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THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
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A polypeptide derived from gp41, a vaccine composition
comprising said polypeptide, and uses for treating
an infection by an HIV virus in an individual
FIELD OF THE INVENTION
The present invention relates to the field of the in vitro diagnosis of
the progression status of an infection of an individual with a virus
belonging to the family of the Human Immunodeficiency Viruses (HIV) as
well as with the therapeutical treatment of this infectious disease.
BACKGROUND OF THE INVENTION
AIDS disease, which is primarily caused by infection of individuals
with a HIV retrovirus, is now the most devastating disease in the whole
world, since the number of individuals which are, to date, infected with
HIV viruses is estimated to about 40 millions of individuals.
During the sole year 2001, 5 millions of individuals were infected
with HIV while 3 millions of individuals have deceased in the same time.
Since the discovery of the main AIDS causative agent in 1983, namely
the HIV virus, extensive efforts have been made in order to understand
the mechanism of action of this virus and to develop accurate methods
for (i) reproducibly diagnosing the infection, as well as (ii) carrying out a
prognosis of the progression of the disease in a given patient.
For surveillance purposes, the United States Centers for Disease
Control (CDC) currently defines AIDS in an adult or adolescent age 13
years or older as the presence of one of 25 AIDS-indicator conditions,
such as KS, PCP or disseminated MAC. In children younger than 13
years, the definition of AIDS is similar to that in adolescents and adults,
except that lymphoid interstitial pneumonitis and recurrent bacterial
infections are included in the list of AIDS-defining conditions (CDC,
1987b). The case definition in adults and adolescents was expanded in
1993 to include HIV infection in an individual with a CD4+ T cell count
less than 200 cells per cubic millimeter (mm3) of blood (CDC, 1992). The
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current surveillance definition replaced criteria published in 1987 that
were based on clinical conditions and evidence of HIV infection but not
on CD4+ T cell determinations (CDC, 1987).
In clinical practice, symptomatology and measurements of immune
function, notably levels of CD4+ T lymphocytes, are used to guide the
treatment of HIV-infected persons
HIV infects and kills CD4+ T lymphocytes in vitro, although
scientists have developed immortalized T-cell lines in order to propagate
HIV in the laboratory (Popovic et al., 1984; Zagury et al., 1986; Garry,
1989; Clark et al., 1991). Several mechanisms of CD4+ T cell killing have
been observed in lentivirus systems in vitro and may explain the
progressive loss of these cells in HIV-infected individuals (reviewed in
Garry, 1989; Fauci, 1993a; Pantaleo et at., 1993a). These mechanisms
include disruption of the cell membrane as HIV buds from the surface
(Leonard et at., 1988) or the intracellular accumulation of heterodisperse
RNAs and unintegrated DNA (Pauza et al., 1990; Koga et at., 1988).
Evidence also suggests that intracellular complexing of CD4 and viral
envelope products can result in cell killing (Hoxie et at., 1986).
In addition to these direct mechanisms of CD4+ T cell depletion,
indirect mechanisms may result in the death of uninfected CD4+ T cells
(reviewed in Fauci, 1993a; Pantaleo et at., 1993a). Uninfected cells often
fuse with infected cells, resulting in giant cells called syncytia that have
been associated with the cytopathic effect of HIV in vitro (Sodroski et al.,
1986; Lifson et al., 1986). Uninfected cells also may be killed when free
gp120, the envelope protein of HIV, binds to their surfaces, marking them
for destruction by antibody-dependent cellular cytotoxicity responses
(Lyerly et al., 1987). Other autoimmune phenomena may also contribute
to CD4+ T cell death since HIV envelope proteins share some degree of
homology with certain major histocompatibility complex type II (MHC-II)
molecules (Golding et at., 1989; Koenig et at., 1988).
=
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A number of investigators have suggested that superantigens,
either encoded by HIV or derived from unrelated agents, may trigger
massive stimulation and expansion of CD4+ T cells, ultimately leading to
depletion or anergy of these cells (Janeway, 1991; Hugin et at., 1991).
The untimely induction of a form of programmed cell death called
apoptosis has been proposed as an additional mechanism for CD4+ T
cell loss in HIV infection (Ameisen and Capron, 1991; Terai et at., 1991;
Laurent-Crawford et al., 1991). Recent reports indicate that apoptosis
occurs to a greater extent in HIV-infected individuals than in non-infected
io persons, both in the peripheral blood and lymph nodes (Finkel et
al.,
1995; Pantaleo and Fauci, 1995b; Muro-Cacho et at., 1995).
It has also been observed that HIV infects precursors of CD4+ T
cells in the bone marrow and thymus and damages the
microenvironment of these organs necessary for the optimal sustenance
is and maturation of progenitor cells (Schnittman et al., 1990b;
Stanley et
at., 1992). These findings may help explain the lack of regeneration of
the CD4+ T cell pool in patients with AIDS (Fauci, 1993a).
Recent studies have demonstrated a substantial viral burden and
active viral replication in both the peripheral blood and lymphoid tissues
20 even early in HIV infection (Fox et al., 1989; Coombs et at., 1989;
Ho et
at., 1989; Michael et al., 1992; Bagnarelli et al., 1992; Pantaleo et al.,
1993b; Embretson et at., 1993; Piatak et al., 1993). One group has
reported that 25 percent of CD4+ T cells in the lymph nodes of HIV-
infected individuals harbor HIV DNA early in the course of disease
25 (Embretson et at., 1993). Other data suggest that HIV infection is
sustained by a dynamic process involving continuous rounds of new viral
infection and the destruction and replacement of over 1 billion CD4+ T
cells per day (Wei et al., 1995; Ho et al., 1995).
Concerning the prognosis of progression of the disease in HIV-
30 infected patients, a first current method consists of evaluating the
increase in the number of HIV viruses which are present in a whole blood
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sample collected from a patient, for example by performing conventional
immunoassays with antibodies specifically directed against HIV proteins,
and more specifically against the HIV capsid glycoprotein gp120.
A second current method for the prognosis of progression of AIDS
in a patient consists of measuring the number of copies of the HIV
genome which is found in a whole blood sample collected from that
patient, for example through performing a quantitative PCR amplification
of the nucleic acids contained in said sample, using one or several
nucleic acid primer(s) that specifically hybridise with the HIV genomic
lo RNA.
These two methods above are useful, since numerous studies
have shown that people with high levels of HIV in their blood stream are
more likely to develop new AIDS-related symptom or die than individuals
with lower levels of the virus.
A third current method for the prognosis of progression of AIDS in
a patient consists of measuring the absolute CD4+ T-cell levels in whole
blood samples from infected patients (HIV patients), for example by
carrying out flow cytometry from a blood sample of that patient, using a
labelled antibody directed against the CD4 antigen.
All of these prognosis methods above can reproducibly be used
but also have their respective technical limits, in relation with, for
example, their biological significance as regards the evolution of the
disease.
The use of antibodies for evaluating the number of HIV viral
particles present in a biological sample form a patient comprise
drawbacks due to the specificity of the antibodies which are used, since it
is well known that the HIV structural proteins produced by distinct HIV
virus isolates significantly differ in their antigenic properties and that
false
negative results may thus be generated.
The measure of the number of copies of the HIV genome in a
biological sample from a patient is indeed indicative that the provirus
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which has integrated within the infected individual's cell genome has
entered into active replication cycles and that the disease is in active
progression. However, this technique does not simultaneously reflect the
patient's immune response against the virus progression.
5 The measure
of the CD4+ T-cell levels in a patient is also
indicative of the disease progression, since the pathogenesis of acquired
immunodeficiency syndrome (AIDS) is largely attribuable to the decrease
in T-lymphocytes bearing the CD4 receptor (CD4+). Progressive
depletion of CD4+ T-lymphocytes is associated with an increase of
clinical complications. Because of this association, the measurement of
CD4+ T-cell levels is used to establish decision points for monitoring the
relevance of treatments against AIDS. CD4+ T-Iymphocyte levels are also
used as prognostic indicators in patients with human immunodeficiency
virus (HIV) disease.
However, the measure of the CD4+ T-cell levels in a patient does
not directly reflect the immunological status of the patient, excepted as
regards the resulting immunodeficiency. Notably the measure of the
CD4+ T-cell levels does not account for the status of the possible
biological effectors that cause or mediate the observed CD4+ depletion,
and thus of the possible biological effectors that cause this observed
patient's immunodeficiency.
Indeed, it may also be mentioned that a forecast of the
progression of AIDS, in a given patient infected with HIV, can also be
carried out through the detection of mutations occurring in the amino acid
sequence of known co-receptors for HIV that are expressed by the
patient's cells, especially CD4+ cells, such as the CCR5 co-receptor,
since it has been observed that HIV-infected people bearing a specific
mutation in one of their two copies encoding the CCR5 co-receptor may
have a slower disease course that people with two normal copies of this
gene.
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However, there remains a need in the art for additional methods
that will allow the one skilled in the art to determine the status of
progression of AIDS in patients who have been infected with a HIV virus
so as to enable a more precise prognosis of the evolution of the disease,
including the occurrence of, or the evolution of, the numerous well known
AIDS-related diseases, and also to enable a more precise monitoring of
the therapeutical treatment which may be the more beneficial to the HIV-
infected patient, once taken into account the progression status of the
AIDS disease. For example, there is a need in the art for novel biological
io markers which are indicative of the progression of AIDS, which should
preferably be of biological relevance as regards the biology of the HIV
infection, such as, for example, novel biological markers of relevance as
regards the immunological status of the patient tested.
Indeed, these novel biological markers might be used in
combination with one or several already known markers such as those
cited above.
Further, there is still a need in the art for novel therapeutically
useful compounds for preventing individuals from the occurrence of AIDS
upon infection with a HIV virus or, more generally, for treating patients
infected with a HIV virus. Particularly, in the definition of novel anti-HIV
multi-therapies or HAART ("Highly Active Anti-retroviral Therapy"), there
is a need to include novel pharmaceutically active compounds that will
specifically be directed against other target molecules than the HIV
protease and the HIV retrotranscriptase and which will act on targets
involved in distinct stages of the disease. Notably, there is a need in the
art for novel compounds of pharmaceutical interest that are biologically
active in HIV-infected patients wherein HIV has begun to actively
replicate, especially in HIV-infected patient which are close to undergo a
decrease in the number of their CD4+ T-cells and who are thus
susceptible to immunodeficiency, as well as in HIV-infected patients for
whom the depletion of their CD4+ 1-cells has already begun.
