JP2003524016A - HLA binding peptides and their uses - Google Patents

Abstract

  (57) [Summary] The present invention relates to immunogenic and immunogenic peptides that can specifically bind the glycoprotein encoded by the HLA allele and induce T cell activation in T cells restricted by this allele. Means and methods are provided for selecting a composition. This peptide is useful for raising an immune response against the desired antigen. The present invention relates to compositions and methods for preventing, treating or diagnosing many pathological conditions such as viral diseases and cancer. In particular, the invention provides novel peptides that can bind to selected major histocompatibility complex (MHC) molecules and induce an immune response.

Description

Detailed Description of the Invention

Citation of Related Application This application is a continuation-in-part application of USSN 08 / 205,713 filed on Mar. 4, 1994. This application is also USSN09 / 017,735, USSN
08 / 753,622, USSN 08 / 822,382, USSN 60/013
, 980, USSN08 / 589,108, USSN08 / 454,033, U
SSN 08 / 349,177, USSN 08 / 073,205, and USSN
08 / 207,146. This application also claims USSN 09 / 017,52
4, USSN08 / 821,739, USSN60 / 013,833, USSN
08 / 758,409, USSN08 / 589,107, USSN08 / 451
, 913, and USSN08 / 347,610, USSN08 / 186,2.
66, USSN08 / 159,399, USSN09 / 116,061, USS
N08 / 103,396, USSN08 / 027,746, and USSN07
/ 926,666. The present invention also relates to USSN 09 / 017,743;
USSN08 / 753,615; USSN08 / 590,298; USSN08
/ 452,843; USSN09 / 115,400; USSN08 / 344,8
24; and USSN 08 / 278,634. This application is also USSN
08 / 197,484 and USSN 08 / 815,396. All of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION The present invention relates to compositions and methods for preventing, treating or diagnosing many pathological conditions such as viral diseases and cancer. In particular, the present invention provides novel peptides capable of binding to 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 predominantly expressed on cells involved in initiating and sustaining the immune response (eg, 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 specific immunogenic peptide presented. Class I MHC molecules are expressed on almost all nucleated molecules and are recognized by cytotoxic T lymphocytes (CTL), which in turn are
Destroy antigen-bearing cells. CTLs are especially important in the fight against tumor rejection and viral infections.

CTLs recognize certain antigens in the form of peptide fragments that bind to MHC class I molecules rather than the intact foreign antigen itself. This antigen usually has to be synthesized endogenously by the cell and some of this protein antigen is broken down in the cytoplasm into small peptide fragments. Some of these smaller peptides translocate to the pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with subunit β2 microglobulin. This peptide-MHC class I complex then translocates to the cell surface for expression and potential recognition by specific CTLs.

Studies of the crystal structure of HLA-A2.1, a human MHC class I molecule, show that the peptide binding groove is created by folding of the α1 and α2 domains of the class I heavy chain (Bjorkman). Et al., Nature 329: 506.
(1987)). However, the identity of the groove-binding peptide was not determined in this study.

Buus et al., Science 242: 1065 (1988) first described a method for acid elution of bound peptides from MHC. Then, Rammensee
And co-workers (Falk et al., Nature 351: 290 (1991)).
Have developed an approach for characterizing naturally processed peptides that bind to class I molecules. Other investigators have described conventional automated sequencing of peptides eluted from type B class I molecules (Jardetzky et al., Nature).
353: 326 (1991)) and mass spectrometry of peptides eluted from class A molecules of type A2.1 (Hunt et al., Science 225: 1261 (19).
92)) successfully achieved direct amino acid sequencing of larger amounts of peptides in various HPLC fractions. A general review of the characterization of MHC class I naturally processed peptides is provided by Rotzschke and Falk (Roetz.
schke and Falk, Immunol. Today 12: 447 (19
91)).

Sette et al., Proc. Natl. Acad. Sci. USA 86:32
96 (1989) showed that MHC allele-specific motifs could be used to predict MHC binding capacity. Schaeffer et al., Proc. Natl. A
cad. Sci. USA 86: 4649 (1989) have shown that MHC binding is associated with immunogenicity. Some authors (De Bruijn et al., Eur. J.
． Immunol. 21: 2963-2970 (1991); Pamer et al.,
Nature 353: 852-955 (1991)) provided preliminary evidence that class I binding motifs could be applied to the identification of potential immunogenic peptides in animal models. Class I motifs specific for many human alleles of a given class I isotype have not yet been described. It is desired that the combined frequencies of these different alleles should be very high enough to cover a large fraction or perhaps most human outbred populations.

Despite the developments in the field, the prior art has not yet provided useful human peptide-based vaccines or therapeutics based on this study. The present invention provides these and other advantages.

SUMMARY OF THE INVENTION The present invention provides a composition comprising an immunogenic peptide having a binding motif for an HLA-A2.1 molecule. The immunogenic peptide that binds to the appropriate MHC allele is preferably 9-10 residues long and contains conserved residues at certain positions, such as positions 2 and 9. In addition, these peptides are in the 1-, 3-, 6- and / or 7-positions for peptides of 9 amino acids length and 1-, 3-, 4-, 5-positions for peptides of 10 amino acids length, Other positions, such as positions 7, 8 and / or 9 do not include a negative binding residue as defined herein. The present invention defines positions within the motif that allow for selection of peptides that bind efficiently to HLA A2.1.

The motif of the invention comprises peptides of 9 amino acids, which are I, V, A,
A first conserved residue at the second position from the N-terminus selected from the group consisting of and T, and a C-terminal position selected from the group consisting of V, L, I, A, and M Has a second conserved residue of. Alternatively, the peptide is V, L,
A first conserved residue at the second position from the N-terminal selected from the group consisting of I, A, and M; and a position at the C-terminal position selected from the group consisting of A and M. It may have 2 conserved residues. When the peptide has 10 residues, the peptide is selected from the group consisting of L, M, I, V, A, and T, and is the first conserved residue at the second position from the N-terminus; and Having a C-terminal second conserved residue selected from the group consisting of V, I, L, A, and M; wherein the first and second conserved residues are: 7 residues apart.

Epitopes on many immunogenic target proteins can be identified using the peptides of the invention. Examples of suitable antigens are prostate specific antigen (PSA), prostate specific membrane antigen (PSM), hepatitis B core antigen and surface antigens (HBVc, HB
Vs), hepatitis C antigen, Epstein-Barr virus antigen, human immunodeficiency virus type 1 (HIV1), Kaposi's herpes simplex virus (KSHV), human papillomavirus (HPV) antigen, Lassa virus, Mycobacterium tuberculosis (mycobac)
terium tuberculosis (MT), mouse p53 (mp53)
), CEA, trypanosome surface antigen (TSA), tyrosinase-related protein (TRP) family members, and Her2 / neu, and. Thus, the peptides are useful in both in vivo and ex vivo therapeutic agents as well as in pharmaceutical compositions for diagnostic applications.

The present invention also provides a composition comprising an immunogenic peptide having a binding motif for MHC class I molecules. The immunogenic peptide is typically about 8 to about 1.
It is between one residue and has conserved residues involved in the binding protein encoded by the appropriate MHC allele. Many allele-specific motifs have been identified.

For example, the motif for HLA-A3.2 is from N-terminal to C-terminal,
Contain the first conserved residue of L, M, I, V, S, A, T, and F at position 2 and the second conserved residue of K, R, or Y at the C-terminus . The other first conserved residue is C, G or D and, alternatively, E. The other second conserved residue is H or F. The first and second conserved residues are preferably 6 to 7 residues apart.

The motif for HLA-A1 extends from the N-terminus to the C-terminus at the first conserved residue of T, S or M, the second conserved residue of D or E, and the conserved residue of Y. Included residues. The other second conserved residue is A, S or T. The first or second conserved residue is contiguous and preferably 6 to 7 residues away from the third conserved residue. The second motif is the first and second
The conserved residues in A are separated by 6 to 7 residues and consist of the first conserved residue in E or D and the second conserved residue in Y.

The motif for HLA-A11 is T at the 2-position from the N-terminus to the C-terminus,
Contains a first conserved residue of V, M, L, I, S, A, G, N, C, D or F, and a second conserved residue of K, R, Y or H . The first and second conserved residues are preferably 6 or 7 residues apart.

The motif for HLA-A24.1 is position 2 from the N-terminus to the C-terminus.
At Y, F, W or M first conserved residue and F, I, W or L C
Includes terminal conserved residues. The first and second conserved residues are preferably separated by 6-7 residues.

Epitopes on many strong target proteins can be identified in this manner. Examples of suitable antigens include prostate specific membrane antigen (PSA), prostate cancer specific antigen (
PSM), hepatitis B core antigen and surface antigens (HBVc, HBVs), hepatitis C antigen, malignant melanoma antigen (MAGE-1), Epstein-Barr virus antigen, human immunodeficiency virus type 1 (HIV1), papilloma Viral antigen, Lassa virus,
Mycobacterium tuberculosis (M
T), p53, and mouse p53 (mp53), CEA, trypanosome surface antigen (TSA) and Her2 / neu, and members of the tyrosinase-related protein (TRP) family. Thus, the peptides are useful in both in vivo and ex vivo therapeutic agents as well as in pharmaceutical compositions for diagnostic applications.

The invention also provides a composition comprising an immunogenic peptide having a binding motif for a non-A-HLA allele. The immunogenic peptide is preferably about 9 to 10 residues long and has a conserved residue at a particular position such as proline at position 2 and an aromatic residue at the carboxylic acid terminus (eg, Y, W, F) or hydrophobic residues (eg, L, I, V, M, or A). In particular, an advantage of the peptides of the invention is their ability to bind two or more different HLA alleles.

Epitopes on many immunogenic target proteins can be identified in this manner.
Examples of suitable antigens are prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigen, malignant melanoma antigen (MAGE).
-1) Epstein-Barr virus antigen, human immunodeficiency virus type 1 (HIV1
), Papillomavirus antigen, Lassa virus, Mycobacterium tuberculosis (mycobacte)
rum tuberculosis) (MT), p53, CEA, Her2 /
neu is an example. Thus, the peptides are useful in both in vivo and ex vivo therapeutic agents as well as in pharmaceutical compositions for diagnostic applications.

DEFINITIONS The term “peptide” is used interchangeably herein with “oligopeptide” and typically depends on the peptide bond between the α-amino group and the carbonyl group of adjacent amino acids. A series of residues (typically L-amino acids) that bind to each other. Oligopeptides of the present invention are less than about 15 residues long, and typically range from about 8
Consists of about 11 residues, preferably 9 or 10 residues.

