U.S. patent application number 11/703058 was filed with the patent office on 2008-01-31 for potentiation of the immune response.
Invention is credited to Brigitte T. Huber, Tracy Schmitz, Robert Underwood.
Application Number | 20080026007 11/703058 |
Document ID | / |
Family ID | 25313198 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080026007 |
Kind Code |
A1 |
Huber; Brigitte T. ; et
al. |
January 31, 2008 |
Potentiation of the immune response
Abstract
A method for stimulating proliferation of T-cells containing
cytoplasmic post-prolyl dipeptidase activity; the method, in one
aspect, involves contacting the T-cells with an organic compound at
a concentration below 10.sup.-8M, wherein the compound is
characterized in that: (a) it is capable of crossing the membrane
of T-cells to enter the cytoplasm, (b) it binds to the dipeptidase
activity at a concentration of below 10.sup.-8M, and thus (c)
stimulates proliferation of the T-cells at that concentration.
Inventors: |
Huber; Brigitte T.; (Boston,
MA) ; Schmitz; Tracy; (Cambridge, MA) ;
Underwood; Robert; (Quincy, MA) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
25313198 |
Appl. No.: |
11/703058 |
Filed: |
February 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10778667 |
Feb 13, 2004 |
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11703058 |
Feb 6, 2007 |
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09491855 |
Jan 26, 2000 |
6692753 |
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10778667 |
Feb 13, 2004 |
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08852395 |
May 7, 1997 |
6040145 |
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09491855 |
Jan 26, 2000 |
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Current U.S.
Class: |
424/206.1 ;
424/204.1; 424/208.1; 424/231.1; 424/234.1; 424/257.1; 424/269.1;
424/272.1 |
Current CPC
Class: |
A61P 37/04 20180101;
Y02A 50/30 20180101; A61K 2039/55511 20130101; Y02A 50/487
20180101; A61K 38/55 20130101; G01N 2333/96433 20130101; Y02A
50/412 20180101; A61P 31/18 20180101; A61K 39/39 20130101; A61P
43/00 20180101; G01N 2333/16 20130101; A61K 2039/55516
20130101 |
Class at
Publication: |
424/206.1 ;
424/204.1; 424/208.1; 424/231.1; 424/234.1; 424/257.1; 424/269.1;
424/272.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 43/00 20060101 A61P043/00 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under NIH
grant No. AI36696, and the Government therefore has certain rights
in this invention.
Claims
1-19. (canceled)
20. A method for stimulating an antigen-specific immune response in
a subject comprising administering to an HIV-negative subject in
need thereof a pathogenic peptide antigen and a compound having the
structure ##STR2## wherein each D.sub.1 and D.sub.2, independently,
is a hydroxyl group or a group which is capable of being hydrolyzed
to a hydroxyl group in aqueous solution at physiological pH; and X
comprises an amino acid or a peptide which mimics the site of the
substrate recognized by a post-prolyl cleaving enzyme.
21. The method of claim 20, wherein the pathogenic peptide antigen
is a viral peptide antigen.
22. The method of claim 21, wherein the viral peptide antigen is
derived from HIV, influenza virus, human papilloma virus, and
herpes virus.
23. The method of claim 20, wherein the pathogenic peptide antigen
is a bacterial peptide antigen.
24. The method of claim 23, wherein the bacterial peptide antigen
is derived from E. coli.
25. The method of claim 20, wherein the pathogenic peptide antigen
is a protozoan peptide antigen.
26. The method of claim 25, wherein the protozoan peptide antigen
is derived from a malaria causing agent or an amoebic dysentery
causing agent.
27. The method of claim 20, wherein the subject has an infection
other than HIV infection.
28. The method of claim 20, wherein the subject is at risk of
developing an infection.
29. The method of claim 27, wherein the infection is selected from
the group consisting of a bacterial infection, a viral infection or
a protozoan infection.
30. The method of claim 28, wherein the infection is selected from
the group consisting of a bacterial infection, a viral infection or
a protozoan infection.
