Note: Descriptions are shown in the official language in which they were submitted.
W092/0~2 PCT/U~9~/0644~
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PATHOGEN-SPECIFIC CTL THERAPY
BACKGROUND OF THE INVENTION
This invention relates to therapies involving
administration of cytotoxic T lymphocytes.
Cytotoxic T cells (CTL's) that specifically lyse
HIV-l i~,fected autologous target cells have been found to
occur at uncommonly high freguency in the blood of HIV-
infected individuals; killing by such cells is
predominantly mediated by CD3+CD8+ effector cells although
cytotoxic CD4+ cells and natural killer cells also play a
role tWalker et al., Nature 328:345, 1987; Plata et al.,
Nature 328:348, 1987; Walker et al., Science 240:64,
1988; Sethi et al., N~ture 335:178, 1988; Xoenig et al.,
Proc. ~atl. Acad. Sci . USA 85:8638, 1988; Nixon et al.,
Nature 336:484, 1988; Tsubota et al., J. Exp. Med .
61~ 421, 1989; Riviere et al., J. Virol. 63:2270, 1989;
Kou~ et al., Blood 73:~909, 1989; Hoffenbach et al., J.
Immunol . 142:452, 1989; Culmann et al., Eur. J . Immunol .
19:2383, 1989; and Hosmalin et al., Proc. Natl . Acad.
Sci. USA 87:2344, 1990). CD8+ T cells recognize
antigenic peptides presented by MHC class I molecules.
To be recognized by a CTL, a peptide must be properly
processed, be capable of binding to MHC strongly enough
to compete with other peptides, and be recognized as a
peptide-MHC complex by T cells in the repertoire. Recent
studies indicate that in some infections only a small
number of peptides meet these criteria and that CTL
specific for these epitopes dominate the lytic response
30 (Braciale et al., Immunol . Rev. 98:95, 1987; Whitton et
al ., J. Virol . 62:687, 1988; Klavinskis et al., J. Virol .
63:4311, 1989; Whitton et al., J. Virol. 62:687, }988;
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W092/OM62 PCT/US91/3~
2 ~ 8 ~ ~
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Braciale et al., Proc. Natl. ~cad. Sci. USA 86:277, 1989;
Townsend et al., Cell 44: 959, 1986).
Hosmalin et al. (1990, supra) and Takahashi et al.
(Proc. Natl. Acad. Sci. USA 85:3105, 1988; ',cience
246:118, 1989; J. Exp. Med. 170:2023, 1989) report that a
small number of epitopes likely dominates the CTL
respon~e to HIV-l in mice. Yamamoto et al. (J. Immunol.
144:3385, 1990) and Miller et al. (Abstract FA 74, sixth
International Conference on AIDS, San Francisco, CA,
1990) report that in SIV-infected macaques, the CTL
response to qaq appears to have a limited epitvpe
specificity. Culmann et al. (1989, supra) report that,
in two human subjects, the CTL response to HIV-encoded
nef protein is predominantly directed against the same 16
15 amino acid region. Koenig et al. (J. Immunol. 145:127,
1990) report that a ten amino acid fragment of the nef
protein is recognized by the CTL's of two out of ten ~IIV-
seropositive individuals.
SUMMARY OF THE INVENTION
In one aspect, the invention features a method of
treating a mammal infected with an intracellular pathogen
involving selecting, from a sample of the mammal's
lymphocytes, a sub-sample which is enriched for cytotoxic
T lymphocytes which recognize a pathogen-specific antigen
and which are capable of lysing pathogen-infected cells
of the patient, and administering to the patient a
therapeutically effective amount of the sub-sample of
cytotoxic T lymphocytes. Preparation of such a pathogen-
specific CTL~enriched sub-sample generally involves
stimulating the proliferation of a mammal's T-lymphocytes
ln vitro, using a nonspecific mitogen, in the presence of
pathogen-infected cells (i.e., cells displaying pathogen-
specific antigens on their surfaces); inclusion of such
pathogen-infected cells results in the selective
expansion of a population of CTL's which are capable of
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wo g2,04462 - 2 0 8 9 ~ 8 8 P~/US91/0644~
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-- 3
targeting and lysing host cells harboring the pathogen.
~reparation of the pathogen-specific CTL-enriched su~-
sample may further involve identifying, for a particular
mammal, those pathogenic-specific antigens which are
capable of eliciting a particularly potent CTL response
and presenting peptides displaying these antigens to the
proliferating T cells in order to further expand the
pathogen-specific CTL population in the sub-sample.
