Note: Descriptions are shown in the official language in which they were submitted.
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HLA BINDING PEPTIDES AND THEIR USES
REFERENCE TO RELATED APPLICATIONS
The present application is a continuation-in-part of USSN 08/205,713 filed
March 4, 1994. The present application is also related to USSN 09/017,735,
TJSSN
08/753,622, USSN 08/822,382, USSN 60/013,980, USSN 08/589,108, USSN
08/454,033,
USSN 08/349,177, USSN 08/073,205, and USSN 08/027,146. The present application
is
also related to USSN 091017,524, USSN 08/821,739, USSN 60/013,833, USSN
08/758,409, USSN 08/589,107, USSN 08/451,913 and to USSN 08/347,610, USSN
08/186,266, USSN 08/159,339, USSN 09/116,061, USSN 08/103,396, USSN
08/027,746,
and USSN 07/926,666. The present application is also related to USSN
09/017,743;
USSN 08/753,615; USSN 08/590,298; USSN 08/452,843; USSN 09/115,400; USSN
08/344,824; and USSN 08/278,634. The present application is also related to
USSN
08/197,484 and USSN 08/815,396. All of the above applications are incorporated
herein
by reference.
BACKGROUND OF THE INVENTION
The present invention relates to compositions and methods for preventing,
treating or diagnosing a number of pathological states such as viral diseases
and cancers.
In particular, it provides novel peptides capable of binding selected major
histocompatibility complex (MHC) molecules and inducing an immune response.
MHC molecules axe classified as either Class I or Class II molecules.
Class II MHC molecules are expressed primarily on cells involved .in
initiating and
sustaining immune responses, such as T lymphocytes, B lymphocytes,
macrophages, etc.
Class II MHC molecules axe recognized by helper T lymphocytes and induce
proliferation
of helper T lymphocytes and amplification of the immune response to the
particular
immunogenic peptide that is displayed. Class I MHC molecules are expressed on
almost
all nucleated cells and are recognized by cytotoxic T lymphocytes (CTLs),
which then
destroy the antigen-bearing cells. CTLs are particularly important in tumor
rejection and
in fighting viral infections.
The CTL recognizes the antigen in the form of a peptide fragment bound
to the MHC class I molecules rather than. the intact foreign antigen itself.
The antigen
must normally be endogenously synthesized by the cell, and a portion of the
protein
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2
antigen is degraded into small peptide fragments in the cytoplasm.Some of
these small
peptides translocate into a pre-Golgi compartment and interact with class I
heavy chains
to facilitate proper folding and association with the subunit j32
microglobulin. The
peptide-MHC class I complex is then routed to the cell surface for expression
and
potential recognition by specific CTLs.
Investigations of the crystal structure of the human MHC class I molecule,
HLA-A2.1, indicate that a peptide binding groove is created by the folding of
the al anal
a2 domains of the class I heavy chain (Bjorkman et al., Nature 329:506 (
1987). In these
investigations, however, the identity of peptides bound to the groove was not
determined.
Buus et al., Science 242:1065 (1988) first described a method for acid
elution of bound peptides from MHC. Subsequently, Rammensee and his coworkers
(Falk et al., Nature 351:290 (1991) have developed an approach to characterize
naturally
processed peptides bound to class I molecules. Other investigators have
successfully
achieved direct amino acid sequencing of the more abundant peptides in various
HPLC
fractions by conventional automated sequencing of peptides eluted from class I
molecules
of the B type (Jardetzky, et al., Nature 353:326 (1991) and of the A2.1 type
by mass
spectrometry (Hunt, et al., Science 225:1261 (1992). A review of the
characterization of
naturally processed peptides in MHC Class I has been presented by Rotzschke
and Falk
(Rotzschlce and Falk, Immunol. Today 12:447 (1991).
Sette et al., Proc. Natl. Acad. Sci. USA 86:3296 (1989) showed that MHC
allele specific motifs could be used to predict MFiC binding capacity.
Schaeffer et al.,
Proc. Natl. Acad. Sci. USA 86:4649 (1989) showed that MHC binding was related
to
immunogenicity. Several authors (De Bruijn et al., Eur. J. Immunol., 21:2963-
2970
(1991); Pamer et al., 991 Nature 353:852-955 (1991)) have provided preliminary
evidence that class I binding motifs can be applied to the identification of
potential
immunogenic peptides in animal models. Class I motifs specific for a number of
human
alleles of a given class I isotype have yet to be described. It is desirable
that the
combined frequencies of these different alleles should be high enough to cover
a large
fraction or perhaps the majority of the human outbred population.
Despite the developments in the art, the prior art has yet to provide a
useful human peptide-based vaccine or therapeutic agent based on this work.
The present
invention provides these and other advantages.
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3
SUMMARY OF THE INVENTION
The present invention provides compositions comprising immunogenic
peptides having binding motifs for HLA-A2.1 molecules. The immunogenic
peptides,
wluch bind to the appropriate MHC allele, are preferably 9 to 10 residues in
length and
comprise conserved residues at certain positions such as positions 2 and 9.
Moreover, the
peptides do not comprise negative binding residues as defined herein at other
positions
such as positions 1, 3, 6 and/or 7 in the case of peptides 9 amino acids in
length and
positions 1, 3, 4, 5, 7, 8 and/or 9 in the case of peptides 10 amino acids in
length. The
present invention defines positions within a motif enabling the selection of
peptides
which will bind efficiently to HLA A2.1.
The motifs of the inventions include peptide of 9 amino acids which have
a first conserved residue at the second position from the N-terminus selected
from the
,group consisting of I, V, A and T and a second conserved residue at the C-
terminal
position selected from the group consisting of V, L, I, A and M.
Alternatively, the
1 S peptide may have a first conserved residue at the second position from the
N-terminus
selected from the group consisting of L, M, I, V, A and T; and a second
conserved residue
at the C-terminal position selected from the group consisting of A and M. If
the peptide
has 10 residues it will~contain a first conserved residue at the second
position from the N-
terminus selected from the group consisting of L, M, I, V, A, and T; and a
second
conserved residue at the C-terminal position selected from the group
consisting of V, I, L,
A and M; wherein the first and second conserved residues are separated by 7
residues.
Epitopes on a number of immunogenic target proteins can be identified
using the peptides of the invention. Examples of suitable antigens include
prostate cancer
specific antigen (PSA), prostate specific membrane antigen (PSM), hepatitis B
core and
surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barn virus
antigens, human
immunodeficiency type-1 virus (HIV1), Kaposi's sarcoma herpes virus (KSHV),
human
papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis (MT),
p53 and
marine p53 (mp53), CEA, trypanosome surface antigen (TSA), members of.the
tyrosinas
related protein (TRP) families, and Her2/neu. The peptides are thus useful in
pharmaceutical compositions fox both in vivo and ex vivo therapeutic and
diagnostic
applications.
The present invention also provides compositions comprising
immunogenic peptides having binding motifs for MHC Class I molecules. The
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4
immunogenic peptides are typically between about 8 and about 11 residues and
comprise
conserved residues involved in binding proteins encoded by the appropriate MHC
allele.
A number of allele specific motifs have been identified.
For instance, the motif for HLA-A3.2 comprises from the N-terminus to
C-terminus a first conserved residue of L, M, I, V, S, A, T and F at position
2 and a
second conserved residue of K, R or Y at the C-terminal end. Other first
conserved
residues are C, G or D and alternatively E. Other second conserved residues
are H or F.
The first and second conserved residues are preferably separated by 6 to 7
residues.
The motif for HLA-A1 comprises from the N-terminus to the C-terminus a
first conserved residue of T, S or M, a second conserved residue of D or E,
and a third
conserved residue of Y. Other second conserved residues are A, S or T. The
first and
second conserved residues are adjacent and are preferably separated from the
third
conserved residue by 6 to 7 residues. A second motif consists.of a frst
conserved residue
lof E or D and a second conserved residue of Y where the first and second
conserved
residues are separated by 5 to 6 residues.
The motif for HLA-A11 comprises from the N-terminus to the C-terminus
a first conserved residue of T, V, M, L, I, S, A, G, N, C D, or F at position
2 and a C-
terminal conserved residue of K, R, Y or H. The first and second conserved
residues are
preferably separated by 6 or 7 residues.
The motif for HLA-A24.1 comprises from the N-terminus to the C-
terminus a first conserved residue of Y, F or W at position 2 and a C terminal
conserved
residue of F, I, W, M or L. The first and second conserved residues are
preferably
separated by 6 to 7 residues.
Epitopes on a number of potential target proteins can be identified in this
manner. Examples of suitable antigens include prostate specific antigen (PSA),
prostate
specific membrane antigen (PSM), hepatitis B core and surface antigens (HBVc,
HBVs)
hepatitis C antigens, malignant melanoma azitigen IMAGE-1) Epstein-Barr virus
antigens, human immunodeficiency type-1 virus (HIV1), papilloma virus
antigens, Lassa
virus, mycobacterium tuberculosis (MT), p53 and marine p53 (mp53), CEA, and
Her2/neu, and members of the tyrosinase related protein (TRP) families. The
peptides are
thus useful in pharmaceutical compositions fox both in vivo and ex vivo
therapeutic and
diagnostic applications.
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The present invention also provides compositions comprising
immunogenic peptides having binding motifs for non-A HLA alieles. The
immunogenic
peptides are preferably about 9 to 10 residues in length and comprise
conserved residues
at certain positions such as proline at position 2 and an aromatic residue
(e.g., Y, W, F) or
5 hydrophobic residue (e.g., L, I, V, M, or A) at the carboxy terminus. In
particular, an
advantage of the peptides of the invention is their ability to bind to two or
more different
HLA alleles.
Epitopes on a number of potential target proteins can be identified in this
manner. Examples of suitable antigens include prostate specific antigen (PSA),
hepatitis
B core and surface antigens (HBVc, HBVs) hepatitis C antigens, malignant
melanoma
antigen IMAGE-1) Epstein-Barr virus antigens, human. immunodeficiency type-1
virus
(HIV 1), papilloma virus antigens, Lassa virus, mycobacterium tuberculosis
(MT), p53,
CEA, and Her2/neu. The peptides are thus useful in pharmaceutical compositions
for
both in vivo and ex vivo therapeutic and diagnostic applications.
Definitions
The term "peptide" is used interchangeably with "oligopeptide" in the
present specification to designate a series of residues, typically L-amino
acids, connected
one to the other typically by peptide bonds between the alpha-amino and
carbonyl groups
of adjacent amino acids. The oligopeptides of the invention are less than
about 15
residues in length anal usually consist of between about 8 and about 11
residues,
preferably 9 or 10 residues.
An "immunogenic peptide" is a peptide which comprises an allele-specific
motif such that the peptide will bind an MHC molecule and induce a CTL
response.
Immunogenic peptides of the invention are capable of binding to an appropriate
HLA-
A2.1 molecule and inducing a cytotoxic T cell response against the antigen
from which
the immunogenic peptide is derived.
Immunogenic peptides are conveniently identified using the algorithms of
the invention. The algorithms are mathematical procedures that produce a score
which
enables the selection of immunogenic peptides. Typically one uses the
algorithmic score
with a "binding threshold" to enable selection of peptides that have a high
probability of
binding at a certain affinity and will in turn be immunogenic. The algorithm
is based
upon either the effects on MHC binding of a particular amino acid at a
particular position
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6
of a peptide or the effects on binding of a particular substitution in a motif
containing
peptide.
A "conserved residue" is an amino acid which occuxs in a significantly
higher frequency than would be expected by random distribution at a particular
position
in a peptide. Typically a conserved residue is one where the MHC structure may
provide
a contact point with the immunogenic peptide. At least one to three or more,
preferably
two, conserved residues within a peptide of defined length defines a motif for
an
immunogenic peptide. These residues are typically in close contact with the
peptide
binding groove, with their side chains buried in specific pockets of the
groove itself.
Typically, an immunogenic peptide will comprise up to three conserved
residues, more
usually two conserved residues.
As used herein, "negative binding residues" are amino acids which if
present at certain positions (for example, positions 1, 3 and/or 7 of a 9-mer)
will result in
7a peptide being a nonbinder or poor binder and in turn fail to be immunogenic
i.e. induce
a CTL response.
The term "motif' refers to the pattern of residues in a peptide of defined
length, usually about 8 to about 11 amino acids, which is recognized by a
particular MHC
allele. The peptide motifs are typically different fox each human MHC allele
and differ in
the pattern of the highly conserved residues and negative residues.
The binding motif for an allele can be defined with increasing degrees of
precision. In one case, all of the conserved residues are present in the
correct positions in
a peptide and there are no negative residues in positions 1,3 and/or 7.
The phrases "isolated" or "biologically pure" refer to material which is
substantially or essentially free from components which normally accompany it
as found
in its native state. Thus, the peptides of this invention do not contain
materials normally
associated with their in situ environment, e.g., MHC I molecules on antigen
presenting
cells. Even where a protein has been isolated to a homogenous or dominant
band, there
are trace contaminants in the range of 5-10% of native protein which co-purify
with the
desired protein. Isolated peptides of this invention do not contain such
endogenous co-
purified protein.
The term "residue" refers to an amino acid or amino acid mimetic
incorporated in an oligopeptide by an amide bond or amide bond mimetic.
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DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. HLA-A2.1 Motif
The present invention relates to the determination of allele-specific peptide
motifs for human Class I MHC (sometimes referred to as HLA) allele subtypes,
in
particular, peptide motifs recognized by HLA-A2.1 alleles. These motifs are
then used to
define T cell epitopes from any desired antigen, particularly those associated
with human
viral diseases, cancers or autoiummune diseases, for which the amino acid
sequence of
the potential antigen or autoantigen targets is known.
Epitopes on a number of potential target proteins can be identified in this
manner. Examples of suitable antigens include prostate specific antigen (PSA),
hepatitis
B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Bare
virus
antigens, melanoma antigens (e.g., MAGE-1), human immunodeficiency virus (HIV)
,antigens, human papilloma virus (HPV) antigens, Lassa virus, mycobacterium
tuberculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2/neu.
Peptides comprising the epitopes from these antigens are synthesized and
then tested for their ability to bind to the appropriate MHC molecules in
assays using, for
example, purified class I molecules and radioiodonated peptides and/or cells
expressing
empty class I molecules by, for instance, immunofluorescent staining and flow
microfluorometry, peptide-dependent class I assembly assays, and inhibition of
CTL
recognition by peptide competition. Those peptides that bind to the class I
molecule are
further evaluated for their ability to serve as targets fox CTLs derived from
infected or
immunized individuals, as well as for their capacity to induce primary in
vitro or in vivo
CTL responses that can give rise to CTL populations capable of reacting with
virally
infected target cells or tumor cells as potential therapeutic agents.
The MHC class I antigens are encoded by the HLA-A, B, and C loci.
HLA-A and B antigens are expressed at the cell surface at approximately equal
densities,
whereas the expression of HLA-C is significantly lower (perhaps as much as 10-
fold
- lower). Each of these loci have a number of alleles. The peptide binding
motifs of the
invention are relatively specific for each allelic subtype.
For peptide-based vaccines, the peptides of the present invention
preferably comprise a motif recognized by an MHC I molecule having a wide
distribution
in the human population. Since the MHC alleles occur at different frequencies
within
different ethnic groups and races, the choice of target MHC allele may depend
upon the
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target population. Table 1 shows the frequency of various alleles at~the HLA-A
locus
products among different races. For instance, the majority of the, Caucasoid
population
can be covered by peptides which bind to four HLA-A allele subtypes,
specifically HLA-
A2.1, Al, A3.2, and A24.1. Similarly, the majority of the Asian population is
encompassed with the addition of peptides binding to a fifth allele HLA-A11.2.
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9 ..
TABLE 1
A Allele/SubtypeN(69)* A(54) 27 4(1038)
Al 10.1(7) 1.8(1) 39.8(199)
5(8) 37.0(20)
11
. 3.3(17)
A2.1 10.1 (7) 0
2
A2
0. 8 (4)
. 1.4(1) 5.5(3)
A2.3
A2.4 - -
1 1.4(1) 0 0.2(0)
A3
.
2 5.7(4) 5.5(3) 21.5(108)
A3
. 0
All.l 7(4) 31 4(17) 8.7(44)
2 ~
Al 1. . 3.7(2) 0
3
X3. 4.3(3) _ 3.9(20)
~4 2.9(2) 27.7(15) 15.3(77)
A24.2 - -
A24.3 - 6.9(35)
A25 1.4(1) _
1 4.3(3) 9.2(5) 5.9(30)
A26
. 7.2(5) _ 1.0(5)
A26.2
A26V - 3.7(2) _
6(8)
1
'- A28.1 10.1 (7) - .