31. The method of claim 20, wherein the compound is administered
orally.
32. The method of claim 20, wherein the compound is selected from
the group consisting of Ala-boroPro, Pro-boroPro, Gly-boroPro and
Lys-boroPro.
33. The method of claim 20, wherein the compound is
Val-boroPro.
34. The method of claim 20, wherein the antigen-specific immune
response is a cytolytic T lymphocyte response.
35. The method of claim 20, wherein X is an amino acid.
36. The method of claim 20, wherein X is a peptide.
Description
RELATED APPLICATIONS
[0001] This application is a continuation and claims priority under
35 U.S.C. .sctn. 120 to application U.S. Ser. No. 09/491,855, filed
January 26, entitled, "Potentiation of the Immune Response,"
pending, which is a divisional application and claims priority
under 35 U.S.C. .sctn. 120 to U.S. Ser. No. 08/852,395, filed May
7, 1997, entitled "Potentiation of the Immune Response," now issued
as U.S. Pat. No. 6,040,145, the entire contents of each of which
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] This invention relates to treatment of viral infections
using organic compounds which interact with T-cell enzymes.
[0004] One of the classic markers of full-blown AIDS resulting from
long-term infection with HIV-1 is a severe depletion of CD4.sup.+
T-cells, which are a key component of the immune system. Attempts
have been made to increase the CD4.sup.+ counts of AIDS patients,
and some of these efforts, notably treatment with protease
inhibitors, have met with considerable success. Other approaches,
e.g., stimulation of the immune response by vaccination with viral
peptides, have been less successful. The reasons for CD4.sup.+
depletion in AIDS, and resistance of CD4.sup.+ cells to stimulation
by some therapies are not fully understood.
SUMMARY OF THE INVENTION
[0005] We have discovered that the activation state of human
T-cells can be affected by compounds which interact with a
cytoplasmic post-prolyl dipeptidase activity which has similarities
to, but is distinct from, the membrane-bound T-cell serine protease
CD26. The compounds useful in the invention are inhibitors of this
activity, which is, in naturally-occurring T-cells in healthy
individuals, involved in protection of T-cells from apoptosis, or
programmed cell death. Thus, in high concentrations, the inhibitors
hasten the death of T-cells, by inhibiting the protective enzyme.
We have discovered, surprisingly, that at low concentrations the
inhibitors exhibit a paradoxical effect: they are potent
stimulators of T-cell activity in HIV-infected individuals. The
concentrations of inhibitor which induce this T-cell stimulatory
response at very low (on the order of 10.sup.-8-10.sup.-12M), and
therefore the inhibitors can be used with minimal side effects,
even if, in larger doses, the inhibitors would be toxic.
[0006] Our hypothesis is that the resistance to full activation
observed in T-cells of HIV-infected individuals involves a blocking
of the cytoplasmic enzymatic activity discussed above. We believe
that this blocking of activation, involving this cytoplasmic
activity, prevents differentiation of T-cells of HIV-infected
individuals into effector cells, eventually leading to T-cell
death.
[0007] Thus, the invention features a method for stimulating
proliferation of T-cells of a human patient suffering from a
disease state characterized by the inability of the patients'
T-cells to respond normally to T-cell proliferation-inducing
stimuli; the method involves contacting the T-cells with an organic
compound at a concentration below 10.sup.-8M, wherein the compound
is characterized in that it binds to the post-prolyl cleaving
dipeptidase activity present in the cytoplasm of human T-cells,
e.g., CD4.sup.+ cells or Jurkat cells.
[0008] Treatment according to the invention can be in vitro or in
vivo. In in vivo therapy, the enzyme-interacting compound of the
invention is administered such that the blood concentration in the
patient (e.g., an HIV-infected patient) is below 10.sup.-11. The
compounds can also be used in vitro at low concentrations to
stimulate proliferation of non-infected, beneficial T-cells, such
as CD4.sup.+ cells and CTL's. In this embodiment, PBMC are isolated
from a patient and incubated with a concentration of lower than
10.sup.-8M of the compound, to bring about proliferation of
T-cells, which are then reinfased into the patient.
