U.S. patent application number 10/121415 was filed with the patent office on 2002-10-31 for hla binding peptides and their uses.
Invention is credited to Kast, W. Martin, Sette, Alessandro, Sidney, John, Southwood, Scott.
Application Number | 20020160960 10/121415 |
Document ID | / |
Family ID | 38324304 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160960 |
Kind Code |
A1 |
Sette, Alessandro ; et
al. |
October 31, 2002 |
HLA binding peptides and their uses
Abstract
The present invention provides the means and methods for
selecting immunogenic peptides and the immunogenic peptide
compositions capable of specifically binding glycoproteins encoded
by HLA alleles and inducing T cell activation in T cells restricted
by the allele. The peptides are useful to elicit an immune response
against a desired antigen.
Inventors: |
Sette, Alessandro; (La
Jolla, CA) ; Sidney, John; (La Jolla, CA) ;
Kast, W. Martin; (Willowbrook, IL) ; Southwood,
Scott; (Santee, CA) |
Correspondence
Address: |
Bruce D. Grant
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130
US
|
Family ID: |
38324304 |
Appl. No.: |
10/121415 |
Filed: |
April 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10121415 |
Apr 11, 2002 |
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09189702 |
Nov 10, 1998 |
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09189702 |
Nov 10, 1998 |
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09098584 |
Jun 17, 1998 |
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09098584 |
Jun 17, 1998 |
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08205713 |
Mar 4, 1994 |
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08205713 |
Mar 4, 1994 |
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08159184 |
Nov 29, 1993 |
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Current U.S.
Class: |
424/185.1 ;
514/21.6 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 38/08 20130101; C07K 14/70539 20130101; C07K 14/005 20130101;
C12N 2770/24222 20130101; C07K 14/4748 20130101; A61P 37/00
20180101; C12N 2730/10122 20130101; A61K 2039/55555 20130101 |
Class at
Publication: |
514/15 |
International
Class: |
A61K 038/08 |
Claims
What is claimed is:
1. A composition comprising a peptide consisting of the sequence
YLQLVFGIEV (SEQ ID NO: 1).
2. A composition comprising an immunogenic peptide having an
HLA-A2.1 binding motif, which immunogenic peptide is YLQLVFGIEV
(SEQ ID NO: 1).
3. A method for inducing a cytotoxic T-cell response against a
preselected antigen in a patient expressing an HLA-A2.1 MHC
product, the method comprising contacting the cytotoxic T cells
from the patient with a composition comprising an immunogenic
peptide of the sequence YLQLVFGIEV (SEQ ID NO: 1).
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 09/189,702 filed Nov. 10, 1998, which is a
continuation-in-part of U.S. application Ser. No. 09/098,584 filed
Jun. 17, 1998, which is a continuation-in-part of 08/205,713 filed
Mar. 4, 1994, which is a continuation-in-part of 08/159,184 filed
Nov. 29, 1993. Each of these applications is incorporated herein by
reference in its entirety.
BACKGROUND
[0002] 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.
[0003] 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 MHC
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 rejection and in fighting viral
infections.
[0004] 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 .beta.2 microglobulin. The peptide-MHC
class I complex is then routed to the cell surface for expression
and potential recognition by specific CTLs.
[0005] 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 .alpha.1 and .alpha.2 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.
[0006] 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).
[0007] Sette et al., Proc. Natl. Acad. Sci. USA 86:3296 (1989)
showed that MHC allele specific motifs could be used to predict MHC
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); Pamner 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.
[0008] 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
[0009] The present invention provides compositions comprising
immunogenic peptides having binding motifs for HLA-A2.1 molecules.
The immunogenic peptides, which 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.
[0010] The motifs of the inventions include peptides 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 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.
[0011] 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-Barr
virus antigens, human imnmunodeficiency type-1 virus (HIV1),
Kaposi's sarcoma herpes virus (KSHV), human papilloma virus (HPV)
antigens, Lassa virus, mycobacterium tuberculosis (MT), p53 and
murine 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 for both in
vivo and ex vivo therapeutic and diagnostic applications.
[0012] The present invention also 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.
[0013] 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.
[0014] 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 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.
[0015] The motif for ULA-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.
[0016] 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.
[0017] The present invention also provides compositions comprising
immunogenic peptides having binding motifs for non-A HLA alleles.
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
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.
[0018] Definitions
[0019] 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 8 and about 11 residues, preferably 9 or 10
residues.
[0020] 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.
[0021] 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 of
a peptide or the effects on binding of a particular substitution in
a motif containing peptide.
[0022] 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. 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.
[0023] As used herein, "negative binding residues" are amino acids
which if present at certain positions (for example, positions 1, 3,
6 and/or 7 of a 9-mer) will result in a peptide being a nonbinder
or poor binder and in turn fail to be immunogenic, i.e., induce a
CTL response.
