Note: Descriptions are shown in the official language in which they were submitted.
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HLA BINDING PEPTIDES AND THEIR USES
The present application is a continuation in part of USSN 08/589,107 and is
related to USSN 60/013,833, USSN 08/451,913 and to USSN 08/347,610, which is a
continuation in part of USSN 08/159,339, which is continuation in part of USSN
08/103,396
which is a continuation in part of USSN 08/027,746 which is a continuation in
part of USSN
07/926,666. The application is also related to USSN 08/186,266, 08/821,739,
and
08/758,409. 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 are 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 MEC
molecules are 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 rej ection 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
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 a 1 and a2
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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 (Rotzschke
and Falk,
Immunol. Today,12:447 (1991).
Sette et al., P~oc. Natl. Acad. Sci. USA, 86:3296 (1989) showed that MHC
allele specific motifs could be used to predict MHC binding capacity.
Schaeffer et al., P~oc.
Natl. Acad. Sci. USA, 86:4649 (1989) showed that MHC binding was related to
immunogenicity. Several authors (De Bruijn et al., Eur. I. 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.
SUMMARY OF THE INVENTION
The present invention provides compositions comprising immunogenic
peptides having binding motifs for MHC Class I molecules. The 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.
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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-A 1 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 axe adjacent and are preferably separated from the third
conserved residue
by 6 to 7 residues. A second motif consists of a first conserved residue of 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-A 11 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
I S 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),
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 usefixl in pharmaceutical compositions for
both ih vitro 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 and
usually consist of between about ~ and about 11 residues, preferably 9 or 10
residues.
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An "immunogenic peptide" is a peptide which comprises an allele-specific
motif such that the peptide will bind the MHC allele and be capable of
inducing a CTL
response. Thus, immunogenic peptides are capable of binding to an appropriate
class I MHC
molecule and inducing a cytotoxic T cell response against the antigen from
which the
immunogenic peptide is derived.
A "conserved residue" is an amino acid which occurs in a significantly higher
frequency than would be expected by random distribution at a particular
position in a peptide
motif. Typically a conserved residue is one at which the immunogenic peptide
may provide a
contact point with the MHC molecule. At least one to three or more, preferably
two,
conserved residues within a peptide of defined length defines a motif for an
irnmunogenic
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 will result in a peptide being a nonbinder or poor binder
and in turn fail to
induce a CTL response despite the presence of the appropriate conserved
residues within the
peptide.
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 for each human MHC allele
and differ in the
pattern of the highly conserved 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 binding residues present.
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 a oligopeptide by an amide bond or amide bond mimetic.
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DESCRIPTION OF THE SPECIFIC EMBODIIVVIENTS
The present invention 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-Barr
virus antigens,
melanoma antigens (e.g., MAGE-1), human irnmunodeficiency virus (HIV) antigens
and
human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis
(MT), p53,
CEA, and Her2lneu.
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, innnunofluorescent staining and flow
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 fox' their capacity to induce primary in
vitro or in vitro
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 occux at different frequencies within
different ethnic
groups and races, the choice of target MHC allele may depend upon the 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
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6
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.
TABLE 1
A Allele/Subtwe N 69)* A 54) C 502)
A1 10.1(7) 1.8(1) 27.4(138)
A2.1 11.5(8) 37.0(20) 39.8(199)
A2.2 10.1 (7) 0 3.3(17)
A2.3 1.4(1) 5.5(3) 0.8(4)
A2.4 _ _ _
A2.5 - _ _
A3.1 1.4(1) 0 0.2(0)
A3.2 5.7(4) 5.5(3) 21.5(108)
A11.1 0 5.5(3) 0
Al 1.2 5.7(4) 31.4(17) 8.7(44)
A11.3 0 3.7(2) 0
A23 4.3(3) - 3.9(20)
A24 2.9(2) 27.7(15) 15.3(77)
A24.2 - - -
A24.3 - - -
A25 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)
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A Allele/Subtwe N 69)* A 54) C1502)
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, Immuhobiology 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 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, are known and readily available. For 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
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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 2 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 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 2
HUMAN CELL LINES (LILA-A SOURCES)
HLA-A allele B cell line
A1 MAT
COX (9022)
STEINLIN
(9087)
A2.1 JY
A3.2 EHM (9080)
H0301 (9055)GM3107
A24.1 KT3(9107),TISI (9042)
A11 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 for 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 3). Thus, for each of the taxgeted 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.
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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, B1.23.2, could be used
in
alternative affinity purification protocols as described in the example
section below.
TABLE 3
ANTIBODY REAGENTS
anti-HLA Name
HLA-A1 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 (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.
Sequencing of the isolated peptides can be performed according to standard
techniques such as Edman degradation (Hunkapiller, M.W., et al., Methods
E~czymol. 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
(e.g., 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
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sequence is known. Typically, identification of potential peptide epitopes is
initially carned
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
molecular
5 binding assay as described, for instance, in the related applications, noted
above. Other
alternatives described in the literature include inhibition of antigen
presentation (Sette, et al.,
J. Immuhol.,141:3893 (1991), iu 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.
Immunol., 21:2963 [1991]).
