e.g. isotype control) is
evidence of efficacious treatment of mycobacterial infection.
Sequence CWU 1
1
10120DNAMus musculus 1cacccttgcc ctcctaaacc 20220DNAMus musculus
2caaggcacag ggtcatcatc 20320DNAMus musculus 3ccagcagctc tctcggaatc
20419DNAMus musculus 4tcatatgtcc cgctggtgc 19521DNAMus musculus
5cttcttaaca gcacgtcctg g 21621DNAMus musculus 6ggtctcagat
ctcgcaggtc a 21724DNAMus musculus 7tgaaagagac cctacatccc ttga
24826DNAMus musculus 8cagaaaattg gaagttggga tatgtt 26921DNAMus
musculus 9cgcaaagacc tgtatgccaa t 211020DNAMus musculus
10gggctgtgat ctccttctgc 20
* * * * *