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SUMMARY OF THE INVENTION
The invention is firstly directed to a polypeptide comprising
the following amino acid sequence:
Xi X2X3X4X5X6SWS NKSX7X8X9XioXi I (I),
wherein X1, X2, X3, X5, X6, X7, X97 X10, and X11 mean, independently one
from each other, any amino acid residue, X4 means any amino acid
residue except A and W, and wherein X8 means any amino acid
residue except E and S.
It also relates to a pharmaceutical composition for preventing or
treating a disease linked to the infection of an individual with a virus of
the HIV family, which comprises an effective amount of a ligand
compound which specifically binds to the polypeptide (I) in combination
with at least one physiologically acceptable excipient.
The invention also deals with in vitro methods for the screening of
compounds for preventing or treating a disease linked with the infection
of an individual with an HIV virus, wherein said method comprises the
steps of :
(i) incubating a candidate compound to be tested with a polypeptide
as described above,
(ii) assaying for the binding of the candidate compound to be tested
with a polypeptide as described above.
The invention is also directed to a vaccine composition comprising
a polypeptide (I) and an immunoadjuvant compound.
It is also directed to methods for treating HIV-infected patients that
make use of the therapeutically active compounds and of the
pharmaceutical compositions that are further described in the present
specification.
DESCRIPTION OF THE FIGURES
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Figure 1. Over-expression of NKp44L after treatment of purified CD4+ T
cells with vaccinia virus expressing several HIV proteins.
Purified CD4+ T cells were infected with 20 pfu/cell of several
recombinant vaccinia virus expressing HIV protein. Two days later, the
cells were washed twice, and stained with anti-NKp44L mAb (grey thick
line), or with IgM isotype control (blak thin line). The cells were analyzed
by flow cytometry. Ul: Uninfected cells., WT: cells infected with wild type
vaccinia virus. Gag, Pol, gp160, gp120, gp41, Tat, Nef : cells infected
with vaccinia virus, expressing respectively Gag, Pol, gp160, gp120,
gp41, Tat, or Nef. The percentage of NKp44L expression was noted for
each panel.
Abscissa : NKp44L expression, Ordinates: Number of cells.
Figure 2. Over-expression of NKp44L after treatment of purified CD4+ T
cells with recombinant gp160 HIV protein.
One million of cells were incubated with 5 ug/m1 of control protein
(Ctl; black circle), or recombinant gp160 protein (gp160-A : black triangle)
; (gp160-B: black square) or without protein (UT: untreated cells) during 2
days in presence of 10 U/ml 1L2.
' 2A) The cells were washed and stained with anti-NKp44L mAb
and CD4 mAb or with isotype controls and analyzed by flow cytometry.
The percentage of NKp44L expression in CD4+ T cells was noted for
each panel. Abscissa : CD4 expression, Ordinates: NKp44L expression.
2B) NK-lysis sensitivity of CD4+ T cells incubated with
recombinant gp160 HIV protein was analyzed for cytotoxic activity with
activated autologous purified NK cells. NK lysis activity was performed at
different effetctor/target (E/T) ratios (Abscissa). Open diamonds with
dotted lines: Unteated cells; Closed bottoms: Control protein-treated
cells; Closed triangles: gp160-A-treated cells; and closed squares:
gpl 60-B-treated cells. Ordinates : Specific NK lysis (%).
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Figure 3. One pool of peptides from the HIV qp41 protein both induced
an higher sensitivity to NK lysis and an over-expression of NKp44L.
One million of purified CD4+ T cells were treated with 5 ug/ml of
pools of peptides from HIV gp41 protein (noted from A to J) or from
gp120 protein (gp120), as control. Each pool of peptides included 10
peptides, as described in Material and Methods section. The cells were
incubed two days in presence-of 10 u/ml IL2, and then washed twice.
3A) NK-lysis sensitivity of CD4+ T cells incubated with the
different pools of peptides was analyzed for cytotoxic activity with
activated autologous purified NK cells. NK lysis activity was performed at
different effetctor/target (E/T) ratios (Abscissa). Ordinates : Specific NK
lysis (%).
3B) The cells were stained with anti-NKp44L mAb and CD4 mAb
or with isotype controls and analyzed by flow cytometry. In this panel of
figures, the results were only done for the untreated cells (none) or the
cells treated with polls of peptides from gp120 or from the gp41 (polls C
and J). The percentage of NKp44L expression in CD4+ T cells was noted
for each panel. For the other pools a low expression of NKp44L, ranged
from 0.2 to 1.3 %, was observed. Abscissa: NKp44L expression,
Ordinates: CD4 expression.
Figure 4. Analysis of each peptide from the pool C derived from HIV
qp41 protein.
One million of purified CD4+ T cells were treated with 5 ug/ml of
peptides from the pool C (see figure 6) (noted from C141 to 0150) or as
controls the peptide gp41-E162 or the peptide gp120-87. The cells were
incubed two days in presence of 10 u/ml IL2, and then washed twice.
4A) Killing pattern of CD4+ T cells incubated with the different
peptides were tested for their sensitivity to NK. Data are shown for an
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E/T ratio of 40/1 with activated autologous purified NK effector cells.
Ordinates : Specific NK lysis (%).
4B) The cells were stained with anti-NKp44L mAb and 004 mAb
or with isotype controls and analyzed by flow cytometry. Ordinates :
5 Expression of NKp44L.
Figure 5. Drastic role of the NH2-SWSNKS-000H motif expressed by
the gp41 HIV protein.
5A) Sequences of the peptide gp41-C147 (wild type : WT) and
10 two different control peptides included some modification just inside
the
"SWSNKS" motif (control 1 : CtI1) or in all of the 15-mers sequence
(control 2: CtI2).
One million of purified CD4+ T cells were treated with 1 ug/ml of highly
purified WT peptide or with the both control peptides (0tI1 and CtI2). The
cells were incubed two days in presence of 10 u/ml IL2, and then washed
twice.
5B) NK-lysis sensitivity of CD4+ T cells incubated with the
different peptides was analyzed for cytotoxic activity with activated
autologous purified NK cells. NK lysis activity was performed at different
effetctor/target (E/T) ratios (Abscissa). Open diamonds with dotted lines:
Unteated cells; Closed bottoms: WT peptide-treated cells; Closed
squares: CtI1-peptide-treated cells, and Closed triangles: CtI2-peptide-
treated cells. Ordinates : Specific NK lysis (%).
5C) The cells were stained with anti-NKp44L mAb and CD4 mAb
or with isotype controls and analyzed by flow cytometry. The percentage
of NKp44L expression in CD4+ T cells was noted for each panel.
Abscissa: NKp44L expression, Ordinates: CD4 expression.
Figure 6. Kinetics studies of NK lysis activity and NKp44L expression
after addition of the "active SWSNKS" peptide.
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One million of purified CD4+ T cells were treated with 1 pg/ml of
highly purified wild type (WT) peptide or with the both control peptides
(CtI1 and CtI2) during several times ranged from 0 to 2880 min. After
incubation, the cells were washed twice and then analyzed for cytotoxic
activity with activated autologous purified NK cells. NK lysis activity was
performed at different effetctor/target (E/T) ratios 9A). NK cytotoxic
activity was performed after pretreatment of cell with 1Oug/m1 of anti-
NK44L mAb (B). Flow cytometry analysis revealed the cell surface
expression of NKp44L (A), and for the intra-cellular expression of
NKp44L (B). Open diamonds with dotted lines: Unteated cells; Closed
bottoms: WT peptide-treated cells; Closed squares: Ct11-peptide-treated
cells, and Closed triangles: CtI2-peptide-treated cells; Open bottoms with
dotted line : WT-peptide-treatment cells after pretreatment with anti-
NKp44L mAb and Open squares with dotted line: CtI1-peptide-treated
cells after pretreatment with anti-Nkp44L mAb.
Figure 7. Cell surface expression of NKp44L of different human cells
Cell surface expression of NKp44L of K562, Jurkat, and resting
PBMC. The cells were incubated with 1 pg/ml of anti-NKp44L mAb anti-
NKp44L mAb (grey thick line) or with the IgM isotype control (black thin),
and analyzed by flow cytometry. Abscissa : NKp44L expression,
Ordinates: number of cells
Figure 8. Identification of #7.1, an anti-NKp44L mAb that specifically
inhibits NKp44-mediated NK lysis.
(A) Cell surface expression of NCR-Ig fusion proteins. Uninfected (UI)
and HIV-1-infected (Sf2) U2 cells were incubated with 10 pg/ml of
NKp30-1g (30-Ig), NKp46-Ig (46-Ig) or NKp44-Ig (44-Ig) fusion proteins
(black lines) or with the Ig control (dotted lines) and analyzed by flow
cytometry. Frequency of NKp44-Ig expression is noted. (B) Cell surface
expression of NKp44L detected with #7.1 mAb. Uninfected (UI) and HIV-
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1-infected (Sf2) U2 cells were incubated with 1 pg/ml of #7.1 mAb (black
line) or with the IgM isotype control (dotted line) and analyzed by flow
cytometry. (C) #7.1 mAb inhibits NKp44-Ig binding. HIV-1-infected U2
cells that had previously been stained with either 10!mg of NKp44-Ig
fusion protein (lower panel; black line) or control NKp46-Ig fusion protein
(upper panel; black line) were incubated with #7.1 mAb (gray line) or IgM
isotype control (dotted line) for 1 lh at 4 C and washed twice in 1% PBS-
BSA. The cells were then analyzed by flow cytometry. Percentage of
cells expressing NKp44L, after NCR-Ig incubation, is noted. (D) Inhibition
of natural cytotoxicity by #7.1 mAb. Purified NK cells, cultured in the
presence of 100 U/ml IL2, were analyzed for cytotoxic activity against
either the uninfected U2 cells and the HIV-1-infected U2 cell line, after
treatment with 101mg/m1 of #7.1 mAb, at different effector/target cell
ratios. Open circles: IgM-isotype control-treated uninfected U2 cells;
Closed circles: #7.1-treated uninfected U2 cells. Open squares: IgM-
isotype controltreated HIV-1-infected U2 cells; Closed squares: #7.1-
treated HIV-1-infected U2 cells. (E) Inhibition of natural cytotoxicity by
#44/8 anti-NKp44 mAb. Purified NK cells, cultured in presence of 100
U/ml IL2, were analyzed for cytotoxic activity against either the
uninfected U2 cells or the HIV-1-infected U2 cell line, after treatment with
10 pg/ml of #44/8 anti-NKp44 mAb, at different effector/target cell ratios.