5(38)
7
A28.2 1.4(1) _ .
1 4(1) _ 1.4(7)
9 1
. . 1.8(1) 5.3 (27)
A2 10.1 (7)
2
A29
. 8.6(6) - 4.9(25)
A30.1
A30.2 1.4(1) . - 0.2(1)
A30.3 7.2(5) - 3.9(20)
A31 4.3(3) 7.4(4) 6.9(35)
A32 2.8(2) _ 7.1 (36)
Aw33.1 8.6(6) - ~ 2.5(13)
Aw33.2 2.8(2) 16.6(9) 1.2(6)
Aw34.1 1.4(1) - -
8(4)
0
Aw34.2 14.5(10) _ .
Aw36 5.9(4) -
Table compiled from B. DuPont, Immunobiolog.~of HLA, Vol. I,
Histocompatibility
Testing 1987, Springer-Verlag, New York 1989.
* N - negroid; A = Asian; C = Caucasoid. Numbers in
parenthesis represent the number of individuals included in the analysis.
The nomenclature used to describe peptide compounds follows the
conventional practice wherein the amino group is presented to the left (the N-
terminus)
and the carboxyl group to the right (the C_terminus) of each amino acid
residue. In the
formulae representing selected specific embodiments of the present invention,
the amino-
and carboxyl-terminal groups, although not specifically shown, are in the form
they
would assume at physiologic pH values, unless otherwise specified. In the
amino acid
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structure formulae, each residue is generally represented by standard three
letter or single
letter designations. The L-form of an amino acid residue is represented by a
capital single
letter or a capital first letter of a three-letter symbol, and the D-form for
those amino acids
having D-fortes is represented by a lower case single letter or a lower case
three letter
5 ' symbol. Glycine has no asymmetric carbon atom and is simply referred to as
"Gly" or G.
The procedures used to identify peptides of the present invention generally
follow the methods disclosed in Falk et al., Nature 351:290 (1991), which is
incorporated
herein by reference. Briefly, the methods involve large-scale isolation of MHC
class I
molecules, typically by immunoprecipitation or affinity chromatography, from
the
10 appropriate cell or cell line. Examples of other methods for isolation of
the desired MHC
molecule equally well known to the artisan include ion exchange
chromatography, lectin
chromatography, size exclusion, high performance ligand chromatography, and a
combination of all of the above techniques.
In the typical case, immunoprecipitation is used to isolate the desired
allele. A number of protocols can be used, depending upon the specificity of
the
antibodies used. For example, allele-specific mAb reagents can be used for the
affinity
purification of the HLA-A, HLA-B1, and HLA-C molecules. Several mAb reagents
for
the isolation of HLA-A molecules are available. The monoclonal BB7.2 is
suitable.for
isolating HLA-A2 molecules. Affinity columns prepared with these mAbs using
standard
techniques are successfully used to purify the respective HLA-A allele
products.
In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C
mAbs, such as W6/32 and B9.12.1, and one anti-HLA-B, C mAb, B 1.23.2, could be
used
in alternative affinity purification protocols as described in previous
applications.
The peptides bound to the peptide binding groove of the isolated MHC
molecules are eluted typically using acid treatment. Peptides can also be
dissociated from
class I molecules by a variety of standard denaturing means, such as heat, pH,
detergents,
salts, chaotropic agents, or a combination thereof.
Peptide fractions are further separated from the MHC molecules by
reversed-phase high performance liquid chromatography (HPLC) and sequenced.
Peptides can be separated by a variety of other standard means well known to
the artisan,
including filtration, ultrafiltration, electrophoresis, size chromatography,
precipitation
with specific antibodies, ion exchange chromatography, isoelectrofocusing, and
the like.
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11
Sequencing of the isolated peptides can be performed according to
standard techniques such as Edman degradation (Hunkapiller, M.W., et al.,
Methods
Enzymol. 91, 399 [1983]). Other methods suitable for sequencing include mass
spectrometry sequencing of individual peptides as previously described (Hunt,
et al.,
Science 225:1261 (1992), which is incorporated herein by reference). Amino
acid
sequencing of bulk heterogenous peptides (~, pooled HPLC fractions) from
different
class I molecules typically reveals a characteristic sequence motif for each
class I allele.
Definition of motifs specific for different class I alleles allows the
identification of potential peptide epitopes from an antigenic protein whose
amino acid
sequence is known. Typically, identification of potential peptide epitopes is
initially
carried out using a computer to scan the amino acid sequence of a desired
antigen for the
presence of motifs. The epitopic sequences are then synthesized. The capacity
to bind
~IIiC Class molecules is measured in a variety of different ways. One means is
a Class I
molecule binding assay as described in the related applications, noted above.
Other
alternatives described in the literature include inhibition of antigen
presentation (Sette, et
al., J. Imlxmnol. 141:3893 (1991), in vitro assembly assays (Townsend, et al.,
Cell 62:285
(1990), and FACS based assays using mutated cells, such as RMA.S (Melief, et
al., Eur.
J. Ixnmunol. 21:2963 (1991)).
Next, peptides that test positive in the MHC class I binding assay are
.assayed for the ability of the peptides to induce specific CTL responses in
vitro. For
instance, antigen-presenting cells that have been incubated with a peptide can
be assayed
for the ability to induce CTL responses in responder cell populations. Antigen-
presenting
cells can be normal cells such as peripheral blood mononuclear cells or
dendritic cells
(Inaba, et al., J. Exp. Med. 166:182 (1987); Boog, Eur. J. Immunol. 18:219
[1988]).
Alternatively, mutant mammalian cell lines that are deficient in their
ability to load class I molecules with internally processed peptides, such as
the mouse cell
lines RMA-S (Karre, et al.. Nature, 319:675 (1986); Ljunggren, et al., Eur. J.
Immunol.
21:2963-2970 (1991)), and the human somatic T cell hybrid, T-2 (Cerundolo, et
al.,
Nature 345:449-452 (1990)) and which have been transfected with the
appropriate human
class I genes are conveniently used, when peptide is added to them, to test
for the capacity
of the peptide to induce in vitro primary CTL responses. Other eukaryotic cell
lines
which could be used include various insect cell lines such as mosquito larvae
(ATCC cell
lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851),
armyworm
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12 . ,
(ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a
Schneider
cell line (see Schneider J Embryol Exp. Morphol. 27:353-365 [1:927]).
Peripheral blood lymphocytes are conveniently isolated following simple
venipuncture or leukapheresis of normal donors or patients and used as the
responder cell
sources of CTL precursors. In one embodiment, the appropriate antigen-
presenting cells
are incubated with 10-100 ~M of peptide in serum-free media for 4 hours under
appropriate cultwre conditions. The peptide-loaded antigen-presenting cells
are then
incubated with the responder cell populations in vitro for 7 to 10 days under
optimized
culture conditions. Positive CTL activation can be determined by assaying the
cultures
for the presence of CTLs that kill radiolabeled target cells, both specific
peptide-pulsed
targets as well as target cells expressing the endogenously processed form of
the relevant
virus or tumor antigen from which the peptide sequence was derived.
Specificity and MHC restriction of the CTL is determined by testing
against different peptide target cells expressing appropriate or inappropriate
human MHC
class I. The peptides that test positive in the MHC binding assays and give
rise to specific
CTL responses are referred to herein as immunogenic peptides.
The immunogenic peptides can be prepared synthetically, or by
recombinant DNA technology or from natural sources such as whole viruses or
tumors.
Although the peptide will preferably be substantially free of other naturally
occurnng host
cell proteins and fragments thereof, in some embodiments the peptides can be
synthetically conjugated to native fragments or particles.
The polypeptides or peptides can be a variety of lengths, either in their
neutral (uncharged) forms or in forms which are salts, and either free of
modifications
such as glycosylation, side chain oxidation, or phosphorylation or containing
these
modifications, subject to the condition that the modification not destroy the
biological
activity of the polypeptides as herein described.
Desirably, the peptide will be as small as possible while still maintaining
substantially all of the biological activity of the large peptide. When
possible, it may be
desirable to optimize peptides of the invention to a length of 9 or 10 amino
acid residues,
commensurate in size with endogenously processed viral peptides or tumor cell
peptides
that are bound to MHC class I molecules on the cell surface.
Peptides having the desired activity may be modified as necessary to
provide certain desired attributes, e.g., improved pharmacological
characteristics, while
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13 ,
increasing or at least retaining substantially all of the biological activity'
of the unmodified
peptide to bind the desired MHC molecule and activate the appropriate T cell.
For
instance, the peptides may be subject to various changes, such as
substitutions, either
conservative or non-conservative, where such changes might provide for certain
advantages in their use, such as improved MHC binding. By conservative
substitutions is
meant replacing an amino acid residue with another which is biologically
and/or
chemically similar, e.g., one hydrophobic residue for another, or one polar
residue for
another. The substitutions include combinations such as Gly, Ala; Val, Ile,
Leu, Met;
Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single
amino acid
substitutions may also be probed using D-amino acids. Such modifications may
be made
using well known peptide synthesis procedures, as described in e.g.,
Merrifield, Science
232:341-347 (1986), Barany and Merrifield, The Peptides, Gross and Meienhofer,
eds.
(N.Y., Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase
Peptide
Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984), incorporated by reference
herein.
The peptides can also be modified by extending or decreasing the
compound's amino acid sequence, e.g., by the addition or deletion of amino
acids. The
peptides or analogs of the invention can also be modified by altering the
order or
composition of certain residues, it being readily appreciated that certain
amino acid
residues essential for biological activity, e.g., those at critical contact
sites or conserved
residues, may generally not be altered without an adverse effect on biological
activity.
The non-critical amino acids need not be limited to those naturally occurring
in proteins,
such as L-a-amino acids, or their D-isomers, but may include non-natural amino
acids as
well, such as ~i-'y-8-amino acids, as well as many derivatives of L-a-amino
acids.
Typically, a series of peptides with single amino acid substitutions are
employed to determine the effect of electrostatic charge, hydrophobicity, etc.
on binding.
For instance, a series of positively charged (e.g., Lys or Arg) or negatively
charged (e.g.,
Glu) amino acid substitutions are made along the length of the peptide
revealing different
patterns of sensitivity towards various MHC molecules and T cell receptors. In
addition,
multiple substitutions using small, relatively neutral moieties such as Ala,
Gly, Pro, or
similar residues may be employed. The substitutions may be homo-oligomers or
hetero-
. oligomers. The number and types of residues which are substituted or added
depend on
the spacing necessary between essential contact points and certain functional
attributes
which are sought (e.g., hydrophobicity versus hydrophilicity). Increased
binding affinity
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14
fox an MHC molecule or T cell receptor may also be achieved by
such'substitutions,
compared to the affinity of the parent peptide. In any event, such
substitutions should
employ amino acid residues or other molecular fragments chosen to avoid, for
example,
steric and charge interference which might disrupt binding.
Amino acid substitutions are typically of single residues. Substitutions,
deletions, insertions or any combination thereof may be combined to arrive at
a final
peptide. Substitutional variants are those in which at least one residue of a
peptide has
been removed and a different residue inserted in its place. Such substitutions
generally
are made in accordance with the following Table 2 when it is desired to finely
modulate
the characteristics of the peptide.
TABLE 2
Original Residue Exemplary Substitution
Ala Ser
~g Lys, His
Asn G~
Asp Glu
Cys Ser
G~ Asn
Glu Asp
Gly Pro
His LYs~ ~'g
Ile Leu; Val
Leu Ile; Val
Lys Arg; His
Met Leu; Ile
Phe Tyr; Trp
Ser T~'
S er
Trp Tyr; Phe
Tyr Trp; Phe
Val Ile; Leu
A
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Pro Gly
Substantial changes in function (e.g., affinity for MHC molecules or T cell
receptors) are made by selecting substitutions that are less conservative than
those in
5 Table 2, i.e., selecting residues that differ more significantly in their
effect on maintaining
(a) the structure of the peptide backbone in the area of the substitution, for
example as a
sheet or helical conformation, (b) the charge or hydrophobicity of the
molecule at the
target site or (c) the bulk of the side chain. The substitutions which in
general are
expected to produce the greatest changes in peptide properties will be those
in which (a)
10 hydrophilic residue, e.g. seryl, is substituted for (or by) a hydrophobic
residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a residue having an
electropositive side chain,
e.g., lysl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g.
glutamyl or aspartyl; or (c) a residue having a bulky side chain, e.g.
phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine.
15 The peptides may also comprise isosteres of two or more residues in the
irnmunogenic peptide. An isostere as defined here is a sequence of two or more
residues
that can be substituted for a second sequence because the steric conformation
of the first
sequence fits a binding site specific for the second sequence. The term
specifically
includes peptide backbone modifications well known to those skilled in the
art. Such
modifications include modifications of the amide nitrogen, the a-carbon, amide
carbonyl,
complete replacement of the amide bond, extensions, deletions or backbone
crosslinks.
See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides
and
Proteins, Vol. VII (Weinstein ed., 1983).
Modifications of peptides with various amino acid mimetics or unnatural
amino acids are particularly useful in increasing the stability of the peptide
in vivo.
Stability can be assayed in a number of ways. For instance, peptidases and
various
biological media, such as human plasma and serum, have been used to test
stability. See,
e.g., Verhoef et al., Eur. J. Drug Metab. Pharmacokin. 11:291-302 (1986). Half
life of
the peptides of the present invention is conveniently determined using a 25%
human
serum (v/v) assay. The protocol is generally as follows. Pooled human serum
(Type AB,
non-heat inactivated) is delipidated by centrifugation before use. The serum
is then
diluted to 25% with Rl'MI tissue culture media and used to test peptide
stability. At
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16
predetermined time intervals a small amount of reaction solution is removed
and added to
either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample
is cooled
(4°C) for 15 minutes and then spun to pellet the precipitated serum
proteins. The
presence of the peptides is then determined by reversed-phase HPLC using
stability-
specific chromatography conditions.
The peptides of the present invention or analogs thereof which have CTL
stimulating activity may be modified to provide desired attributes other than
improved
serum half life. For instance, the ability of the peptides to induce CTL
activity can be
enhanced by linkage to a sequence which contains at least one epitope that is
capable of
inducing a T helper cell response. Particularly preferred immunogenic
peptides/T helper
conjugates are linked by a spacer molecule. The spacer is typically comprised
of
relatively small, neutral molecules, such as amino acids or amino acid
mimetics, which
are substantially uncharged under physiological conditions. The spacers are
typically
selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino
acids or neutral
polar amino acids. It will be understood that the optionally present spacer
need not be
comprised of the same residues and thus may be a hetero- or homo-oligomer.
When
present, the spacer will usually be at least one or two residues, more usually
three to six
residues. Alternatively, the CTL peptide may be linked to the T helper peptide
without a
spacer.
2p The immunogenic peptide may be linked to the T helper peptide either
directly or via a spacer either at the amino or carboxy terminus of the CTL
peptide. The
amino terminus of either the imxriunogenic peptide or the T helper peptide may
be
acylated. Exemplary T helper peptides include tetanus toxoid 830-
843,.influenza 307-
319, malaria circumsporozoite 382-398 and 378-389.
In some embodiments it may be desirable to include in the pharmaceutical
compositions of the invention at least one component which primes CTL. Lipids
have
been identified as agents capable of priming CTL in vivo against viral
antigens. For
example, palmitic acid residues can be attached to the alpha and epsilon amino
groups of
a Lys residue and then linked, e.g., via one or more linking residues such as
Gly, Gly-
Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated
peptide can then
be injected directly in a micellar form, incorporated into a liposome or
emulsified in an
adjuvant, e.g., incomplete Freund's~adjuvant. In a preferred embodiment a
particularly
effective immunogen comprises palmitic acid attached to alpha and epsilon
amino groups
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17
of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of
the
immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli
lipoproteins, such as tripalinitoyl-S-glycerylcysteinlyseryl-serine (P3CSS)
can be used to
prime virus specific CTL when covalently attached to an appropriate peptide.
See, Deres
et al., Natuxe 342:561-564 (1989), incorporated herein by reference. Peptides
of the
invention can be coupled to P3CSS, for example, anal the lipopeptide
administered to an
individual to specifically prime a CTL response to the target antigen.