[0009] We believe that administration of low concentrations of the
inhibitors of the invention may have an allosteric effect such that
the T-cell cytoplasmic enzyme, which is a multimeric (i.e.,
multiple subunit) enzyme, exhibits an increased affinity of the
enzyme for its natural substrate or ligands, allowing the
previously blocked T-cell to proceed to full activation, and hence
survival, proliferation, and interleukin-2 production. Stimulation
of the T-cell immune response in HIV-infected patients according to
the invention yields increased numbers of immune effector cells,
which can fight both HP, itself and other opportunistic
pathogens.
[0010] Treatment according to the invention has the advantages of
specificity and low toxicity, not just because of the low
concentrations of inhibitor which can be used, but also because, in
T-cells of patients not infected with a virus such as HIV, the
inhibitors have no discernable effect. Furthermore, treatment
according to the invention advantageously does not necessarily
require in vitro manipulation of the T-cells from HIV-infected
patients. Furthermore, no immunization is required, and treatment
will be effective even where HIV proteins have mutated because the
therapy targets a cellular enzyme. The fact that, in T-cells
treated according to the invention in vitro, no increase in the
level of the HIV protein p24 is observed, probably indicates that
the T-cells which are infected with HIV are not stimulated by the
low dose inhibitor treatment of the invention.
[0011] The invention also permits immunization of HIV-infected
patients with, e.g., HIV peptides. Under normal circumstances, such
patients cannot be vaccinated because of the defect in the T-cell
stimulation pathway. Use of inhibitors in low doses as adjuvants
can render T-cells responsive to vaccination with HIV antigens, in
particular peptides.
[0012] Treatment of HIV-infected patients with low doses of
inhibitors according to the invention can also enhance the activity
of other AIDS drugs, in particular protease inhibitors. We have
found that treatment according to the invention generally fails to
bring about an increase in CD4.sup.- count in patients whose
CD4.sup.- count is already very low, i.e., below about 400. In such
patients, the CD4.sup.- count can be increased to above this level
using known protease inhibitors, and the newly generated CD4.sup.-
T-cells resulting from such treatment are particularly susceptible
to the stimulatory effects of treatment according to the invention,
leading to an optimal combination AIDS therapy. Preferably, the
drugs are administered orally.
[0013] The low dose administration of inhibitors of the invention
can also be used to produce an adjuvant effect in HIV-negative
individuals, who are to be immunized with peptides or other viral
antigens; this mode of vaccination can be used for prophylaxis for
HIV, as well as any other viral pathogen. Ordinarily, meaningful
cytolytic T-lymphocyte ("CTL") responses, both in vitro and in
vivo, have been difficult to achieve with peptide immunization. The
invention should make it possible to produce significant CTL
responses to viral peptides, e.g., peptides from influenza, HIV,
human papilloma virus, and herpes peptides. This adjuvant effect
can also be used to stimulate CTL responses to peptide antigens
from other pathogens as well, e.g., pathogenic bacteria such as
toxigenic E. coli, and protozoan pathogens such as the pathogens
which are the causative agents of malaria and amoebic dysentery.
The compounds, when used as adjuvants, are preferably administered
orally.
[0014] The invention provides a new and highly advantageous method
of potentiating the immune response in both HIV infected and
uninfected patients, in methods employing extremely low
concentrations of inhibitors which, at these concentrations,
exhibit a paradoxical effect (i.e., they act as stimulatory rather
than inhibitory molecules, as they would at higher concentrations).
The very low concentrations employed according to the invention
allows treatment with minimal side reactions and toxicity. The
specificity of the treatment of the invention also avoids such
adverse effects, which are seen, for example, in treatment with
immune stimulatory compounds such as interleukin-2.
[0015] Other features and advantages of the invention will be
apparent from the following detailed description thereof, and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a pair of graphs showing the lymphocyte
stimulatory effect of treatment of the invention on peripheral
blood mononuclear cells (PBMC) from HIV-infected and uninfected
patients.