“Immunogenic peptide” means that the peptide binds to MHC molecules and
A peptide containing an allele-specific motif that induces a response. The immunogenic peptides of the invention can bind to the appropriate HLA-A2.1 molecule and induce a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived.

Immunogenic peptides are conveniently identified using the algorithm of the invention. This algorithm is a mathematical procedure that yields a score that allows the selection of immunogenic peptides. Typically, using an algorithm score with a "coupling threshold",
It has a high probability of binding with a particular affinity, which allows the selection of peptides that will be immunogenic. This algorithm is based on the effect of specific amino acids at specific positions on the peptide on MHC binding or on the binding of specific substitutions in peptide-containing motifs.

A “conserved residue” is an amino acid that is present at a particular position in a peptide at a significantly higher frequency than would be expected by a random distribution. Typically, conserved residues are those whose MHC structure may provide a point of contact with the immunogenic peptide. At least 1 to 3 or more (preferably 2) conserved residues within the defined length define the motif for the immunogenic peptide. These residues typically make intimate contact with the groove to which the peptide binds, where their side chains are buried in specific pockets of the groove itself. Typically, an immunogenic peptide comprises up to 3 conserved residues, more usually 2 conserved residues.

As used herein, a “negative binding residue” does not bind when present at a particular position (eg, 9-mer position 1, 3, and / or 7), or It is a poorly bound peptide and then does not become immunogenic (ie, CTL
Give rise to peptides (which do not induce a response).

The term “motif” refers to a 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. This peptide motif is usually different in each human MHC allele,
And differ in the pattern of highly conserved and negative residues.

Binding motifs for alleles can be defined with increasing degrees of accuracy. In one case, all conserved residues are at the exact position of the peptide and there are no negative residues at positions 1, 3 and / or 7.

The phrases “isolated” or “biologically pure” refer to material that is substantially or essentially free of the components normally associated with it, as found in its native form. Therefore, the peptides of the invention do not include substances that normally associate with their in situ environment (eg, MHC I molecules on antigen presenting cells). 5-10 of native protein, even when the protein is isolated homogeneously or in dominant bands
There are trace contaminants that have been purified with the desired protein in the% range. The isolated polypeptide of the present invention does not include the protein as an exogenous co-purified protein.

The term “residue” refers to an amino acid or amino acid mimetic that is incorporated into an oligopeptide by an amide bond or an amide bond mimetic.

Description of the Preferred Embodiments I. HLA-A2.1 Motif The present invention provides an allele-specific peptide motif for the human class I MHC (sometimes referred to as HLA) allele subtype, In particular, HLA-A2.1
It relates to the determination of peptide motifs recognized by alleles. These motifs are then T cell epitopes derived from any desired antigen, particularly those associated with human viral disease, cancer or autoimmune disease, where the amino acid sequences of potential antigens or autoantigen targets are known. Used to specify

Epitopes on many potential target proteins can be identified in this manner.
Examples of suitable antigens include prostate cancer specific antigen (PSA), hepatitis B core antigen and surface antigens (HBVc, HBVs), hepatitis C antigen, Epstein-Barr virus antigen, melanoma antigen (eg MAGH-1. ), Human immunodeficiency virus (
HIV) antigen, human papillomavirus (HPV) antigen, Lassa virus, Mycobacterium tuberculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2 / neu.

Peptides containing epitopes from these antigens were synthesized and then purified, eg, by immunofluorescence staining and flow microfluorescence measurements, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition, eg, purification. Their ability to bind the appropriate MHC molecules is tested in an assay using cells expressing class I molecules and radioiodinated peptides, and / or empty class I molecules. These peptides that bind to class I molecules, for their ability to function as targets for CTLs from infected or immunized individuals,
And, as potential therapeutic agents, early in vitro or in vivo CTL that can give rise to CTL populations capable of reacting with virus-infected target cells or tumor cells
They will be further evaluated for their ability to induce a response.

MHC class I antigens are encoded by the HLA-A, HLA-B, and HLA-C loci. The HLA-A and HLA-B antigens are expressed on the cell surface at a suitable isobaric density, whereas HLA-C expression is significantly lower (probably 1
0 times lower). Each of these loci has many alleles. The peptide binding motifs of the invention are relatively specific for each allelic subtype.

For peptide-based vaccines, the peptides of the invention preferably include a motif recognized by MHC I molecules with a wide distribution in the human population. Because MHC alleles are present at different frequencies within different ethnic groups and races, the choice of target MHC allele depends on its target population. Table 1 shows the frequencies of various allelic products of the HLA-A locus among different races. For example, most Caucasian populations have four HLA-A allele subtypes (specifically,
It is covered by peptides that bind to HLA-A2.1, A1, A3.2 and A24.1). Similarly, most Asian populations have a fifth allele, HLA-A.
A peptide bond to 11.2 was added and included.

[0034]

[Table 1] DuPont, Immunobiology of HLA, Volume 1, His
tocompatibility Testing 1987, Springe
Table compiled from r-Verlag, New York 1989 ^* N-Negroid; A = Asian; C = Caucasian. Numbers in brackets represent the number of individuals included in this analysis.

The nomenclature used to describe peptide compounds is that the amino group is to the left of each amino acid residue (N-terminal) and the carboxyl group is to the right of each amino acid residue (C-terminal).
Follow conventional practice, represented in. In the formulas representing selected particular embodiments of the invention, the amino and carboxyl end groups are in the form envisaged at physiological pH values, unless specified otherwise, unless specified otherwise. In the structural formulas of amino acids, each residue is generally represented by the standard three-letter or one-letter code. The L-form of amino acid residues is represented by the one-letter capital letter or the first letter capital letter of the three-letter code, and the D form for those amino acids that have the D form is either the lower case one letter or the lower case three. It is represented by a letter notation.
Glycine has no asymmetric carbon atoms and is referred to as the unit "Gly" or G.

The procedure used to identify the peptides of the invention is generally described by Falk et al.
Follow the method described in Nature 351: 290 (1991), incorporated herein by reference. In summary, this method typically involves MHC from a suitable cell or cell line by immunoprecipitation or affinity chromatography.
Including large scale isolation of class I molecules. Examples of other methods for isolating a desired MHC molecule, equally well known to those skilled in the art, include ion exchange chromatography, lectin chromatography, size exclusion chromatography, high performance liquid chromatography, and the methods described above. Includes all combinations of technologies.

[0037] Typically, immunoprecipitation is used to isolate the desired allele. Many protocols can be used depending on the specificity of the antibody used. For example, allele-specific mAb reagents can be used for affinity purification of HLA-A, HLA-B1, and HLA-C molecules. Several mAb reagents for isolation of HLA-A molecules are available (see Table 2). Therefore, for each of the targeted HLA-A alleles, reagents are available that can be used for the direct isolation of HLA-A molecules. Affinity columns prepared with these mAbs using standard techniques have been successfully used to purify the respective HLA-A allele product.

In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C mA
b (eg W6 / 32 and B9.12.1), and one anti-HLA-B,
The C mAb, B1.23.2, can be used in the alternative affinity purification protocol described in previous applications.

The peptide bound in the peptide binding groove of the isolated MHC molecule is typically
Elute 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 combinations thereof.

The peptide fraction was subjected to MH by reverse phase high performance liquid chromatography (HPLC).
It is further separated from the C molecule and sequenced. Peptides include a variety of other standard means well known to those of skill in the art (filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectric focusing, etc.). ).

Sequencing of the isolated peptides can be performed according to standard techniques such as Edman degradation (Hunkapiller et al., Methods Enzymol. 91, 399 (1983)). Other methods suitable for sequencing are mass spectrometric sequencing of individual peptides as previously described (Hunt et al., Science 225: 1261 (1992), incorporated herein by reference). including. Amino acid sequencing of large amounts of heterogeneous peptides from various Class I molecules (eg, pooled HPLC fractions) typically shows characteristic sequence motifs for each Class I allele.

The definition of motifs specific for various class I alleles allows the identification of potential peptide epitopes from antigenic proteins whose amino acid sequences are known. Typically, identification of potential peptide epitopes is first carried out using a computer to scan the amino acid sequence of the desired antigen for the presence of the motif. The epitope sequence is then synthesized. The ability 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 above-referenced application. Other alternatives described in the literature include inhibition of antigen presentation (Sette et al., J. Immunol. 14).
1: 3893 (1991)), in vitro assembly assay (Townsen).
d et al., Cell 62: 285 (1990)) and mutated cells (ell
s) (eg RMA-S) using FACS-based assays (Melie).
f et al., Eur. J. Immunol. 21: 2963 (1991)).

Peptides that test positive in the MHC class I binding assay are then assayed for their ability to induce a specific CTL response in vitro. For example, antigen presenting cells incubated with the peptides can be assayed for their ability to induce a CTL response in a responding cell population. 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).
Boug, Eur. J Immunol. 18: 219 (1988)).

Alternatively, a mutant mammalian cell line lacking the ability to load a class I molecule with an internally processed peptide (eg mouse cell line RMA-S (
Kaerre et al., Nature, 319: 675 (1986); Ljunggr.
en et al., Eur. J. Immunol. 21: 2963-2970 (1991).
), And human somatic T cell hybrid T-2 (Cerundolo et al., Na
345: 449-452 (1990)), a mutant mammalian cell line transfected with the appropriate human class I gene shows that when the peptide is added to these cells, the peptide undergoes primary CTL in vitro. It is conveniently used to test for the ability to induce a response. Other eukaryotic cell lines that may be used include mosquito larvae (ATCC cell line CCL 125, 126, 1660, 1,
591, 6585, 6586), silkworm (ATTC CRL 8851), armyworm (ATCC CRL 1711), moth (ATCC CCL 80), and Drosophila cell line (eg, Schneider cell line (Schneid)).
er, J.I. Embryol. Exp. Morphol. 27: 353-365 (
1927))).

Peripheral blood lymphocytes are conveniently isolated after convenient venipuncture or leukapheresis of normal donors or patients and used as a source of responding cells for CTL precursors. In one embodiment, suitable antigen presenting cells are incubated with 10-100M of peptide in serum-free medium for 4 hours under suitable culture conditions. The peptide-loaded antigen presenting cells are then incubated with the responding cell population in vitro for 7-10 days under optimized culture conditions. Positive CTL activation results in cultures of both the specific peptide-pulsed target, as well as target cells expressing the endogenously processed form of the relevant viral or tumor antigen from which this peptide sequence was derived. , Can be determined by assaying for the presence of CTL that kills radiolabeled target cells.