Open circles: IgG1-isotype control-treated uninfected U2 cells; Closed
circles: #44/8 anti-NKp44 mAbtreated uninfected U2 cells. Open squares:
IgG1-isotype control-treated HIV-1 infected U2 cells; Closed squares:
#44/8 anti-NKp44 mAb-treated HIV-1 infected U2 cells.
Figure 9. Critical role of the NH2-SWSNKS-COOH motif from the
qp41 HIV protein.
(E) NKp44L effects were inhibited with anti-gp41-C146 polyclonal
antibody. One million purified CD4+ T cells from two infected patients
(#CG: 322 CD4+ cells/mm3 and #BT: 208 CD4+ cells/mm3) were treated
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overnight with several concentrations of anti-gp41-C146 antibody. The
cells were stained with the anti-NKp44L mAb and analyzed by flow
cytometry. (F) Inhibition of NK lysis activity in the presence of anti-gp41-
C146 antibody. CD4+ T cells from #CG and #BT samples, incubated with
several concentrations of anti-gp41-C146 antibody, were then analyzed
for cytotoxic activity with IL2-activated autologous purified NK cells. Open
circles: Untreated cells; Closed squares, triangles, and diamonds: CD4+
T cells treated with 1, 10, and 20 mg/ml of anti-gp41-C146 antibody,
respectively. Peptide C146 is a polypeptide consisting of the aminoacid
io sequence of formula (II).
DETAILED DESCRIPTION OF THE INVENTION
a) Previous findings of the inventors
It has previously been found by the inventors, that a specific
protein, termed NKp44L is expressed by the CD4+ T-cells form HIV-
infected individuals whereas this protein is not expressed by the CD4+ T-
cells from individuals which are not infected with HIV. The NKp44L
protein is not expressed (i) in peripheral blood mononuclear cells (PBMC)
from HIV-infected patients that do not express the CD3 antigen, (ii) in
PBMC form HIV-infected patients that express the CD3 antigen but not
the CD4 antigen, nor (iii) in PBMC from HIV-infected patients expressing
the CD8 antigen. Particularly, the expression level of the NKp44L protein
is further enhanced in activated CD4+ 1-cells, such as PHA-activated
CD4+ T-cells, from HIV-infected individuals.
Further, it has been previously found by the inventors that an
increasing expression level of the NKp44L protein is correlated with the
decrease in the number of CD4+ T-cells which is observed in HIV-
infected patients, thus in patients undergoing a progression of AIDS.
Consequently, the expression level of the NKp44L protein is indicative of
the immunological status of an HIV-infected patient.
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it has also been found that CD4+ T-cells from HIV-infected
patients, and especially CD4+ T-cells that express the NKp44L protein,
consist of specific targets for their cytolysis by Natural Killer (NK) cells,
particularly activated NK cells, and especially autologous NK cells from
the same patient.
Importantly, the present inventors have shown that the NK cells of
an HIV-infected individual are activated specifically, through a non-MHC
dependent triggering mechanism, by the autologous CD4+ T-cells that
io express the NKp44L protein.
Further, the expression level of the NKp44L protein consists of a
novel biological marker of the state of advancement of the HIV infection
endowed with a very high biological significance, since it has been shown
by the inventors that NKp44L expressed by the CD4+ T-cells triggers the
autologous NK cells and activate these NK cells for specific cytolysis of
the CD4+ T-cells, through a non-MHC dependent recognition of the CD4+
T-cells by the activated NK cells. In this particular context, the NKp44L
protein expressed by the CD4+ T-cells of the HIV-infected patient activate
the NK cells through the specific binding of the NKp44L protein to its
specific receptor counterpart which is expressed at the membrane
surface of the NK cells, namely the NKp44 receptor protein which has
already been described by Canton' et al. (1999) and by Vitale et al.
(1998).
Further, the NKp44L protein has formerly been isolated by another
inventive entity and this protein has already been shown to be expressed
in various kinds of tumour cell lines. Still further, the NKp44L expressed
by certain tumour cells has been shown to be a ligand that specifically
binds to the NKp44 receptor protein cited above, which receptor protein
is expressed by the NK cells, including the activated NK cells. It has also
been formerly shown by this other inventive entity that the NKp44
receptor protein that is expressed by the activated NK cells might be
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responsible for at least part of the tumour cells cytolysis effected by the
activated NK cells (unpublished information).
Taken together, the results obtained by the inventors have allowed
them to carry out various methods which make use of the NKp44L
5 protein as a
novel biologically relevant marker of the disease progression
for individuals that are already diagnosed as having been infected by
HIV.
b) Findings according to the invention
10 The
inventors have now surprisingly found that a specific
polypeptide, derived from the gp41 protein from HIV, markedly enhances
the expression of the Nkp44L protein of sequence SEQ ID N 1, at the
membrane surface of CD4+ T-cells.
The NKp44L protein is encoded by a nucleic acid of sequence
15 SEQ ID N 3.
It has also been determined according to the present invention
that the lysis by the NK cells of the CD4+ T-cells from patients infected
with HIV depends on that specific HIV polypeptide.
HIV-1 gp41 is composed of three domains, an extracellular
domain (ectodomain), a transmembrane domain and an intracellular
domain (endodomain). The gp41 ectodomain contains three major
functional regions, i.e., the fusion peptide located at the N-terminus of
gp41, followed by two 4-3 heptad repeats adjacent to the N- and C-
terminal portions of the gp41 ectodomain, designated NHR (N-terminal
heptad repeat) and CHR (C-terminal heptad repeat), respectively. The N-
and C-terminal repeats are also named as "HR1" and "HR2".
Both NHR and CHR regions function as essential structures
required for conformational changes during the process of membrane
fusion between HIV-1 and CD4+ T cells.
Surprinsingly, the inventors have found that a short peptide,
derived from the gp41 protein, which is located between the well-known
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HR1 and HR2 regions, induces the surface expression of NKp44L on
CD4+T cells.
In other words, the inventors have identified a short peptide
derived from the gp41 protein of HIV, which is responsible for the
NKp44L surface expression and thus also for the lysis of CD4+ T cells by
the endogenous NK cells.
These results obtained by the inventors have allowed them to
carry out screening methods, which make use of a specific peptide
derived from gp41 as a new target for therapeutical agents, distinct from
the well known HR1 and HR2 regions.
Importantly, the present inventors have also shown that the protein
NKp44L is expressed on tumor cell surface and that this expression of
NKp44L is induced or enhanced by said short peptide derived from gp41.
Thus according to the invention, said short peptide derived from
gp41 can be used for expressing NKp44L at the surface of tumor cells
and then induce their specific lysis by NK cells.
Accordingly, the invention concerns therapeutical methods, and
pharmaceutical compositions, comprising a polypeptide as briefly
described above, for manufacturing anti-cancer pharmaceutical
compositions.
In another aspect, the inventors have found that antibodies
directed against a polypeptide derived from the gp41 protein, are
produced during HIV infection of an individual.
More precisely, It has been found, according to the invention a
statistically significant correlation between the expression level of
antibodies directed against a polypeptide derived from gp41, collected
from HIV-infected individuals and the level of CD4+ T cells.
It has also been found that these antibodies inhibits the
cytotoxicity of NK cells towards CD4+ T cells, in patients infected by HIV.
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Accordingly, the invention concerns therapeutical methods, and
vaccine compositions, comprising a polypeptide as briefly described
above.
It has also been found according to the invention that the level of
the antibodies described above, decreases during the progression of HIV
infection, especially as regards the development of the patient's
immunodeficiency caused by the progressive depletion of his CD4+ T
cells.
This result obtained by the inventors has allowed them to carry out
methods which make use of the level of antibodies directed against a
polypeptide derived from gp41, as a novel biologically relevant marker of
the disease progression for individuals that are already diagnosed as
having been infected by HIV.
Polvpeptides according to the invention
Accordingly, an object of the invention is a polypeptide comprising
the following amino acid sequence:
XiX2X3X4X5X6SWSNKSX7X8X9XioXi (I),
wherein X1, Xz X3, X5, X6, X7, X9, X10, and X11 mean, independently one
from each other, any amino acid residue, X4 means any amino acid
residue except A and W, and wherein X8 means any amino acid residue
except E and S.
The term "any amino acid residue" designates any amino acid
residue selected from the group consisting of the following amino acids :
A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
Said polypeptide is also referred to as a polypeptide of formula (1).
The invention encompasses further polypeptides comprising the following
amino acid sequence:
PWASNASWSNKSLDDIW (II).
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In certain embodiments said polypeptide has a length of at least
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or 200 amino
acid residues.
A polypeptide, as defined above, is preferably derived from the
gp41 protein and possesses at least 39, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140 or 150 consecutive amino acids of gp41 protein from HIV-1
and comprises the amino acid sequence of formula (I) above.
The polypeptide of formula (I) can be produced by recombinant
DNA techniques, for example on the basis of the DNA sequence of gp41
io protein from HIV1, by using any of a variety of expression vectors known
to those of ordinary skill in the art. Expression may be achieved in any
appropriate host cell that has been transformed or transfected with an
expression vector containing a DNA molecule that encodes a
recombinant polypeptide. Suitable host cells include prokaryotes, yeast,
and higher eukaryotic cells, such as mammalian cells and plant cells.