In preferred embodiments, the sub-sample is
prepared by incubating the sample with a mitogen which is
capable of inducing lymphocyte proliferation (preferably,
phytohemagglutinin); the sample is further incubated with
IL-2; the sample is further contacted with a pathogen-
specific antigen recognized by CTL's of the mammal and
which is capable of inducing a CT~ response in the
mammal; the method further involves administering to the
mammal a therapeutically effective amount of a pathoyen-
specific antigen recognized by the CTL's of the mammal
and which is capable of stimulating a cytotoxic T
lymphocyte response in the mammal.
In another aspect, the invention features a method
of treating a mammal infected with an intracellular
pathogen involving administering to the mammal a
therapeutically effective amount of a pathogen-specific
antigen which is recognized by the CTL's of the mammal
and which is capable of stimulating a cytotoxic T
lymphocyte response in the mammal.
In preferred embodiments of both aspects, the
anti~en is displayed on the surface of an autologous
antigen-presenting cell (preferably a B-lymphocyte); the
mammal is a human; the intracellular pathogen is a virus
(preferably a human immunodeficiency virus, a human T
cell leukemia virus, or a Herpes virus, preferably, an
Epstein-Barr virusj, a mycobacterium, a protozoan, or a
3S fungus; and the pathogen-specific antigen ls
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WO 92/0~62 r ~ ~ PCT/US91/0~
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20~9~88 ~- I
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immunodominant. In the case of a patient infected with a
human immunodeficiency virus, the pathogen-specific
antigen is preferably an HIV-encoded proteiri (for t
example, the product of the env or the pol gene), or a
5 CTL-stimulatory fragment thereof.
By "intracellular pathogen" is meant a disease-
causing organism which resides, during at least a part of
its life cycle, within a host cell. By "enriched for
cytotoxic T lymphocytes" is meant that the sub-sample has
10 a substantially greater number of pathogen-specific
cytotoxic T lymphocytes (i.e., T lymphocytes which
recognize and destroy cells bearing foreign antigens, in
this case, pathogen-specific antigens, on their surfaces)
than a freshly isolated sample of the patient's
15 lymphocytes. By "pathogen-specific antigen" is meant a
protein produced upon infection by a pathogen which is
recognized (i.e., responded to) as foreign by cells, in
this case, cytotoxic T cells, of the patient's immune
system. By "lyse" is meant to destroy or disintegrate,
~ 20 for example, a host cell harboring a pathogen. By
"pathogen-infected cells" is meant those host cells
harboring a pathogen, either in an active or a latent
state. By "mitogen" is meant a substance that stimulates
mitosis and, thus, cell proliferation. By "CTL response"
25 is meant the proliferation of CTL's in response to, and
specific for, a stimulatory antigen. By "autologous" is
meant occurring in the same patient. By "antigen-
presenting cell" is meant any cell capable of displaying
on its cell surface an antigen, or an immunogenic
30 fragment theraof. By "human immunodeficiency virus" is
meant, without limitation, HIV-1 and HIV-2; By "human T
cell leukemia virus1' is meant, without limitation, HTLV-
I and HTLV-II; by "Herpes virus" is meant, without
limitation, HerPes simPlex type 1 and type 2, Herpes
35 zoster, and cytomegalovirus as well as Epstein-Barr
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W09~/04462 PCT/~S91/064~ 1
5 8
virus. By "virus" is also meant, without limitation,
Papillomavirus, Creutzfeldt-Jakob virus, and feline
leukemia virus. By "mycobacterium" is meant, without
limitation, Mycobacterium leprae, MYcobacterium
tuberculosis. By "protozoan" is meant, without
limitation, Toxo~lasma qondii, Giardia lamblla,
Trypanosoma cruzi, organisms of the genus Leishmania, and
organisms of the genus Plasmodium which cause malaria.
By fungus is meant, without limitation, Pneumocvstis
carinii, Candida albicans, and Candida tropicalls. By
"CTL-stimulatory fragment" is meant a peptide which is
capable of stimulating antigen-specific CTL
proliferation. By "immunodominant" is meant (an antigen)
capable of eliciting an unusually potent CTL response.