CTL specificity and MHC restriction are related to appropriate or inappropriate human MHC class I
It is determined by testing against various peptide target cells expressing M
Peptides that test positive in the HC binding assay and produce a specific CTL response are referred to herein as immunogenic peptides.

The immunogenic peptide may be synthesized synthetically or by recombinant DNA technology.
Alternatively, it can be prepared from natural sources such as whole virus or whole tumor. Although these peptides are preferably substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments, these peptides are synthetically derived from naturally occurring fragments or particles. Can be conjugated to.

These polypeptides or peptides can be of varying lengths, either in their neutral (uncharged) form or in their salt form, and glycosylated,
These modifications are not included, provided that they do not include modifications such as side chain oxidation, or phosphorylation, or that the modifications do not destroy the biological activity of the polypeptide as described herein. Can either include.

Desirably, these peptides are as small as possible while still retaining substantially all of the biological activity of the larger peptide. Where possible, the peptides of the invention are size balanced with endogenously processed viral or tumor cell peptides that bind to MHC class I molecules on the cell surface,
It may be desirable to optimize for a length of 9 or 10 amino acid residues.

A peptide having the desired activity binds to the desired MHC molecule and has the appropriate T
It activates cells, while increasing or at least maintaining substantially all of the biological activity of the unmodified peptide, while still achieving certain desired properties (eg, improved pharmacological characteristics). It can be modified as needed to provide. For example, these peptides may be subjected to a variety of alterations (eg, either conservative or non-conservative substitutions), where such alterations have certain advantages (eg, improvements) in their use. MHC binding). A conservative substitution causes an amino acid residue to be replaced by another amino acid residue that is biologically and / or chemically similar (eg, one hydrophobic residue to another or one polar residue to another). Stuff) is meant to be replaced. Such substitutions include Gly, Ala; Val, Il
e, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Ly
s, Arg; and combinations such as Phe, Tyr. The effects of single amino acid substitutions can also be probed with D amino acids. Such modifications are
Incorporated herein by reference, for example, Merrifield, Scie
232: 341-347 (1986), Barany and Merri.
field, The Peptides, Gross and Meienhofe
r Ed. (NY, Academic Press), pp. 1-284 (1979).
;; and Stewart and Young, Solid Phase Pep.
Tide Synthesis, (Rockford, Ill., Pierce
), 2nd edition (1984), using well-known peptide synthesis procedures.

These peptides can also be modified by extending or decreasing the amino acid sequence of the compound, for example by the addition or deletion of amino acids. The peptides or analogs of the invention can be modified by altering the order or composition of specific residues, such that certain amino acid residues essential for biological activity (eg, amino acid residues at important contact sites or conserved It will be readily understood that the selected residues) may generally not be altered without adversely affecting biological activity. The unimportant amino acids need not be limited to the naturally occurring amino acids in the protein (eg, L-α-amino acids) or their D isomers, as long as they are unnatural amino acids (eg, β-γ-δ-amino acids). ) As well as many derivatives of L-α-amino acids.

[0052] Typically, a series of peptides with single amino acid substitutions are used to determine the effects of electrostatic charge, hydrophobicity, etc. on binding. For example, the substitution of a series of positively charged amino acids (eg Lys or Arg) or negatively charged amino acids (eg Glu) results in lengths of peptides that show different patterns of sensitivity towards various MHC molecules and T cell receptors. It is done in line with it. In addition, small,
Multiple substitutions with relatively neutral moieties (eg, Ala, Gly, Pro, or similar residues) can be used. These substitutions can be homo-oligomers or hetero-oligomers. The number and type of residues that are replaced or added will depend on the spacing required between the requisite contacts and the particular functional property sought (eg, hydrophobic vs. hydrophilic). Increased binding affinity for the MHC molecule or T cell receptor relative to the affinity of the parent peptide can also be achieved by such substitutions. Regardless, such substitutions should use, for example, amino acid residues or other molecular fragments selected to avoid steric and charge interference that can disrupt binding.

Amino acid substitutions are typically single residue substitutions. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final peptide. Substitutional variants are those in which at least one residue in the peptide has been removed and a different residue inserted in its place. Such substitutions are generally made according to Table 3 below, where it is desired to fine tune the characteristics of the peptide.

[0054]

[Table 2] Substantial alterations in function (eg, affinity for MHC molecules or T cell receptors) can be achieved by selecting less conservative substitutions than those in Table 3, ie, (a) the peptide backbone in the region of substitution. Residues that are significantly different in structure (eg, sheet or helix structure, etc.), (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining bulk of the side chains are selected. It is done by Substitutions that are generally expected to produce the greatest change in peptide properties are: (a) a hydrophilic residue (eg, ceryl) is replaced with a hydrophobic residue (eg, leucyl, isoleucyl, phenylalanyl, valyl, or alanyl). Substituted (or substituted with a hydrophobic residue); (b) Residue having a positive side chain (eg, lysyl, arginyl, or histidyl) is a negative residue (eg, glutamyl or aspartyl). ) Is substituted (or is substituted by a negative residue); or (c) a residue having a bulky side chain (eg, phenylalanine) has no side chain (eg, glycine). ) Is substituted (or is substituted by a residue having no side chain).

These peptides may also have an isotope of two or more residues in the immunogenic peptide. An isostere as defined herein may replace the second sequence because the steric conformation of the first sequence matches the specific binding site for the second sequence. It is a sequence of two or more residues. The term specifically includes peptide backbone modifications well known to those of skill in the art. Such modifications include modifications of the amide nitrogen, alpha carbon, amide carbonyl, complete substitution, extension, deletion of amide bonds, or backbone bridging. In general, Spatola, Chemistry and Bioc
hemistry of Amino Acids, Peptides and
See Proteins, Volume VII (Edited by Weinstein, 1983).

Modification of peptides with various amino acid mimetics or unnatural amino acids is particularly useful in increasing peptide stability in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media (
Human plasma and serum, for example) have been used to test stability. For example, Verhoef et al., Eur. J. Drug Metab Pharmac
okin. 11: 291-302 (1986). The half-life of the peptides of the invention is conveniently determined using a 25% human serum (v / v) assay. This protocol is generally as follows. Pooled human serum (type AB, not heat inactivated) was delipidated by centrifugation before use.
) Will be done. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid solution or ethanol. Cool cloudy reaction sample for 15 minutes (4 ° C)
And then spin to pellet the precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability specific chromatographic conditions.

The peptides of the invention or their analogs having CTL stimulating activity can be modified to provide desired properties other than improved serum half-life. For example, the ability of the peptide to induce CTL activity can be enhanced by linking sequences that include at least one epitope that can induce a T helper cell response. Particularly preferred immunogenic peptide / T helper conjugates are linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules (eg, substantially uncharged amino acids or amino acid mimetics under physiological conditions). The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of non-polar amino acids or neutral polar amino acids. It is understood that the optionally present spacers need not be composed of the same residues and can therefore be hetero-oligomers or homo-oligomers. When present, the spacer is usually at least 1 or 2 residues, more usually 3-6 residues. Alternatively, the CTL peptide can be linked to the T helper peptide without a spacer.

The immunogenic peptide can be linked to the T helper peptide either directly or via a spacer, either at the amino-terminus or the carboxy-terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoites 382-398 and 37.
8 to 389.

In some embodiments, it may be desirable to include in the pharmaceutical composition of the invention at least one component that aids in the priming of CTLs. Lipids have been identified as agents that can help prime CTLs against viral antigens in vivo. For example, a palmitic acid residue can be attached to the alpha and epsilon amino groups of a Lys residue and then, for example, one or more linking residues (eg, Gly, Gl.
y-Gly-, Ser, Ser-Ser, etc.) to the immunogenic peptide. The lipidated peptide can then be incorporated into liposomes or emulsified in an adjuvant such as Incomplete Freund's Adjuvant and injected directly in micellar form. In a preferred embodiment, a particularly effective immunogen comprises palmitic acid linked to the alpha and epsilon amino groups of Lys,
This Lys is linked to the amino terminus of the immunogenic peptide via a bond (eg Ser-Ser).

As another example of lipid priming of the CTL response, E. E. coli lipoprotein (eg tripalmitoyl-S-glyceryl silteinyl seryl (glyce
rylcysteinlyseryl) -serine (P3CSS)) can be used to prime virus-specific CTL when covalently linked to the appropriate peptide. Deres et al., Nature, incorporated herein by reference.
e 342: 561-564 (1989). The peptide of the present invention is
For example, P ₃ CSS can be bound and the lipopeptide can be administered to an individual to specifically prime a CTL response against a target antigen. Furthermore, also the induction of neutralizing antibodies, since P ₃ may be primed with CSS, which is conjugated to peptides representing appropriate epitope, the two compositions can be combined, humoral and cell-mediated to infection Both responses can be elicited more efficiently.

Furthermore, the additional amino acids facilitate the linking of one peptide to another, linking to a carrier support or larger peptides,
It may be added to the terminus of the peptide, such as to modify the physical or chemical properties of the peptide or oligopeptide. Amino acids such as tyrosine, cysteine, lysine, glutamic acid, or aspartic acid can be introduced at the C-terminus or N-terminus of the peptide or oligopeptide. In some cases, modifications at the C-terminus may change the binding characteristics of the peptide. Furthermore, these peptide or oligopeptide sequences are modified by terminal NH ₂ acetylation (eg alkanoyl (C ₁ -C ₂₀ ) or thioglycolyl acetylation), terminal carboxylamidation (eg ammonia, methylamine) and the like. It may differ from the native sequence depending on what has been done. In some examples, these modifications may provide a site for attachment 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, these peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. For example, Stewart and Y
Oung, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co. (1984).

Alternatively, recombinant DNA technology may be used in which the nucleotide sequence encoding the immunogenic peptide of interest is inserted into an expression vector and transformed or transfected into a suitable host cell, It is then cultured under conditions suitable for expression. These procedures are generally described in Sambr, which is hereby incorporated by reference.
Look et al., Molecular Cloning, A Laboratory.
Manual, Cold Spring Harbor Press, Cold
It is known in the art as generally described in Spring Harbor, New York (1982). Thus, fusion proteins containing one or more peptide sequences of the present invention can be used to present suitable T cell epitopes.