[0024] 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 and negative residues.
[0025] 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, 6 and/or 7.
[0026] 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 homogeneous 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.
[0027] The term "residue" refers to an amino acid or amino acid
mimetic incorporated in an oligopeptide by an amide bond or amide
bond mimetic.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present invention relates to the determination of
allele-specific peptide motifs for human Class I MHC (sometimes
referred to as HLA) allele subtypes, and 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 autoimmune
diseases, for which the amino acid sequence of the potential
antigen or autoantigen targets is known.
[0029] Epitopes on a number of potential target proteins can be
identified. 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 antigen (MAGE-1), Epstein-Barr virus antigens,
human immunodeficiency type-1 virus (HIV1), papilloma virus
antigens, Lassa virus, mycobacterium tuberculosis (MT), p53 and
murine p53 (mp53), CEA, Her2/neu and members of the tyrosine kinase
related protein families (TKP). The peptides are thus useful in
pharmaceutical compositions for both in vivo and ex vivo
therapeutic and diagnostic applications.
[0030] 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 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 infected target cells or tumor cells as
potential therapeutic agents.
[0031] 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 relatively specific [sic] for each allelic
subtype.
[0032] 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 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,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, as shown in Table 1.
1 TABLE 1 A Allele/Subtype 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 A11.2 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) 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. *N-Negroid; A = Asian; C = Caucasoid. Numbers in
parenthesis represent the number of individuals included in the
analysis.
[0033] 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 having D-forms 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.
[0034] 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.
[0035] 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 commercial sources, such as American
Type Culture Collection ("Catalogue of Cell Lines and Hybridomas,"
6th edition (1988) Rockville, Md., U.S.A.); National Institute of
General Medical Sciences 1990/1991 Catalog of Cell Lines (NIGMS)
Human Genetic Mutant Cell Repository, Camden, N.J.; and ASHI
Repository, Bingham and Women's Hospital, 75 Francis Street,
Boston, Mass. 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.
2TABLE 2 HUMAN CELL LINES (HLA-A SOURCES) HLA-A allele B cell line
A1 MAT COX (9022) STEINLIN (9087) A2.1 JY A3.2 EHM (9080) HO301
(9055)GM3107 A24.1 T3(9107),TISI (9042) A11 BVR (GM6828A) WT100
(GM8602)WT52 (GM8603)
[0036] 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). Monoclonal antibody BB7.2 is suitable for isolating HLA-A2
molecules. Thus, for 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.
3TABLE 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
[0037] 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, B1.23.2, could be used in alternative affinity
purification protocols as described in previous applications.
[0038] 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.
[0039] 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.
[0040] 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
(e.g., pooled HPLC fractions) from different class I molecules
typically reveals a characteristic sequence motif for each class I
allele.
[0041] 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 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 alternatives described in the literature include
inhibition of antigen presentation (Sette, et al., J. Immunol.
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. Immunol. 21:2963 (1991)).
[0042] 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]).
[0043] 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
(Krre, 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
(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]).
[0044] 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 .mu.M 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. 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.
[0045] 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.
[0046] 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
and fragments thereof, in some embodiments the peptides can be
synthetically conjugated to native fragments or particles.
[0047] 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.
[0048] 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.
[0049] 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 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.
[0050] 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-.alpha.-amino acids, or their
D-isomers, but may include non-natural amino acids as well, such as
.beta.-.gamma.-.delta.-amino acids, as well as many derivatives of
L-.alpha.-amino acids.
[0051] 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 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.
[0052] 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.
4 TABLE 4 Original Residue Exemplary Substitution Ala Ser Arg Lys,
His Asn Gln Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Lys; Arg
Ile Leu; Val Leu Ile; Val Lys Arg; His Met Leu; Ile Phe Tyr; Trp
Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val Ile; Leu Pro Gly
[0053] 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. 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.
[0054] 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 .alpha.-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).
[0055] 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
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 (40.degree. 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.
[0056] 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.
[0057] 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.
Exemplary T helper peptides include tetanus toxoid 830-843,
influenza 307-319, malaria circumsporozoite 382-398 and
378-389.
[0058] 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 of Lys,
which is attached via linkage, e.g. Ser-Ser, to the amino terminus
of the immunogenic peptide.
[0059] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) 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 P.sub.3CSS, 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 P.sub.3CSS 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.
[0060] 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-NH.sub.2 acylation, e.g., by alkanoyl
(C.sub.1-C.sub.20) 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.
[0061] 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.
[0062] 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, N.Y. (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.
[0063] 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 base(s) 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.
[0064] 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.
[0065] 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 .mu.g to about 5000 .mu.g of peptide
for a 70 kg patient, followed by boosting dosages of from about 1.0
.mu.g to about 1000 .mu.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.
[0066] 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.