[0017] FIG. 2 is a graph illustrating the T-cell stimulatory
effects of two inhibitory compounds used according to the
invention.
[0018] FIGS. 3, 4, and 9 are graphs showing stimulatory effects of
treatment according to the invention in lymphocytes of HIV-infected
patients, compared to treatment using two control compounds.
[0019] FIG. 5 is a graph illustrating a stimulatory effect of an
inhibitor according to the invention on PBMC in vitro, showing the
correlation with CD4.sup.+ counts. The data are plotted as the
natural 10 g of the stimulation index (vertical dimension) versus
the natural log of the CD4.sup.- count of the patient (horizontal
dimension).
[0020] FIG. 6 is a histogram demonstrating that an inhibitor
according to the invention induces dose-dependent apoptosis in
resting T-cells (these dosages are higher than the extremely low
doses used according to the invention).
[0021] FIG. 7 is a histogram demonstrating that an inhibitor
according to the invention induces, at higher doses than in the
invention, dose-dependent apoptosis in both CD26.sup.+ and
CD26.sup.+ populations of PBMC.
[0022] FIG. 8 is a graph showing that an inhibitor of CD26
inhibited the cytoplasmic enzyme as well.
DETAILED DESCRIPTION
Therapeutic Compounds
[0023] Any organic compound can be used according to the invention
which exhibits the following properties: (1) it is capable of
crossing the membrane of human T-cells to reach the cytoplasm,
where the compound can (2) intereact with the cytoplasmic
dipeptidase present in the T-cells, in order to (3) stimulate
activation/proliferation of T-cells (and most preferably CD4.sup.+
cells or CTL's) at concentrations below 10.sup.-8M.
[0024] A simple screening method is described below for the
identification of compounds which are candidate therapeutic
compounds according to the invention.
Substrate and Enzyme Preparation
[0025] The first step is to provide a cytoplasmic enzyme
preparation. The preparation need not be a pure enzyme sample; a
crude cytoplasmic extract is sufficient to screw compounds for the
desired activity. The extract can be prepared from any human T-cell
line which is negative for CD26; an example of such a suitable cell
line is the commercially available Jurkat cell line.
[0026] A suitable enzyme-containing cell extract can be prepared as
follows. First, Jurkat cells (10.sup.6-10.sup.11 cells) are grown
and a cell pellet is obtained by centrifugation. The cell pellet is
stored in frozen condition.
[0027] For use in the assay, the frozen pellet is thawed by the
addition of ice cold lysis buffer, in the amount of approximately 1
ml per 10.sup.8 cells. The liquified material is homogenized with
ten strokes of a Dounce homogenizer, and then clarified by
centrifugation at 1500 g. The supernatant is removed (and saved),
and the 1500 g. pellet is resuspended in lysis buffer and
homogenized with ten strokes of a Dounce homogenizer. Clarification
is again carried out by centrifugation at 1500 g, 4.degree. C.
[0028] The 1500 g supernatants are then combined, and EDTA is added
to 5 mM. The resultant liquid is centrifuged at 75,000 g at
4.degree. C. for twenty minutes, and the supernatant is then
removed and centrifuged at 175,000 g. at 4.degree. C., for 60
minutes. The resultant supernatant, containing the cytosolic
extract, is the DPPV activity-containing preparation used in the
assay, described below, for candidate therapeutic compounds of the
invention.
[0029] The assay is based on our observation that the T-cell
cytoplasmic enzyme of interest is a post-prolyl cleaving serine
protease. We therefore chose as a reporter substrate a compound
which contains proline in the penultimate position; any of a number
of substrates meeting this requirement can be used. In the assay
described herein, we employed a fluorescent cleavage assay using
the substrate AlaProAFC. Alternatively, a colorimetric assay can be
carried out using as a substrate Gly-Pro-pNA. The choice of
terminal amino acid is not critical, provided that the substrate
contain a fire: terminal amino group.