Coding sequences for peptides of the lengths contemplated herein may be found in chemical techniques (eg, Matteucci et al., J. Am. Chem. Soc. 103: 31).
85 (1981) phosphotriester method), so that modifications can be conveniently made by substituting the bases encoding the native peptide sequence with appropriate bases. The coding sequence may then be provided with a suitable linker and ligated into expression vectors commercially available in the art and used to transform a suitable host to transform the desired fusion protein. Can be produced. Large numbers of such vectors and suitable host systems are currently available. For expression of the fusion protein, the coding sequence includes an operably linked start and stop codon, a promoter region and a terminator region, to provide an expression vector for expression in the desired cellular host. And usually a replication system is provided. 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 vector is transformed into a suitable bacterial host. Of course, yeast or mammalian cell hosts, with appropriate vectors and control sequences, can also be used.

The peptides of the invention and their pharmaceutical and vaccine compositions are useful for administration to mammals, particularly humans, to treat and / or prevent viral infections and cancer. Examples of diseases that can be treated or prevented using the immunogenic peptides of the present invention include prostate cancer, hepatitis B, hepatitis C, HPV infection, AID
S, kidney cancer, cervical cancer, lymphoma, CMV, malaria, and condyloma acuminata (
sondryroma 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. Individuals in the latent or acute phase of infection can be treated with immunogenic peptides separately, or in combination with other treatments, as appropriate. In therapeutic applications, the composition is in an amount sufficient to elicit an effective CTL response against a viral or tumor antigen, and to cure or at least partially arrest symptoms and / or complications,
Administered to the patient. An amount adequate to accomplish this is defined as a "therapeutically effective dose." An effective amount for this use depends on, for example, the peptide composition, mode of administration, stage and severity of the disease being treated, weight and general health of the patient, and the judgment of the prescribing physician, Generally, for the first immunization, which is for therapeutic or prophylactic administration, about 1.0 μ for a 70 kg patient.
specific CTL in the blood of patients ranging from g to about 50,000 μg of peptide
Measuring activity is followed by an additional dosage of about 1.0 μg to about 10,000 μg of the peptide, which follows a boosting regimen of weeks to months, depending on the patient's response and condition. It should be borne in mind that the peptides and compositions of the present invention can generally be used in severe disease states (ie, life threatening or potentially life threatening situations). In such cases, it is possible to administer a substantial excess of these peptide compositions, taking into account the minimization of exogenous substances and the relatively non-toxic nature of the peptides, and this is a treatment. May be felt desirable by the physician.

For therapeutic use, administration should begin at the first sign of viral infection, or at tumor detection or surgical removal, or shortly after diagnosis in the case of acute infection. This is followed by additional doses, at least until the symptoms are substantially alleviated, and for the period thereafter. In chronic infection, a loading dose followed by an additional dose may be required.

Treatment of infected individuals with the compositions of the present invention may accelerate resolution of the infection in acutely infected individuals. For individuals susceptible to (or predisposed to) a chronic infection, this composition is particularly useful in a method for preventing the progression of an acute to chronic infection. If the susceptible individuals are identified prior to or during infection, for example as described herein, the composition can be targeted to these individuals, and It may minimize the need for administration to large populations.

The peptide composition may also be used for the treatment of chronic infections and for stimulating the immune system to eliminate virally infected cells in the carrier. Cytotoxic T
It is important to provide in the formulation and mode of administration a sufficient amount of immunopotentiating peptides to effectively stimulate a fine response. Thus, for treatment of chronic infections, a typical dose is about 1.0 μg to about 5 for a 70 kg patient per dose.
It is in the range of about 50,000 μg, preferably about 5 μg to 10,000 μg. An immunizing dose, followed by boosting doses at established intervals of, for example, 1 to 4 weeks, may possibly be required over an extended period of time to effectively immunize an individual. In the case of chronic infection, administration should be continued at least until clinical symptoms or laboratory tests show that the viral infection has been eliminated or substantially reduced, and for the period thereafter.

Pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, or topical administration. Preferably, the pharmaceutical composition is administered parenterally (eg intravenously, subcutaneously, intracutaneously or intramuscularly). Accordingly, the present invention provides a composition for parenteral administration comprising a solution of the immunogenic peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as 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 solution can be packaged or lyophilized for use as a lyophilized preparation that is combined with a sterile solution prior to administration. The composition comprises pharmaceutically acceptable auxiliary substances (eg, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc.) required to approach physiological conditions, such as sodium acetate, sodium lactate. , Sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.).

The concentration of the CTL stimulating peptide of the present invention in the pharmaceutical formulation can vary very widely. That is, less than about 0.1% by weight, usually from about 2% by weight, or at least about 2% to as high as 20% to 50% or more by weight, and the particular mode of administration selected. Is mainly selected according to fluid volume, viscosity, etc.

The peptides of the present invention can also be administered via liposomes to target the peptide to specific cellular tissues, such as lymphoid tissues, or selectively targeted to infected cells. , And increase the half-life of the peptide. As the liposome, emulsion, foam, micelle, insoluble monolayer, liquid crystal, phospholipid dispersion,
A lamella layer etc. are mentioned. In these preparations, the peptide to be delivered is
As part of a liposome, alone or in combination with, for example, a molecule that binds to a receptor commonly found in lymphoid cells (eg, a monoclonal antibody that binds the CD45 antigen), or other therapeutic or immunogenic composition. It is taken in with the thing. Thus, liposomes, either filled or decorated with the desired peptides of the invention, can be directed to sites of lymphoid cells, where:
The liposomes then deliver the selected therapeutic / immunogenic peptide composition.
Liposomes for use in the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipid is generally guided by considerations of, for example, liposome size, acid lability, and liposome stability in the bloodstream. Incorporated herein by reference, eg, Szoka et al., An.
n. Rev. Biophys. Bioeng. 9: 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,02.
Various methods are available for the preparation of liposomes, as described in No. 8, and No. 5,019,369.

Ligands to be incorporated into liposomes for targeting immune cells can include, for example, antibodies or fragments thereof specific for the cell surface determinants of cells of the desired immune system. Liposomal suspensions containing the peptides can be administered intravenously, topically, topically, etc., at doses that vary, among other things, depending on the mode of administration, the peptide to be delivered, and the stage of the disease to be treated.

For solid compositions, conventional non-toxic solid carriers may be used. Examples of the solid carrier include pharmaceutical grades of mannitol, lactose,
Includes starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like. For oral administration, a pharmaceutically acceptable non-toxic composition may comprise any commonly used excipient (eg, the carriers listed above), and generally 10-95% (more preferably, It is formed by incorporating active ingredient (i.e., one or more peptides of the invention) at a concentration of 25% to 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, of course, must be non-toxic,
And preferably it must be soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms (eg, caproic acid with aliphatic polyhydric alcohols or their cyclic anhydrides,
Octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid). Mixed esters, such as mixed or natural glycerides, can be used. The surfactant may make up 0.1% to 20% by weight of the composition,
Preferably, it constitutes 0.25% to 5% by weight. The balance of the composition is usually propellant. If desired, a carrier can also be included, eg, with lecithin for intranasal delivery.

In another aspect, the invention relates to a vaccine comprising as an active ingredient an immunogenicly effective amount of an immunogenic peptide described herein. The peptide may be linked to its own carrier or introduced into the host (including human) as a homopolymer or heteropolymer of active peptide units. Such polymers have the advantage of increased immunological response, and when various peptides are used to make the polymer, antibodies and / or CTL that react with various antigenic determinants of viral or tumor cells. Have the additional ability to induce Useful carriers are well known in the art, and these carriers include, for example:
Examples include thyroglobulin, albumin (eg, human serum albumin), tetanus toxoid, polyamino acid (eg, polylysine, polyglutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccine may also include a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline or saline, and more typically an adjuvant. Adjuvants such as incomplete Freud's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are substances well known in the art. Further, as disclosed herein, the CTL response can be primed by conjugating the peptides of the invention to lipids (eg, P3CSS). Upon immunization with a peptide composition, as described herein, via injection, aerosol, oral, transdermal, or other suitable routes, the host's immune system will detect the desired antigen. Responds to the vaccine by producing large amounts of CTL specific for, and the host is at least partially immune to subsequent infection or is at least partially resistant to the development of chronic infection. Becomes

Vaccine compositions containing the peptides of the invention elicit an immune response against an antigen in patients predisposed to, or otherwise at risk for, viral infection or cancer, It is therefore administered to enhance the patient's own immune response capacity. Such an amount is defined as "immunogenically effective dose." In this application, the exact amount will also depend on the health and weight of the patient, the mode of administration, the nature of the formulation, etc., but will generally be in the range of about 1.0 μg to about 50,000 μg / 70 kg patient. And, more commonly, in the range of about 10 μg to about 500 μg per 70 kg body weight.

In some cases, it may be desirable to combine the peptide vaccines of the invention with a vaccine that induces a neutralizing antibody response against the virus of interest, particularly the viral envelope antigen.

Nucleic acids encoding one or more peptides of the invention may also be administered to a patient, for therapeutic or immunization purposes. A number of methods are conventionally used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, by Wolff et al., Science 247: 1465-.
1468 (1990) and US Pat. Nos. 5,580,859 and 5,
589,466. Nucleic acids are also disclosed, for example, in US Pat.
It may be administered using ballistic delivery, as described in No. 253. Particles composed of only DNA can be administered. Alternatively, the DNA can be attached to particles such as gold particles. Nucleic acids can also be complexed and delivered to cationic compounds such as cationic lipids. Gene delivery methods mediated by lipids are described, for example, in WO 96/18372; WO 9
3/24640; Mannino and Gould-Fogerite, Bio.
Techniques 6 (7): 682-691 (1988); Rose US Patent No. 5,279,833; WO 91/06309; and Felgn.
er et al., Proc. Natl. Acad. Sci. USA 84: 7413-7.
414 (1987). The peptides of the invention can also be expressed by an attenuated viral host such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences encoding the peptides of the invention. Upon introduction into an acutely or chronically infected host, or into an uninfected host, this recombinant vaccinia virus expresses an immunogenic peptide, thereby inducing a CTL response in the host. Vaccinia vectors and methods useful in immunization protocols are described, for example, in US Pat. No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described by Stover et al. (Na), which is incorporated herein by reference.
true 351: 456-460 (1991)). A wide variety of other vectors useful in therapeutic administration or immunization of the peptides of the invention (eg, Sa
lmonella typhi vectors, etc.) will be apparent to those of skill 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. The amino acid sequence of this epitope is back-translated to create a DNA sequence (minigene) encoding the selected CTL epitope for expression in human cells. A human codon usage table is used to guide the codon choice for each amino acid. DNA sequences encoding these epitopes are added directly to create contiguous polypeptide sequences. Additional elements can be incorporated into the design of the minigene to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: helper T lymphocyte epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Further, MHC presentation of CTL epitopes can be improved by including synthetic (eg, polyalanine) or naturally occurring flanking sequences flanking the CTL epitope.