10 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 ih 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 hybridoma, 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), sillcworm (ATTC CRL 8851), armyworm (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 [1927]). That have been transfected with
the
appropriate human class I MHC allele encoding genes and the human B2
microglobulin
genes.
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 wM of peptide in serum-free media for 4 hours under
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.
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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 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 isolated from natural sources such as whole viruses or
tumors. Although
the peptide will preferably be substantially free of other naturally occurring
host cell proteins
and fragments thereof, in some embodiments the peptides card 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,
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
cell line
(see Schneider J. Emb~yol. Exp. Morphol., 27:353-365 [1927]). That have been
transfected
with the appropriate human class I MHC allele encoding genes and the human B2
microglobulin genes.
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 wM of peptide in serum-free media for 4 hours under
appropriate
culture conditions. The peptide-loaded antigen-presenting cells are then
incubated with the
responder cell populations ih 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
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12
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 are referred to herein as immunogenic peptides.
The immunogenic peptides can be prepared synthetically, or by recombinant
DNA technology or isolated from natural sources such as whole viruses or
tumors. Although
the peptide will preferably be substantially free of other naturally occurring
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 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
nonconservative, 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 Mernfield, The
Peptides, Gross
and Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart and
Young,
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Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (194),
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 noncritical 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 is
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 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 4 when it is desired to finely modulate
the
characteristics of the peptide.
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TABLE 4
Original Residue Exemplary Substitution
Ala Ser
Arg Lys
Asn Gln; His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn; Gln
Ile Leu; Val
Leu Ile; Val
Lys Arg
Met Leu; Ile
Phe Met; Leu; Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr 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 4,
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) hydrophilic
residue, e.g.,
Beryl, 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.,
lysyl, arginyl, or
histidyl, is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or
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(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
5 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 crossliinlcs. See, eg
nerallx, Spatola,
10 Chemistry and Biochemistry ofAmino Acids, peptides ahd Proteihs, 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
ih vitro. Stability
can be assayed in a number of ways. For instance, peptidases and various
biological media,
15 such as human plasma and serum, have been used to test stability. See.,
e.g., Verhoef et al.,
Eur. J. Drug Metab Pharmacokih, 11:291-302 (1986). Half life of the peptides
of the present
invention is conveniently determined using a 25% human serum (vlv) 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 RPMI 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 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
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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.
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
acylated.
In some embodiments it may be desirable to include in the pharmaceutical
compositions of the invention at least one component which assists in priming
CTL. Lipids
have been identified as agents capable of assisting the priming CTL ih vitro
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 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., 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 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, glutarnic 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-C2o) or thioglycolyl
acetylation, terminal-
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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, 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 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
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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
prescribing physician, but generally range for the initial immunization (that
is for therapeutic
or prophylactic administration) from about 1.0 wg to about 5000 ~,g of peptide
for a 70 kg
patient, followed by boosting dosages of from about 1.0 ~.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
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.
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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 p.g to 1000 p,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, 0.9% saline, 0.3% glycine, 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 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
target the peptides to a particular cells tissue, such as lymphoid tissue.
Liposomes are also
useful in increasing the half life of the peptides. Liposomes include
emulsions, foams,
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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
5 with other therapeutic or immunogenic compositions. Thus, liposomes filled
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
10 of lipids is generally guided by consideration of, e.g., liposome sue, 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., AfZn. Rev. Biophys. Bioehg.,
9:467 (1980), U.S.
Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated
herein by
reference.
15 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, ihte~ alia, the manner of administration, the peptide being
delivered, and the
20 stage of the disease being treated.
For solid compositions, conventional nontoxic solid Garners may be used
which include, for example, pharmaceutical grades of mannitol, 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 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 atoms,
such as caproic,
octanoic, lauric, palmitic, steamic, linoleic, linolenic, olesteric and oleic
acids with an
aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as
mixed or natural
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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.
In another aspect the present invention is directed to vaccines which contain
as
S 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 carrier 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
bovine serum
albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid),
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, 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 laxge 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 p.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.
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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. 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
nucleci 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) BioTechhiques 6(7): 682-691; Rose
U.S. Pat
No. 5,279,833; WO 91106309; and Felgner et al. (1987) Proc. Natl. 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
Calinette
Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460
(1991)) which is
incorporated herein by reference. A wide variety of other vectors useful for
therapeutic
administration or immunization of the peptides of the invention, e.g.,
Salmonella t~hi'
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 andlor 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
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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 Iigase. 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 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 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 bicistronic 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
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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
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 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 ih vitro
transfection. A plasmid expressing green fluorescent protein (GFP) can be co-
transfected to
allow enrichment of transfected cells using fluorescence activated cell
sorting (FACS).
These cells are then chromium-51 labeled and used as target cells for epitope-
specific CTL
lines. Cytolysis, detected by 51 Cr release, indicates production of MIiC
presentation of
minigene-encoded CTL epitopes.
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In vitro 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 ira vitro
induction of
10 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
15 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
20 cell). In order to optimize the i~c 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 culture,
of sufficient quantity to become loaded onto the human Class I molecules to be
expressed on
25 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.