[0030] In the assay we carried out, we employed a fluorescence
spectrometer for excitation at 400 nm and emission at 505 nm. The
spectrometer was calibrated for fluoresence intensity of 0.000=10
mM HEPES, pH 7.4; and fluoresence intensity of 1.000=10 Mm HEPES, 1
.mu.M AFC.
[0031] To carry out the assay, between 10 and 100 .mu.l of enzyme
extact, above, is diluted to 1 ml with 10 mM HEPES, pH 7.4,
containing 10 mM Ala-Pro-AFC. At least one extract/substrate sample
is run without test compound, to provide a standard for comparison
with the test sample.
[0032] In the test samples, multiple samples are run containing
varying concentrations, down to 10.sup.-8M, of the test compound.
The sample (with or without test compound) is placed in a cuvette,
and inserted into a fluoresent spectrometer. Enzymatic activity is
measured as the accumulation of fluoresence intensity (i.e.,
substrate cleavage product) over time (1 min.). A compound is
identified as an inhibitor if the accumulative fluoresence is
decreased as a result of the presence of the inhibiting
compound.
[0033] Once a compound has been identified as an enzymatic
inhibitor, as described above, further assays are carried out to
determine whether the compound is capable of moving across the
T-cell membrane into the cytoplasm; this is an assay which can be
carried out using well-known techniques.
[0034] If desired, additional in vitro assays can be carried out
using candidate compounds of the invention, prior to their use in
vivo. One such assay employs the candidate compound at a very low
concentration, in a test designed to determine whether at low
concentrations the compound can stimulate the proliferation of PBMC
from HIV infected patients in vitro. As is shown in the data of
FIG. 4, stimulation can be measured by, e.g., incorporation of a
labelled nucleotide.
[0035] The compounds can also be tested at higher doses to
determine whether they exhibit the opposite effect of
proliferation, as above, i.e., dose-dependent apoptosis caused by
enzyme inhibition, as in the experiments of FIG. 6.
Candidate Compounds
[0036] As is discussed above, compounds which are potentially
capable of apoptosis induction at high doses and proliferation
induction at low doses are those which, at normal or high doses,
inhibit cytoplasmic T-cell dipeptidase, and can cross the T-cell
membrane into the T-cell cytoplasm, where the enzyme interaction
occurs. The compounds thus should be organic compounds which have a
free amino group at the amino terminus; a proline or proline analog
at the penultimate position; and an enzyme binding site which
mimics the post-prolyl cleavage site of cytoplasmic
dipeptidase.
[0037] A number of known classes of compounds can be screened and
used according to the invention. Once such class are CD26 (i.e.,
DPPIV) inhibitors, including those described in Bachovchin et al.
U.S. Pat. No. 4,935,493, hereby incorporated by reference. In the
'493 patent, there are described compounds having the structure:
##STR1## where each D1 and D2, independently, is a hydroxyl group
or a group which is capable of being hydrolyzed to a hydroxyl group
in aqueous solution at physiological pH; and X comprises an amino
acid or a peptide which mimics the site of the substrate recognized
by a post-prolyl cleaving enzyme.
[0038] The compounds in the '493 patent are inhibitors of CD26, and
are also candidate inhibitors of the invention. As is discussed
above, because of the low concentrations of compounds used
according to the invention, it is acceptable to use, in the
invention, a compound which interacts not only with the cytoplasmic
enzyme, but also CD26.
[0039] The class of compounds described in the '493 patent are also
discussed and exemplified in Takacs et al. U.S. patent application
Ser. No. 07/923,337, corresponding to PCT Application No.
WO94/03055, hereby incorporated by reference. In this application,
one of the families of molecules in the '493 patent is described as
the "Xaa-boroPro molecules." exemplified by Ala-boroPro,
Pro-boroPro, and Gly-boroPro. These Xaa-boroPro molecules are all
candidate compounds for use in the methods of the present
invention. Two of these compounds are used in some of the examples
described below; those compounds are Lys-boroPro ("KPB"), and
Val-boroPro ("VBP").