The minigene sequence is converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified, and annealed under suitable conditions using well known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic minigene, which encodes a CTL epitope polypeptide, can then be cloned into the 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 cell. Several vector elements are required: a promoter with a downstream cloning site for insertion of the minigene; a polyadenylation signal for efficient termination of transcription; E. coli origin of replication; and E. c
oli selectable marker (eg ampicillin resistance or kanamycin resistance). A number of promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. See US Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Further 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 can be incorporated into the transcribed region of the minigene. Inclusion of mRNA stabilizing sequences can also be considered to increase expression of the minigene. It has recently been proposed that immunostimulatory sequences (ISS or CpG) play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the coding sequence of the minigene, if found to enhance immunogenicity.

In some embodiments, a bioistronic that enables production of a minigene-encoded epitope and a second protein included to enhance or reduce immunogenicity. Expression vectors can be used. Examples of proteins or polypeptides that may beneficially enhance the immune response when co-expressed include cytokines (eg IL2, IL12, GM
-CSF), cytokine-inducing molecules (eg LeIF), or costimulatory molecules. Helper (HTL) epitopes can be linked to intracellular targeting signals and expressed separately from CTL epitopes. This is CT
Allows the HTL epitope to be directed to a cell compartment different from the L epitope. If required, this may facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically reducing the immune response by co-expression of immunosuppressive molecules (eg TGF-β) may be beneficial in certain diseases.

Once the expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is suitable for E. coli
The strain is transformed and the DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, and all other elements contained in the vector are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells with the correct plasmid can be stored as a master cell bank and a working cell bank.

A therapeutic amount of plasmid DNA was prepared from E. It is produced by fermentation in E. coli and subsequent purification. Aliquots from the working cell bank are used to inoculate a fermentation medium (eg, Terrific Broth) and grown to saturation in shake flasks or bioreactors according to well-known techniques. Plasmid DNA can be purified using standard bioseparation techniques, such as the solid phase anion exchange resin provided by Quiagen. If required, supercoiled DNA can be isolated from open circular and linear forms using gel electrophoresis or other techniques.

Purified plasmid DNA can be prepared for injection using various formulations. The simplest of these is sterile phosphate buffered saline (PB).
Reconstitution of lyophilized DNA in S). Various methods have been described,
And new technology may become available. As mentioned above, the nucleic acid is conveniently formulated with a cationic lipid. In addition, glycolipids, fusogenic
enic) liposome, protective, interactive, non-condensed
ing) (PINC) generically, peptides and compounds also affect purified plasmid DNA to affect variables such as stability, intramuscular dispersion, and trafficking to specific organs or cell types. Can be complexed.

Sensitization of target cells can be used as a functional assay for the expression and MHC class I presentation of CTL epitopes encoded by minigenes. 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 depends on the final formulation. Electroporation is "naked" despite the fact that cationic lipids allow direct in vitro transfection.
It can be used for DNA. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of the transfected cells using fluorescence activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for the epitope-specific CTL line. Cytolysis detected by release of 51Cr indicates production of MHC presentation of the CTL epitope encoded by the minigene.

In vivo immunogenicity is a second consideration for functional testing of minigene DNA formulations.
Is the approach. Transgenic mice expressing the appropriate human MHC molecule are immunized with the DNA product. The dose and route of administration depends on the formulation (
For example, 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 tested. These effector cells (CTL
) Is assayed for cytolysis of chromium-51 labeled target cells loaded with peptide using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to the epitope encoded by the minigene is
Demonstrate DNA vaccine function for in vivo induction of TL.

Antigenic peptides can also be used to induce CTL ex vivo. The resulting CTLs 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 to peptide vaccine approaches. Ex vivo CTL responses to specific pathogens (infectious agents or tumor antigens) indicate that in tissue culture, the patient's CTL progenitor cells (CTLp) together with a source of antigen presenting cells (APCs) and an appropriate immunogenic peptide. Induced by incubating.
After a suitable incubation time (typically 1 to 4 weeks), at which time these CTLp are activated and mature and proliferate to effector CTL, these cells are injected into the patient. Brought back, where they destroy their specific target cells (infected cells or tumor cells).

These peptides may also find use as diagnostic reagents. For example, the peptides of the invention can be used to determine the susceptibility of a particular individual to a treatment regimen with a peptide or related peptides, and thus in modifying existing treatment protocols or in prognosing for affected individuals. It can be useful in making decisions. Furthermore, these peptides can also be used to predict which individuals are at significant risk of developing a chronic infection.

The following examples are provided by way of illustration only and not by way of limitation.

Example 1 Isolation of Class I antigens was performed as described in the related application above. The naturally processed peptides were then isolated and sequenced as described in the above-referenced application. Allele-specific motifs and algorithms were determined and a quantitative binding assay was performed.

Using the motifs identified above for the HLA-A2.1 allele, amino acid sequences from multiple antigens were analyzed for the presence of these motifs. Table 3
Provides the results of these searches. The letter "J" represents norleucine.

The above examples are provided to illustrate the present invention and not to limit its scope.
Other modifications of the invention will be readily apparent to those skilled in the art and are covered by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

[0096]

[Table 3] II. Non-HLA-A2 Motif The present invention also relates to the determination of allele-specific peptide motifs for the human class I MHC (sometimes referred to as HLA) allele subtype. These motifs are then T cell epitopes derived from any desired antigen, particularly those associated with human viral disease, cancer or autoimmune disease, where the amino acid sequences of potential antigens or autoantigen targets are known. Used to specify

Epitopes on many potential target proteins can be identified in this manner.
Examples of suitable antigens are prostate cancer specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigen, Epstein-Barr virus antigen, melanoma antigen (eg MAGE-1. ), Human immunodeficiency virus (
HIV) antigen, human papillomavirus (HPV) antigen, Lassa virus, Mycobacterium tuberculosis (MT), p53, CEA, and Her2 / neu.

Peptides containing epitopes from these antigens were synthesized and then purified, eg, by immunofluorescence staining and flow microfluorescence measurements, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition, eg, purification. Their ability to bind the appropriate MHC molecule is tested in an assay using cells expressing class I molecules and radioiodinated peptides, and / or empty class I molecules. These peptides that bind to class I molecules, for their ability to function as targets for CTLs from infected or immunized individuals,
And, as potential therapeutic agents, early in vitro or in vivo CTL that can give rise to CTL populations capable of reacting with virus-infected target cells or tumor cells
They will be further evaluated for their ability to induce a response.

MHC class I antigens are encoded by the HLA-A, HLA-B, and HLA-C loci. The HLA-A and HLA-B antigens are expressed on the cell surface at a suitable isobaric density, whereas HLA-C expression is significantly lower (probably 1
0 times lower). Each of these loci has many alleles. The peptide binding motifs of the invention are relatively specific for each allelic subtype.

For peptide-based vaccines, the peptides of the invention preferably include a motif recognized by MHC I molecules with a wide distribution in the human population. Since MHC alleles occur at different frequencies within different ethnic groups and races, the choice of target MHC allele depends on its target population. Table 4 shows the frequencies of various allelic products of the HLA-A locus among different races. For example,
Most Caucasian populations have four HLA-A allele subtypes (especially H
It can be covered by peptides that bind LA-A2.1, A1, A3.2 and A24.1). Similarly, most Asian populations have a fifth allele, HLA-A.
A peptide bond to 11.2 was added and included.

[0101]

[Table 4] DuPont, Immunobiology of HLA, Volume 1, His
tocompatibility Testing 1987, Springe
Table compiled from r-Verlag, New York 1989 ^* N-Negroid; A = Asian; C = Caucasian. Numbers in brackets represent the number of individuals included in this analysis.

The nomenclature used to describe peptide compounds is that the amino group is to the left of each amino acid residue (N-terminal) and the carboxyl group is to the right of each amino acid residue (C-terminal).
Follow conventional practice, represented in. In the formulas representing selected particular embodiments of the invention, the amino and carboxyl end groups are in the form envisaged at physiological pH values, unless specified otherwise, unless specified otherwise. In the structural formulas of amino acids, each residue is generally represented by the standard three-letter or one-letter code. The L-form of amino acid residues is represented by the capital one-letter code or the first letter capitalization of the three-letter code, and the D form for these amino acids is represented by the lowercase one-letter code or the lowercase three-letter code. To be done. Glycine has no asymmetric carbon atoms and is referred to as the unit "Gly" or G.

The procedures used to identify the peptides of the invention are generally described by Falk et al.,
Follow the method described in Nature 351: 290 (1991), incorporated herein by reference. In summary, this method typically involves MHC from a suitable cell or cell line by immunoprecipitation or affinity chromatography.
Including large scale isolation of class I molecules. Examples of other methods for isolating a desired MHC molecule, equally well known to those skilled in the art, include ion exchange chromatography, lectin chromatography, size exclusion chromatography, high performance liquid chromatography, and the methods described above. Includes all combinations of technologies.

Numerous cells with defined MHC molecules, especially MHC class I molecules, are known and readily available. For example, the human EBV transformed B cell line has been shown to be an excellent source for preparative isolation of class I and class II MHC molecules. Well characterized cell lines are available from personal and commercial sources (eg, American Type Culture C).
collection (“Catalog of Cell Lines a
nd Hybridomas ", 6th edition (1988) Rockville, Ma.
ryland, U.S.A. S. A. ); National Institute of
General Medical Sciences 1990/1991
Catalog of Cell Lines (NIGMS) Human Ge
net Mutant Cell Repository, Camden,
NJ; and ASHI Repository, Brigham and Wo
men's Hospital, 75 Frances Street, Bos
Ton, MA 02115). Table 5 lists some B cell lines that are suitable for use as a source for the HLA-A allele.
All of these cell lines can be grown in large batches and are therefore useful for large scale production of MHC molecules. Those skilled in the art will recognize that these are merely exemplary cell lines,
And many other cell sources can be used. Similar EBV B cell lines homozygous for HLA-B and similar EBV B cell lines homozygous for HLA-C were prepared for the HLA-B allele and HLA-C, respectively.
It can serve as a source for alleles.

[0105]

[Table 5] In the typical case, immunoprecipitation is used to isolate the desired allele. Many protocols can be used depending on the specificity of the antibody used. For example, allele-specific mAb reagents can be used for affinity purification of HLA-A, HLA-B, and HLA-C molecules. Several mAb reagents for isolation of HLA-A molecules are available (see Table 6). Therefore, the targeted H
For each of the LA-A alleles, reagents are available that can be used for direct isolation of HLA-A molecules. Affinity columns prepared with these mAbs using standard techniques have been successfully used to purify the respective HLA-A allele product.