Further, as the
induction of neutralizing antibodies can also be primed with P3CSS conjugated
to a
peptide which displays an appropriate epitope, the two compositions can be
combined to
more effectively elicit both humoral and cell-mediated responses to infection.
In addition, additional amino acids can be added to the termini of a peptide
to provide for ease of linking peptides one to another, for coupling to a
carrier support, or
'larger peptide, for modifying the physical or chemical properties of the
peptide or
oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine,
glutamic or
aspartic acid, or the like, can be introduced at the C- or N-terminus of the
peptide or
oligopeptide. Modification at the C terminus in some cases may alter binding
characteristics of the peptide. In addition, the peptide or oligopeptide
sequences can
differ from the natural sequence by being modified by terminal-NH2 acylation,
e.g., by
allcanoyl (C1-Cao) or thioglycolyl acetylation, terminal-carboxyl amidation,
e.g.,
ammonia, methylamine, etc. In some instances these modifications may provide
sites for
linking to a support or other molecule.
The peptides of the invention can be prepared in a wide variety of ways.
Because of their relatively short size, the peptides can be synthesized in
solution or on a
solid support in accordance with conventional techniques. Various automatic
synthesizers are commercially available and can be used in accordance with
known
protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis,
2d. ed.,
Pierce Chemical Co. (1984), supra.
Alternatively, recombinant DNA technology may be employed wherein a
nucleotide sequence which encodes an immunogenic peptide of interest is
inserted into an
expression vector, transformed or transfected into an appropriate host cell
and cultivated
under conditions suitable for expression. These procedures are generally known
in the
art, as described generally in Sambrook et al., Molecular Cloning A Laboratory
Manual,
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18
Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), which is
incorporated
herein by reference. Thus, fusion proteins which comprise one or more peptide
sequences of the .invention can be used to pxesent the appropriate T cell
epitope.
As the coding sequence for peptides of the length contemplated herein can
be synthesized by chemical techniques, for example, the phosphotriester method
of
Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981), modification can be made
simply
by substituting the appropriate bases) for those encoding the native peptide
sequence.
The coding sequence can then be provided with appropriate linkers and ligated
into
expression vectors commonly available in the art, and the vectors used to
transform
suitable hosts to produce the desired fusion protein. A number of such vectors
and
suitable host systems are now available. Fox expression of the fusion
proteins, the coding
sequence will be provided with operably linked start and stop codons, promoter
and
terminator regions and usually a replication system to provide an expression
vector for
expression in the desired cellular host. For example, promoter sequences
compatible with
bacterial hosts are provided in plasmids containing convenient restriction
sites for
insertion of the desired coding sequence. The resulting expression vectors are
transformed into suitable bacterial hosts. Of course, yeast or mammalian cell
hosts may
also be used, employing suitable vectors and control sequences.
The peptides of the present invention and pharmaceutical and vaccine
compositions thereof are useful for administration to mammals, particularly
humans, to
treat and/or prevent viral infection and cancer. Examples of diseases which
can be treated
using the immunogenic peptides of the invention include prostate cancer,
hepatitis B,
hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV and
condlyloma acuminatum.
Fox pharmaceutical compositions, the immunogenic peptides of the
invexition are administered to an individual already suffering from cancer or
infected with
the virus of interest. Those in the incubation phase or the acute phase of
infection can be
treated with the immunogeni.c peptides separately or in conjunction with other
treatments,
as appropriate. In therapeutic applications, compositions are administered to
a patient in
an amount sufficient to elicit an effective CTL response to the virus or tumor
antigen and
to cure or at least partially arrest symptoms andlor complications. An amount
adequate to
accomplish this is defined as "therapeutically effective dose." Amounts
effective for this
use will depend on, e.g., the peptide composition, the manner of
administration, the stage
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19
and severity of the disease being treated, the weight and general state of
health of the
patient, and the judgment of the prescribing physician, but generally range
for the initial
immunization (that is for therapeutic or prophylactic administration) from
about 1.0 ~g to
about 5000 ~.g of peptide for a 70 kg patient, followed by boosting dosages of
from about
1.0 p.g to about 1000 ~.g of peptide pursuant to a boosting regimen over weeks
to months
depending upon the patient's response and condition by measuring specific CTL
activity
in the patient's blood. It must be kept in mind that the peptides and
compositions of the
present invention may generally be employed in serious disease states, that
is, life-
threatening or potentially life threatening situations. In such cases, in view
of the
minimization of extraneous substances and the relative nontoxic nature of the
peptides, it
is possible and may be felt desirable by the treating physician to administer
substantial
excesses of these peptide compositions.
For therapeutic use, administration should begin at the first sign of viral
infection or the detection or surgical removal of tumors or shortly after
diagnosis in the
case of acute infection. This is followed by boosting doses until at least
symptoms are
substantially abated and for a period thereafter. In chronic infection,
loading doses
followed by boosting doses may be required.
Treatment of an infected individual with the compositions of the invention
may hasten resolution of the infection in acutely infected individuals. For
those
individuals susceptible (or predisposed) to developing chronic infection the
compositions
axe particularly useful in methods for preventing the evolution from acute to
chronic
infection. Where the susceptible individuals are identified prior to or during
infection, fox
instance, as described herein, the composition can be targeted to them,
minimizing need
for administration to a larger population.
The peptide compositions can also be used for the treatment of chronic
infection and to stimulate the immune system to eliminate virus-infected cells
in Garners.
It is important to provide an amount of immuno-potentiating peptide in a
formulation and
mode of administration sufficient to effectively stimulate a cytotoxic T cell
response.
Thus, for treatment of chronic infection, a representative dose is in the
range of about 1.0
~.g to about 5000 ~,g, preferably about 5 ~,g to 1000 ~,g for a 70 kg patient
per dose.
Immunizing doses followed by boosting doses at established intervals, e.g.,
from one to
four weeks, may be required, possibly for a prolonged period of time to
effectively
immunize an individual. In the case of chronic infection, administration
should continue
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until at least clinical symptoms or laboratory tests indicate that the viral
infection has
been eliminated or substantially abated and for a period thereafter.
The pharmaceutical compositions for therapeutic treatment are intended
for parenteral, topical, oral or local administration. Preferably, the
pharmaceutical
5 compositions are administered parenterally, e.g., intravenously,
subcutaneously,
intradermally, or intramuscularly. Thus, the invention provides compositions
for
parenteral administration which comprise a solution of the immunogenic
peptides
dissolved or suspended in an acceptable carnet, preferably an aqueous carrier.
A variety
of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline,
0.3% glycine,
10 hyaluronic acid and the like. These compositions may be sterilized by
conventional, well
known sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions
may be packaged for use as is, or lyophilized, the lyophilized preparation
being combined
with a sterile solution prior to administration. The compositions may contain
?pharmaceutically acceptable auxiliary substances as required to approximate
15 physiological conditions, such as pH adjusting and buffering agents,
tonicity adjusting
agents, wetting agents and the like, for example, sodium acetate, sodium
lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine
oleate, etc.
The concentration of CTL stimulatory peptides of the invention in the
20 pharmaceutical formulations can vary widely, i.e., from less than about
0.1%, usually at
or at least about 2% to as much as 20% to 50% or more by weight, and will be
selected
primarily by fluid volumes, viscosities, etc., in accordance with the
particular mode of
administration selected.
The peptides of the invention may also be administered via liposomes,
which serve to target the peptides to a particular tissue, such as lymphoid
tissue, or
targeted selectively to infected cells, as well as increase the half life of
the peptide
composition. Liposomes include emulsions, foams, micelles, insoluble
monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the like. In
these
preparations the peptide to be delivered is incorporated as part of a
liposome, alone or in
conjunction with a molecule which binds to, e.g., a receptor prevalent among
lymphoid
cells, such as monoclonal antibodies which bind to the CD45 antigen, or with
other
therapeutic or immunogenic compositions. Thus, liposomes either filled or
decorated
with a desired peptide of the invention can be directed to the site of
lymphoid cells, where
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21
the liposomes then deliver the selected therapeutic/immunogenic peptide
compositions.
Liposomes for use in the invention are formed from standard vesicle-forming
lipids,
which generally include neutral and negatively charged phospholipids and a
sterol, such
as cholesterol. The selection of lipids is generally guided by
consideration'of, e.g.,
liposome size, acid lability and stability of the liposomes in the blood
stream. A variety
of methods are available for preparing liposomes, as described in, e.g., Szoka
et al., Ann.
Rev Biophys. Bioen~. 9:467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728,
4,837,028,
and 5,019,369, incorporated herein by reference.
For targeting to the immune cells, a ligand to be incorporated into the
liposome can include, e.g., antibodies or fragments thereof specific for cell
surface
determinants of the desired immune system cells. A liposome suspension
containing a
peptide may be administered intravenously, locally, topically, etc. in a dose
which varies
according to, inter alia, the manner of administration, the peptide being
delivered, and the
stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used
which include, for example, pharmaceutical grades of mann.itol, lactose,
starch,
magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose,
magnesium
carbonate, and the like. For oral administration, a pharmaceutically
acceptable nontoxic
composition is formed by incorporating any of the normally employed
excipients, such as
those carriers previously listed, and generally 10-95% of active ingredient,
that is, one or
more peptides of the invention, and more preferably at a concentration of 25%-
75%.
For aerosol administration, the immunogenic peptides are preferably
supplied in rinely divided form along with a surfactant and propellant.
Typical
percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The
surfactant
must, of course, be nontoxic, and preferably soluble in the propellant.
Representative of
such agents are the esters or paxtial esters of fatty acids containing from 6
to 22 carbon
atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,
linolenic, olesteric and
oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters,
such as mixed or natural glycerides may be employed. The surfactant may
constitute
0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the
composition is ordinarily propellant. A carrier can also be included, as
desired, as with,
e.g., lecithin for intranasal delivery.
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22
In another aspect the present invention is directed to vaccines which
contain as an active ingredient an immunogenically effective amount of an
immunogenic
peptide as described herein. The peptides) may be introduced into a host,
including
humans, linked to its own earner or as a homopolymer or heteropolymer of
active peptide
units. Such a polymer has the advantage of increased immunological reaction
and, where
different peptides are used to make up the polymer, the additional ability to
induce
antibodies and/or CTLs that react with different antigenic determinants of the
virus or
tumor cells. Useful carriers are well known in the art, and include, e.g.,
thyroglobulin,
albumins such as human serum albumin, tetanus toxoid, polyamino acids such as
poly(lysixie:glutamic acid), influenza, hepatitis B virus core protein,
hepatitis B virus
recombinant vaccine and the like. The vaccines can also contain a
physiologically
tolerable (acceptable) diluent such as water, phosphate buffered saline, or
saline, and
further typically include an adjuvant. Adjuvants such as incomplete Freund's
adjuvant,
aluxninum phosphate, aluminum hydroxide, or alum are materials well known in
the art.
And, as mentioned above, CTL responses can be primed by conjugating peptides
of the
invention to lipids, such as P3CSS. Upon immunization with a peptide
composition as
described herein, via injection, aerosol, oral, transdermal or other route,
the immune
system of the host responds to the vaccine by producing large amounts of CTLs
specific
for the desired antigen, and the host becomes at least partially immune to
later infection,
or resistant to developing chronic infection.
Vaccine compositions containing the peptides of the invention axe
administered to a patient susceptible to or otherwise at risk of viral
infection or cancer to
elicit an immune response against the antigen and thus enhance the patient's
own immune
response capabilities. Such an amount is defined to be an "immunogenically
effective
dose." In this use, the precise amounts again depend on the patient's state of
health and
weight, the mode of administration, the nature of the formulation, etc., but
generally
range from about 1.0 ~g to about 5000 ~,g per 70 kilogram patient, more
commonly from
about 10 ~.g to about 500 ~,g mg per 70 kg of body weight.
In some instances it may be desirable to combine the peptide vaccines of
the invention with vaccines which induce neutralizing antibody responses to
the virus of
interest, particularly to viral envelope antigens.
For therapeutic or immunization purposes, nucleic acids encoding one or
more of the peptides of the invention can also be administered to the patient.
A number
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23
of methods are conveniently used to deliver the nucleic acids to the patient.
For instance,
the nulceic acid can be delivered directly, as "naked DNA". This approach is
described,
for instance, in Wolff et. al., Science 247: 1465-1468 (1990) as well as U.S.
Patent Nos.
5,580,859 and 5,589,466. The nucleic acids can also be administered using
ballistic
delivery as described, for instance, in U.S. Patent No. 5,204,253. Particles
comprised
solely of DNA can be administered. Alternatively, DNA can be adhered to
particles, such
as gold particles. The nucleic acids can also be delivered complexed to
cationic
compounds, such as cationic lipids. Lipid-mediated gene delivery methods are
described,
for instance, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988)
BioTechniques 6(7): 682-691; Rose U.S. Pat No. 5,279,833; WO 91/06309; and
Felgner
et al. (1987) Proc. lVatl. Acad. Sci. USA 84: 7413-7414. The peptides of the
invention
can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox.
This
approach involves the use of vaccinia virus as a vector to express nucleotide
sequences
that encode the peptides of the invention. Upon introduction into an acutely
or
chronically infected host or into a noninfected host, the recombinant vaccinia
virus
expresses the immunogenic peptide, and thereby elicits a host CTL response.
Vaccinia
vectors and methods useful in immunization protocols are described in,
e.g.,U.S. Patent
No. 4,722,848, incorporated herein by reference. Another vector is BCG
(Bacille
Cahnette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-
460
(1991)) which is incozporated herein by reference. A wide variety of other
vectors useful
for therapeutic administration or immunization of the peptides of the
invention, e.g.,
Salmonella typhi vectors and the like, will be apparent to those skilled in
the art from the
description herein.
A preferred means of administering nucleic acids encoding the peptides of
the invention uses minigene constructs encoding multiple epitopes of the
invention. To
create a DNA sequence encoding the selected CTL epitopes (minigene) for
expression in
human cells, the amino acid sequences of the epitopes are reverse translated.
A human
codon usage table is used to guide the codon choice for each amino acid. These
epitope-
encoding DNA sequences are directly adjoined, creating a continuous
polypeptide
sequence. To optimize expression and/or immunogenicity, additional elements
can be
incorporated into the minigene design. Examples of amino acid sequence that
could be
reverse translated and included in the minigene sequence include: helper T
lymphocyte
epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention
signal. In
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24
addition, MHC presentation of CTL epitopes may be improved by including
synthetic
(e.g: poly-alanine) or naturally-occurring flanking sequences adjacent to the
CTL
epitopes.
The minigene sequence is converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the minigene.
Overlapping
oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified
and
annealed under appropriate conditions using well known techniques. he ends of
the
oligonucleotides are joined using T4 DNA ligase. This synthetic minigene,
encoding the
CTL epitope polypeptide, can then cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are
included in the vector to ensure expression in the target cells. Several
vector elements are
required: a promoter with a down-stream cloning site for minigene insertion; a
polyadenylation signal for efficient transcription termination; an E. coli
origin of
replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin
resistance).
Numerous promoters can be used for this purpose, e.g., the human
cytomegalovirus
(hCMV) promoter. See, U.S. Patent Nos. 5,580,859 and 5,589,466 for other
suitable
promoter sequences.
Additional vector modifications may be desired to optimize minitgene
expression and immunogenicity. In some cases, introns are required for
efficient gene
expression, and one or more synthetic or naturally-occurring introns could be
incorporated into the transcribed region of the minigene. The inclusion of
mRNA
stabilization sequences can also be considered for increasing minigene
expression. It has
recently been proposed that immunostimulatory sequences (ISSs or CpGs) play a
role in
the immunogenicity of DNA vaccines. These sequences could be included in the
vector,
outside the minigene coding sequence, if found to enhance immunogenicity.
In some embodiments, a bioistronic expression vector, to allow production
of the minigene-encoded epitopes and a second protein included to enhance or
decrease
immunogenicity can be used. Examples of proteins or polypeptides that could
beneficially enhance the immune response if co-expressed include cytokines
(e.g., IL2,
IL12, GM-CSF), cytokine-inducing molecules (e.g. LeIF) or costimulatory
molecules.