In the method of the present invention, the CTL's
of the enriched sub-sample or CTL's expanded in the host
following administration of pathogen-specific CTL-
stimulatory peptide or peptide-bearing antigen-presenting
cells recognize and selectively target pathogen-infected
cells. Because such pathogen-infected cells represent a
small percentage ~ the total cell population, this
method minimizes side effects, such as immunosuppression,
which may result from other forms of therapy such as
those which destroy or impair the function of host cells
which are either infected or at risk of being infected by
the pathogen. Moreover, the pathogen-specific CTL
population may be administered to the mammal free of
lymphokines, thereby avoiding the vascular-leak syndrome
generally associated with lymphokine therapies, at least
in humans and mice. Finally, the pathogen-specific CTL
population is generated from a mammal's lymphocyte
sample. This is an important feature of the invention
because antigens capable of inducing an effective CTL
response (i.e., inducing significant CTL proliferation)
may vary, and, for example, in the case of HIV-1, do vary
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W092/U~62 . PCT/US9~t~
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from one individual to the next. By beginning with a
mammal's own lymphocytes, it is possible to enrich for
CT~'s which recognize and lyse cells bearing pathogen-
specific antigens which are immunodominant for that
particular human or mammal, thereby maximizing a human~s
or mammal's CT~ response to a pathogenic infection.
Other feature~ and advantages of the invention
will be apparent from the following description of the
preferred embodiments, and from the claims~
DESCRIPTION OF THE PREFERRED EMBODIMEN~S
The drawing will first briefly be described.
DRAWING
Fig. 1 is a diagrammatic representation of a set
of truncated HIV-1 envelope and reverse transcriptase
proteins, from which can be derived peptides which are
useful as candidate HIV-1-specific, CTL-stimulatory
antigens.
Fig. 2 is a schematic representation of the HIV-
l-specific epitopes recognized as immunodominant by the
CTL's of eight independent HIV-1-seropositive
individuals.
EXAMPLE
There now follows a description of a method for
generating, from a sample of an HIV-infected patient's
lymphocytes, a sub-sample enriched in cytotoxic T
lymphocytes (CTL) which recognize an HIV-l-specific
antigen and which lyse cells displaying this antigen on
their cell surfaces. The method generally involves
establishing a T cell line (i.e., population) from a
sample of the patient's lymphocytes and stimulating
proliferation of this T cell line with a nonspecific
mitogen. Because HIV-1 infected cells (i.e., cells
displaying HIV-1 antigens on their surface) are naturally
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W092/0~62 PCT/US91/0~
2089~8~
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included in the sample in infected individuals and
because the culture conditions enhance viral replication,
the final preparation is enriched for cytotoxic T
lymphocytes, which recognize and lyse cells displaying an
HIV-1 specific antigen. The method may further involve
identifying, for a particular patient, an immunodominant
HIV-1 specific antigen(s) which is(are) capable of
inducing a CTL response and presenting a peptide(s)
displaying this epitope to the proliferating T cell line
to further enrich the sub-sample for HIV-1 specific
CTL's.
T-LYmphocvte Culture
Heparinized whole blood was obtained from HIV-1
infected patients, i.e., patients seropositive for HIV-1
as assayed by Western blot ar:lysis, and peripheral blood
lymphocytes (PBLs) were isolated by Ficoll-Hypaque
density centrifugation. Cells were cultured at 5xlO5~ml
in RPMI 1640 supplemented with 15% fetal calf serum
(Hazleton), 2mM HEPES, 2mM glutamine, 100 U/ml
penicillin, lOO ~g/ml streptomycin, and 50~M ~-
mercaptoethanol, and T cell lines were established by
addition of 2 ~g/ml PHA-P (Difco) and 200 U/ml rhu IL2
(Cetus). Twice a week cultures were adjusted to 5xlOs/ml
with fresh IL-2-containing media.
Using this method, T cell lines were generated
from ten CDC group II, five group III and three CDC group
IV individuals. These cell lines grew vigorously for
over a month without further stimulation, even from
patients with AIDS, although those cells grew less
vigorously. After 6 weeks, most cell lines with no
further exogenous stimulation had stopped dividing. No T
cell line could be generated from one hemophiliac patient
with longstanding AIDS and profound immunodeficiency
(absolute CD4 count 20/mm3, CD4/CD8 ratio 0.03).