Preferably, the host cells employed are E. coil, yeast or a mammalian cell
line such as COS or CHO. Supernatants from suitable host/vector
systems which secrete recombinant protein or polypeptide into culture
media may be first concentrated using a commercially available filter.
Following concentration, the concentrate may be applied to a suitable
purification matrix such as an affinity matrix or an ion exchange resin.
Finally, one or more reverse phase HPLC steps can be employed to
further purify a recombinant polypeptide.
When polypeptide of formula (I) comprises less than about 100
amino acids, and generally less than about 50 amino acids, it can be
generated by synthetic means, using techniques well known to those of
ordinary skill in the art. For example, such polypeptides may be
synthesized using any of the commercially available solid-phase
techniques, such as the Merrifield solid-phase synthesis method, where
amino acids are sequentially added to a growing amino acid chain. See
Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for
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automated synthesis of polypeptides is commercially available from
suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,
Calif.), and may be operated according to the manufacturer's
instructions.
Preferably, a polypeptide of formula (I) consists of the following
amino acid sequence: PWASNASWSNKSLDDIW (II).
The induction of NKp44L expression on CD4+ T-cells surface
induced by the polypypetide of amino acid sequence (II) is illustrated in
examples 1-3 below.
The high kinetics of the induction of the NKp44L expression at the
cell surface is compatible with the induction of a translocation of a pre-
synthesised NKp44L intracellular protein, from the cytoplasm towards the
cell surface.
Pharmaceutical compositions according to the invention
Another object of the invention is a pharmaceutical composition for
preventing or treating a disease linked to the infection of an individual
with a virus of the HIV family, which comprises an effective amount of a
ligand compound which specifically binds to the polypeptide of formula
(I), in combination with at least one physiologically acceptable excipient.
By "physiologically acceptable excipient or carrier" is meant solid
or liquid filler, diluent or substance which may be safely used in systemic
or topical administration. Depending on the particular route of
administration, a variety of pharmaceutically acceptable carriers well
known in the art include solid or liquid fillers, diluents, hydrotropes,
surface active agents, and encapsulating substances. The amount of
carrier employed in conjunction with the F(ab)<sub>2</sub> fragments to provide
practical quantity of material per unit dose of composition.
Pharmaceutically acceptable carriers for systemic administration
that may be incorporated in the composition of the invention include
sugar, starches, cellulose, vegetable oils, buffers, polyols and alginic
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acid. Specific pharmaceutically acceptable carriers are described in the
following documents, all incorporated herein by reference; U.S. Pat. No.
4,401,663, Buckwalter et al. issued August 30, 1983; European Patent
Application No. 089710, LaHann et al. published Sept. 28, 1983; and
5 European Patent Application No. 0068592, Buckwalter et al. published
Jan. 5, 1983. Preferred carriers for parenteral administration include
propylene glycol, pyrrolidone, ethyl oleate, aqueous ethanol, and
combinations thereof.
Representative carriers include acacia, agar, alginates,
10 hydroxyalkylcellulose, hydroxypropyl
methylcellulose,
carboxymethylcellulose, carboxymethylcellulose sodium, carrageenan,
powdered cellulose, guar gum, cholesterol, gelatin, gum agar, gum
arabic, gum karaya, gum ghatti, locust bean gum, octoxynol 9, oleyl
alcohol, pectin, poly(acrylic acid) and its homologs, polyethylene glycol,
15 polyvinyl alcohol, polyacrylamide, sodium lauryl sulfate,
poly(ethylene
oxide), polyvinylpyrrolidone, glycol monostearate, propylene glycol
monostearate, xanthan gum, tragacanth, sorbitan esters, stearyl alcohol,
starch and its modifications. Suitable ranges vary from about 0.5% to
about 1%.
20 For formulating a pharmaceutical composition according to the
invention, the one skilled in the art will advantageously refer to the last
edition of the European pharmacopoeia or of the United States
pharmacopoeia.
Preferably, the one skilled in the art will refer to the fourth edition
"2002" of the European Pharmacopoeia, or also to the edition USP 25-
NF20 of the United States Pharmacopoeia.
The weight amount of therapeutically active compound that is
contained in each dose of the pharmaceutical composition of the
invention will depend on the molecular weight of said therapeutically
active compound as well as on the weight amount that is effective in
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blocking the cytolysis of the CD4+ T-cells by the NK cells in an HIV-
infected patient.
For determining the appropriate amount of the therapeutically
active compound, in a dose of a pharmaceutical composition of the
invention, the one skilled in the art firstly determines the in vitro CD4+ T-
cell cytolysis inhibiting ability of various weight amounts or concentrations
of said therapeutically active compound, for example by performing the
screening method of the invention which are describe below, and then
retain or select the given amount or concentration of said therapeutically
io active compound that blocks cytolysis.
Then, the one skilled in the art transposes said retained or
selected amount or concentration to the in vivo human situation, so that
the concentration of said therapeutically active compound in the blood of
a patient to which the pharmaceutical composition of the invention has
been administered is identical to the concentration that blocks cytolysis in
vitro.
Preferably, the ligand compound consists of an antibody directed
to the polypeptide according to the invention.
Preferably, the ligand compound, or the pharmaceutical
composition containing it, can be combined with a compound that inhibits
the membrane fusion between HIV and CD4+ T cells. Such compounds
are, for example, peptides derived from the HR1 or HR2 region of the
gp41 protein and more precisely peptides referred to as T20, T21 or
those described in US patent application 6,623,741.
The invention concerns also an antibody directed against a
polypeptide of formula (I).
This invention is also directed to the use of a ligand compound
which specifically binds to the polypeptide of formula (I), for
manufacturing a pharmaceutical composition for preventing or treating a
disease linked to the infection of an individual with a virus of the HIV
family.
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Additionally, The inventors have also shown that the protein
NKp44L is expressed on tumor cell surface, such as Jurkat cells and
K562 cells.
They have also shown that a polypeptide of formula (I) induces the
cell surface expression, of NKp44L by tumor cells, which then render
these polypeptide-treated tumor cells susceptible to specific lysis by the
NK cells.
Accordingly, the invention concerns methods and pharmaceutical
compositions, comprising a polypeptide of formula (I), for treating cancer.
The invention concerns a pharmaceutical composition for treating
a cancer, which comprises an effective amount of an antigenic
compound comprising or consisting of a polypeptide of formula (I), in
combination with at least one physiologically acceptable excipient.
Preferably, the physiologically acceptable excipients used to carry
is out the pharmaceutical composition described above are the same than
those that are described in the first part of the specification concerning
ligands of NKp44L.
The invention concerns also a pharmaceutical composition for
treating a cancer, which comprises an effective amount of an antigenic
compound comprising or consisting of a polypeptide of formula (I), fused
to a targeting cancer cells, in combination with at least one
physiologically acceptable excipient.
Preferably, said compound, which targets cancer cells, consists of
an antibody directed to an antigen specific of cancer, such as SOP-I,
NY-ESO-1, or SSX-2 specific of breast cancer, SSX-2, NY-ESO-1, or
MAGE-3 specific of melanoma, described in US patent 6,338,947; or
antigens specific of renal cancers such as those described in US patent
6,440,663; KH-1 and N3 specific of colon cancer, described in US patent
6,238,668.
Immunogenic and vaccine compositions according to the invention
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The inventors have found that antibodies directed against a
polypeptide derived from the gp41 protein, i.e. the polypeptide of formula
(I), are produced during HIV infection of an individual.
More precisely, It has been found, according to the invention a
statistically significant correlation between the expression level of
antibodies directed the polypeptide of formula (I), collected from HIV-
infected individuals and their level of CD4+ T cells.
It has also been found that these antibodies inhibits the
cytotoxicity of NK cells towards CD4+ T cells, in patients infected by HIV.
Further, the inventors have surprisingly found that antibodies
screened for their capacity to inhibit CD4+ T cells NK lysis, react
specifically with NKp44L on HIV-1 infected cells (example 5).
More precisely, when cells chronically infected by HIV-1 are
pretreated with the antibody described above, their NK-mediated lysis
decreased sharply (Fig. 9E), and treatment of NK cells by an anti-NKp44
mAb produced the same effect (Fig. 9F).
NKp44 is a protein having the amino acid sequence SEQ ID N 2,
encoded by a nucleic acid of sequence SEQ ID N 4.
It has also been found that an antibody directed against a
polypeptide of formula (I) above, inhibits NKp44L expression onto CD4+T
cells surface, and by the way, decreases sensitivity to NK lysis.
On the opposite, such a result is not obtain with antibodies
directed against other polypeptides derived from gp41 protein, for
example polypeptides T20 or T21, derived from HR1 or HR2 domains of
gp41.
More precisely, it has been found that NKp44L expression was
substantially lower in purified CD4+ T cells from two HIV-1 infected
patients that were incubated with an antibody directed to the
polynucleotide (1), than in purified and then untreated cells or in those
treated with a control Ab. (example 6, figure 9)
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It has also been found that the serum from patients infected by
HIV, when depleted in antibodies directed against polypeptide (I) is not
able to decrease the CD4+ T cells NK lysis.
These results strongly suggest that the polypeptide of formula (I)
play a key role in inducing NKp44L expression during HIV infection and
that the gp41 protein participates in the selective destruction of CD4+ T
cells by activated NK cells.
Without wishing to be bound to any particular theory, the inventors
believe that HIV-1 has acquired the ability to use NK cells to disarm the
host immune system by selectively triggering CD4+T cells.
Hence, the blockage of NKp44L expression by antibodies directed
against polypeptide (I) could be applied to counteract these deleterious
effects.
To illustrate this hypothesis, the inventors have found that there
exists an inverse relationship between NKp44L expression at the CD4+ T
cell surface and the level of antibodies directed against the polypeptide
derived from gp41, of formula (I).
Accordingly, another object of the invention is a vaccine
composition comprising a polypeptide of formula (I) and an
immunoadjuvant compound.