[0067] 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.
[0068] 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 .mu.g
to about 5000 .mu.g, preferably about 5 .mu.g to 1000 .mu.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.
[0069] 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 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.
[0070] 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.
[0071] 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. Bioeng.
9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369, incorporated herein by reference.
[0072] 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.
[0073] For solid compositions, conventional nontoxic solid carriers
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%.
[0074] 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, 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.
[0075] 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
peptide(s) 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 anti genic 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(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, 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 P.sub.3CSS. 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.
[0076] 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 .mu.g to about 5000 .mu.g per 70
kilogram patient, more commonly from about 10 .mu.g to about 500
.mu.g mg per 70 kg of body weight.
[0077] 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.
[0078] 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. Pat. 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. Pat. 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 Feigner 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. Pat. 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 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 typhi vectors and the like, will be apparent to
those skilled in the art from the description herein.
[0079] 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 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.
[0080] 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. The 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.
[0081] 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. Pat. Nos. 5,580,859 and 5,589,466 for other
suitable promoter sequences.
[0082] 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.
[0083] 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-.beta.) may
be beneficial in certain diseases.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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 sorting (FACS). These cells are then .sup.51Cr labeled and
used as target cells for epitope-specific CTL lines. Cytolysis,
detected by .sup.51Cr release, indicates production of MHC
presentation of minigene-encoded CTL epitopes.
[0088] 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, .sup.51Cr 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.
[0089] 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).
[0090] 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 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 .mu.g/ml peptide.
[0091] 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 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 lymphocyte:stimulator 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.
[0092] 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 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.
[0093] 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.
[0094] 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 .alpha.1 and .alpha.2 domains, and 3) a
non-covalently associated non-polymorphic light chain, .beta..sub.2
microglobulin. Removing the bound peptides and/or dissociating the
.beta..sub.2 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.
[0095] Two possible ways to free up MHC class I molecules of bound
peptides include lowering the culture temperature from 37.degree.
C. to 26.degree. C. overnight to destabilize .beta..sub.2
microglobulin 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.degree. 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 MHC molecules by the cold temperature procedure.
[0096] 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.degree. 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.
[0097] 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.
[0098] 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.times.10.sup.6 to about 1.times.10.sup.12, more preferably about
1.times.10.sup.8 to about 1.times.10.sup.11, and even more
preferably, about 1.times.10.sup.9 to about 1.times.10.sup.10
activated CD8+ cells are utilized for adult humans, compared to
about 5.times.10.sup.6-5.times.10.sup.7 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.
[0099] Methods of re-introducing cellular components are known in
the art and include procedures such as those exemplified in U.S.
Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to
Rosenberg. For example, administration of activated CD8+ cells via
intravenous infusion is appropriate.
[0100] 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.
[0101] 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.
[0102] 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, A11, and A24.1. These
motifs are described previously. The motifs described in Tables
5-8, below, are defined from pool sequencing data of naturally
processed peptides as described in the related applications.
5TABLE 5 Summary HLA-A3,2 Allele-Specific Motif Position Conserved
Residues 1 -- 2 V,L,M 3 Y,D 4 -- 5 -- 6 -- 7 I 8 Q,N 9 K 10 K
[0103]
6TABLE 6 Summary HLA-A1 Allele-Specific Motif Position Conserved
Residues 1 -- 2 S,T 3 D,E 4 P 5 -- 6 -- 7 L 8 -- 9 Y 10 K
[0104]
7TABLE 7 Summary HLA-A11 Allele-Specific Motif Position Conserved
Residues 1 -- 2 T,V 3 M,F 4 -- 5 -- 6 -- 7 -- 8 Q 9 K 10 K
[0105]
8TABLE 8 Summary HLA-A24.1 Allele-Specific Motif Position Conserved
Residues 1 -- 2 Y 3 I,M 4 D,E,G,K,P 5 L,M,N 6 V 7 N,V 8 A,E,K,Q,S 9
F,L 10 F,A
[0106] 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.
[0107] The following example is offered by way of illustration, not
by way of limitation.
EXAMPLE 1
Identification of Immunogenic Peptides
[0108] Using the motifs identified above for various MHC class I
allele amino acid sequences from multiple pathogens and
tumor-related proteins were analyzed for the presence of these
motifs. Screening was carried out as described in related
applications. Multiple immunogenic peptides were identified using
these procedures. One peptide consisted of the sequence YLQLVFGIEV
(SEQ ID NO: 1), a 10-mer derived from the MAGE-2 antigen,
designated as peptide 1090.01. This peptide conformed with the
HLA-A2.1 motif.
[0109] 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.
Sequence CWU 1
1
1 1 10 PRT Artificial Sequence Immunogenetic peptide 1 Tyr Leu Gln
Leu Val Phe Gly Ile Glu Val 1 5 10
* * * * *