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W092/0~62 2 ~ 8 9 ~`8 8 ;; PCT/US91/0~
After 2-3 weeks of culture, T cell lines were
screened for cytotoxicity against autologous B cell lines
infected with vaccinia virus containing the HIV-1 env
gene or a fragment of the Pol gene (i.e., constructs
vPEl6 and vCF21, respectively, described in Walker et
al., 1987, supra; Walker et al., 1988, suPra); a B cell
line infected with a vaccinia virus containing the lacZ
gene was used as a control (i.e., construct vSC8,
described in Chakrabarti et al., Nature 320:535, 1986;
Flexner et al., Virol. 166:339, 1988). Autologous EBV-
transformed B cell lines were generated from the
patient's peripheral blood lymphocyte samples by standard
techniques using B95-8 marmoset cell line supernatant.
Env vaccinia (vPEl6)-infected cells expressed the gpl20
and gpl60 !env) proteins from isolate HIV-lII1B; pol-
vaccinia (vCF21)-infected cells expressed all but the
last 22 amino acids of the reverse transcriptase (RT)
protein from isolate HIV-lHXB 2. Expression of HIV-1
proteins was verified by radioimmunoprecipitation of
infected cell lines with ~HIV serum as described in Essex
et al. (Science 220, 859, 1983). Vaccinia virus was
prepared and titered by plaque forming assay on CV-1
cells as described in Mackett, M., et al. ("The
construction and characterization of vaccinia virus
recombinants expressing foreign genes", in D. Glover,
ed., DNA cloning: A practical approach, Vol II, IRL
Press, Oxford, 1985). Env-vaccinia infected cells were,
in some cases, also titered by assaying syncytia
formation upon cocultivation with C8166 cells (as
described in Salahuddin et al., Virology 129, 51, 1983).
For vaccinia virus infection, 1-4 pfu/cell of virus was
added to 5x105 exponentially growing B cells in 500 ul of
media in a 24 well microtiter plate. Cells were
incubated, with rocXing, at 37C over CO2 for 30 min; an
additional 1 ml of media was added to each well, and
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W092/0~62 2 0 8 9 ~ 8 8 PCT/US91/0~1
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cells were further incubated, without rock:ing, for 16 hr.
Cytotoxicity was measured using a 5l-Chromium (Cr)
release assay as follows. B cells (i.e., target cells)
we~-e pelleted and resuspended in 200ul of serum-
con~aining media to whlch was added lOOuCi of Na2(5lCrO4)(Dupont). A~ter incubation for 1 hr at 37~C over CO2
with occasional mixing, targets were washed 3 times and
resuspended at 105 cells/ml. lO~ labelled targets were
added to triplicate wells of U bottom microtiter plates.
For peptide experiments, the labelled targets were
incubated with peptide at a final concentration of 50
~g/ml for 30 min. at 37C over CO2 before adding effector
cells. Effector cells were suspended at various E:T
ratios in lOOul of media and added to target cells;
plates were incubated at 37C over CO2 for 4 hr. For
each target, spontaneous elease (SR) was determined from
wells to which 100 ul of media was added, and total
release (TR) was calculated from wells containing lOOul
1% NP40. Supernatants (75ul)-were collected from each
well and were counted on a gamma counter after addition
of 75ul of 1% NP40. Percent specific cytotoxicity was
calculated from the average cpm as [(average cpm -
SR)/(TR - SR)] x 100. Spontaneous release below 15% of
total release was considered acceptable.
Using such an assay, it was demonstrated that 12
out of 18 PHA-stimulated T cell lines directed
significant cytolysis of either RT or env-expressing
tarqets (defined as specific lysis of HIV-1 expressing
targets minus lysis of the lacZ expressing target of
>10%, at E:T of 25:1). Five of the ten group II cell
lines lysed RT-bearing targets, four lysed env-bearing
targets; two of the five group III cell lines lysed RT
and env-targets and of the three group IV cell lines, two
lysed RT and all lysed env targets. In addition, it was
' ' . ~''" ~ " ' '.' ' ;, '" .' '. ' '"
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W092/0~62 -~ ~ PCT/US91/0~1
20~9~
-- 10 --
demonstrated that such T cell lines directed as much as
40% HIV-1-specific cell lysis above background (i.e.,
lacZ target) lysis in a 4 hr assay using an
effector:target (E:T) ratio of 6:1. This was in contrast
to autologous, freshly isolated peripheral blood
lymphocytes which, from some patients, showed little or
no HIV-specific lysis.