In addition to allele-specific mAbs, a broadly reactive anti-HLA-A, B, C mA
b (eg W6 / 32 and B9.12.1), and one anti-HLA-B,
The C mAb, B1.23.2, can be used in an alternative affinity purification protocol described in the Examples section below.

[0107]

[Table 6] The peptide bound in the peptide binding groove of the isolated MHC molecule is typically
Elute 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 combinations thereof.

The peptide fraction was separated by MH by reverse phase high performance liquid chromatography (HPLC).
It is further separated from the C molecule and sequenced. Peptides include a variety of other standard means well known to those of skill in the art (filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectric focusing, etc.). ).

Sequencing of the isolated peptides was performed by Edman degradation (Hunkapiller,
M. W. Et al., Methods Enzymol. 91, 399 (1983)). Other suitable methods for sequencing are mass spectrometry sequencing of individual peptides as previously described (Hunt et al., Science 225: 1261 (199, incorporated herein by reference).
2)) is included. Amino acid sequencing of large amounts of heterogeneous peptides from various Class I molecules (eg, pooled HPLC fractions) typically shows characteristic sequence motifs for each Class I allele.

The definition of motifs specific for various class I alleles allows the identification of potential peptide epitopes from antigenic proteins whose amino acid sequences are known. Typically, identification of potential peptide epitopes is first carried out using a computer to scan the amino acid sequence of the desired antigen for the presence of the motif. The epitope sequence is then synthesized. The ability 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 above-referenced application. Other alternatives described in the literature include inhibition of antigen presentation (Sette et al., J. Immunol. 14).
1: 3893 (1991)), in vitro assembly assay (Townsen).
d et al., Cell 62: 285 (1990)) and mutated cells (ell
s) (eg RMA-S) using FACS-based assays (Melie).
f et al., Eur. J. Immunol. 21: 2963 (1991)).

Peptides that test positive in the MHC class I binding assay are then assayed for their ability to induce a specific CTL response in vitro. For example, antigen presenting cells incubated with the peptides can be assayed for their ability to induce a CTL response in a responding cell population. 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).
Boug, Eur. J Immunol. 18: 219 (1988)).

Alternatively, a mutant mammalian cell line lacking the ability to load a class I molecule with an internally processed peptide (eg mouse cell line RMA-S (
Kaerre et al., Nature, 319: 675 (1986); Ljunggr.
en et al., Eur. J. Immunol. 21: 2963-2970 (1991).
), And human somatic T cell hybrid T-2 (Cerundolo et al., Na
345: 449-452 (1990)), a mutant mammalian cell line transfected with the appropriate human class I gene shows that when the peptide is added to these cells, the peptide undergoes primary CTL in vitro. It is conveniently used to test for the ability to induce a response. Other eukaryotic cell lines that may be used include mosquito larvae (ATCC cell line CCL 125, 126, 1660, 1,
591, 6585, 6586), silkworm (ATTC CRL 8851), armyworm (ATCC CRL 1711), moth (ATCC CCL 80), and Drosophila cell line (eg, Schneider cell line (Schneid)).
er, J.I. Embryol. Exp. Morphol. 27: 353-365 (
1927))).

Peripheral blood lymphocytes are conveniently isolated after convenient venipuncture or leukapheresis of normal donors or patients and used as a source of responsive cells for CTL precursors. In one embodiment, suitable antigen presenting cells are incubated with 10-100M of peptide in serum-free medium for 4 hours under suitable culture conditions. The peptide-loaded antigen presenting cells are then incubated with the responding cell population in vitro for 7-10 days under optimized culture conditions. Positive CTL activation results in cultures of both the specific peptide-pulsed target, as well as target cells expressing the endogenously processed form of the relevant viral or tumor antigen from which this peptide sequence was derived. , Can be determined by assaying for the presence of CTL that kills radiolabeled target cells.

CTL specificity and MHC restriction are related to appropriate or inappropriate human MHC class I
It is determined by testing against various peptide target cells expressing M
Peptides that test positive in the HC binding assay and produce a specific CTL response are referred to herein as immunogenic peptides.

The immunogenic peptide can be synthesized synthetically or by recombinant DNA technology.
Alternatively, it can be prepared from natural sources such as whole virus or whole tumor. Although these peptides are preferably substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments, these peptides are synthetically derived from naturally occurring fragments or particles. Can be conjugated to.

These polypeptides or peptides can be of varying lengths, either in their neutral (uncharged) form or in their salt form, and glycosylated,
These modifications are not included, provided that they do not include modifications such as side chain oxidation, or phosphorylation, or that the modifications do not destroy the biological activity of the polypeptide as described herein. Can either include.

Desirably, these peptides are as small as possible while still retaining substantially all of the biological activity of the larger peptide. Where possible, the peptides of the invention are size balanced with endogenously processed viral or tumor cell peptides that bind to MHC class I molecules on the cell surface,
It may be desirable to optimize for a length of 9 or 10 amino acid residues.

A peptide with the desired activity binds to the desired MHC molecule and has the appropriate T
It activates cells, while increasing or at least maintaining substantially all of the biological activity of the unmodified peptide, while still achieving certain desired properties (eg, improved pharmacological characteristics). It can be modified as needed to provide. For example, these peptides may be subjected to a variety of alterations (eg, either conservative or non-conservative substitutions), where such alterations have certain advantages (eg, improvements) in their use. MHC binding). A conservative substitution causes an amino acid residue to be replaced by another amino acid residue that is biologically and / or chemically similar (eg, one hydrophobic residue to another or one polar residue to another). Stuff) is meant to be replaced. Such substitutions include Gly, Ala; Val, Il
e, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Ly
s, Arg; and combinations such as Phe, Tyr. The effects of single amino acid substitutions can also be probed with D amino acids. Such modifications are
Incorporated herein by reference, for example, Merrifield, Scie
232: 341-347 (1986), Barany and Merri.
field, The Peptides, Gross and Meienhofe
r Ed. (NY, Academic Press), pp. 1-284 (1979).
;; and Stewart and Young, Solid Phase Pep.
Tide Synthesis, (Rockford, Ill., Pierce
), 2nd edition (1984), using well-known peptide synthesis procedures.

These peptides can also be modified by extending or decreasing the amino acid sequence of the compound, for example by the addition or deletion of amino acids. The peptides or analogs of the invention may also be modified by altering the order or composition of particular residues, such that certain amino acid residues essential for biological activity (e.g., amino acid residues at important contact sites or It is readily understood that the conserved residues) may generally not be altered without adversely affecting biological activity. The unimportant amino acids need not be limited to the naturally occurring amino acids in the protein (eg, L-α-amino acids) or their D isomers, as long as they are unnatural amino acids (
(Eg β-γ-δ-amino acids) as well as many derivatives of L-α-amino acids.

Typically, a series of peptides with single amino acid substitutions are used to determine the effects of electrostatic charge, hydrophobicity, etc. on binding. For example, the substitution of a series of positively charged amino acids (eg Lys or Arg) or negatively charged amino acids (eg Glu) results in lengths of peptides that show different patterns of sensitivity towards various MHC molecules and T cell receptors. It is done in line with it. In addition, small,
Multiple substitutions with relatively neutral moieties (eg, Ala, Gly, Pro, or similar residues) can be used. These substitutions can be homo-oligomers or hetero-oligomers. The number and type of residues that are replaced or added will depend on the spacing required between the requisite contacts and the particular functional property sought (eg, hydrophobic vs. hydrophilic). Increased binding affinity for the MHC molecule or T cell receptor relative to the affinity of the parent peptide can also be achieved by such substitutions. Regardless, such substitutions should use, for example, amino acid residues or other molecular fragments selected to avoid steric and charge interference that can disrupt binding.

Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final peptide. Substitutional variants are those in which at least one residue in the peptide has been removed and a different residue inserted in its place. Such substitutions are generally made according to Table 2 below, where it is desired to fine tune the characteristics of the peptide.

[0122]

[Table 7] Substantial alterations in function (eg, affinity for MHC molecules or T cell receptors) can be achieved by selecting less conservative substitutions than those in Table 2, ie (a) the peptide backbone in the region of substitution. Residues that are significantly different in structure (eg, sheet or helix structure, etc.), (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining bulk of the side chains are selected. It is done by Substitutions that are generally expected to produce the greatest change in peptide properties are: (a) a hydrophilic residue (eg, ceryl) is replaced with a hydrophobic residue (eg, leucyl, isoleucyl, phenylalanyl, valyl, or alanyl). (B) a residue having an electropositive side chain (eg, lysyl, arginyl, or histidyl) is an electronegative residue (eg, substituted with a hydrophobic residue); , Glutamyl or aspartyl) (or those substituted by a negative residue); or (c) a residue having a bulky side chain (eg, phenylalanine) having no side chain. (For example,
Glycine) is substituted (or is substituted by a residue having no side chain).

These peptides may also have isosteres of two or more residues in the immunogenic peptide. An isostere as defined herein may replace the second sequence because the steric conformation of the first sequence matches the specific binding site for the second sequence. It is a sequence of two or more residues. The term specifically includes peptide backbone modifications well known to those of skill in the art. Such modifications include modifications of the amide nitrogen, alpha carbon, amide carbonyl, complete substitution, extension, deletion of amide bonds, or backbone bridging. In general, Spatola, Chemistry and Bioc
hemistry of Amino Acids, Peptides and
See Proteins, Volume VII (Edited by Weinstein, 1983).

Modification of peptides with various amino acid mimetics or unnatural amino acids is particularly useful in increasing peptide stability in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media (
Human plasma and serum, for example) have been used to test stability. For example, Verhoef et al., Eur. J. Drug Metab Pharmac
okin. 11: 291-302 (1986). The half-life of the peptides of the invention is conveniently determined using a 25% human serum (v / v) assay. This protocol is generally as follows. Pooled human serum (type AB, not heat inactivated) was delipidated by centrifugation before use.
) Will be done. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid solution or ethanol. Cool cloudy reaction sample for 15 minutes (4 ° C)
And then spin to pellet the precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability specific chromatographic conditions.