A further object of the invention is an immunogenic composition
comprising a polypeptide of formula (I), in combination with at least one
physiologically acceptable excipient.
By "immunogenic composition" it is herein intended a substance
which is able to induce an immune response in an individual, and for
example to induce the production of antibodies directed against the
polypeptide of formula (I).
Preferably, said immunoadjuvant compound is selected in the
group consisting of Freund complete adjuvant, Freund incomplete
adjuvant, aluminium hydroxide, calcium phosphate, aluminium
phosphate, potassium phosphate, Cholera toxin (CT) and its B subunit
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(CTB), toxins from Bordetefia pertusssis (PT), labile toxin (LT) from
Escherichia coil, monophosphoryl lipid A, CpG oligonucleotides,
imidazoquinolones, oil in water emulsions, comprising squalene and
synthetic copolymers, muramyl dipeptides and their derivatives, saponins
5 and immunostimulating complexes (ISCOMs), and
dimethyldioctadecylammonium bromide or chloride (DDA).
For example, to promote a Th2-type immune response, said
immunoadjuvant can be selected in the group comprising: aluminium
hydroxide, aluminium phosphate, potassium phosphate, calcium
10 phosphate, or bacteria toxins such as Cholera toxin (CT) and its B
subunit (CTB), toxins from Bordetella pertusssis (PT), or labile toxin (LT)
from Escherichia coll. When a Th1-type response is searched, said
immunoadjuant can be selected in the group comprising:
monophosphoryl lipid A, CpG oligonucleotides, imidazoquinolones. To
15 stimulate preferably an antibody response, oil-based adjuvants such as
oil in water emulsions, comprising squalene and synthetic copolymers
can be used as an immunoadjuvant or also muramyl dipeptides and their
derivatives. To promote a mucosal immune response, polysaccharides
such as dextran, mannans, glucans, or chitosans can be used as an
20 immunoadjuvant. To enhance antibody production and stimulate T
cytotoxic lymphocytes, saponins and immunostimulating complexes
(ISCOMs) can be used as an immunoadjuvant. To induce a humoral and
cellular response, a Freund complete adjuvant can be used as an
immunoadjuvant. To induce an inflammatory response and a high level of
25 antibodies, a Freund uncomplete adjuvant can be used as an
immunoadjuvant. To obtain a delayed hypersensitivity lipophilic amines
such as dimethyldioctadecylammonium bromide or chloride (DDA) can
be used as an immunoadjuvant.
In order to enhance the immunogenicity of the vaccine
composition according to the invention, the polypeptide of formula (I),
can comprise from 2 to 12 peptides of formula "SWSNKS".
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In particular, said antigenic polypeptide, can have the following
formula (III) :
NH2-PepNt-[(l)0-PepXn]n-PepCt- COOH (III),
wherein:
- "PepNt" consists of a polypeptide having an amino acid length varying
from 0 to 100 amino acid residues and located at the N-terminal end of
the polypeptide of formula (III);
- "[(I),-PepXn]" consists of a polypeptide unit wherein:
- "(1)1" to - "(On" each consists of, one independently from each other, a
polypeptide of formula "SWSNKS", with n being an integer from 1 to
12; and
- "PepXi" to "PepXn" each consists of, one independently from the
other, a spacer polypeptide having an amino acid length varying from
0 to 30 amino acid residues, with n being an integer from Ito 12;
is - n is
the number of [(l)n-PepXn] polypeptide units in said polypeptide,
with n being an integer from 1 to 12; and
- "PepCt" consists of a polypeptide having an amino acid length varying
from 0 to 100 amino acid residues and located at the C-terminal end of
the polypeptide of formula (III).
Said antigenic polypeptide can be covalently linked through an
amino acid residue to a carrier protein or a synthetic polymer.
In order to enhance peptide immunogenicity, the peptide of
formula (I) can be covalently linked ("conjugated") to a larger molecule
which serves as a carrier.
Attachment of the peptide to the carrier can be by one of several
methods, including linking through a peptide Lys using glutaraldehyde
(Reichlin, Methods Enzymol. 70: 159-165, 1980) or DCC procedures (for
example, Atassi et al., Biochem. Biophys. Acta 670: 300-302, 1981),
through a Peptide Asp or Glu using DCC (Bauminger et al., Methods
Enzymol 70: 151-159, 1980), through a peptide Tyr using bis-diazotized
benzidine (Walter et al., Proc. Nat. Acad. Sci. USA 77: 5197-5200, 1980),
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through photochemical attachment sites (Parker et al., Cold Spring
Harbor Symposium - Modern Aoproaches to Vaccines, Ed. Chanock &
Lerner, Cold Spring Harbor Press, New York, 1984), or through a peptide
Cys (Liu et al., Biochem. 18: 690-697, 1979).
Peptide carrier conjugates can be separated from excess free
peptide by dialysis or gel filtration. The level of loading of the peptide on
the carrier can be determined either using a radioactive tracer to
establish the loading level in a particular procedure, or by quantitative
amino acid analysis of the conjugate, in comparison with the unloaded
carrier. It is convenient, when using the latter technique, to incorporate a
unique non-natural amino acid into the peptide, at the N-terminal or C-
terminal side, such as Nle, which can then serve as a quantitative marker
for peptide incorporation, as measured by amino acid analysis of the
conjugate. This Nle can also function as a spacer between the antigenic
site and any amino acid incorporated to facilitate attachment, such as
Cys, Lys, or Tyr, as described above.
Preferably, said carrier protein is selected from the group
consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin,
or diphtheria toxoid.
In a vaccine composition according to the invention, said synthetic
polymer can be a multiple branch peptide construction comprising a core
matrix comprised of lysine residues.
Radially branched systems using lysine skeletons in polymers
have been used by J. P. Tam [Proc. Natl. Acad. Sci. U.S.A., 85, 5409-
5413 (1988)] to develop antigens without the use of carriers. Those
antigens were designed to generate vaccines against a variety of
diseases. Specifically, antigens for generating vaccines against HIV
infection are described by Tam in PCT patent application ser. no.
W093/03766, and in US patent Application US5,229,490.
The core matrix is preferably a dendritic polymer which is
branched in nature, preferably with each of the branches thereof being
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identical. The core matrix is based on a core molecule which has at least
two functional groups to which molecular branches having terminal
functional groups are covalently bonded. Exemplary for use to form the
core matrix is lysine. A central lysine residue is bonded to two lysine
residues, each through its carboxyl group, to one of the amino groups of
the central lysine residue. This provides a molecule with four amino
groups, which may be a core matrix for a structure comprising four
polypeptides of formula (I). The manufacture of the above structures, has
been know in the art. See, e.g., Tam et al., J. lmmun. 148, 914-920
to (1992) and Wang et al., Science, 254, 285-288 (1991).
Additionally, spacers between said polypeptide and said carrier
protein or synthetic polymer can be added. A peptide linker sequence
may be employed to separate the first and second polypeptide
components by a distance sufficient to ensure that each polypeptide folds
into its secondary and tertiary structures. Such a peptide linker sequence
is incorporated into the fusion protein using standard techniques well
known in the art. Suitable peptide linker sequences may be chosen
based on the following factors: (1) their ability to adopt a flexible
extended conformation; (2) their inability to adopt a secondary structure
that could interact with functional epitopes on the first and second
polypeptides; and (3) the lack of hydrophobic or charged residues that
might react with the polypeptide functional epitopes. Preferred peptide
linker sequences contain Gly, Asn and Ser residues. Other near neutral
amino acids, such as Thr and Ala may also be used in the linker
sequence. Amino acid sequences which may be usefully employed as
linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985;
Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat.
No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may
generally be from 1 to about 50 amino acids in length. Linker sequences
are not required when the first and second polypeptides have non-
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essential N-terminal amino acid regions that can be used to separate the
functional domains and prevent steric interference.
The invention concerns also a vaccine composition comprising a
polypeptide comprising the amino acid sequence SWSNKS, said
polypeptide, being covalently linked through an amino acid residue to a
carrier protein or to a synthetic polymer.
Preferably, said carrier protein is selected from the group
consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin,
or diphtheria toxoid.
The synthetic polymer can be a multiple branch peptide
construction comprising a core matrix comprised of lysine residues.
Spacers between said polypeptide and said carrier protein or synthetic
polymer can be introduced.
Prefrably, In the vaccine composition cited immediately above,
there are spacers between said polypeptide and said carrier protein or
synthetic polymer.
This invention is also directed to the use of an antibody directed
against a polypeptide of formula (I) for manufacturing a pharmaceutical
composition for treating a disease linked to the infection of an individual
with a virus of the HIV family.
Additional compounds
In a preferred embodiment, the vaccine composition according to
the invention, further comprises one or more components selected from
the group consisting of surfactants, absorption promoters, water
absorbing polymers, substances which inhibit enzymatic degradation,
alcohol, organic solvents, oils, pH controlling agents, preservatives,
osmotic pressure controlling agents, propellants, water and mixture
thereof.
Examples of appropriate supplementary carriers include, but are
not limited to, sterile water, saline, buffers, phosphate-buffered saline,
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buffered sodium chloride, vegetable oils, Minimum Essential Medium
(MEM), MEM with HEPES buffer, etc.
Optionally, the vaccine composition of the invention may contain
conventional, secondary adjuvants in varying amounts depending on the
5 adjuvant and the desired result. The customary amount ranges from
about 0.02% to about 20% by weight, depending upon the other
ingredients and desired effect.
Examples of suitable secondary adjuvants include, but are not
limited to, stabilizers; emulsifiers; aluminum hydroxide; aluminum
io phosphate; pH adjusters such as sodium hydroxide, hydrochloric acid,
etc.; surfactants such as Tween® 80 (polysorbate 80, commercially
available from Sigma Chemical Co., St. Louis, Mo.); liposomes; iscom
adjuvant; synthetic glycopeptides such as murannyl dipeptides; extenders
such as dextran or dextran combinations, for example, with aluminum
is phosphate; carboxypolymethylene; bacterial cell walls such as
mycobacterial cell wall extract; their derivatives such as Corynebacterium
parvum; Propionibacterium acne; Mycobacterium bovis, for example,
Bovine Calmette Guerin (BCG); vaccinia or animal poxvirus proteins;
subviral particle adjuvants such as orbivirus; cholera toxin; N,N-
20 dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine
(avridine);
monophosphoryl lipid A; dimethyldioctadecylammonium bromide (DDA,
commercially available from Kodak, Rochester, N.Y.); synthetics and
mixtures thereof. Desirably, aluminum hydroxide is admixed with other
secondary adjuvants or an immunoadjuvant such as Quil A.