Prior to enhanced HIV-1-specific killing, the PHA-
stimulated T cell lines acquired NK-like activity,
presumably in response to the supraphysiological
concentration of IL-2; this effect declined over time in
culture.
CTL immunodominant peptides
To selectively enhance the prollferation of HIV-
1-specific CTL clones, the cultured T cell line was
further stimulated with a peptide which included an
immunodominant HIV-1-specific epitope recognized by the
patient's CTL.
To identify such immunodominant epitopes for a
particular patient, his/her established T cell line was
screened after 2-5 weeks in culture for cytotoxicity
against autologous B cell targets infected with the
vaccinia virus constructs encoding nested truncations of
env and RT proteins; cytotoxicity was assayed by 5 lCr-
releaseO The epitope was further defined by assayingcytotoxicity (again, by 5lCr-release) against autologous
targets which presented short overlapping peptides
spanning the region identified as immunodominant using
truncated proteins. A representative set of vaccinia
vectors expressing nested truncations of the above env
and RT isolates (i.e., for env, vPE16, vPEl7, vPE18,
vPE8, vPE20, vPE21, vPE22; for RT, vCF32-vCF37, described
in Hosmalin et al., 1990, su~ra) are shown in Fig. 1.
Fig. 2 shows that cell lines established from t:he
PBLs of eight independent patients recognize different
. .
W092/04462 PCT/US91/0~1
~' 2~89488
immunodominant env and RT epitopes. In particular, in
four cell lines studied, response to a single lO~ amino
acid region (i.e., amino acids 747 to 851) of the env
glycoprotein unequivocally dominated cytolysis; in one
cell line, an additional region (i.e., l to 204) was also
recognized. To further refine the commonly recognized
104 amino acid epitope, a set of seven 22-amino acid
peptides with 8-amino acid overlaps spanning this region
was synthesized by standard techniques (by scientists
other than the named inventors) and used to sensitize
autologous 5lCr-labelled targets. Three of the four T
cell lines that recognized determinants in this region
responded to different peptide epitopes. For one
subject, a peptide concentration of 0.2 ~g/ml was needed
to begin to sensitize targets and the response plateaued
at 12 ~g/ml. For this subject, three separate T cell
lines generated over a period of six months consistently
recognized the same single dominant epitope. The T cell
line from another subject, analyzed with a set of 20-
amino acid peptides with lO-amino acid overlaps (obtained
from the MRC AIDS Reagent Project), also recognized a
single peptide (i.e., amino acids 219 to 238).
Because the HIV-l isolate used as a source of
candidate CTL-stimulatory antigens is likely different
from the HIV-l isolates harbored by the patient and
because at least some ~IV-l proteins vary considerably in
amino acid sequence between isolates, it is likely that
viral antigens identified by this method, e.g., those
described above, are relatively invariant.
Following identification of immunodominant
epitopes, peptides bearing these epitopes were used to
stimulate CTL proliferation as follows. T cell lines
(approximately 3-4 weeks after initiation of culture)
were harvested and resuspended in fresh IL-2-containing
media at lo6 cells/ml. Cell lines were selected with
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W092/0~62 PCT/US91/0~
2~9~88: - ~
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either peptide alone (by adding an e~ual volume of
peptide-containing media to a final concentration of 1-
50 ~g/ml) or peptide presented by autologous antigen-
presenting B cells. For the latter, B cell lines were
irradiated (5000 rad), resuspended at 106/ml and
incubated with peptide at 1-50 ~g/ml at 37C with
occasional mixing. After 2 hr, the incubated cells were
pelleted and added to the T cell line in an equal volume
of fresh media. Twice weekly, T cells were counted and
fresh media added to maintain a cellular concentration of
5xlO5/ml. After 10 days, treated T cell lines were
tested for cytotoxicity against vaccinia-infected
targets.