Resting or precursor CD8 + cells are then incubated in culture with the
appropriate stimulator cells for a time period sufficient to activate the CDS
+ 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
individual's
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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.
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
MLIC/peptide complexes per APC is crucial for the stimulation of CTL,
particularly in
primary immune responses. While small amounts of peptideMMRIC complexes per
cell are
sufficient to render a cell susceptible to lysis by CTL, or to stimulate 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 induction 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, ~3amicroglobulin. Removing
the bound
peptides and/or dissociating the (32microglobulin 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.
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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 destablize
(32microglobulin and stripping the endogenous peptides from the cell using a
mild acid
treatment. The methods release previously bound peptides into the
extracellular 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
(e.g., resting
PBMC) would not produce high amounts of empty surface MLIC 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 1-
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 for
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 ih
vitro and ih vitro uses, as well as with the amount and type of cells that are
the ultimate target
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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 108 to about
I X 1011, and even more preferably, about 1 X io~ to about 1 X 101°
activated CD8 + cells are
S utilized for adult humans, compared to about 5 X 106 - 5 X 10$ cells used in
mice.
Preferably, as discussed above, the activated CD8 + cells are harvested from
the cell culture prior to administration of the CD8 + cells to the individual
being treated. 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.
IS 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
monoclonal 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, Al, A1 l, and A24.1. These motifs are described on
page 3, above.
The motifs described in Tables 5-8, below, are defined from pool sequencing
data of
naturally processed peptides as described in the related applications.
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TABLE 5
Summary
HLA-A3.2 Allele-Snecific
Motif
Conserved
PositionResidues
1 -
2 V,L,M
3 Y,D
4 -
-
6 -
7 I
8 Q,N
9 K
K
TABLE 6
Summary
HLA-A 1 Allele-Specific
Motif
Conserved
Position Residues
1 -
2 S,T
3 D,E
4 P
5 -
6 -
7 L
8 _
9 Y
5
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TABLE 7
Summary
HLA-Al l Allele-Specific
Motif
Conserved
PositionResidues
1 -
2 T,V
3 M,F
4 -
5 -
6 -
7 _
Q
9 K
10 K
Table ~
Summary
HLA-A24.1 Allele-Specific Motif
Conserved
Position Residues
1 -
2 Y
3 I,M
4 D,E,G,K,P
5 L,M,N
6 V
7 N,V
A,E,K,Q,S
9 F,L
10 F,A
5
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Example 1
Identification of immunoge~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 carned out described in the related
applications. Tables 9-10
provide the results of searches of the antigens.
Table 9
SEO ID NO: AA Sequence Source
1 9 FLWTQSLRR Lassa np 10
2 9 GIPYCNYSK Lassa Josiah(GP)
360
3 9 GWRVWDVK Lassa Josiah(NP)
159
4 9 MLRLFDFNK Lassa gp 312
5 9 RTRDIYISR Lassa gp 248
6 9 SAVIDALPR Lassa Josiah(NP)
447
7 9 SVLRAVLPR Lassa Josiah(NP)
548
8 9 VTFLLLCGR Lassa Josiah(GP)
47
9 10 AMSCDFNGGK Lassa gp 152
10 GSYIALDSGR Lassa gp 198
11 10 KSFLWTQSLR Lassa Josiah(NP)
8
12 10 LTYSQLMTLK Lassa np 365
13 10 RTRDIYISRR Lassa gp 248
14 9 YRHLLCLER Hu TRP-2
10 GTYEGLLRRR Hu TRP-2
16 9 LLRRNQMGR Hu TRP-2
17 9 FQNSTFSFR Hu TRP-2
18 9 LLAFLQYRR Hu TRP-2
10 The peptides listed in Table 10 were identified as described above and are
grouped according to pathogen or antigen from which they were derived.
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Table 10
HBV
SEQ ID NO Sequence Source
20 HTLWKAGILYK POL.149
21 SVVRR.AFPH POL.524
22 AFTFSPTYK POL.655
23 SSAGPCALR X.64
24 CALRFTSAR X.69
HCV
SEQ ID NO Sequence Source
25 WMNSTGFTK NS1/E2.557
26 LGFGAYMSK NS3.1267
27 VAGALVAFK NS4.1864
HIV 1
SEQ ID NO Sequence Source
28 MVHQAISPR GAG.174
29 QMVHQAISPR GAG.173
30 TIKTGGQLK POL.92
31 TLFCASDAK ENV.63
32 TTLFCASDAK ENV.62
33 RIVELLGRR ENV.958
34 VTIKIGGQLK POL.91
35 VMIVWQVDR VIF.7
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CEA
SEQ ID NO Sequence Source
36 HLFGYSWYK CEA.61
37 HTQVLFT_A_K_ CEA.636
38 TISPSYTYYR CEA.419
39 RTLTLFNVTR CEA.554
40 ISPSYTYYR CEA.420
41 TISPLNTSYR CEA.241
42 RTLTLLSVTR CEA.376
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 are encompassed by the appended claims. All publications, patents, and
patent
applications cited herein are hereby incorporated by reference.
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