25 Examples of suitable stabilizers include, but are not limited to,
sucrose, gelatin, peptone, digested protein extracts such as NZ-Amine or
NZ-Amine AS. Examples of emulsifiers include, but are not limited to,
mineral oil, vegetable oil, peanut oil and other standard, metabolizable,
non-toxic oils useful for injectables or intranasal vaccines compositions.
30 These adjuvants are identified herein as "secondary" merely to
contrast with the above-described immunoadjuvant compounds.
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Conventional preservatives can be added to the vaccine
composition in effective amounts ranging from about 0.0001% to about
0.1% by weight. Depending on the preservative employed in the
formulation, amounts below or above this range may be useful. Typical
preservatives include, for example, potassium sorbate, sodium
metabisulfite, phenol, methyl paraben, propyl paraben, thimerosal, etc.
The choice of inactivated, modified or other type of vaccine
composition and method of preparation of the improved vaccine
composition formulation of the present invention are known or readily
io determined by those of ordinary skill in the art.
When the polypeptide (I) is not covalently bound to an
immunoadjuvant, in the vaccine composition acording to the invention, a
pharmacologically effective amount of the immunoadjuvant compound
described above may be given, for example orally, parenterally or
otherwise, concurrently with, sequentially to or shortly after the
administration of the polypeptide of formula (I).
As a general rule, the vaccine composition of the present invention
is conveniently administered orally, parenterally (subcutaneously,
intramuscularly, intravenously, intradermally or intraperitoneally),
intrabuccally, intranasally, or transdermally.
In vitro screening and diagnosis methods according to the
invention
a) screening methods
The inventors have surprisingly found that a specific polypeptide,
derived from the gp41 protein of HIV, markedly enhances the expression
of the Nkp44L protein on CD4+ T-cells surface.
The invention also concerns a first method for the in vitro
screening of compounds for preventing or treating a disease linked with
the infection of an individual with an HIV virus, comprising the steps of:
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(i) incubating a candidate compound to be tested with a polypeptide
of formula (I),
(ii) assaying for the binding of the candidate compound to be tested
with a polypeptide of formula (I).
The binding of the candidate compound to the polypeptide of
formula (I) can be carried on by the one skilled in the art, for example by
using a Two-hybrid system. Other means, known from the one skilled in
the art can be used for the binding assays such as the use of bio sensor
techniques (Edwards and Leatherbarrow (1997) or also by Szabo et al.
(1995)), affinity chromatography, or High Throughput Screening (HTS),
(Leblanc et al 2002).
The candidate compounds, which may be screened according to
the screening method above, may be of any kind, including, without
being limited to, natural or synthetic compounds or molecules of
biological origin such as polypeptides.
Preferably, step (ii) consists of subjecting to a gel migration assay
the mixture obtained at the end of step (i) and detecting the complexes
formed between the candidate compound and the polypeptide of formula
(I).
The gel migration assay can be carried out as known by the one
skilled in the art.
The detection of the complexes formed between the complexes
formed between the candidate compound and the polypeptide according
to the invention can be easily observed by determining the stain position
(protein bands) corresponding to the proteins analysed since the
apparent molecular weight of a protein changes if it is part of a complex
with another protein.
On one hand, the stains (protein bands) corresponding to the
proteins submitted to the gel migration assay can be detected by specific
antibodies for example antibodies specifically directed against a
polypeptide of formula (I). One the other hand, a polypeptide of formula
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(I) can be tagged for an easier detection of the protein/candidate
compound on the gel. For example, the polypeptide according to the
invention can be fused to GST, HA, a poly-Histidine chain, or other
detectable molecules in order to facilitate the identification of the
different
proteins on the gel.
The invention further concerns a second method for the in vitro
screening of compounds for preventing or treating a disease linked to the
infection of an individual with an HIV virus, comprising the steps of:
a) (i) bringing into contact a first CD4+ T-cell culture with a candidate
compound, and HIV virus;
(ii) bringing into contact a second CD4+ T-cell culture with HIV virus,
in the absence of said candidate compound ; and
b) detecting the presence of NKp44L at the CD4+ T-cells surface issued
from the culture (i) and (ii).
The detection of the presence of NKp44L at the CD4+ T-cells
surface can be carried out as known by the one skilled in the art, for
instance by a cytofluorometric analysis as it is described in the part
Material and methods, corresponding to the example 6.
Preferably, the method described above, comprises an additional
step (c) which consists of selecting positively the candidate compound as
a therapeutical agent when the level of expression of NKp44L at the
CD4+ T-cells surface issued from the culture (ii) is higher than the level
of expression of NKp44L at the CD4+ T-cells surface issued from the
culture (i).
The comparison of the level of expression of NKp44L at the
CD4+ T-cells surface can be assessed by counting the number of CD4+
T cells expressing NKp44L on their surface, using a fluorescence
activated cell sorter (FACS), as described in the corresponding Material
and Methods section.
Alternatively, the detection of the presence of NKp44L at the
CD4+ T-cells surface can be carried out indirectly, by measuring the NK
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lysis activity of CD4+ T cells, as it is described in the section Material and
Methods.
This particular embodiment of the step (b) of the screening
method above, despite it consists of an in vitro method, has the technical
advantage to directly reflect the therapeutical potential of the candidate
compound by directly evidencing the biological activity of said candidate
compound, as regards preventing the CD4+ T-cells cytolysis by the
activated NK cells.
The activated NK cells may consist of cells from a NK cell line,
io such as the NK92 cell line described by Gong et al. (1994) or may
consist of a primary culture of normal human purified NK cells.
The CD4+ T-cells that express the NKp44L protein may consist of
CD4+ T-cells, eventually under the form of a cell line, that have been
transfected with a vector that allow the expression by said cells of the
is NKp44L protein, or may consist of CD4+ T-cells that were initially
purified
from a blood sample of an HIV-infected patient.
In a specific embodiment of the screening method above, the
activated NK cells and the CD4+ T-cells are autologous in that they both
come from the same HIV-infected patient.
20
Preferably, the cytolysis measure consists of the conventional
technique wherein the CD4+ T-cells, which are the target cells, are
initially rendered radioactive with 51Cr, and wherein the cytolysis value
consists of the percentage of cell lysis, as measured by the amount of
51Cr that is released in the cell culture medium by the lysed CD4+ T-cells.
25 Most
preferably, the cytolysis value is obtained by assaying the
cytolytic activity of the NK cells at increasing effector (NK cells) to target
(CD4+ T-cells) ratios, for example from 1:1 to 50:1 effector: target cell
ratios.
Candidate compounds for use in the screening methods described
30 immediately above can be selected among the candidate compounds
which binds to one or several polypeptides of formula (I).
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Accordingly, the invention also concerns a method for the in vitro
screening of compounds for preventing or treating a disease linked with
the infection of an individual with an HIV virus, comprising the steps of:
(I)
submitting a candidate compound to the first screening method
5 above, and
(ii) submitting a candidate compound positively selectionned at step
(i) to the second screening method described immediately above.
b) Diagnosis methods of the invention
10 The
inventors have found that, that antibodies directed against
polypeptide (I) are produced during HIV infection.
It has also been found according to the invention a statistically
significant correlation between the expression level of antibodies directed
against polypeptide (I) collected from HIV-infected individuals and the
15 level of CD4 T cells.
it has been found according to the invention that the level of these
antibodies decrease during the progression of HIV infection, especially
as regards the development of the patient's immunodeficiency caused by
the progressive depletion of his CD4+ T cells.
20 Besides, the
inventors have found that this kind of kinetics is
specific for antibodies directed against polypeptide of formula (I) and is
not observed with antibodies directed against peptides T20 or T21
derived from HR1 and HR2 domains of gp41.
It flows from the experimental results obtained by the inventors
25 which are
briefly described above that the level of antibodies directed
against a polypeptide (I), contained in the serum of an individual, reveals
itself to consist of an accurate biological marker of the progression status
of the infection of an individual with an HIV virus. Further, the expression
level said antibodies consists of a novel biological marker of the state of
30 advancement
of the HIV infection endowed with a very high biological
significance, since it has been shown by the inventors that the level of
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antibodies directed against polypeptide (I) is more important at the
beginning of infection of a patient by HIV.
Accordingly, the invention also concerns a method for the in vitro
assessment of the progression status of the infection of an individual with
an HIV virus, wherein said method comprises the step of detecting in a
sample from said individual, antibodies directed against a polypeptide of
formula (I).
As used herein an "HIV" virus consists of either an HIV-1 or an HIV-2
virus, and more particularly any virus strain or isolate of an HIV-1 or an
HIV-2 virus.
As used herein, the "assessment of the progression status" of the
infection consists of raw experimental data indicative of the
immunological status of the HIV-infected patient tested; Hence, as
already mentioned above, there is a statistically relevant correlation
between the amount of antibodies directed against the polypeptide of
formula (I) and the CD4+ T cells count.
The sole detection of antibodies directed against a polypeptide of
formula (I) might not be sufficient for a global accurate clinical diagnosis,
or prognosis, of the progression status of the disease within the patient
tested. Thus, the detection of antibodies directed against a polypeptide of
formula (I) might be completed by, or combined with, other diagnosis or
prognosis markers of the disease, for example one of the prior art
markers that have previously been cited in the present specification.
The detection of antibodies directed against a polypeptide of
formula (I) can be achieved using known techniques such as ELISA or
RIA tests.