Helper (HTL) epitopes could be joined to intracellular targeting signals and
expressed
separately from the CTL epitopes. This would allow direction of the HTL
epitopes to a
cell compartment different than the CTL epitopes. If required, this could
facilitate more
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25 ',
efficient entry of HTL epitopes into the MHC class II pathway, thereby
improving CTL
induction. In contrast to CTL induction, specifically decreasing the immune
response by
co-expression of immunosuppressive molecules (e.g. TGF-Vii) may be beneficial
in certain
diseases.
Once an expression vector is selected, the minigene is cloned into the
polylinker region downstream of the promoter. This plasmid is transformed into
an
appropriate E. coli strain, and DNA is prepared using standard techniques. The
orientation and DNA sequence of the minigene, as well as all other elements
included in
the vector, are confirmed using restriction mapping and DNA sequence analysis.
Bacterial cells harboring the correct plasmid can be stored as a master cell
bank and a
working cell bank.
Therapeutic quantities of plasmid DNA are produced by fermentation in E.
coli, followed by purification. Aliquots from the working cell bank are used
to inoculate
~~fermentation medium (such as Ternfic Broth), and grown to saturation in
shaker flasks or
a bioreactor according to yvell known techniques. Plasmid DNA can be purified
using
standard bioseparation technologies such as solid phase anion-exchange resins
supplied
by Quiagen. If required, supercoiled DNA can be isolated from the open
circular and
linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of
formulations. The simplest of these is reconstitution of lyophilized DNA in
sterile
phosphate-buffer saline (PBS). A variety of methods have been described, and
new
techniques may become available. As noted above, nucleic acids are
conveniently
formulated with cationic lipids. In addition, glycolipids, fusogenic
liposomes, peptides
and compounds referred to collectively as protective, interactive, non-
condensing (PINC)
could also be complexed to purified plasmid DNA to influence variables such as
stability,
intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression
and MHC class I presentation of minigene-encoded CTL epitopes. The plasmid DNA
is
introduced into a mammalian cell line that is suitable as a target for
standard CTL
chromium release assays. The transfection method used will be dependent on the
final
formulation. Electroporation can be used for "naked" DNA, whereas cationic
lipids allow
direct in vitro transfection. A plasmid expressing green fluorescent protein
(GFP) can be
co-transfected to allow enrichment of transfected cells using fluorescence
activated cell
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26
sorting (FACS). These cells are then chromium-51 labeled and used as~target
cells for
epitope=specific CTL lines. Cytolysis, detected by SlCr release, indicates
production of
MHC presentation of minigene-encoded CTL epitopes.
In vivo immunogenicity is a second approach for functional testing of
minigene DNA formulations. Transgenic mice expressing appropriate human MHC
molecules are immunized with the DNA product. The dose and route of
administration
are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-complexed
DNA).
Twenty-one days after immunization, splenocytes are harvested and restimulated
for 1
week in the presence of peptides encoding each epitope being tested. These
effector cells
(CTLs) are assayed for cytolysis of peptide-loaded, chromium-51 labeled target
cells
using standard techniques. Lysis of target cells sensitized by MHC loading of
peptides
corresponding to minigene-encoded epitopes demonstrates DNA vaccine function
for in
vivo induction of CTLs.
Antigenic peptides may be used to elicit CTL ex vivo,'as well. The
resulting CTL, can be used to treat chronic infections (viral or bacterial) or
tumors in
patients that do not respond to other conventional forms of therapy, or will
not respond to
a peptide vaccine approach of therapy. Ex vivo CTL responses to a particular
pathogen
(infectious agent or tumor antigen) are induced by incubating in tissue
culture the
patient's CTL precursor cells (CTLp) together with a source of antigen-
presenting cells
(APC) and the appropriate immunogenic peptide. After an appropriate incubation
time
(typically 1-4 weeks), in which the CTLp are activated and mature and expand
into
effector CTL, the cells are infused back into the patient, where they will
destroy their
specific target cell (an infected cell or a tumor cell).
The peptides may also find use as diagnostic reagents. For example, a
peptide of the invention may be used to determine the susceptibility of a
particular
individual to a treatment regimen which employs the peptide or related
peptides, and thus
may be helpful in modifying an existing treatment protocol or in determining a
prognosis
for an affected individual. In addition, the peptides may also be used to
predict which
individuals will be at substantial risk for developing chronic infection.
The following example is offered by way of illustration, not by way of
limitation.
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EXAMPLE 1
Class I antigen isolation was carried out as described
in the related
applications,
noted above.
Naturally
processed
peptides
were then
isolated
and
sequenced
as described
there.
An allele-specific
motif and
algorithms
were determined
S and quantitative
binding
assays
were carried
out.
Using the motifs identified above for the HLA-A2.1
allele amino acid
sequences from a number of antigens were analyzed for the presence
of these motifs.
Table 3
provides
the results
of these
seaxches.
The letter
"J" represents
norleucine.
The above examples are provided to illustrate the invention
but not to limit
its scope.Other variants of the invention will be readily apparent
to one of ordinary skill
in the art
and are
encompassed
by the,appended
claims.
All publications,
patents,
and
patent applications
cited herein
are hereby
incorporated
by reference.
Table 3
''P-eptide AA. Sequence Source A*0201
17.0317 9 LQIGNIISI F1u.24 0.0130
38.0103 9 NLSLSCHAA CEA.432 0.0110
1233.11 9 YLSGANLNV CEA.605V9 0.0690
1295.03 9 SMPPPGTRV p53.149M2 0.0290
1295.04 9 SLPPPGTRV p53.149L2 0.0410
1317.24 9 KTCPVQLWV p53.139 0.0069
1323.02 9 KLLPENNVV p53.24V9 0.0130
1323.04 9 ALNKMFBQV p53.129B7V9 0.0260
1323.06 9 KLBPVQLWV p53.139L2B3 0.1100
1323.08 9 BLTIHYNYV p53.229B1L2V9 0.0430
1323.18 10 LLPPQHLIRV p53.188L2 0.0061
1323.29 11 YMCNSSCMGGM p53.236 0.0075
1323.31 11 YLCNSSCMGGV p53.236L2V11 0.2300
1323.34 11 KLYQGSYGFRV p53.101L2V11 0.0620
1324.07 9 CQLAKTCPV p53.135 0.0240
1325.01 9 RLPEAAPPV p53.65L2 0.0640
1325.02 9 GLAPPQHLV p53.187V9 0.0130
1325.04 9 KMAELVHFL MAGE3.112M2 0.2100
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Peptide AA Seauence Source ~ . A*0201
~
1325.05 9 KLAELVHFL MA.GE3.112L2 0.2500
1326.01 9 CLLAKTCPV p53.135L2 0.0400
1326.02 9 KLSQHMTEV p53.164L2 0.0410
1326.04 9 ELAPVVAPV p53.68L2V9 0.0860
1326.06 10 QLAKTCPVQV p53.136 0.0320
1326.08 9 HLTEVVRRV p53.168L2 0.0180
1329.01 11 KTYQGSYGFR.L 0.0028
1329.03 10 WVPYEPPEV p53.216 0.0081
1329.14 9 BQLAKTBPV p53.135B1B7 0.0490
1329.15 9 BLLAKTBPV p53.135B1L2B7 0.1100
1330.01 9 QIIGYVIGT CEA.78 0.0160
1330.02 9 QLIGYVIGV CEA.78L2V9 0.5300
~~1330.059 YVCGIQNSV CEA.569 0.0510
1330.06 9 YLCGIQNSV CEA.569L2 0.1000
1330.07 9 ATVGIMIGV CEA.687 0.1400
1330.08 9 ~ ALVGIMIGV CEA.687L2 0.5000
1330.09 10 VLYGPDDPTI CEA.411 0.0170
1330.10 10 VLYGPDDPTV ~ CEA.411V10 0.0310
1331.02 9 DLMLSPDDV p53.42V9
1331.03 9 ALMLSPDDI p53.42A1
1331.04 9 ALMLSPDDV p53.42A1V9
1331.05 9 DLMLSPADI p53.42A7
1331.06 9 DLMLSPADV p53.42A7V9
1331.07 9 DLMLSPDAI p53.42A8
1331.08 9 DLMLSPDAV p53.42A8V9
38.0007 9 AILTFGSFV KSHV.89 0.0850
38.0009 9 ~ HLRDFALAV KSHV.106 0.0183
38.0015 9 ALLGSIALL KSHV.155 0.0470
38.0018 9 . ALLATILAA KSHV.161 0.0490
38.0019 9 LLATILAAV KSHV.162 0.1600
38.0022 9 RLFADELAA KSHV.14 0.0150
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29
Peptide AA. Seguence Source ~ ~ A*0201
.
38.0024 9 YLSKCTLAV KSHV.65 0.2000
38.0026 9 LVYHIYSKI KSHV.153 0.0457
38.0029 9 SMYLCILSA KSHV.208 . 0.0250
38.0030 9 YLCILSALV KSHV.210 0.3500
38.0033 9 VMFSYLQSL KSHV.268 0.5000
~~
38.0035 9 RLHVYAYSA KSHV.285 0.0270
38.0039 9 GLQTLGAFV KSHV.98 0.0110
38.0040 9 FVEEQMTWA KSHV.105 0.0380
38.0041 9 QMTWAQTVV KSHV.109 0.0110
38.0042 9 IILDTAIFV KSHV.130 0.6800
38.0043 9 AIFVCNAFV KSHV.135 0.0910
38.0046 9 AMGNRLVEA KSHV.172 0.0200
~~38.00479 RLVEACNLL KSHV.176 0.0180
38.0059 9 TLSIVTFSL KSHV.198 0.2200
38.0063 9 KLSVLLLEV KSHV.292 0.1400
38.0064 9 LLLEVNRSV KSHV.296 0.0270
38.0068 9 FVSSPTLPV KSHV.78 0.0350
38.0070 9 AMLVLLAEI KSHV.281 0.0820
38.0075 9 QMARLAWEA KSHV.1116 0.0990
38.0131 10 VLAIEGIFMA KSHV.10 0.0730
38.0132 10 . YLYHPLLSPI KSHV.27 0.1400
38.0134 10 SLFEAMLANV KSHV.49 0.9500
38.0135 10 STTGINQLGL KSHV.62 0.0710
38.0137 10 LAILTFGSFV KSHV.88 0.0160
38.0139 10 ALLGSIALLA KSHV.155 0.0360
38.0141 10 ALLATILAAV KSHV.161 0.1100
38.0142 10 LLATILAAVA KSHV.162 0.0110
38.0143 10 RLFADELAAL KSHV.14 0.1800
38.0148 , 10 YLSKCTLAVL KSHV.65 0.0300
38.0150 10 LLVYHIYSKI KSHV.152 0.0130
38.0151 10 SMYLCILSAL KSHV.208 0.0360
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p-eptide AA Seguence Source ~ ~ A*0201
.
38.0153 10 HLHRQMLSFV KSHV.68 0.0160
38.0163 10 LLCGKTGAFL KSHV.167 0.0100
38.0164 10 ETLSIVTFSL KSHV.197 O.OI80
39.0063 9 VMCTYSPPL mp53.119 1.4000
39.0065 9 KLFCQLAKT mp53.129 0.0160
39.0067 9 ATPPAGSRV mp53.146 0.0130 .
39.0133 10 FLQSGTAKSV mp53.110 0.0180
39.0169 10 CMDRGLTVFV KSHV.311 0.0120
39.0170 10 ~ VLLTfWWRWRL KSHV.327 0.1500
40.0070 9 GVFTGLTHI HCV.1565 0.0110
40.0072 9 QMWKCLIRL HCV.1611 0.0620
40.0074 9 IMTCMSADL HCV.1650 0.0121
40.0076 9 ALAAYCLST HCV.1674 0.2500
40.0080 9 VLSGKPAII HCV.1692 0.0150
40.0082 9 FISGIQYLA HCV.1773 ' 0.1000
40.0134 10 YIMTCMSADL HCV.1649 0.0300
40.0137 10 AIASLMAFTA HCV.1791 0.0580
40.0138 10 GLAGAAIGSV HCV.1838 0.0320
41.0058 8 MIGVLVGV CEA.692 0.0120
41.0061 9 VLPLAYISL TRP 1 0.0110
41.0062 9 SLGCIFFPL TRP 1 0.9700
41.0063 9 PLAYISLFL TRP 1 0.0220
41.0065 9 LMLFYQVWA TRP1 0.0270
41.0071 9 I~ISIYNYFV TRP 1 0.2300
41.0072 9 TTIS~~'NYFV TRP 1 0.0600
41.0075 9 FVWTHYYSV TRPl 1.5000
41.0077 9 FLTVJHRYHL TRP1 0.5500
41.0078 9 LTWHRYHLL TRP1 0.1600
41.0082 9, MLQEPSFSL TRP1 0.6900
41.0083 9 SLPYWNFAT TRP 1 0.0110
41.0088 9 RLPEPQDVA TRP1 0.0180
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peptide AAA Seauence Source ~ . ~ A*0201
41.0090 9 VTQCLEVRV TRP 1 0.0160
41.0096 9 LLHTFTDAV TRP 1 0.2700
41.0100 9 NMVPFWPPV TRP1 0.6200
41.0104 9 AW GALLLV TRP 1 0.0210
41.0105 9 AWAALLLV TRP1 0.0390
41.0108 9 LLVA.AIFGV TRP 1 1.9000
41.0112 9 SMDEANQPL TRP 1 0.0770
41.0114 9 VLPLAYISV TRP1 0.1100
41.0115 9 SLGCIFFPV TRP1 3.2000
41.0116 9 PLAYISLFV TRP1 0.0310
41.0117 9 LLLFQQARV TRP1 0.1100
41.0118 ~9 LMLFYQVW TRP1 2.4000
41.0119 9 LLPSSGPGV TRP1 0.3700
41.0121 9 NLSIYNYFV TRP 1 0.9700
41.0122 9. NLSVYNYFV TRP1 0.8700
41.0123 9 FLWTHWSV TRP1 5.6000
41.0124 9 SLKI~TFLGV TRP 1 0.0224
41.0125 9 FLTWHRYHV TRP1 0.3800
41.0129 9 MLQEPSFSV TRP1 1.6000
41.0130 9 SLPYWNFAV TRP1 0.5700
41.0131 9 ALGKNVCDV TRP1 0.0160
41.0132 9 SLLISPNSV TRP1 0.1300
41.0133 9 SLFSQWRW TRP1 0.0740
41.0134 9 TLGTLCNSV TRP1 0.0330
41.0136 9 RLPEPQDW TRP1 0.1000
41.0137 9 VLQCLEVRV TRP1 0.0360
41.0138 9 SLNSFRNTV TRP1, 0.0140
41.0139 9 SLDSFRNTV TRP1 0.0440
41.0141 9 FLNGTGGQV TRP 1 0.0220
41.0142 9 VLLHTFTDV TRP1 0.0180
41.0145 9 ALVGALLLV TRP1 0.2600
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~
peptide AA Sequence ~ . ~ A*0201
Source
41.0146 9 ALVAALLLV TRPl 0.5800
41.0147 9 LLVALIFGV TRP 1 1.0000
41.0148 9 YLIRARRSV TRP 1 0.0170
41.0149 9 SMDEANQPV TRP1 0.1600
41.0151 10 SLGCIFFPLL, TRP1 0.1800
41.0157 10 GMCCPDLSPV TRP1 0.0950
41.0160 ~10 AACNQKILTV TRPl 0.0120
41.0162 10 FLTWHRYHLL TRP1 0.0830
41.0166 10 SLHNLAHLFL TRP 1 0.3900
41.0174 10 LLLVAAIFGV TRP1 0.3000
41,0177 10 LLVAAIFGVA TRP1 0.0820
41.0178 10 ALIFGTASYL TRP1 0.0230
41.0180 10 SMDEANQPLL TRP 1 0.0250
41.0181 10 LLTDQYQCYA TRPl 0.0320
41.0183 10 SLGCIFFPLV TRP1 0.3200
41.0186 10 FLMLFYQVWV TRP1 0.8100
41.0189 10 ALCDQRVLIV TRP1 0.0530
41.0190 10 ALCNQKILTV TRP 1 0.0770
41.0191 10 FLTVVI-~RYHLV TRP 1 0.0510
41.0197 10 SLFINLAHLFV TRP l 0.5000
41.0198 10 NLAHLFLNGV TRP1 0.4100
41.0199 10 NMVPFWPPVV TRPl 0.2800
41.0201 10 ILVVAALLLV TRP1 0.0190
41.0203 10 LLVALIFGTV TRP1 0.1200
41.0205 10 ALIFGTASYV TRP1 0.0900
41.0206 10 SMDEANQPLV TRP1 0.0350
41.0207 10 LLTDQYQCYV TRP1 0.2100
41.0212 11 . LLIQNIIQNDT CEA.107 0.0140
41.0214 11 IIQNDTGFYTL CEA.112 0.0130
41.0221 11 TLFNVTRNDTA CEA.201 0.0110
41.0235 11 LTLLSVTRNDV CEA.378 0.0150
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p~ ~ Seguence Source ~ ~ . A*0201
41.0243 11 GLYTCQANNSA CEA.473, 0.0290
41.0268 11 ATVGIMIGVLV CEA.687 0.0160
44.0075 11 GLVPPQHLIRV mp53.184.V3 0.0370
44.0087 11 GLAPPVHLIRV mp53.184.V6 0.0330
44.0092 11 GLAPPEHLIRV, mp53.184.E6 0.1600
1227.10 9 ILIGVLVGV CEA.691.L2 0.2300
1234.26 10 YLIMVKCWMV Her2/neu.952.L20.3800
V10
1295.06 9 LLGRDSFEV mp53.261 0.2000
1319.01 9 FMYSDFHFI FIu.RRP2.446 0.4400
1319.06 9 NMLSTVLGV Flu.RRP2.446 0.1700
1319.14 9 SLENFRAYV FIu.RRP2.446 0.0430
1325.06 KMAELVHFV Mage3.112 0.1900
1325.07 KLAELVHFV Mage3.112 0.3500
1334.01 VLIQRNPQV Her2/neu.153.V90.0910
1334.02 VLLGVVFGV Her2/neu.665.L22.1000
.