In one specific example, T cell line 132, derived
from a patient with generalized lymphadenopathy, was
shown to recognize both an N-terminal and a C-terminal
epitope in env but none in RT. To selectively enhance
the growth of HIV-1-specific CTL clones with peptide, a
three week T cell line from this individual was incubated
either directly with the C-terminal env peptide that it
recognized (i.e., amino acids 802-823 of sequence,
Y~WNLLQYWSQELKNSAVNLLN) or with irradiated autologous B
cells preincubated with this peptide and washed to remove
unbound peptide. In both instances, there was
substantial enhancement of ~IV-l specific cytotoxicity to
the extent that the killing curves for the peptide-
selected cell lines resembled those obtainable from env-
specific clones. Peptide concentrations of 1 ~g/ml (350
nM) and 50 ~g/ml (1.75 uM) were equally effective; the
lower concentration fell on the linear part of the
peptide dose response curve for sensitizing tarqets for
cytotoxicity; the higher concentration fell on the
plateau. The threshold for a response was 0.2 ~g/ml or
70nM. Cell lines stimulated with peptide grew less well
than untreated cells, possibly because of lysis of
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WO9~/0~62 PCT/US~1/0~
~ 208948`8'
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peptide-presenting T cells; the decline in cell numbers
was greater at the higher peptide concentration. Cell
lines treated with peptide-presenting cells experienced a
dramatic burst of cell growth and increased in number 8-
fold above untreated cells. They also developedcytotoxicity against EBV-transformed autologous B cells.
TheraPv
Sub-samples enriched for pathogen-specific CTL's
are administered to a pathogen-infected patient as
follows. Cells are washed with PBS to remove culture
medium and are infused back into the patient by the
standard techniques developed for cancer therapy by
Rosenberg (see, e.g., Rosenberg et al., N. Eng. J. Med .
319:1676, 1988). Typically, infusion is performed
intravenously using 109 - 1011 cells, and the procedure
takes approximately 30 minutes. If necessary, treatment
can be repeated. Therapy can be administered soon a~ter
pathogen infection or upon onset of symptoms. In
addition, one or more PBL samples isolated from a
pathogen-infected, asymptomatic individual, or a CTL-
enriched sub-sample prepared following pathogen
infection, may be stored, frozen in liquid nitrogen,
until such time as that patient requires therapy.
Because the CTL's of the enriched sub-sample
recognize and selectively target pathogen-infected cells
and because such pathogen-infected cells represent a
small percentage of the total cell population, this
method minimizes side effects resulting from generalized
cell damage. In the specific example of an HIV-infected
patient, the enriched CTL sub-sample would target HIV-
infected CD4 lymphocytes, monocytes and macrophages,
leaving other cells of the immune system (including
uninfected CD4-bearing lymphocytic and monocytic cells)
intact and thus reducing the risk of immunosuppression.
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W092/0~62 PCT/US91/0~
20~9`~8~ @~
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This method also avoids the side effects, e.g., the
vascular-leak syndrome associated with lymphokine
therapy.
CTL-stimulatory pathogen~specific antigens (e.~.,
peptides including immunodominant epitopes) are
administered in therapeutic amounts to a pathogen-
infected patient either as a purified peptide or as a
processed antigen presented on the surface of an
irradiated autologous antigen-presenting cell (e.g.,
irradiated autologous B cells or autologous PBLs,
incubated with peptide antigen as described in the above
example). Because such CT~-stimulatory pathogen-
specific antigens expand CTL populations which recognize
and selectively lyse pathogen-infected cells, this method
minimizes generalized cell damage. Typically, such a
peptide antigen would be mixed with a pharmaceutically
acceptable carrier (e.g., physiological saline) and
administered to a patient by the standard procedures,
e.g., intravenous injection. Alternatively, irradiated
antigen-presenting cells would be infused back into a
patient by ~he standard techniques of Rosenberg (supra)
as described above. Such CTL-stimulatory pathogen-
specific antigens may be administered (as described
above) at any time following infusion with a pathogen-
~5 specific CTL-enriched sub-sample to further stimulate the
pathogen-specific CTL response.
When appropriate, lymphokines such as IL-2 or IL-
4 may be co-administered with either pathogen-specific
CTL-stimulatory peptides or sub-samples of pathogen-
specific CTL-enriched lymphocytes to further enhance
lymphocyte proliferation. To minimize the side effects
often associated with this treatment, a patient may be
treated with antihistamines, aspirin or acetaminophen,
prior to administration of lymphokines. In addition, a
patient may be treated with cyclophosphamide prior to
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W092/0~6~ 2 0 ~ 91 8 B PCT/US9l/0~l
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administration of pathogen-specific CTL-stimulatory
peptides or pathogen-specific CTL's.
Pathogen-specific CTL's can combat pathogen
infection by recognizing and lysing cells infected with
pathogen, thereby preventing further spread of infection.