Methods of treatment according to the invention
This invention also deals with a method for preventing or for
treating a disease linked to the infection of an individual with a virus of
the HIV family, wherein said method comprises a step of administering to
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a patient in need of such treatment an effective amount of an
immunogenic or a vaccine composition comprising a polypeptide of
formula (i) in combination with an immunoadjuvant coompound.
In a specific embodiment of the invention, said method comprises
the following steps:
- preparing a purified batch (HPLC, > 95%) of polypeptide
comprising the aminoacid sequence (II), linked to diphteria toxoid,
- Injecting diphteria toxoid alone to a first "control" group of
mammals
- Injecting the polypeptide comprising the aminoacid sequence (II),
linked to diphteria toxoid, to a second "test" group of mammal,
Preferably, when the mammals used are of the Macaca mulatta
species, they received 0,5 mg per mammal, and per injection of diphteria
toxoid, linked or not, to the polypeptide cited above, followed by 3 new
injections 3, 6 or 9 weeks later, in combination with Freund's incomplete
adjuvant,
- infecting the two groups of mammals with SHIV33, two weeks
after the last injection, with 1 ml of a dilution at 50 TCID50/ml.
-Collecting samples consisting of serum and PBMC (peripheral
blood mononuclear cells) at day 0, and 1 week after each injection
(before infection) and then every week after infection by SHIV,
-analysing the collected samples.
These analyses can comprises, without limitation, the control of the
general aspect of the sample, the measurement of the amount of CD4+ T
cells using a FAGS, the measurement of the viral load by RT-PCR, the
determination of the seroconversion by detecting antibodies directed
against Env protein by EIA technique, the detection of antibodies directed
against polypeptide of formula (II) by ELISA.
These analyses can comprise also the study of the effects of
antibodies produced by said mammals on cells infected by HIV, and for
example chronically infected cells. These cells are for example, U2 cells
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infected by HIV-sf2, Jurkat cells infected by HIV-BRU, or CD4+ T cells
infected by BRU or collected from HIV infected patients.
Further, these analyses can comprise the measurement of
NKp44L expression level, by FACS, the analysis of P24 amount, by EIA,
or the analysis of NK cytotoxic activity of several strains of NK cells, such
as autologous or allogenic NK92, NK3.3, or NKL cells, using a 51Cr test
as described below.
A further object of the invention consists of a method for
preventing or for treating a disease linked to the infection of an individual
io with a virus
of the HIV family, wherein said method comprises a step of
administering to a patient in need of such treatment an effective amount
of an antibody directed against the polypeptide of formula (1)
The invention concerns also the use of a ligand compound which
specifically binds to the polypeptide of formula (1), for manufacturing a
pharmaceutical composition for preventing or treating a disease linked to
the infection of an individual with a virus of the HIV family.
The invention also deals with the use of a polypeptide of formula
(I), for manufacturing a vaccine composition for treating a disease linked
to the infection of an individual with a virus of the HIV family.
The present invention is further illustrated by, without in any way
being limited to, the following examples.
EXAMPLES
A. General material and methods
A.1 HIV-1 infected donors.
Blood samples of 25 HIV-1-infected patients were obtained from
consenting donors at HOpital Pitie-Salpetriere. Bio-clinical examinations
included routine determinations of the viral load, total blood and CD4+ T
lymphocyte counts.
As control group, Blood samples from 20 uninfected donors were
obtained by leukapheresis from the blood bank (HOpital Pitie-Salpetriere).
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A.2 Cytofluorometric analysis
A three-colors FACS analysis was performed on freshly harvested
PBMC. lsotype-matched immunoglobulin served as the negative control
(BD). Cells were incubated 1h at 4 C, with the appropriate cocktail of
antibodies. Anti-CD3; anti-CD4; anti-CD8, anti-CD56, anti-NKp44, anti-
NKp46 or anti-NKp44L mAb. Erythrocytes were lysed using the FACS
lysing solution (BD). A minimum of 20,000 leucocytes was analyzed on a
FACScan, as previously described.
To measure the expression of cell surface activation markers,
PBMC were stained with PE- or FITC-conjugated anti-HLA-DR, anti-
CD69, anti-CD25, or anti-CD71 (all from BD) and analyzed by FACS.
A.3 Purification of T CD4+ cells expressing NKp44L
CD4+ T cell subset sorting was performed using the
RosetteSepCD4+ enrichment kit (StemCell). CD4+ T expressing NKp44L
were positively selected by a two step magnetic separation, CD4+ T cells
were incubated with 10 pg/ml of anti-NKp44L for 1-h at RT, followed by
treatment with goat anti IgM mouse-coated Dynabeads (Dynal) at a
bead-to-cell ratio of 10:1 for 30 min at RT. The cell fraction purity was
determined by FACS analysis.
A.4 Isolation of primary NK cells and NK cytotoxicity assays.
NK lines were generated from PBMC, and then purified using the
StemSep cell separation system and the NK cell enrichment antibody
cocktail (StemCell technologies). NK purified cells were cultured in
MyeloCult H5100 medium (StemCell technologies) supplemented with
100 units rhIL-2 (Boheringer). The purity of these preparations was
evaluated by flow cytometry after staining with anti-CD3 (BD), anti-CD56
(BD), anti-NKp44, and anti-NKp46 mAbs.
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The cytolytic activity was assayed in 4-h 61Cr-release assay as
previously described. Briefly, the target cells were labeled for 2-h at 37 C
with 100 pCi per 106 cells Na61Cr (Amersham), and washed twice with
culture medium. The target cells were then distributed in round-bottomed
5 96-well
microtiter plates (4 x 103 cells per well), and the effector cells
were added at several E/T rario. The plates were incubated 4-h at 37 C.
The supernatant were then collected and 61Cr-release was measured in
a gamma counted. In experiments in which Abs were included, these
were added to final concentration of 20 pg/ml. The relative specific 61Cr-
io release was
calculated according to conventional methods. Values for
spontaneous 61Cr-release, which are deducted in the calculation, were
between 10 and 20% of the total incorporated radioactivity. The results
are presented after subtraction of the nonspecific lysis obtained with
control targets. Each point represents the average of triplicate values.
15 The range of the triplicates was always within 5% of their mean.
A.5 Statistical analysis
Correlation analyses were performed using Sperman's non-
parametric rank correlation analysis. All calculations were performed
20 using the GraphPad Prism.
B. Material and Methods of the examples -6
B.1 Purification of T CD4+ cells
25 CD4+ T cell
subset sorting was performed using the
RosetteSepCD4+ enrichment kit (StemCell). The cell fraction purity was
determined by FACS analysis.
B.2 Cytofluorometric analysis
30 A two-colors
FACS analysis was performed on purified CD44- T
cells. lsotype-matched immunoglobulin served as the negative control
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(BD). Cells were incubated 1h at 4 C, with the appropriate cocktail of
antibodies. anti-CD4 or anti-NKp44L mAb. A minimum of 20,000 CD4+ T
cells was analyzed on a FACScan, as previously described. The intra-
cellular expression of NKp44L was realized as previously described,
briefly, the cells were incubated in 4% PFA buffer for 20 min, then
washed in stained in presence of 0.1% saponin/PBS/1% BSA buffer at
4 C. the cells were then analyzed by FACS.
B.3 Isolation of primary NK cells and NK cytotoxicity assays.
NK lines were generated from PBMC, and then purified using the
StemSep cell separation system and the NK cell enrichment antibody
cocktail (StemCell technologies). NK purified cells were cultured in
MyeloCult H5100 medium (StemCell technologies) supplemented with
100 units rhIL-2 (Boheringer). The purity of these preparations was
evaluated by flow cytometry after staining with anti-CD3 (BD), anti-CD56
(BD), anti-NKp44, and anti-NKp46 mAbs.
The cytololytic activity was assayed in 4-h 61Cr-release assay as
previously described. Briefly, the target cells were labeled for 2-h at 37 C
with 100 pCi per 106 cells Na61Cr (Amersham), and washed twice with
culture medium. The target cells were then distributed in round-bottomed
96-well microtiter plates (4 x 103 cells per well), and the effector cells
were added at several E/T rario. The plates were incubated 4-h at 37 C.
The supernatant were then collected and 61Cr-release was measured in
a gamma counted. In experiments in which Abs were included, these
were added to final concentration of 20 pg/ml. The relative specific 61Cr-
release was calculated as previously described. Values for spontaneous
61Cr-release, which are deducted in the calculation, were between 10
and 20% of the total incorporated radioactivity. The results are presented
after subtraction of the nonspecific lysis obtained with control targets.
Each point represents the average of triplicate values. The range of the
triplicates was always within 5% of their mean.
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B.4 Recombinant vaccinia virus expression HIV-1 protein.
Purified CD4+ T cells were infected with wild type vaccinia virus
(WT) or with the various recombinant vaccinia virus at a multiplicity
infection of 20 PFU/cell were used as target cells. Recombinant vaccinia
viruses for HIV-1-LAI Gag, Pol, Env, Nef, Tat and Vif proteins were
provided by Transgene (Strasbourg, France).
B.5 Peptides and pools of peptides.
The synthetic 15-mers peptides were purchased from Epytop
(Nimes, France) or kindly provided by Agence Nationale de la Recherche
sur le SIDA. All were more than 80% pure as shown by HPLC profiles.
Pools of peptides included around 10 different peptides and each peptide
overlap the previous continuous peptide for 11 residues.
B6. mAbs screened for their capacity to inhibit NK lysis
11. Anti-NKp44 (44/8; IgG1) and anti-NKp44L mAb (#7.1; IgM) were
obtained from 5-weekold BalB/c mice immunized with the ClonaCell-HY
hybridoma cloning kit, according to the manufacturer's instructions
(StemCell Technologies Inc.). The anti-mouse-peroxidase hybridoma
screening reagent (Roche) and ELISA were used to select antibodies.
Anti-NKp44 (44/8) mAb was prepared by immunizing the mice with
NKp44-Ig protein, after specific deletion of the IgG1 human Fc fragment.