V9
1334.03 SLISAVVGV Her2/neu.653.L20.7000:
V9
1334.04 YMIMVKBWMI Her2/neu.952.B70.2700
1334.05 YLIMVKBWMV Her2/neu.952.L20.6900
B7V10
1334.06 KLWEELSVV Mage3.220.L2V 0.4500
9
1334.08 AMBRWGLLV Her2/neu.5.M2B 0.1400
3V9
1345.01 9 IJIGVLVGV CEA.691.J2 . 0.0570
1345.02 9 ATVGIJIGV CEA.687.J6 0.1595
1345.03 9 SJPPPGTRV p53.149.J2 0.0545
1345.04 10 LVFGIELJEV MAGE3.160.J8 0.7650
918.12 8 ILGFVFTL Flu.M1.59 0.7900
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peptide AA Seauence Source ~ ~ ~ A*0201
1095.22 9 KIFGSLAFL Her2lneu,
1090.01 10 YLQLVFGIEV MAGE2
1126.01 9 MMNDQLMFL PSM
1126.02 10 ALVLAGGFFL PSM
1126.03 9 WLCAGALVL PSM
1126.05 9 MVFELANSI PSM
1126.06 10 RMMNDQLMFL PSM
1126.09 9 LVLAGGFFL PSM
1126.10 9 VLAGGFFLL PSM
1126.12 9 LLHETDSAV PSM
1126.14 9 LMYSLVHNL PSM
1126.16 10 QLMFLERAFI PSM
1126.17 9 LMFLERAFI PSM
1126.20 10 KLGSGNDFEV PSM
1129.01 10 LLQERGVAYI PSM
1129.04 10 GMPEGDLVYV PSM
1129.05 10 FLDELKAENI PSM
1129.08 ' 9 ALFDIESKV PSM
1129.10 10 GLPSIPVHPI PSM
II. Non-HLA-A2 Motifs
The present invention also relates to the determination of allele-specific
peptide motifs for human Class I MHC (sometimes referred to as HLA) allele
subtypes.
These motifs are then used to define T cell epitopes from any desired antigen,
particularly
those associated with human viral diseases, cancers or autoimmune diseases,
for which
the amino acid sequence of the potential antigen or autoantigen targets is
known.
Epitopes on a number of potential target proteins can be identified in this
manner. Examples of suitable antigens include prostate specific antigen (PSA),
hepatitis
B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Ban
virus
antigens, melanoma antigens (e.g., MAGE-1), human immunodeficiency virus (HIV)
antigens and human papilloma virus (HPV) antigens, Lassa virus, mycobacterium
tuberculosis (MT), p53, CEA, and Her2/neu.
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Peptides comprising the epitopes from these antigens are synthesized and
then tested for their ability to bind to the appropriate MHC molecules in
assays using, for
example, purified class I molecules and radioiodonated peptides and/or cells
expressing
empty class I molecules by, for instance, immunofluorescent staining and flow
5 microfluorimetry, peptide-dependent class I assembly assays, and inhibition
of CTL
recognition by peptide competition. Those peptides that bind to the class I
molecule are
further evaluated for their ability to serve as targets for CTLs derived from
infected or .
immunized individuals, as well as for their capacity to induce primary in
vitro or in vivo
CTL responses that can give rise to CTL populations capable of reacting with
virally
10 infected taxget cells or tumor cells as potential therapeutic agents.
The MHC class I antigens are encoded by the HLA-A, B, and C loci.
HLA-A and B antigens are expressed at the cell surface at approximately equal
densities,
whereas the expression of HLA-C is significantly lower (perhaps as much as 10-
fold
lower). Each of these loci have a number of alleles. The peptide binding
motifs of the
15 invention are relatively specific for each allelic subtype.
For peptide-based vaccines, the peptides of the present invention
preferably comprise a motif recognized by an MHC I molecule having a wide
distribution
in the human population. Since the NIHC alleles occur at different frequencies
within
different ethnic groups and races, the choice of target MM~IC allele may
depend upon the
20 target population. Table 4 shows the frequency of various alleles at the
HLA-A locus
products among different races. For instance, the majority of the Caucasoid
population
can be covered by peptides which bind to four HLA-A allele subtypes,
specifically HLA-
A2.1, A1, A3.2, and A24.1. Similarly, the majority of the Asian population is
encompassed with the addition of peptides binding to a fifth allele HLA-A11.2.
TABLE 4
A Allele/Subtype N 69 * A 54 C 502
A1 10.1(7) 1.8(1) 27.4(138)
~.1 11.5(8) 37.0(20) 39.8(199)
A2.2 10.1(7) 0 3.3(17)
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36
~.3 1.4(1) 5.5(3) , 0.8(4) ' , .
~.4 - _ _
A2.5 _ _ _
A3.1 1.4(1) 0 0.2(0)
A3.2 5.7(4) 5.5(3) 21.5(108)
All.l 0 5.5(3) 0
A11.2 , 5.7(4) 31.4(17) 8.7(44)
A11.3 0 3.7(2) 0
~3 4.3(3) - 3.9(20)
~4 2.9(2) 27.7(15) 15.3(77)
A24.2 - , _ _
A24.3 - - -
~5 1.4(1) - 6.9(35)
A26.1 4.3(3) 9.2(5) 5.9(30)
A26.2 7.2(5) - 1.0(5)
A26V - 3.7(2) -
A28.1 10.1(7) _ 1.6(8)
A28.2 1.4(1) - 7.5(38)
~A29.1 1.4(1) - 1.4(7)
A29.2 10.1(7) 1.8(1) 5.3(27)
A30.1 8.6(6) - 4.9(25)
A30.2 1.4(1) - 0.2(1)
A30.3 7.2(5) - 3.9(20)
A31 4.3(3) 7.4(4) 6.9(35)
A32 2.8(2) - 7.1(36)
Aw33.1 8.6(6) - 2.5(13)
Aw33.2 2.8(2) 16.6(9) 1.2(6)
Aw34.1 1.4(1) - -
Aw34.2 14.5(10) - 0.8(4)
Aw36 5.9(4) - -
Table compiled from B. DuPont, Immunobiology of HLA, Vol. I,
Histocompatibility
Testing 1987, Springer-Verlag, New York 1989.
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WO 01/62776 PCT/US00/04655
37
* N - negroid; A = Asian; C = Caucasoid. Numbers in
parenthesis represent the number of individuals included in the~analysis.
The nomenclature used to describe peptide compounds follows the
conventional practice wherein the amino group is presented to the left (the N-
terminus)
anal the carboxyl group to the right (the C-terminus) of each amino acid
residue. In the
formulae representing selected specific embodiments of the present invention,
the amino-
and carboxyl-terminal groups, although not specifically shown, axe in the form
they
would assume at physiologic pH values, unless otherwise specified. In the
amino acid
structure formulae, each residue is generally represented by standard three
letter or single
letter designations. The L-form of an amino acid residue is represented by a
capital single
letter or a capital first letter of a three-letter symbol, and the D-form for
those amino acids
is represented by a lower case single letter or a lower case three letter
symbol. Glycine
has no asymmetric carbon atom and is simply referred to as "Gly" or G.
The procedures used to identify peptides of the present invention generally
follow the methods disclosed in Falk et al., Nature 351:290 (1991), which is
incorporated
herein by reference. Briefly, the methods involve large-scale isolation of MHC
class I
molecules, typically by immunoprecipitation or affinity chromatography, from
the
appropriate cell or cell line. Examples of other methods for isolation of the
desired MHC
molecule equally well known to the artisan include ion exchange
chromatography, lectin
chromatography, size exclusion, high performance ligand chromatography, and a
combination of all of the above techniques.
A large number of cells with defined MHC molecules, particularly MHC
Class I molecules, axe known and readily available. Fox example, human EBV-
transformed B cell lines have been shown to be excellent sources for the
preparative
isolation of class I and class II MHC molecules. Well-characterized cell lines
are
available from private and commercial sources, such as American Type Culture
Collection ("Catalogue of Cell Lines and Hybridomas," 6th edition (1988)
Rockville,
Maryland, U.S.A.); National Institute of General Medical Sciences 1990/1991
Catalog of
Cell Lines (NIGMS) Human Genetic Mutant Cell Repository, Camden, NJ; and ASHI
Repository, Bingham and Women's Hospital, 75 Francis Street, Boston, MA 02115.
Table 5 lists some B cell lines suitable for use as sources for HLA-A alleles.
All of these
cell lines can be grown in large batches and are therefore useful for large
scale production
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38
of MHC molecules. One of skill will recognize that these are merely exemplary
cell lines
and that many other cell sources can be employed. Similar EBV.B cell lines
homozygous
for HLA-B and HLA-C could serve as sources for HLA-B and HLA-C alleles,
respectively.
TABLE 5
HUMAN CELL LINES (HLA-A SOURCES)
I-iLA-A allele B cell line
A1 ' MAT
COX (9022)
STEINLIN
(9087)
A2.1
A3.2 EHM (9080)
H0301 (9055)GM3107
A24.1 T3(9107),TISI (9042)
Al l BVR (GM6828A)
WT100 (GM8602)WT52
(GM8603)
In the typical case, immunoprecipitation is used to isolate the desired
allele. A number of protocols can be used, depending upon the specificity of
the
antibodies used. For example, allele-specific mAb reagents can be used fox the
affinity
purification of the HLA-A, HLA-B, and HLA-C molecules. Several mAb reagents
for ,
the isolation of HLA-A molecules are available (Table 6). Thus, fox each of
the targeted
HLA-A alleles, reagents are available that may be used for the direct
isolation of the
HLA-A molecules. Affinity columns prepared with these mAbs using standard
techniques are successfully used to purify the respective HLA-A allele
products.
In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C
mAbs, such as W6132 and B9.12.1, and one anti-HLA-B, C mAb, B 1.23.2, could be
used
in alternative affinity purification protocols as described in the example
section below.
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39
TABLE 6
ANTIBODY REAGENTS
anti-HLA Name
HLA-A l 12/ 18
HLA-A3 GAPA3 (ATCC, HB122)
HLA-11,24.1 A11.1M (ATCC, HB164)
HLA-A,B,C W6/32 (ATCC, HB95)
monomorphic . B9.12.1 (INSERM-CNRS)
HLA-B,C B.1.23.2 (INSERM-CNRS)
monomorphic
The peptides bound to the peptide binding groove of the isolated MHC
molecules are eluted typically using acid treatment. Peptides can also be
dissociated from
class I molecules by a variety of standard denaturing means, such as heat, pH,
detergents,
salts, chaotropic agents, or a combination thereof.
Peptide fractions are further separated from the MHC molecules by
reversed-phase high performance liquid chromatography (HI'LC) and sequenced.
Peptides can be separated by a variety of other standard means well known to
the artisan,
including filtration, ultrafiltration, electrophoresis, size chromatography,
precipitation
with specific antibodies, ion exchange chromatography, isoelectrofocusing, and
the like.
Sequencing of the isolated peptides can be performed according to
standard techniques such as Edman degradation (Hunkapiller, M.W., et al.,
Methods
Enzymol. 91, 399 [1983]). Other methods suitable for sequencing include mass
spectrometry sequencing of individual peptides as previously described (Hunt,
et al.,
Science 225:1261 (1992), which is incorporated herein by reference). Amino
acid
sequencing of bulk heterogenous peptides (~, pooled HPLC fractions) from
different
class I molecules typically reveals a characteristic sequence motif for each
class I allele.
Definition of motifs specific for different class I alleles allows the
identification of potential peptide epitopes from an antigenic protein whose
amino acid
sequence is known. Typically, identification of potential peptide epitopes is
initially
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carried out using a computer to scan the amino acid sequence of a desired
antigen for the
presence of motifs. The epitopic sequences are then synthesized. The capacity
to bind
MHC Class molecules is measured in a variety of different ways. One means is a
Class I
molecule binding assay as described in the related applications, noted above.
Other
5 alternatives described in the literature include inhibition of antigen
presentation (Sette, et
al., J. I-~ 141:3893 (1991), in vitro assembly assays (Townsend, et al., Cell
62:285
(1990), and FACS based assays using mutated ells, such as RMA.S (Melief, et
al., Eur. J.
Immunol. 21:2963 (1991)).
Next, peptides that test positive in the MHC class I binding assay are
10 assayed for the ability of the peptides to induce specific CTL responses in
vitro. For
instance, Antigen-presenting cells that have been incubated with a peptide can
be assayed
for the ability to induce CTL responses in responder cell populations. Antigen-
presenting
cells can be normal cells such as peripheral blood mononuclear cells or
dendritic cells
~(Inaba, et al., J. Exp. Med. 166:182 (1987); Boog, Eur. J. Immunol. 18:219
[1988]).
15 Alternatively, mutant mammalian cell lines that are deficient in their
ability to load class I molecules with internally processed peptides, such as
the mouse cell
lines RMA-S (Karre, et al.. Nature, 319:675 (1986); Ljunggren, et al., Eur. J.
Immunol.
21:2963-2970 (1991)), and the human somatic T cell hybrid, T-2 (Cerundolo, et
al.,
Nature 345:449-452 (1990)) and which have been transfected with the
appropriate human
20 class I genes are conveniently used, when peptide is added to them, to test
for the capacity
of the peptide to induce in vitxo primary CTL responses. Other eukaryotic cell
lines
which could be used include various insect cell lines such as mosquito larvae
(ATCC cell
lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851),
armyworm
(ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a
Schneider
25 cell line (see Schneider J. Embryol. Exp. Morphol. 27:353-365 [1927]).
Peripheral blood lymphocytes are conveniently isolated following simple
venipuncture or leukapheresis of normal donors or patients and used as the
responder cell
sources of CTL precursors. In one embodiment, th.e appropriate antigen-
presenting cells
are incubated with 10-100 p,M of peptide in serum-free media for 4 hours under
30 appropriate culture conditions. The peptide-loaded antigen-presenting cells
are then
incubated with the responder cell populations in vitro for 7 to 10 days under
optimized ,
culture conditions. Positive CTL activation can be determined by assaying the
cultures
for the presence of CTLs that kill radiolabeled target cells, both specific
peptide-pulsed
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41
targets as well as target cells expressing endogenously processed form of the
relevant
virus or tumor antigen from which the peptide sequence was derived.
Specificity and MHC restriction of the CTL is determined by testing
against different peptide target cells expressing appropriate or inappropriate
human MHC
class I. The peptides that test positive in the MHC binding assays and give
rise to specific
CTL responses axe referred to herein as immunogenic peptides.
The immunogenic peptides can be prepared synthetically, or by
recombinant DNA technology or from natural sources such as whole viruses or
tumors.
Although the peptide will preferably be substantially free of other naturally
occurring host
cell proteins anal fragments thereof, in some embodiments the peptides can be
synthetically conjugated to native fragments or particles.