EXAMPLE 1
[0040] Peripheral blood mononuclear cells (PBMC) were obtained by
standard methods from HIV-infected individuals, and from uninfected
individuals. Varying dosages of KBP or VBP were contacted with the
PBMC in vitro, and stimulation of proliferation was measured by
incorporation of .sup.3H thymidine (cpm). The results of these
experiments are shown in FIG. 1: very low doses of the Val-boroPro
and Lys-boroPro stimulated proliferation of PBMC from HIV-infected
patients, but not PBMC from uninfected patients.
[0041] As shown in FIG. 1, at no concentration of the boroPro
enzyme inhibitor did it affect the PBMC from uninfected
individuals. The inhibitor, at moderate concentrations, also did
not cause proliferation of PBMC from HIV-infected individuals, but
it did cause marked proliferation at very low concentrations
(10.sup.-9 and 10.sup.-10). These results are consistent with our
hypothesis, discussed above, that, at low concentrations, these
enzyme inhibitors exhibit a paradoxical effect: rather than
inhibiting the apoptosis-controlling cytoplasmic T-cell enzyme,
they interact with that enzyme in a manner which blocks
inactivation and causes proliferation of T-cells.
[0042] Concordant results are shown in FIG. 2, a histogram showing
that low doses of Lys-boroPro and Val-boroPro cause proliferation
of PBMC of HIV-infected patients, while higher doses (10.sup.-4M)
do not have this effect.
[0043] The same results are shown in FIGS. 3, 4, 9, and 10 which
also present data for two control compounds OKT3, and PHA, both of
which are non-specific mitogens.
[0044] Referring to FIG. 5, data are presented in a form which
shows that low concentrations of the inhibitors of the invention
have little effect on the PBMC of HIV-infected patients whose CD4
counts are lower than about 400 (the clinical indication for AIDS).
In the graph of FIG. 5, the natural log of the stimulation index
(the vertical axis) is plotted against the natural log of the CD4
count of the patients; as shown, above a count of 400 there is
particularly significant stimulation of proliferation.
[0045] FIG. 6 is a graph demonstrating that purified T-cells are
highly sensitive to cytoplasmic T-cell dipeptidase inhibitors in
moderate concentrations. CD19.sup.- B cells and CD4.sup.-/CD8.sup.-
T-cells were isolated to high purity and incubated overnight in
Val-boroPro. The amount of cell death was determined by 7AAD flow
cytometry analysis. Data represent % of cell death from duplicate
samples. These data are consistent with our hypothesis that the
inhibitors, in moderate concentration, inhibit a cytoplasmic enzyme
which ordinarily protects against apoptosis.
[0046] FIG. 7 presents data demonstrating that CD26.sup.- and
CD26.sup.- PBMC are equally susceptible to T-cell cytoplasmic
enzyme inhibitor-induced death, where the inhibitor is administered
in moderate concentrations. CD26.sup.- and CD26.sup.- populations
were incubated overnight in the presence or absence of various
concentrations of Val-boroPro. The amount of cell death was
determined by 7AAD flow cytometry analysis. Data represent mean %
of death from duplicate samples. These data indicate that
apoptosis-inhibiting T-cell cytoplasmic enzyme is present in both
CD26.sup.- and CD26.sup.- T-cells.
[0047] FIG. 8 presents data showing the effects of an inhibitor
useful in the invention, Val-boroPro. The experiments were carried
out using two preparations: purified DPPIV (i.e., CD26), and Jurkat
T-cell cytoplasmic extract, described above (Jurkat cells contain
the cytoplasmic T-cell enzyme, but do not bear CD26 on their
surfaces). These preparations were incubated with varying
concentrations of Val-boroPro, and enzymatic activity was
determined by measuring the accumulation of the fluorescent
cleavage product 7-amino-4-trifluoromethylcoumarin (AFC) released
from the substrate Ala-ProAFC upon enzymatic cleavage. Val-boroPro
inhibited both the enzyme DPPIV and the cytoplasmic T-cell enzyme
in the Jurkat preparation.
[0048] Other embodiments are within the following claims.
* * * * *