The peptides of the invention or their analogs having CTL stimulating activity can be modified to provide desired properties other than improved serum half-life. For example, the ability of the peptide to induce CTL activity can be enhanced by linking sequences that include at least one epitope that can induce a T helper cell response. Particularly preferred immunogenic peptide / T helper conjugates are linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules (eg, substantially uncharged amino acids or amino acid mimetics under physiological conditions). The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of non-polar amino acids or neutral polar amino acids. It is understood that the optionally present spacers need not be composed of the same residues and can therefore be hetero-oligomers or homo-oligomers. When present, the spacer is usually at least 1 or 2 residues, more usually 3-6 residues. Alternatively, the CTL peptide can be linked to the T helper peptide without a spacer.

The immunogenic peptide can be linked to the T helper peptide either directly or via a spacer, either at the amino-terminus or the carboxy-terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoites 382-398 and 37.
8 to 389.

In some embodiments, it may be desirable to include in the pharmaceutical composition of the invention at least one CTL priming component. Lipids have been identified as agents capable of priming CTLs against viral antigens in vivo. For example, a palmitic acid residue can be attached to the alpha and epsilon amino groups of a Lys residue, and then, eg, one or more linking residues (eg, Gly, Gly-Gly-
, Ser, Ser-Ser, etc.) to an immunogenic peptide.
The lipidated peptide can then be directly injected in micellar form, incorporated into liposomes, or emulsified in an adjuvant, such as Freund's incomplete adjuvant. In a preferred embodiment, a particularly effective immunogen is Ly.
It comprises palmitic acid linked to the α-amino and ε-amino groups of s, the Lys being linked to the amino terminus of the immunogenic peptide via a bond (eg Ser-Ser).

As another example of lipid priming of the CTL response, E. E. coli lipoprotein (eg tripalmitoyl-S-glyceryl silteinyl seryl (glyce
rylcysteinlyseryl) -serine (P3CSS)) can be used to prime virus-specific CTL when covalently linked to the appropriate peptide. Deres et al., Nature, incorporated herein by reference.
e 342: 561-564 (1989), incorporated by reference. The peptides of the invention, for example, be coupled to P 3 ^CSS, and the lipopeptide administered to an individual, capable of specifically priming the CTL response to the target antigen. Furthermore, also the induction of neutralizing antibodies, since P ³ may be primed with CSS, which is conjugated to peptides representing appropriate epitope, the two compositions can be combined, humoral and cell-mediated to infection Both responses can be elicited more efficiently.

Furthermore, the additional amino acids facilitate the linking of one peptide to another, linking to a carrier support or larger peptides,
It may be added to the terminus of the peptide, such as to modify the physical or chemical properties of the peptide or oligopeptide. Amino acids such as tyrosine, cysteine, lysine, glutamic acid, or aspartic acid can be introduced at the C-terminus or N-terminus of the peptide or oligopeptide. In some cases, modifications at the C-terminus may change the binding characteristics of the peptide. Furthermore, these peptide or oligopeptide sequences are modified by terminal NH ₂ acetylation (eg alkanoyl (C ₁ -C ₂₀ ) or thioglycolyl acetylation), terminal carboxylamidation (eg ammonia, methylamine) and the like. It may differ from the native sequence depending on what has been done. In some examples, these modifications may provide a site for attachment 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, these peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. For example, Stewart and Y
Oung, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co. (1984) (supra).

Alternatively, recombinant DNA technology may be used in which the nucleotide sequence encoding the immunogenic peptide of interest is inserted into an expression vector and transformed or transfected into a suitable host cell, It is then cultured under conditions suitable for expression. These procedures are generally described in Sambr, which is hereby incorporated by reference.
Look et al., Molecular Cloning, A Laboratory.
Manual, Cold Spring Harbor Press, Cold
It is known in the art as generally described in Spring Harbor, New York (1982). Thus, fusion proteins containing one or more peptide sequences of the present invention can be used to present suitable T cell epitopes.

Coding sequences for peptides of the lengths contemplated herein can be found in chemical techniques (eg, Matteucci et al., J. Am. Chem. Soc. 103: 31).
85 (1981) phosphotriester method), so that modifications can be conveniently made by substituting the bases encoding the native peptide sequence with appropriate bases. The coding sequence may then be provided with a suitable linker and ligated into expression vectors commercially available in the art and used to transform a suitable host to transform the desired fusion protein. Can be produced. Large numbers of such vectors and suitable host systems are currently available. For expression of the fusion protein, the coding sequence includes an operably linked start and stop codon, a promoter region and a terminator region, to provide an expression vector for expression in the desired cellular host. And usually a replication system is provided. 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 vector is transformed into a suitable bacterial host. Of course, yeast or mammalian cell hosts, with appropriate vectors and control sequences, can also be used.

The peptides of the invention and their pharmaceutical and vaccine compositions are useful for administration to mammals, particularly humans, to treat and / or prevent viral infections and cancer. Examples of diseases that can be treated or prevented using the immunogenic peptides of the present invention include prostate cancer, hepatitis B, hepatitis C, AIDS, kidney cancer,
Cervical cancer, lymphoma, CMV, malaria, and condyloma acuminata (sondly)
Loma 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. Individuals in the latent or acute phase of infection can be treated with immunogenic peptides separately, or in combination with other treatments, as appropriate. In therapeutic applications, the composition is in an amount sufficient to elicit an effective CTL response against a viral or tumor antigen, and to cure or at least partially arrest symptoms and / or complications,
Administered to the patient. An amount adequate to accomplish this is defined as a "therapeutically effective dose." An effective amount for this use depends on, for example, the peptide composition, mode of administration, stage and severity of the disease being treated, weight and general health of the patient, and the judgment of the prescribing physician, Generally, for the first immunization, which is for therapeutic or prophylactic administration, about 1.0 μ for a 70 kg patient.
g to about 5000 μg of peptide, depending on the response and condition of the patient by measuring specific CTL activity in the blood of the patient, according to a boosting regimen of weeks to months, of about 1. An additional dosage of 0 μg to about 1000 μg of peptide follows. The peptides and compositions of the invention are generally used in severe disease states (
That is, it must be kept in mind that it can be used in life-threatening or potentially life-threatening situations. In such cases, it is possible to administer a substantial excess of these peptide compositions, taking into account the minimization of exogenous substances and the relatively non-toxic nature of the peptides, and this is a treatment. May be felt desirable by the physician.

For therapeutic use, administration should begin at the first sign of viral infection, or at tumor detection or surgical removal, or shortly after diagnosis in the case of acute infection. This is followed by additional doses, at least until the symptoms are substantially alleviated, and for the period thereafter. In chronic infection, a loading dose followed by an additional dose may be required.

Treatment of infected individuals with the compositions of the present invention may accelerate resolution of the infection in acutely infected individuals. For individuals susceptible to (or predisposed to) a chronic infection, this composition is particularly useful in a method for preventing the progression of an acute to chronic infection. If the susceptible individuals are identified prior to or during infection, for example as described herein, the composition can be targeted to these individuals, and It may minimize the need for administration to large populations.

The peptide composition may also be used for the treatment of chronic infections and for stimulating the immune system to eliminate virally infected cells in the carrier. Cytotoxic T
It is important to provide in the formulation and mode of administration a sufficient amount of immunopotentiating peptides to effectively stimulate a fine response. Thus, for treatment of chronic infections, a typical dose is about 1.0 μg to about 5 for a 70 kg patient per dose.
000 μg, preferably in the range of about 5 μg to 1000 μg. Immunization dose,
Subsequent booster doses at established intervals of, for example, 1 to 4 weeks may be needed, perhaps over an extended period of time, to effectively immunize the individual. In the case of chronic infection, administration should be continued at least until clinical symptoms or laboratory tests show that the viral infection has been eliminated or substantially reduced, and for the period thereafter.

Pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, or topical administration. Preferably, the pharmaceutical composition is administered parenterally (eg intravenously, subcutaneously, intracutaneously or intramuscularly). Accordingly, the present invention provides a composition for parenteral administration comprising a solution of the immunogenic peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solution can be packaged or lyophilized for use as a lyophilized preparation that is combined with a sterile solution prior to administration. The composition comprises pharmaceutically acceptable auxiliary substances (eg, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc.) required to approximate physiological conditions such as sodium acetate, lactic acid. Sodium, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.).

The concentration of the CTL stimulating peptide of the present invention in the pharmaceutical formulation can vary widely. That is, less than about 0.1% by weight, usually from about 2% by weight, or at least about 2% to as high as 20% to 50% or more by weight, and the particular mode of administration selected. Is mainly selected according to fluid volume, viscosity, etc.

The peptides of the invention can also be administered via liposomes to target the peptide to specific cellular tissues, such as lymphoid tissues, or selectively targeted to infected cells, And increase the half-life of the peptide composition. Examples of liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, and lamella layers. In these preparations, the peptide to be delivered is, as part of a liposome, alone or with a molecule that binds to a receptor that is common among lymphoid cells (eg, a monoclonal antibody that binds to the CD45 antigen). Incorporated in combination or with other therapeutic or immunogenic compositions. Thus, either liposomes filled or modified with the desired peptides of the present invention can be directed to sites of lymphoid cells, where the liposomes are then selected for therapeutic / immunogenic activity. Deliver the peptide composition. Liposomes for use in the present invention are formed from standard vesicle-forming lipids,
It generally contains neutral and negatively charged phospholipids and sterols (
For example, cholesterol). The choice of lipid is generally guided by considerations of, for example, liposome size, acid lability, and liposome stability in the bloodstream. Incorporated herein by reference, eg, Szoka et al., Ann.
． Rev. Biophys. Bioeng. 9: 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
And various methods as described in U.S. Pat. No. 5,019,369 are available for the preparation of liposomes.

Ligands to be incorporated into liposomes for targeting immune cells can include, for example, antibodies or fragments thereof specific for the cell surface determinants of cells of the desired immune system. Liposomal suspensions containing the peptides can be administered intravenously, topically, topically, etc., at doses that vary, among other things, depending on the mode of administration, the peptide to be delivered, and the stage of the disease to be treated.

For solid compositions, conventional non-toxic solid carriers may be used. Examples of the solid carrier include pharmaceutical grades of mannitol, lactose,
Includes starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like. For oral administration, a pharmaceutically acceptable non-toxic composition may comprise any commonly used excipient (eg, the carriers listed above), and generally 10-95% (more preferably, It is formed by incorporating active ingredient (i.e., one or more peptides of the invention) in a concentration of 25% to 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, of course, must be non-toxic,
And preferably it must be soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms (eg, caproic acid with aliphatic polyhydric alcohols or their cyclic anhydrides,
Octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid). Mixed esters, such as mixed or natural glycerides, can be used. The surfactant may make up 0.1% to 20% by weight of the composition,
Preferably, it constitutes 0.25% to 5% by weight. The balance of the composition is usually propellant. If desired, a carrier can also be included, eg, with lecithin for intranasal delivery.