The polypeptides or peptides can be a variety of lengths, either in their
neutral (uncharged) forms or in forms which are salts, and either free of
modifications
such as glycosylation, side chain oxidation, or phosphorylation or containing
these
modifications, subject to the condition that the mbdification not destroy the
biological
activity of the polypeptides as herein described.
Desirably, the peptide will be as small as possible while still maintaining
substantially all of the biological activity of the large peptide. When
possible, it may be
desirable to optimize peptides of the invention to a length of 9 or 10 amino
acid residues,
commensurate in size with endogenously processed viral peptides or tumor cell
peptides
that are bound to MHC class I molecules on the cell surface.
Peptides having the desired activity may be modified as necessary to
provide certain desired attributes, e.g., improved pharmacological
characteristics, while
increasing or at least retaining substantially all of the biological activity
of the unmodified
peptide to bind the desired MHC molecule and activate the appropriate T cell.
For
instance, the peptides may be subject to various changes, such as
substitutions, either
conservative or non-conservative, where such changes might provide fox certain
advantages in their use, such as improved MHC binding. By conservative
substitutions is
meant replacing an amino acid residue with another which is biologically
and/or
chemically similar, e.g., one hydrophobic residue for another, or one polar
residue for
another. The substitutions include combinations such as Gly, Ala; Val, Ile,
Leu, Met;
Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single
amino acid
substitutions may also be probed using D-amino acids. Such modifications may
be made
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42
using well known peptide synthesis procedures, as described in e.g.,
Merrifield, Science
232:341-347 (1986), Barany and Merri.field, The Peptides Gross, and
Meienhofer, eds.
(N.Y., Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase
Peptide
Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984), incorporated by reference
herein.
The peptides can also be modified by extending or decreasing the
compound's amino acid sequence, e.g., by the addition or deletion of amino
acids. The
peptides or analogs of the invention can. also be modified by altering the
order or
composition of certain residues, it being readily appreciated that certain
amino acid
residues essential for biological activity, e.g., those at critical contact
sites or conserved
residues, may generally not be altered without an adverse effect on biological
activity.
The non-critical amino acids need not be limited to those naturally occurring
in proteins,
such as L-a-amino acids, or their D-isomers, but may include non-natural amino
acids as
well, such as (3-'y-8-amino acids, as well as many derivatives of L-a-amino
acids.
Typically, a series of peptides with single amino acid substitutions are
employed to determine the effect of electrostatic charge, hydrophobicity, etc.
on binding.
For instance, a series of positively charged (e.g., Lys or Arg) or negatively
charged (e.g.,
Glu) amino acid substitutions are made along the length of the peptide
revealing different
patterna of sensitivity towards various MHC molecules and T cell receptors. In
addition,
multiple substitutions using small, relatively neutral moieties such as Ala,
Gly, Pro, or
~ similar residues may be employed. The substitutions may be homo-oligomers or
hetero-
oligomers. The number and types of residues which are substituted or added
depend on
the spacing necessary between essential contact points and certain functional
attributes
which are sought (e.g., hydrophobicity versus hydrophilicity). Increased
binding affinity
for an MHC molecule or T cell receptor may also be achieved by such
substitutions,
compared to the affinity of the parent peptide. In any event, such
substitutions should
employ amino acid residues or other molecular fragments chosen to avoid, for
example,
steric and charge interference which might disrupt binding.
Amino acid substitutions are typically of single residues. Substitutions,
deletions, insertions or any combination thereof may be combined to arrive at
a final
peptide. Substitutional variants are those in which at least one residue of a
peptide has
been removed and a different residue inserted in its place. Such substitutions
generally
are made in accordance with the following Table 2 when it is desired to finely
modulate
the characteristics of the peptide.
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43
TABLE 2
Original Residue Exemplary Substitution
Ala Ser
~.g Lys, His
Asn ' c
Asp Glu
Cys Ser
Glu Asp
Gly Pro
His Lys; Arg
Ile Leu; Val
Leu Ile; Val
Lys Arg; His
Met Leu; Ile
Phe Tyr; Trp
S er T~'
T~ S er
Tyz.; Phe
T . Trp; Phe
Val Ile; Leu
Pro Gly
Substantial changes in function (e.g., affinity for MHC molecules or T cell
receptors) are made by selecting substitutions that are less conservative than
those in
Table 2, i.e., selecting residues that differ more significantly in their
effect on maintaining
(a) the structure of the peptide backbone in the area of the substitution, for
example as a
sheet or helical conformation, (b) the charge or hydrophobicity of the
molecule at the
target site or (c)~the bulls of the side chain. The substitutions which in
general are
expected to produce the greatest changes in peptide properties will be those
in which (a)
hydrophilic residue, e.g. seryl, is substituted for (or by) a hydrophobic
residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a residue having an
electropositive side chain,
e.g., lysl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g.
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44
glutamyl or aspartyl; or (c) a residue having a bulky side chain, e.g.
phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine. ,
The peptides may also comprise isosteres of two or more residues in the
immunogenic peptide. An isostere as defined here is a sequence of two or more
residues
that can be substituted for a second sequence because the steric conformation
of the first
sequence fits a binding site specific for the second sequence. The term
specifically
includes peptide backbone modifications well known to those skilled in the
art. Such
modifications include modifications of the amide nitrogen, the a-carbon, amide
carbonyl,
complete replacement of the amide bond, extensions, deletions or backbone
crosslinks.
See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides
and
Proteins, Vol. VII (Weinstein ed., 1983).
Modifications of peptides with various amino acid mimetics or unnatural
amino acids are particularly useful in increasing the stability of the peptide
in vivo.
Stability can be assayed in a number of ways. For instance, peptidases and
various
biological media, such as human plasma and serum, have been used to test
stability. See,
e.g., Verhoef et al., Eur. J. Dru.~~ Metab. Pharmacokin. 11:291-302 (1986).
Half life of the
peptides of the present invention is conveniently determined using a 25% human
serum
(v/v) assay. The protocol is generally as follows. Pooled human serum (Type
AB, non-
heat inactivated) is delipidated by centrifugation before use. The serum is
then diluted to
25% with R.PMI tissue culture media and used to test peptide stability. At
predetermined
time intervals a small amount of reaction solution is removed and added to
either 6%
aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled
(4°C) for 15
minutes and then spun to pellet the precipitated serum proteins. The presence
of the
peptides is then determined by reversed-phase HPLC using stability-specific
chromatography conditions.
The peptides of the present invention or analogs thereof which have CTL
stimulating activity may be modified to provide desired attributes other than
improved
serum half life. For instance, the ability of the peptides to induce CTL
activity can be
enhanced by linkage to a sequence which contains at least one epitope that is
capable of
inducing a T helper cell response. Particularly preferred immunogenic
peptides/T helper
conjugates are linked by a spacer molecule. The spacer is typically comprised
of
relatively small, neutral molecules, such as amino acids or amino acid
mimetics, which
are substantially uncharged under physiological conditions. The spacers are
typically
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selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino
acids or neutral
polar amino acids. It will be understood that the optionally present spacer
need not be
comprised of the same residues and thus may be a hetero- or homo-oligomer.
When
present, the spacer will usually be at least one or two residues, more usually
three to six
5 residues. Alternatively, the CTL peptide may be linked to the T helper
peptide without a
spacer.
The immunogenic peptide may be linked to the T helper peptide either
directly or via a spacer either at the amino or carboxy terminus of the CTL
peptide. The
amino terminus of either the immunogenic peptide or the T helper peptide may
be
10 acylated. Exemplary T helper peptides include tetanus toxoid 830-843,
influenza 307-
319, malaria circumsporozoite 382-398 and 378-389
In some embodiments it may be desirable to include in the pharmaceutical
compositions of the invention at least one component which primes CTL. Lipids
have
been identified as agents capable of priming CTL in vivo against viral
antigens. For
15 example, palinitic acid residues can be attached to the alpha and epsilon
amino groups of
a Lys residue and then linked, e.g., via one or more linking residues such as
Gly, Gly-
Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated
peptide can then
be injected directly in a micellar form, incorporated into a liposome or
emulsified in an
adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment a
particularly
20 effective immunogen comprises palmitic acid attached to alpha and epsilon
amino groups
of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of
the
immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli
lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can
be used to
25 . prime virus specific CTL when covalently attached to an appropriate
peptide. See, Deres
et al., Nature 342:561-564 (1989), incorporated herein by reference. Peptides
of the
invention can be coupled to P3CSS, for example, and the lipopeptide
administered to an
individual to specifically prime a CTL response to the target antigen.
Further, as the
induction of neutralizing antibodies can also be primed with P3CSS conjugated
to a
30 peptide which displays an appropriate epitope, the two compositions can be
combined to
more effectively elicit both humoral and cell-mediated responses to infection.
In addition, additional amino acids can be added to the termini of a peptide
to provide for ease of linking peptides one to another, for coupling to a
carrier support, or
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46
larger peptide, for modifying the physical or chemical.properties of the
peptide or
oligopeptide, or the like. Amino acids such as tyrosine, cysteine; lysine,
glutamic or
aspartic acid, or the like, can be introduced at the C- or N-terminus of the
peptide or
oligopeptide. Modification at the C terminus in some cases may alter binding
characteristics of the peptide. In addition, the peptide or oligopeptide
sequences can
differ from the natural sequence by being modified by terminal-NH2-acylation,
e.g., by
alkanoyl (C1-CZO) or thioglycolyl acetylation, terminal-carboxyl amidation,
e.g.,
ammonia, methylamine, etc. In some instances these modifications may provide
sites fox
linking to a support or other molecule.
The peptides of the invention can be prepared in a wide variety of ways.
Because of their relatively short size, the peptides can be synthesized in
solution or on a
solid support in accordance with conventional techniques. Various automatic .
,synthesizers axe commercially available and can be used in accordance with
known
protocols. See, for example, Stewaxt and Young, Solid Phase Peptide~Synthesis,
2d. ed.,
Pierce Chemical Co. (1984), supra.
Alternatively, recombinant DNA technology may be employed wherein a
nucleotide sequence which encodes an immunogenic peptide of interest is
inserted into an
expression vector, transformed or transfected into' an appropriate host cell
and cultivated
under conditions suitable for expression. These procedures are generally known
in the
art, as described generally in Sambrook et al., Molecular Cloning A Laboratory
Manual,
Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), which is
incorporated
herein by reference. Thus, fusion proteins which comprise one or more peptide
sequences of the invention can be used to present the appropriate T cell
epitope.
As the coding sequence for peptides of the length contemplated herein can
be synthesized by chemical techniques, for example, the phosphotriester method
of
Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981), modification can be made
simply
by substituting the appropriate bases) for those encoding the native peptide
sequence.
The coding sequence can then be provided with appropriate linkers and ligated
into
expression vectors commonly available in the art, and the vectors used to
transform
suitable hosts to produce the desired fusion protein. A number of such vectors
and
suitable host systems are now available. For expression of the fusion
proteins, the coding
sequence will be provided with operably linked start and stop codons, promoter
and
terminator regions and usually a replication system to provide an expression
vector for
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47
expression in the desired cellular host. For example, promoter sequences
compatible with
bacterial hosts are provided in plasmids containing convenient restriction
sites for
insertion of the desired coding sequence. The resulting expression vectors are
transformed into suitable bacterial hosts. Of course, yeast or mammalian cell
hosts may
also be used, employing suitable vectors and control sequences.
The peptides of the present invention and pharmaceutical and vaccine
compositions thereof are useful for administration to mammals, particularly
humans, to
treat and/or prevent viral infection and cancer. Examples of diseases which
can be treated
using the immunogenic peptides of the invention include prostate cancer,
hepatitis B,
hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV and
condlyloma acuminatum.
For pharmaceutical compositions, the immunogenic peptides of the
invention are administered to an individual already suffering from cancer or
infected with
the virus of interest. Those in the incubation phase or the acute phase of
infection can be
treated with the immunogenic peptides separately or in conjunction with other
treatments,
as appropriate. In therapeutic applications, compositions are administered to
a patient in
an amount sufficient to elicit an effective CTL response to the virus or tumor
antigen and
to cure or at least partially arrest symptoms and/or complications. An amount
adequate to
accomplish this is defined as "therapeutically effective dose." Amounts
effective for this
use will depend on, e.g., the peptide composition, the manner of
administration, the stage
and severity of the disease being treated, the weight and general state of
health of the
patient, and the judgment of the prescxibing physician, but generally range
for the initial
immunization (that is for therapeutic or prophylactic administration) from
about 1.0 ~g to
about 5000 ~g of peptide for a 70 kg patient, followed by boosting dosages of
from about
1.0 ~,g to about 1000 p.g of peptide pursuant to a boosting regimen over weeks
to months
depending upon the patient's response and condition by measuring specific CTL
activity
in the patient's blood. It must be kept in mind that the peptides and
compositions of the
present invention may generally be employed in serious disease states, that
is, life-
threatening or potentially life threatening situations. In such cases, in view
of the
minimization of extraneous substances and the relative nontoxic nature of the
peptides, it
is possible and may be felt desirable by the treating physician to administer
substantial
excesses of these peptide compositions.
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48
For therapeutic use, administration should begin at the.first sign of viral
infection or the,detection or surgical removal of tumors or shortly after
diagnosis in the
case of acute infection. This is followed by boosting doses until at least
symptoms are
substantially abated and for a period thereafter. In chronic infection,
loading doses
followed by boosting doses may be required.
Treatment of an infected individual with the compositions of the invention
may hasten resolution of the infection in acutely infected individuals. For
those
individuals susceptible (or predisposed) to developing chronic infection the
compositions
are particularly useful in methods for preventing the evolution from acute to
chronic
infection. Where the susceptible individuals are identified prior to or during
infection, for
instance, as described herein, the composition can be targeted to them,
minimizing need
for administration to a larger population.
The peptide compositions can also be used for the treatment of chronic
infection and to stimulate the immune system to eliminate virus-infected cells
in carriers.
It is important to provide an amount of immuno-potentiating peptide in a
formulation and
mode of administration sufficient to effectively stimulate a cytotoxic T cell
response.
Thus, for treatment of chronic infection, a representative dose is in the
range of about 1.0
~,g to about 5000 ~.g, preferably about 5 ~g to 1000 ~,g for a 70 kg patient
per dose.
Immunizing doses followed by boosting doses at established intervals, e.g.,
from one to
four weeks, may be required, possibly for a prolonged period of time to
effectively
immunize an individual. In the case of chronic infection, administration
should continue
until at least clinical symptoms or laboratory tests indicate that the viral
infection has
been eliminated or substantially abated and for a period thereafter.
The pharmaceutical compositions for therapeutic treatment are intended
for parenteral, topical, oral or local administration. Preferably, the
pharmaceutical
compositions are administered parenterally, e.g., intravenously,
subcutaneously,
intradermally, or intramuscularly. Thus, the invention provides compositions
for
parenteral administration which comprise a solution of the immunogenic
peptides
dissolved or suspended in an acceptable carrier, preferably an aqueous
carrier. A variety
of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline,
0.3% glycine,
hyaluronic acid and the like. These compositions may be sterilized by
conventional, well
lrnown sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions
may be packaged for use as is, or lyophilized, the lyophilized preparation
being combined
CA 02400215 2002-08-12
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49
with a sterile solution prior to administration. The compositions may,contain
pharmaceutically acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering agents, tonicity
adjusting
agents, wetting agents and the like; for example, sodium acetate, sodium
lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine
oleate, etc.
The concentration of CTL stimulatory peptides of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than about 0.1%,
usually at
or at least about 2% to as much as 20% to 50% or more by weight, and will be
selected
primarily by fluid volumes, viscosities, etc., in accordance with the
particular mode of
administration selected.
The peptides of the invention may also be administered via liposomes,
which serve to target the peptides to a particular tissue, such as lymphoid
tissue, or
targeted selectively to infected cells, as well as increase the half life of
the peptide
composition. Liposomes include emulsions, foams, micelles, insoluble
monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the like. In
these
preparations the peptide to be delivered is incorporated as part of a
liposome, alone or in
conjunction with a molecule which binds to, e.g., a receptor prevalent among
lymphoid
cells, such as monoclonal antibodies which bind to the CD45 antigen, or with
other
therapeutic or immunogenic compositions. Thus, liposomes either filled or
decorated
with a desired peptide of the invention can be directed to the site of
lymphoid cells, where
the liposomes then deliver the selected therapeutic/immunogenic peptide
compositions.