To obtain #7.1 mAb (IgM), mice were immunized with acid-treated U2-sf2
cells. These cells were prepared as previously described (S. Sumitran-
Karuppan , E. Moller, Transpl. lmmunol. 4, 163, 1996.). Briefly, one
million U2-Sf2 cells were washed 3 times in PBS and treated for 5 min on
ice in an acid buffer prepared by mixing equal volumes of 0.263 M citric
acid and 0.123 M Na2HPO4 containing 1% (w/v) BSA. After three more
washes, the cells were resuspended in PBS and then irradiated and
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injected into the mice. All hybridomas were analyzed with 51Cr cytotoxic
assays with untreated or acid-treated 721.221 cells as targets, and the
specificity of the anti-NKp44 hybridoma was analyzed by ELISA with
different fusion proteins. The #7.1 mAb (IgM) mAb was purified on a
mannan-binding protein (MBP) column (Pierce), after ammonium sulfate
precipitation (50% saturated solution); the anti-NKp44 mAb was purified
on a protein A/G column (Pierce). The purity of each purified mAb was
confirmed on SDS -PAGE.
C. RESULTS
Example 1 Effects of several HIV viral proteins on NKp44L
expression
The effect of HIV viral protein on NKp44L expression was
examined using infection with recombinant vaccinia virus expressing HIV
viral protein. As shown in Figure 1, the expression of NKp44L was
markedly enhanced in CD4+ T cells infected with vaccinia virus
expressing the gp160 (33,9%) or the gp41 HIV Env proteins (35,6%). In
contrast, neither other HIV proteins tested, like Gag, Pal, Tat, nef, vif, or
gp120, influenced the cell surface expression of NKp44L protein.
Furthermore, the role of the Env protein to enhance the expression of
NKp44L was confirmed in a non-viral system. Purified CD4+ T cells were
treated with recombinant gp160 protein provided of two different origins,
and as shown in Figure 2A, these gp160 recombinant proteins influenced
the expression of NKp44L. Indeed, 10,7% and 9,6% of CD4+ T cells
expressed NKp44L after treatment with the gp160-A and gp160-B,
respectively. On the other hand, no effect was observed with untreated
cells or cells incubated with a control protein. All together, these results
show that the recombinant gp160 protein markedly enhances the cell
surface expression of NKp44L on CD4+ T-cells surface.
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Example 2 qpi60 induces the NK lysis of CD4+ T cells.
Comparison of NK lysis activity from the untreated cells, the cells
treated with the control protein or the cells treated with the both
recombinant gp160 proteins (Figure 2B), shows that target cell lysis was
increased in the presence of CD4+ T cells cultured with recombinant
gp160 protein. The use of two different types of recombinant gp160
proteins indicates that the procedure used to induce over-expression of
NKp44L and increased of NK lysis activity had no influence on the
pa outcome of the experiments. Together, these results indicate that gp41
HIV Env protein was required for the over-expression of NKp44L
correlated with a strong increased of NK lysis activity.
Example 3 identification of the peptide motif of the qp41 Env protein
involved in the increased of NK lysis activity,
The effect of pool of overlap peptides prepared, as described in the
Materials & Methods section, has been tested, to include all of the gp41
protein. CD4+ T cells were incubated with 5 pg of each pool of peptides
and tested against activated NK cells. As shown in Figure 3, NK lysis
was increased in cells incubated with the pool of peptides named gp41C,
but not with all of the other pool of peptides. The expression of NKp44L
in purified CD4+ T cells treated with each of the pool of peptides has
been tested. This receptor was only detectable in cells treated with pool
gp41C, and the percentage of positive cells was 13,3%. In no instance,
NKp44L was detected on the cells incubated with the other pool of
peptides tested. This suggested that one or several peptide motifs
included in the pool gp41C was directly implicated in the increased of NK
lysis via the over-expression of NKp44L. Repeated experiments with all
of the peptides included in the pool gp41C was then tested. As shown in
Figure 4A, the NK cytotoxic activity was strongly increased in presence of
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the peptides gp41-C145, gp41-C146, and gp41-C147. By contrast, with
the other peptides tested, the NK lysis activity remained low, closed to
the background. In parallel, the expression of NKp44L was increased
after pretreatment of CD4+ T cells with the peptides gp41-C145, gp41-
5 0146, and gp41-0147, with a percentage of positive cells varied between
22 and 16%, but not with the other peptides (less than 7% of positive
cells) (Figure 4B). These results indicated that a peptide specific to gp41
Env HIV protein could increased a NK lysis activity. Additional support to
this conclusion come from that the continuous peptides named gp41-
10 0145, gp41-C146, and gp41-C147 included a common peptide motif
NH2-SWSNKS-COOH, This motif specific to the gp41 HIV-1 protein was
strongly conserved. After having shown that some continuous peptides of
the gp41 are some major mediators of NK lysis, it was important to
assess if the peptide motif NH2-SWSNKS-000H was directly implicated
15 in the NK lysis of CD4+ T cells. Preliminary experiment with this 6-mers
peptide shown any increased of cell surface expression of NKp44L or NK
cytotoxic activity, suggesting that this sequence is too small or too rapidly
attack by some peptidases. However, to test this hypothesis, two 15-
mers peptides derived from gp41-C146 (WT) included some mutation
20 inside the NH2-SWSNKS-000H motif (CtI1) or in all of the 15-mers
sequence (CtI2) have been constructed (figure 5A). As shown in Figure
5B, the NK cytotoxic activity was strongly increased in presence of the
peptide WT. By contrast, with untreated cells (none) or the treated with
the both control peptides. Similar pattern was observed concerning the
25 cell surface expression of NKp44L, indeed, in cells treated with the WT
peptide, approximately 17,4% of CD4 T cells expressed this marker. By
contrast, the percentage of NKp44L+ cells was less than 4% in untreated
cells or cells treated by the control peptides. These results show that the
NH2-SWSNKS-000H motif included in the gp41 protein is strongly
30 implicated in the NK lysis of CD4+ T cells.
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The effect of gp41 peptide is time dependant (Figure 6). NK lysis activity
started after 30 min of incubation with the WT peptide and approached a
maximum closed to 4-days. On the other hand, no significant effect is
observed after treatment with the untreated cells or the cells treated with
the control peptides. Furthermore, the increased of NK lysis activity is
strongly inhibited after pretreatment of cells with anti-NKp44L mAb,
confirming that the NK activity is directly correlated with an increase of
cell surface expression of NKp44L, in cells treated with the WT peptide
(Figure 6C). However, kinetic study of the cell surface expression of
NKp44L revealed that this receptor was rapidly expressed at the cell
surface, indeed after 10 min of treatment with WT peptide, around 10%
of CD4+ T cells expressed this protein, and the maximun of expression
(approximately 30%) was obtained 4-days after treatment. The very fast
cell surface expression of NKp44L suggested an absence of new
synthesis of NKp44L, and suggested that this protein was present inside
the CD4+ T cells cultured with IL2. This hypothesis was confirmed by an
intracellular staining of NKp44L. As show in Figure 6D, high expression
of NKp44L was detectable inside the cells, and this independently of the
presence of peptides.
Example 4 Cell surface expression of NKp44L of different human
cells
=
As shown on figure 7, the surface expression of NKp44L on K562,
Jurkat, and resting PBMC has been tested. The cells were incubated
with 1 pg/ml of anti-NKp44L mAb anti-NKp44L mAb (grey thick line) or
with the IgM isotype control (black thin), and analyzed by flow cytometry.
It is clearly shown that, contrary to PBMC, tumor cells, like jurkat, and
K562 cells express NKp44L on their surface.
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Exemple 5 Identification of #7.1, an anti-NKp44L mAb that
specifically inhibits NKp44- mediated NK lysis.
To study the related function and expression of this ligand during HIV-1
infection, a library of mAbs screened for their capacity to inhibit NK lysis
has been tested. One of them, the #7.1 mAb, revealed an epitope
expressed on NKp44L. Specific staining with this mAb was found in HIV-
infected U2 cells only, not in uninfected U2 cells (Fig.8B), and it yielded a
level of staining similar to that of the NKp44-Ig fusion protein (compare
Fig. 8A and 8B). Inhibition of the #7.1 mAb staining by this fusion protein
confirmed that #7.1 mAb specificaly interact with a NKp44 ligand
(NKp44L) (Fig. 80). A control experiment showed no effect with the
NKp46-Ig fusion protein (Fig. 8C). Furthermore, when U2 cells
chronically infected by the HIV-1 Sf2 strain were pretreated with the #7.1
mAb, their NK-mediated lysis decreased sharply (as much as 40%)
(Fig.8D). Treatment of NK cells by the anti-NKp44 mAb produced the
same effect (Fig. 8E).Note that in both series of experiments the
magnitude of the lysis obtained in the presence of anti-NKp44 or 7.1 mAb
was similar to that observed with uninfected cells. We obtained similar
results with Jurkat cells chronically infected by the HIV-1 BRU strain
(data not shown). Together, these data strongly suggest that NKp44L is
expressed during HIV-1 infection and that #7.1 mAb reacts specifically
with NKp44L on HIV-1 infected cells.
Exemple 6 Critical role of the NH2-SWSNKS-COOH motif from the
qp41 HIV protein
A rabbit anti-gp41-C146 peptide polyclonal Ab has been tested for its
capacity of inhibition of NKp44L expression and for sensitivity to NK lysis.
Briefly, highly purified peptide (gp41-C146 peptide) (>95%) was linked to
the KLH and injected in several rabbits. Serum titres were determined by
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ELISA on PeptiPlaks. Antibodies were purified by chromatography.
NKp44L expression was substantially lower (7.8%) in purified CD4+ T
cells from two HIV-1 infected patients that were incubated with purified
anti-gp41-C146 polyclonal Ab than in purified and then untreated cells
(27.2%) or in those treated with a control Ab (27.9%) (Fig. 9E). This
effect was confirmed by the drastic inhibition of NK activity in the
presence of the anti-gp41-C146 polyclonal Ab(Fig.9F). These results
strongly suggest that the gp41 NH2-SWSNKS-COOH motif plays a key
role in inducing NKp44L expression during HIV infection and that the
gp41 protein participates in the selective destruction of CD4+ T cells by
activated NK cells.
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