Moreover, certain pathogen-specific CTL's, e.g., CTL's
specific for Epstein-Barr virus, can be used to prevent
or to treat a virus-induced lymphoma in a patient
infected with EBV alone or in a patient infected with EBV
and a human immunodeficiency virus.
OTHER ~MBODIMENTS
Other embodiments are within the following claims.
For example, PBLs may be grown in human serum-containing
medium or, alternatively, in serum-free medium (e.g., AIM
V, Gibco). Mitogens other than PHA (e.g., concanavalin
A, anti-CD3 monoclonal antibody, or anti-T cell receptor
monoclonal antibody) and lymphokines other than IL-2
(e.g., IL-4) may be used to stimulate lymphocyte
proliferation. Any expression vector capable of
~ 20 transfecting or infecting an antigen-presenting cell may
be used in this invention.
CTL-stimulatory antigens may be included in HIV-
l-encoded proteins other than env and RT, e.g., they may
be included in the gag or nef proteins. To identify such
antigens in gag, nef, or in any ~IV-1-encoded protein, a
complete set of candidate peptides ~ould be prepared by
fragmenting HIV-1 cDNA, cloning each fragment into a
vaccinia expression vector (or any appropriate expression
vector as defined above), and testing for CTL-stimulatory
capability as described in the above example.
Alternatively, candidate peptides may be synthesized in
vitro and tested for CTL-stimulatory activity as
described in the above example. An epitope domain could
be further refined by expressing sub-fragments of the
cloned DNA or by synthesizing sub-fragments of the
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W092/0~62 P~T/US91/0~
20~9~ 8~ 16 -
candidate peptides, whlch span the immunogenic re~ion.
Large numbers of fragments could be tested simultaneously
by attaching candidate peptides to a series of microtiter
wells, adding 5lCr-labelled autologous target cells,
adding an aliquot of a patient's T cells to each well,
and screening for cytotoxicity. Fragments shown to
encode a CTL-stimulatory peptide would be administered
directly to the patient (as a peptide or presented on the
surface of an irradiated, autologous antigen-presenting
cell) or would be used to selectively expand an HIV-1-
specific CTL population in a sample of a patient's
peripheral blood lymphocytes. Any isolate of HIV-1 may
be used as a source of candidate viral-specific antigens,
and a patient infected with any isolate of HIV-1 (e.g.,
HIV-lMN) may be treated usiny the methods of this
invention. Proteins or protein fragments homologous to
HIV-1-encoded proteins may also be useful in this
invention if such proteins or fragments elicit an HIV-1-
specific CTL response; such proteins may be coded for by
other primate lentiviruses, e.g., HTLV-I and HTLV-II as
well as the simian immunodeficiency viruses. T cell
lines or pathogen-infected patients may be presented
simultaneously with more than one pathogen-specific CTL-
stimulatory epitope; such epitopes may be resident in the
same or in different proteins.
Similarly, any pathogen-infected mammal
(particularly, domesticated animals and livestock) may be
treated with a therapeutic amount of a CTL-stimulatory
pathogen-specific antigen or with a sub-sample of the
mammal's lymphocytes enriched for CTL's which recognize
and lyse cells bearing such an antigen, using the methods
described above. Moreover, these methods can be used to
identify CTL-stimulatory antigens of, and to treat human
patients or mammals infected with, other pathogenic
viruses including, but not limited to, HIV-2, human T-
.
.
W092/0~62 PCT/US91tO~a ~
2089~88
cell leukemia viruses, Herpes viruses (e.g., Epstein-
Barr virus) as well as any intracellular disease-causing
mycobacterium, protozoan, or fungus. Generally, sub-
samples of pathogen-specific CTL's would be prepared as
described above for HIV-l, i.e., by culturing a patient's
lymphocytes in the presence of pathogen-infected cells or
in the presence of one or more CTL-stimulatory pathogen-
specific epitopes. In the case of an intracellular
pathogen which does not reside in peripheral blood
lymphocytes, a sample of the patient's or mammal's cells
which harbor pathogen would be isolated by standard
techni~ues and co-cultured with a sample of the patient's
or mammal's lymphocytes to produce a pathogen-specific
CTL-enriched lymphocyte sub-sample.
Pathogen-specific antigens may be presented to T
cells in processed form on the surface of an antigen-
presenting cell other than a B-lymphocyte, e.g., on the
surface of an autologous PBL or an autologous cell of
monocytic lineage.