Liposomes for use in the invention are formed from standard vesicle-forming
lipids,
which generally include neutral and negatively charged phospholipids and a
sterol, such
as cholesterol. The selection of lipids is generally guided by consideration
of, e.g.,
liposome size, acid lability and stability of the liposomes in the blood
stream. A variety
of methods are available for preparing liposomes, as described in, e.g., Szoka
et al., Ann.
Rev: Biophys. Bioen~. 9:467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728,
4,837,028,
and 5,019,369, incorporated herein by reference.
For targeting to the immune cells, a ligand to be incorporated into the
liposome can include, e.g., antibodies or fragments thereof specific for cell
surface
determinants of the desired immune system cells. A liposome suspension
containing a
peptide may be administered intravenously, locally, topically, etc. in a dose
which vanes
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according to, inter alia, the manner of administration, the peptide being
delivered, and the
stage of the disease being treated.
Fox solid compositions, conventional nontoxic solid carriers may be used
which include, for example, pharmaceutical grades of mannitol, lactose,
starch,
5 magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose,
magnesium
carbonate, and the like. For oral administration, a pharmaceutically
acceptable nontoxic
composition is formed by incozporating any of the normally employed
excipients, such as
those carriers previously listed, and generally 10-95% of active ingredient,
that is, one or
more peptides of the invention, and more preferably at a concentration of 25%-
75%.
10 For aerosol administration, the immunogenic peptides are preferably
supplied in finely divided form along with a surfactant and propellant.
Typical
percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The
surfactant
must, of course, be nontoxic, and preferably soluble in the propellant.
Representative of
such agents are the esters or partial esters of fatty acids containing from 6
to 22 carbon
15 atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,
linolenic, olesteric and
oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters,
such as mixed or natural glycerides may be employed. The surfactant may
constitute
0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the
composition is ordinarily propellant. A carrier can also be included, as
desired, as with,
20 e.g., lecithin for intranasal delivery.
In another aspect the present invention is directed to vaccines which
contain as an active ingredient an immunogenically effective amount of an
immunogenic
peptide as described herein. The peptides) may be introduced into a host,
including
humans, linked to its own Garner or as a homopolymer or heteropolymer of
active peptide
25 units. Such a polymer has the advantage of increased immunological reaction
and, where
different peptides are used to make up the polymer, the additional ability to
induce
antibodies and/or CTLs that react with different antigenic determinants of the
virus or
tumor cells. Useful carriers are well known in the art, and include, e.g.,
thyroglobulin,
albumins such as human serum albumin, tetanus toxoid, polyamino acids such as
30 poly(lysine:glutamic acid), influenza, hepatitis B virus core protein,
hepatitis B virus
recombinant vaccine and the like. The vaccines can also contain a
physiologically
tolerable (acceptable) diluent such as water, phosphate buffered saline, or
saline, and
further typically include an adjuvant. Adjuvants such as incomplete Freund's
adjuvant,
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S1 ,
aluminum phosphate, aluminum hydroxide, or alum are materials well known in
the art.
And, as mentioned above, CTL responses can be primed by conjugating peptides
of the
invention to lipids, such as P3CSS. Upon immunization with a peptide
composition as
described herein, via injection, aerosol, oral, transdermal or other route,
the immune
system of the host responds to the vaccine by producing large amounts of CTLs
specific
for the desired antigen, and the host becomes at least partially immune to
later infection,
or resistant to developing chronic infection.
Vaccine compositions containing the peptides of the invention are
administered to a patient susceptible to or otherwise at risk of viral
infection or cancer to
elicit an immune response against the antigen and thus enhance the patient's
own immune
response capabilities. Such an amount is defined to be an "immunogenically
effective
dose." In this use, the precise amounts again depend on the patient's state of
health and
weight, the mode of administration, the nature of the formulation, etc., but
generally
range from about 1.0 ~.g to about 5000 ~.g per 70 kilogram patient, more
commonly from
. about 10 ~.g to about 500 ~,g mg per 70 kg of body weight.
In some instances it may be desirable to combine the peptide vaccines of
the invention with vaccines which induce neutralizing antibody responses to
the virus of
interest, particularly to viral envelope antigens.
For therapeutic or immunization purposes, nucleic acids encoding one or
more of the peptides of the invention can also be administered to the patient.
A number
of methods are conveniently used to deliver the nucleic acids to the patient.
Fox instance,
the nulceic acid can be delivered directly, as "naked DNA". This approach is
described,
for instance, in Wolff et. al., Science 247:1465-1468 (1990) as well as U.S.
Patent Nos.
5,580,859 and 5,589,466. The nucleic acids can also be administered using
ballistic
delivery as described, for instance, in U.S. Patent No. 5,204,253. Particles
comprised
solely of DNA can be administered. Alternatively, DNA can be adhered to
particles, such
as gold particles. The nucleic acids can also be delivered complexed to
cationic
compounds, such as cationic lipids. Lipid-mediated gene delivery methods are
described,
for instance, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988)
BioTechniques 6(7):682-691; Rose U.S. Pat No. 5,279,833; WO 91/06309; and
Felgner et
al. (1987) Proc. Nat!. Acad. Sci. USA 84: 7413-7414. The peptides of the
invention can
also be expressed by attenuatedwiral hosts, such as vaccinia or fowlpox. This
approach
involves the use of vaccinia virus as a vector to express nucleotide sequences
that encode
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52 ,
the peptides of the invention. Upon introduction into an acutely or
chronically infected
host or into a noninfected host, the recombinant vaccinia virus expresses the
immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors
and
methods useful in immunization protocols are described in, e.g.,U.S. Patent
No.
4,722,848, incorporated herein by reference. Another vector is BCG (Bacille
Calmette
Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460
(1991)) which
is incozporated herein by reference. A wide variety of other vectors useful
for therapeutic
administration or immunization of the peptides of the invention, e.g.,
Salmonella typhi
vectors and the like, will be apparent to those skilled in the art from the
description
herein.
A preferred means of administering nucleic acids encoding the peptides of
the invention uses minigene constructs encoding multiple epitopes of the
invention. To
create a DNA sequence encoding the selected CTL epitopes (minigene) for
expression in
_ human cells, the amino acid sequences of the epitopes are reverse
translated. A human
codon usage table is used to guide the codon choice fox each amino acid. These
epitope-
encoding DNA sequences axe directly adjoined, creating a continuous
polypeptide
sequence. To optimize expression and/or immunogenicity, additional elements
can be
incorporated into the minigene design. Examples of amino acid sequence that
could be
reverse translated and included in the minigene sequence include: helper T
lymphocyte
epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention
signal. In
addition, MHC presentation of CTL epitopes may be improved by including
synthetic
(e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the
CTL
epitopes.
The minigene sequence is converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the minigene.
Overlapping
oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified
and
annealed under appropriate conditions using well known techniques. he ends of
the
oligonucleotides are joined using T4 DNA ligase. This synthetic minigene,
encoding the
CTL epitope polypeptide, can then cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are
included in the vector to ensure expression in the target cells. Several
vector elements axe
required: a promoter with a down-stream cloning site for minigene insertion; a
polyadenylation signal for efficient transcription termination; an E. coli
origin of
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53
replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin
resistance).
Numerous promoters can be used for this purpose, e.g., the human
cytomegalovirus
(hCMV) promoter. See, U.S. Patent Nos. 5,580,859 and 5,589,466 fox other
suitable
promoter sequences.
Additional vector modifications may be desired to optimize minigene
expression and immunogenicity. In some cases, introns are required for
efficient gene
expression, and one or more synthetic or naturally-occurring introns could be
incorporated into the transcribed region of the minigene. The inclusion of
mRNA
stabilization sequences can also be considered fox increasing minigene
expression. It has
recently been proposed that immunostimulatory sequences (ISSs or CpGs) play a
role in
the immunogenicity of DNA vaccines. . These sequences could be included in the
vector,
outside the minigene coding sequence, if found to enhance immunogenicity
In some embodiments, a bioistronic expression vector; to allow production
. ~of the minigene-encoded epitopes and a second protein included to enhance
or decrease
. immunogenicity can be used. Examples of proteins or polypeptides that could
beneficially enhance the immune response if co-expressed include cytokines
(e.g., IL2,
IL12, GM-CSF), cytokine-inducing molecules (e.g. LeIF) or costimulatory
molecules.
Helper (HTL) epitopes could be joined to intracellular targeting signals and
expressed
separately from the CTL epitopes. This would allow direction of the HTL
epitopes to a
cell compartment different than the CTL epitopes. If required, this could
facilitate more
efficient entry of HTL epitopes into the MHC class II pathway, thereby
improving CTL
induction. In contrast to CTL induction, specifically decreasing the immune
response by
co-expression of immunosuppressive molecules (e.g. TGF-(3) may be beneficial
in certain
diseases.
Once an expression vector is selected, the minigene is cloned into the
polylinker region downstream of the promoter. This plasmid is transformed into
an
appropriate E. coli strain, and DNA is prepared using standard techniques. The
orientation and DNA sequence of the minigene, as well as all other elements
included in
the vector, are confirmed using restriction mapping and DNA sequence analysis.
Bacterial cells harboring the correct plasmid can be stored as a master cell
bank and a
working cell bank.
Therapeutic quantities of plasmid DNA are produced by fermentation in E.
coli, followed by purification. Aliquots from the working cell bank are used
to inoculate
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54
fermentation medium (such as Terrific Broth), and grown to saturation in
shaker flasks or
a bioreactor according to well known techniques. Plasmid DNA can be purified
using
standard bioseparation technologies such as solid phase anion-exchange resins
supplied
by Quiagen. If required, supercoiled DNA can be isolated from the open
circular and
linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of
formulations. The simplest of these is reconstitution of lyophilized DNA in
sterile
phosphate-buffer saline (PBS). A variety of methods have been described, and
new
techniques may become available. As noted above, nucleic acids are
conveniently
formulated with cationic lipids. In addition, glycolipids, fusogenic
liposomes, peptides
and compounds referred to collectively as protective, interactive, non-
condensing (PINC)
could also be complexed to purified plasmid DNA to influence variables such as
stability,
intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional ass~.y for expression
. and MHC class I presentation of minigene-encoded CTL epitopes. The plasmid
DNA is
introduced into a mammalian cell line that is suitable as a target for
standard CTL
chromium release assays. The transfection method used will be dependent on the
final
formulation. Electroporation can be used for "naked" DNA, whereas cationic
lipids allow
direct in vitro ixansfection. A plasmid expxessing green fluorescent protein
(GFP) can be
co-transfected to allow enrichment of transfected cells using fluorescence
activated cell
sorting (FRCS). These cells are then chromium-51 labeled and used as target
cells for
epitope-specific CTL lines. Cytolysis, detected by SlCr release, indicates
production of
MHC presentation of minigene-encoded CTL epitopes.
I~ vivo immunogenicity is a second approach for functional testing of
minigene DNA formulations. Transgenic mice expressing appropriate human MHC
molecules are immunized with the DNA product. The dose and route of
administration
are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-complexed
DNA).
Twenty-one days after immunization, splenocytes are harvested anal
restimulated for 1
week in the presence of peptides encoding each epitope being tested. These
effector cells
(CTLs) are assayed for cytolysis of peptide-loaded, chromium-51 labeled target
cells
using standard techniques. Lysis of target cells sensitized by MHC loading of
peptides
corresponding to minigene-encoded epitopes demonstrates DNA vaccine function
for in
vivo induction of CTLs.
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Antigenic peptides may be used to elicit CTL ex vivo,'a's well. The
resulting CTL, can be used to treat chronic infections (viral or bacterial) or
tumors in
patients that do not respond to other conventional forms of therapy, or will
not respond to
a peptide vaccine approach of therapy. Ex vivo CTL responses to a particular
pathogen
5 (infectious agent or tumor antigen) are induced by incubating in tissue
culture the
patient's CTL precursor cells (CTLp) together with a source of antigen-
presenting cells
(APC) and the appropriate immunogenic peptide. After an appropriate incubation
time
(typically 1-4 weeks), in which the CTLp axe activated and mature and expand
into
effector CTL, the cells are infused back into the patient, where they will
destroy their
10 specific target cell (an infected cell or a tumor cell). In order to
optimize the ~in vitro
conditions for the generation of specific cytotoxic T cells, the culture of
stimulator cells is
maintained in an appropriate serum-free medium.
Prior to incubation of the stimulator cells with the cells to be activated,
e.g., precursor CD8+ cells, an amount of antigenic peptide is added to the
stimulator cell
15 ' culture, of sufficient quantity to become loaded onto the human Class I
molecules to be
expressed on the surface of the stimulator cells. In the present invention, a
sufficient
amount of peptide is an amount that will allow about 200, and preferably 200
or more,
human Class I MHC molecules loaded with peptide to be expressed on the surface
of
each stimulator cell. Preferably, the stimulator cells are incubated with
>20~g/ml peptide.
20 Resting or precursor CD8+ cells are then incubated in culture with the
appropriate stimulator cells for a time period sufficient to activate the CD8+
cells.
Preferably, the CD8+ cells are activated in an antigen-specific manner. The
ratio of
resting or precursor CD8+ (effector) cells to stimulator cells may vary from
individual to
individual and may further depend upon variables such as the amenability of an
25 individual's lymphocytes to culturing conditions and the nature and
severity of the disease
condition or other condition for which the within-described treatment modality
is used.
Preferably, however, the lymphocyteatimulator cell ratio is in the range of
about 30:1 to
300:1. The effector/stimulator culture may be maintained for as long a time as
is
necessary to stimulate a therapeutically useable or effective number of CD8+
cells.
30 The induction of CTL in vitro requires the specific recognition of peptides
that are bound to allele specific MHC class I molecules on APC. The number of
specific
MHC/peptide complexes per APC is crucial for the stimulation of CTL,
particularly in
primary immune responses. While small amounts of peptide/MHC complexes per
cell
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56
are sufficient to render a cell susceptible to lysis by CTL, or to stimul~.te
a secondary CTL
response, the successful activation of a CTL precursor (pCTL) during primary
response
requires a significantly higher number of MHC/peptide complexes. Peptide
loading of
empty major histocompatability complex molecules on cells allows the induction
of
primary cytotoxic T lymphocyte responses. Peptide loading of empty major
histocompatability complex molecules on cells enables the iri~duction of
primary cytotoxic
T lymphocyte responses.
Since mutant cell lines do not exist for every human MHC allele, it is
advantageous to use a technique to remove endogenous MHC-associated peptides
from
the surface of APC, followed by loading the resulting empty MHC molecules with
the
immunogenic peptides of interest. The. use of non-transformed (non-
tumorigenic), non-
infected cells, and preferably, autologous cells of patients as APC is
desirable for the
design of CTL induction protocols directed towards development of ex vivo CTL
therapies. This application discloses methods for stripping the endogenous MHC-
associated peptides from the surface of APC followed by the loading of desired
peptides.
A stable MHC class I molecule is a trimeric complex formed of the
following elements: 1) a peptide usually of 8 - 10 residues, 2) a
transmembrane heavy
polymorphic protein chain which bears the peptide-binding site in its al and
a2 domains,
and 3) a non-covalently associated non-polymorphic light chain, (32
microglobulin.
Removing the bound peptides and/or dissociating the ~i2 microglobulin from the
complex
renders the MHC class I molecules nonfunctional and unstable, resulting in
rapid
degradation. All MHC class I molecules isolated from PBMCs have endogenous
peptides
bound to them. Therefore, the first step is to remove all endogenous peptides
bound to
MHC class I molecules on the APC without causing their degradation before
exogenous
peptides can be added to them.
Two possible ways to free up MHC class I molecules of bound peptides
include lowering the culture temperature from 37°C to 26°C
overnight to destabilize (32
microglobulin and stripping the endogenous peptides from the cell using a mild
acid
treatment. The methods release previously bound peptides into the
extracellulax
environment allowing new exogenous peptides to bind to the empty class I
molecules.
The cold-temperature incubation method enables exogenous peptides to bind
efficiently
to the MHC complex, but requires an overnight incubation at 26°C which
may slow the
cell's metabolic rate. It is also likely that cells not actively synthesizing
MHC molecules
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5~
(e.g., resting PBMC) would not produce high amounts of empty surface MHC
molecules
by the cold temperature procedure.
Harsh acid stripping involves extraction of the peptides with trifluoroacetic
acid, pH 2, or acid denaturation of the immunoaffinity purified class I-
peptide complexes.
These methods are not feasible for CTL induction, since it is important to
remove the
endogenous peptides while preserving APC viability and an optimal metabolic
state
which is critical for antigen presentation. Mild acid solutions of pH 3 such
as glycine or
citrate-phosphate buffers have been used to identify endogenous peptides and
to identify
tumor associated T cell epitopes. The treatment is especially effective, in
that only the
MHC class I molecules are destabilized (and associated peptides released),
while other
surface antigens remain intact, including MHC class II molecules. Most
importantly,
treatment of cells with the mild acid solutions do not affect the cell's
viability or
metabolic state. The mild acid treatment is rapid since the stripping of the
endogenous
peptides occurs in two minutes at 4°C and the APC is ready to perform
its function after
the appropriate peptides are loaded. The technique is utilized herein to make
peptide-
specific APCs for the generation of primary antigen-specific CTL. The
resulting APC are
efficient in inducing peptide-specific CD8+ CTL.
Activated CD8+ cells may be effectively separated from the stimulator
cells using one of a variety of known methods. For example, monoclonal
antibodies
specific fox the stimulator cells, for the peptides loaded onto the stimulator
cells, or for
the CD8+ cells (or a segment thereof) may be utilized to bind their
appropriate
complementary ligand. Antibody-tagged molecules may then be extracted from the
stimulator-effector cell admixture via appropriate means, e.g., via well-known
immunoprecipitation or immunoassay methods.
Effective, cytotoxic amounts of the activated CD8+ cells can vary between
in vitro and in vivo uses, as well as with the amount and type of cells that
are the ultimate
target of these killer cells. The amount will also vary depending on the
condition of the
patient and should be determined via consideration of all appropriate factors
by the
practitioner. Preferably, however, about 1 X 106 to about 1 X 1012, more
preferably about
1 X 10$ to about 1' X 1011, and even more preferably, about 1 X 109 to about 1
X lOlo
activated CD8+ cells are utilized for adult humans, compared to about 5 X 106 -
5 X 10'
cells used in mice.
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58
Preferably, as discussed above, the activated CD8+ cell's are harvested
from the cell culture prior to administration of the CD8+ cells to, the
individual being
txeated. It is important to note, however, that unlike other present and
proposed treatment
modalities, the present method uses a cell culture system that is not
tumorigenic.
Therefore, if complete separation of stimulator cells and activated CD8+ cells
is not
achieved, there is no inherent danger known to be associated with the
administration of a
small number of stimulator cells, whereas administration of mammalian tumor-
promoting
cells may be extremely hazardous.
Methods of re-introducing cellular components are known in the art and
include procedures such as those exemplified in U.S. Patent No. 4,844,893 to
Honsik, et
al. and U.S. Patent No. 4,690,915 to Rosenberg. For example, administration of
activated
CD8+ cells via intravenous infusion is appropriate.
The immunogenic peptides of this invention may also.be used to make
~inonoclonal antibodies. Such antibodies may be useful as potential diagnostic
or
therapeutic agents.
The peptides may also find use as diagnostic reagents. For example, a
peptide of the invention may be used to determine the susceptibility of a
particular
individual to a treatment regimen which employs the peptide or related
peptides, and thus
may be helpful in modifying an existing treatment protocol or in determining a
prognosis
for an affected individual. In addition, the peptides may also be used to
predict which
individuals will be at substantial risk for developing chronic infection.
To identify peptides of the invention, class I antigen isolation, and
isolation and sequencing of naturally processed peptides was carried out as
described in
the related applications. These peptides were then used to define specific
binding motifs
for each of the following alleles A3.2, A1, A1 l, and A24.1. These motifs are
described
on page 3, above. The motifs described in Tables 8-11, below, are defined from
pool
sequencing data of naturally processed peptides as described in the related
applications.
TABLE 8
Summary
HLA-A3 2 Allele-Specific Motif
Position Conserved Residues
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59 .
2 V,L,M
3 Y,D
4 -
-
G -
I
g Q,N
9 K
K
TABLE 9
.>
Summary
HLA-Al Allele-Specific Motif
Position Conserved Residues
1 -
2 S,T
3 ~ D,E
4 P
5 ' -
6 -
7 L
8 _
9 Y
10 K
TABLE 10
Sunnmary
HLA-Al 1 Allele-Specific Motif
Position Conserved Residues
1 _
2 T,V
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- 60
MF , ,
4 -
-
6 -
8 . . Q
9 K
K
TABLE 11
Summary
HLA-A24.1 Allele-Specific Motif
Position Conserved Residues
1 -
y
3 I,M
q. D,E,G,K,P
L,M,N
6 V
N,V
g A,E,K,Q,S
9 F,L
10 F,A
Example 2
5 Identification of immunogenic peptides
Using the motifs identified above for various MHC class I allele amino
acid sequences from various pathogens and tumor-related proteins were analyzed
for the
presence of these motifs. Screening was carried out described in the related
applications.
Table 12 provides the results of searches of the antigens.
Table 12
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61
PeptideAA Sequence Source A*0301 ' ' A*1101
28.071910 ILEQWVAGRK HDV.nuc.l6 0.0170 0.0012
28.072710 LSAGGKNLSK HDV.nuc.115 0.0097 0.0150
1259.02I1 STDTVDTVLEK Flu.HA.29 0.0001 0.0670
1259.049 GIAPLQLGK FIu.HA.63 0.6100 0.2000
1259.0610 VTAACSHAGK FIu.HA..I49 0.0380 0.0490
1259.089 GIHHPSNSK Flu.HA.195 0.1300 O.OI40
1259.1010 h:IvINY~YWTLLK FIu.HA.243 2.5000 2.3000
1259.1211 ITNKVNSVIEK FIu.HA.392 0.0200 0.0670
1259.1311 KMNIQFTAVGK FIu.HA.402 0.0280 0.0092
1259.149 NIQFTAVGK FIu.HA.404 0.0017 0.0330
1259.1611 AVGKEFNKLEK Flu.HA.409 0.0210 0.0460
~
1259.1911 KVKSQLKNNAK FIu.HA.465 0.0470 0.0031
,
1259.201I SVRNGTYDYPK Flu.HA.495 0.0410 ~ 0.1400
1259.219 SIIPSGPLK FIu.VMTI.I3 0.7800 8.8000
~
1259.2510 RMVLASTTAK FIu.VMT1.1780.5500 0.0350
1259.269 MVLASTTAK FIu.VMT1.1791.7000 1.4000
1259.2810 RMGVQMQRFK FIu.VMTI.2430.1000 0.0059
1259.3310 ATEIRASVGK FIu.VNUC.22 0.1400 0.3000
1259.3711 TMVMELVRMIK FIu.VNUC.1880.0890 0.0310
1259.4310 RVLSFIKGTK FIu.VNUC.3420.8000 0.0830
F119.019 MSLQRQFLR ORF3P 0.2000 0.7200
F119.029 LLGPGRPYR TRP.197 0.0190 0.0091
FI19.039 LLGPGRPYK TRP.197K9 2.2000 0.6800
34.00198 RVYPELPK CEA.139 0.0130 0.0440
34.00208 TVSAELPK CEA.495 0.0037 0.0320
34.00218 TVYAEPPK CEA.317 0.0160 0.0220
34.00298 TINYTLWR MAGE2.74 0.0140 0.0550
34.00308 LVHFLLLK MAGE2.I16 0.0290 0.1500
34.00318 SVFAHPRK MAGE2.237 0.1410 0.0810
34.00438 KVLHHMMVK MAGE3.285 0.0580 0.0190
34.00508 RVCACPGR p53.273 0.3500 0.0490
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62
34.00518 KMFCQLAK p53.132 0.3800 0.3600
'~ ~
34.00628 RAHSSHLK p53.363 0.5500 0.0071
34.01489 FVSNLATGR CEA.656 0.0019 0.0490
34.01529 RLQLSNGNK CEA.546 0.0250 0.0110
34.01539 RINGIPQQK CEA.628 0.0400 0.0780
34.01549 KIRKYTMRK HER2/neu.6810.0620 0.0055
34.01559 LVHFLLLKK MAGE2.116 0.5220 1.4000
34.01569 SMLEVFEGK MAGE2.226 0.0950 1.6000
34.01579 SSFSTTINK MAGE2.69 0.1600 2.0000
34.01589 TSYVKVLHK MAGE2.281 0.5300 0.1500
34.01599 VIFSKASEK ~ MAGE2.149 0.4900 0.0530
34.01609 GSVVGNWQK MAGE3.130 0.0040 0.2060
34.01619 SSLPTTMNK MAGE3.69 0.6180 0.7100
'
34.01629 SVLEVFEGK MAGE3.226 0.1330 0.9000
34.01719 SSBMGGMNK p53.240 0.5440 1.1000
34.01729 SSCMGGMNK p53.240 0.0090 0.0490
34.021110 RTLTLFNVTK CEA.554 0.2200 1.3000
34.021210 TISPLNTSYK CEA.241 0.1800 0.0330
34.021410 STTINYTLWK MAGE2.72 0.0870 0.6500
34.021510 ASSLPTTMNK MAGE3.68 0.0420 0.0270
34.022510 KTYQGSYGFK p53.101 0.4900 0.4200
34.022610 WR.RBPHHEK p53.172 0.1800 0.2100
34.022810 GLAPPQHLIK p53.187 0.0570 0.0160
34.022910 NSSCMGGMNK p53.239 0.0071 0.0290
34.023010 SSBMGGMNRK p53.240 0.0420 0.1600
34.023210 RVCACPGRDK p53.273 0.0190 0.0250
34.029511 KTITVSAELPK CEA.492 0.3600 0.1600
34.029611 TTITVYAEPPK CEA.314 0.0200 0.0280
34.029811 PTISPSYTYYR CEA.418 (0.0002)0.1300
34.030111 GLLGDNQVMPK MAGE2.188 0.0780 0.0047
34.030611 MVELVHFLLLK MAGE2.113 0.0200 0.0120
34.030811 FSTT1NYTLWR MAGE2.71 0.0110 0.0170
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63
34.031111 GLLGDNQIIVVIPK MA.GE3.188 0.1300 '~ '
0.0570
34.031711 RLGFLHSGTAK p53.110 0.0430 0.0001
34.031811 ALI~1KMFCQLAK p53.129 0.4400 0.0420
34.032311 RVCACPGRDRR p53.273 0.0290 0.0290
34.032411 LSQETFSDLWK p53.14 (0.0009)0.0470
34.032811 R.AHSSHLKSKK p53.363 ' 0.02700.0038
34.032911 VTCTYSPALNK p53.122 0.0700 0.1200
34.033011 GTRVRAMAIYK p53.154 1.1000 0.3300
34.033211 STSRHKKLMFK p53.376 0.3100 0.1300
40.01079 LAA.RNVLVK Her2/neu.846 0.0580 0.0285
40.01099 MALESILRR ~ Her2/neu.889 0.0034Ø0237
40.014510 ISWLGLRSLR Her2/neu.450 0.0410 0.0027
40.014710 GSGAFGTVYK Her2/neu.727 0.0660 0.1300
40.015310 ASPLDSTFYR Her2/neu.997 0.0003 ~ 0.0670
Example 3
Identification of immunogenic peptides
Using the B7-like supermotifs identified in the related applications
described above, sequences from various pathogens and tumor-related proteins
were
analyzed for the presence of these motifs. Screening was carned out described
in the
related applications. Table 13 provides the results of searches of the
antigens.
Table 13
P eptide Sequence Source
40.0013 SPGLSAGI CEA.680I8
40.0022 KPYDGIPA Her2/neu.921
40.0023 KPYDGIPI Her2lneu.921I8
40.0050 APRMPEAA p53.63 .
40.0051 APRMPEAI p53.63I8
40.0055 APAAPTPI p53.76I8
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40.0057 APTPAAPI p53.79I8 ' '
40.0059 TPAAPAPI p53.81I8
40:0061 APAPAPSI p53.84I8
40.0062 ' SPAL p53.127 '
40.0063 SPALNKMI p53.127I8
40.0117 SPSAPPHRI CEA.3I9
40.0119 PPHRW CIPI CEA.7I9
40.0120 GPAYSGREI ~ CEA.92
40.0156 MPNQAQMRILI Her2/neu.706I10
1040.0157 MPYGCLLDHVI Her2/neu.801I10
40.0161 APPHRWCIPW . CEA.6
40.0162 APPHRWCIPI CEA.6I10
40.0163 IPWQRLLLTA CEA.13
740.0164 IPWQRLLLTI CEA.13I10
1540.0166 LPQHLFGYSI CEA.58I10
40. 0201 RPR~'RELV SEF Her2/neu. 966
40.0202 RPRFRELVSEI Her2/neu.966I11
40.0205 PPSPREGPLPA Her2/neu.1149
40.0206 PPSPREGPLPI Her2/neu.1149I11
2040.0207 GPLPAARPAGA Her2/neu.1155
40.0208 GPLPAARPAGI Her2lneu.1155I11
40.0231 APAPAAPTPAA p53.74
40.0232 APAPAAPTPAI p53.74I11
40.0233 APAAPTPAAPA p53.76
2S40.0234 APAAPTPAAPI p53.76I11
45.0003 IPWQRLLI CEA.13.I8
45.0004 LPQHLFGI CEA.58.I8
45.0007 RPGVNLSI CEA.428.I8
45.0010 IPQQHTQI CEA.632.I8
3045.0011 TPNNNGTI CEA.646.I8
45.0016 CPLHNQEI Her2/neu.315.I8
45.0017 KPCARVCI Her2/neu.336.I8
45.0019 WPDSLPDI Her2/neu.415.I8
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45.0023 SPYVSRLI Her2lneu.779.I8 '
45 .0024 VPIKWMAI Her2/neu. 8 84.I8
45.0026 RPRFRELI Her2/neu.966.I8
45.0028 AP GAGGMI Her2/neu.103 6.I8
45.0031 SPGKNGVI Her2/neu.1174.I8
45.0037 SPQGASSI MAGE3.64.I8
45.0038 YPLWSQSI ~MAGE3.77.I8
45.0044 SPLPSQAI p53.33.I8
45.0046 MPEAAPPI p53.66.I8
~
45.0047 APAPSWPI ' p53.86.I8
45.0051 KPVEDKDAI CEA.155.I9
45.0054 IPQQHTQVI CEA.632.I9
45.0060 APPVAPAPI p53.70.I9
45.0062 APAAPTPAI p53.76.I9
I5 45.0064 PPGTRVRAI p53.152.I9
45.0065 APPQHLIRI p53.189.I9
45.0071 IPQQHTQVLI CEA.632.I10
45.0072 SPGLSAGATI CEA.680.I10
45.0073 SPMCKGSRCI Her2/neu.196.I10
45.0074 MPNPEGRYTI Her2/neu.282.I10
45.0076 CPLHNQEVTI Her2/neu.31 S.I10
45.0079 KPDLSYMPII Her2/neu.605.I10
45.0080 TPSGAMPNQI Her2/neu.701.I10
45.0084 GPASPLDSTI Her2/neu.995.I10
45.0091 APPVAPAPAI p53.70.I10
45.0092 APAPAAPTPI p53.74.I10
45.0093 APTPAAPAPI p53.79.I10
45.0094 APSWPLSSSI p53.88.I10
45.0103 APT.ISPLNTSI CEA.239.I11
45.0108 SPSYTYYRPGI CEA.421.I11
45.0117 CPSGVKPDLSI Her2/neu.600.I11
45.0118 SPLTSIISAVI Her2lneu.649.I11
45.0119 IPDGENVKIPI Her2/neu.740.I11
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45.0124 SPLDSTFYRSI Her2/neu.998.I11 '
45.0128 LPAARPAGATI Her2/neu.I157:I1I
45.0134 HPRKLLMQDLI MA.GE2.241.I11
45.013 5 GPR.ALIETSYI MAGE2.274.I11
45.0139 GPR.ALVETSYI MAGE3.274.I11
45.0140 APRMPEAAPPi p53.63.I11
45.0141 VPSQKTYQGSI p53.97.I11
1145.10 FPHCLAFAY HBV POL 541 analog
1145.09 FPVCLAFSY HBV POL 541 analog
26.0570 YPALMPLYACI HBV.pol.645
The above description is provided to illustrate the invention but not to limit
its scope. Other variants of the invention will be readily apparent to, one of
ordinary skill
in the art and axe encompassed by the appended claims. All publications,
patents, and
patent applications cited herein axe hereby incorporated by reference.