In another aspect, the present invention relates to a vaccine, as described herein, which comprises as an active ingredient an immunogenicly effective amount of an immunogenic peptide. The peptide may be introduced into the host (including human) either linked to its own carrier or as a homopolymer or heteropolymer of active peptide units. Such polymers have the advantage of increased immunological response, and when different peptides are used to make up the polymer, antibodies and / or CTLs that react with different antigenic determinants of viral or tumor cells. Has the additional ability to induce. Useful carriers are well known in the art and include, for example, thyroglobulin, albumin (eg, human serum albumin), tetanus toxoid, polyamino acids (eg, polylysine: glutamic acid), influenza, hepatitis B virus core protein, type B. Including hepatitis virus recombinant vaccine. These vaccines may also include a physiologically acceptable (acceptable) diluent such as water, phosphate buffered saline, or saline, and may more typically include an adjuvant. . Incomplete Freund's adjuvant, aluminum phosphate (a
Luminum), aluminum hydroxide, or adjuvants such as alum are substances well known in the art. Also, as described above, CTL responses can be primed by conjugating the peptides of the invention to lipids (eg, P ₃ CSS). Upon immunization via injection, aerosol, oral, transdermal, or other routes with a peptide composition as described herein, the host's immune system will produce large amounts of specific antigens of the desired antigen. Responding to the vaccine by producing a CTL of
The host then becomes at least partially immune to subsequent infections or resistant to the development of chronic infections.

Vaccine compositions containing the peptides of the invention elicit an immune response against the antigen in patients who are susceptible to, or otherwise at risk of, viral infection or cancer, It is therefore administered to enhance the patient's own immune response capacity. Such an amount is defined as "immunogenically effective dose." In this application, the exact amount will also depend on the health and weight of the patient, the mode of administration, the nature of the formulation, etc., but will generally be in the range of about 1.0 μg to about 5000 μg / 70 kg patient. , More commonly, in the range of about 10 μg to about 5000 μg per 70 kg body weight.

In some cases, it may be desirable to combine the peptide vaccines of the invention with a vaccine that induces a neutralizing antibody response against the virus of interest, especially the viral envelope antigen.

For therapeutic or immunization purposes, nucleic acids encoding one or more peptides of the invention can also be administered to a patient. A number of methods are conventionally used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, by Wolff et al., Science 247: 1465-.
1468 (1990) and US Pat. Nos. 5,580,859 and 5,
589,466. Nucleic acids are also disclosed, for example, in US Pat.
It may be administered using ballistic delivery, as described in No. 253. Particles composed of only DNA can be administered. Alternatively, the DNA can be attached to particles such as gold particles. Nucleic acids can also be complexed and delivered to cationic compounds such as cationic lipids. Gene delivery methods mediated by lipids are described, for example, in WO 96/18372; WO 9
3/24640; Mannino and Gould-Fogerite, Bio.
Techniques 6 (7): 682-691 (1988); Rose US Patent No. 5,279,833; WO 91/06309; and Felgn.
er et al., Proc. Natl. Acad. Sci. USA 84: 7413-7.
414 (1987). The peptides of the invention can also be expressed by an attenuated viral host such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences encoding the peptides of the invention. Upon introduction into an acutely or chronically infected host, or into an uninfected host, this recombinant vaccinia virus expresses an immunogenic peptide, thereby inducing a CTL response in the host. Vaccinia vectors and methods useful in immunization protocols are described, for example, in US Pat. No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described by Stover et al. (Na), which is incorporated herein by reference.
true 351: 456-460 (1991)). A wide variety of other vectors useful in therapeutic administration or immunization of the peptides of the invention (eg, Sa
lmonella typhi vectors, etc.) will be apparent to those of skill 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. The amino acid sequence of this epitope is back-translated to create a DNA sequence (minigene) encoding the selected CTL epitope for expression in human cells. A human codon usage table is used to guide the codon choice for each amino acid. DNA sequences encoding these epitopes are added directly to create contiguous polypeptide sequences. Additional elements can be incorporated into the design of the minigene to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: helper T lymphocyte epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Further, MHC presentation of CTL epitopes can be improved by including synthetic (eg, polyalanine) or naturally occurring flanking sequences flanking the CTL epitope.

Minigene sequences are converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified, and annealed under suitable conditions using well known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic minigene, which encodes a CTL epitope polypeptide, can then be cloned into the 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 cell. Several vector elements are required: a promoter with a downstream cloning site for insertion of the minigene; a polyadenylation signal for efficient termination of transcription; E. coli origin of replication; and E. c
oli selectable marker (eg ampicillin resistance or kanamycin resistance). A number of promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. See US Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Further 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 can be incorporated into the transcribed region of the minigene. Inclusion of mRNA stabilizing sequences can also be considered to increase expression of the minigene. It has recently been proposed that immunostimulatory sequences (ISS or CpG) play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the coding sequence of the minigene, if found to enhance immunogenicity.

In some embodiments, a bicistronic expression that enables the production of a minigene-encoded epitope and a second protein included to enhance or reduce immunogenicity. Vectors can be used. Examples of proteins or polypeptides that may beneficially enhance the immune response when co-expressed include cytokines (eg IL2, IL12, GM-C).
SF), cytokine-inducing molecules (eg LeIF), or costimulatory molecules. Helper (HTL) epitopes can be linked to intracellular targeting signals and expressed separately from CTL epitopes. This allows the targeting of the HTL epitope to a cell compartment distinct from the CTL epitope. If required, this may facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically reducing the immune response by co-expression of immunosuppressive molecules (eg TGF-β) may be beneficial in certain diseases.

Once the expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is suitable for E. coli
The strain is transformed and the DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, and all other elements contained in the vector are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells with the correct plasmid can be stored as a master cell bank and a working cell bank.

A therapeutic amount of plasmid DNA can be obtained from E. It is produced by fermentation in E. coli and subsequent purification. Aliquots from the working cell bank are used to inoculate a fermentation medium (eg, Terrific Broth) and grown to saturation in shake flasks or bioreactors according to well-known techniques. Plasmid DNA can be purified using standard bioseparation techniques, such as the solid phase anion exchange resin provided by Quiagen. If required, supercoiled DNA can be isolated from open circular and linear forms using gel electrophoresis or other techniques.

Purified plasmid DNA can be prepared for injection using various formulations. The simplest of these is sterile phosphate buffered saline (PB).
Reconstitution of lyophilized DNA in S). Various methods have been described,
And new technology may become available. As mentioned above, the nucleic acid is conveniently formulated with a cationic lipid. In addition, glycolipids, fusogenic
enic) liposome, protective, interactive, non-condensed
ing) (PINC) generically, peptides and compounds also affect purified plasmid DNA to affect variables such as stability, intramuscular dispersion, and trafficking to specific organs or cell types. Can be complexed.

Sensitization of target cells can be used as a functional assay for expression and MHC class I presentation of CTL epitopes encoded by minigenes. 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 depends on the final formulation. Electroporation is "naked" despite the fact that cationic lipids allow direct in vitro transfection.
It can be used for DNA. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of the transfected cells using fluorescence activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for the epitope-specific CTL line. Cytolysis detected by release of 51Cr indicates production of MHC presentation of the CTL epitope encoded by the minigene.

In vivo immunogenicity is the second choice for functional testing of minigene DNA formulations.
Is the approach. Transgenic mice expressing the appropriate human MHC molecule are immunized with the DNA product. The dose and route of administration depends on the formulation (
For example, 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 tested. These effector cells (CTL
) Is assayed for cytolysis of chromium-51 labeled target cells loaded with peptide using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to the epitope encoded by the minigene is
Demonstrate DNA vaccine function for in vivo induction of TL.

Antigenic peptides can also be used to induce CTL ex vivo. The resulting CTLs 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 to peptide vaccine approaches. Ex vivo CTL responses to specific pathogens (infectious agents or tumor antigens) indicate that in tissue culture, the patient's CTL progenitor cells (CTLp) together with a source of antigen presenting cells (APCs) and an appropriate immunogenic peptide. Induced by incubating.
After a suitable incubation time (typically 1 to 4 weeks), at which time these CTLp are activated and mature and proliferate to effector CTL, these cells are injected into the patient. Brought back, where they destroy their specific target cells (infected cells or tumor cells). To optimize in vitro conditions for the generation of specific cytotoxic T cells, cultures of stimulator cells are maintained in a suitable serum free medium.

Prior to incubation of the stimulator cells with cells to be activated (eg precursor CD8 + cells), sufficient to be loaded with human class I molecules to be expressed on the surface of the stimulator cells. An amount of antigenic peptide is added to the stimulated cell culture. In the present invention, a sufficient amount of peptide allows about 200, and preferably 200 or more human class I MHC molecules to be loaded with the peptide to be expressed on the surface of each stimulator cell. Is the amount. Preferably, the stimulator cells are 20 μg / m
Incubated with more than 1 peptide.

Resting CD8 + cells or progenitor CD8 + cells are then incubated in culture with the appropriate stimulator cells for a time sufficient to activate the CD8 + cells. Preferably, CD8 + cells are activated in an antigen-specific manner. The ratio of resting CD8 + cells or progenitor CD8 + (effector) cells to stimulator cells may vary from individual to individual, and the individual's adaptability to culturing conditions for lymphocytes, as well as the disease state or other conditions for which the described treatment modalities are used. May further depend on variables such as the nature and severity of the condition. However, preferably, the lymphocyte: stimulator cell ratio is in the range of about 30: 1 to 300: 1. Effector / stimulation cultures
It can be maintained for a period of time necessary to stimulate a therapeutically useful or effective number of CD8 + cells.

In vitro induction of CTL is associated with allele-specific MHC class I on APC.
It requires specific recognition of the peptide bound to the molecule. The number of specific MHC / peptide complexes per APC is critical for CTL stimulation, especially for the primary immune response. A small amount of peptide / MHC complex per cell is sufficient to sensitize cells to lysis by CTLs, or sufficient to stimulate a secondary CTL response, but during the primary response Successful activation of (pCTL) requires a significantly higher number of MHC / peptide complexes. Peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses. Peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses.

Since the mutant cell line is not present in all human MHC alleles,
It is advantageous to use a technique that removes the endogenous MHC binding peptide from the surface of the APC, and then load the resulting empty MHC molecule with the immunogenic peptide of interest.
Of non-transformed (non-tumorigenic) non-infected cells of the patient, and preferably autologous cells,
Use as an APC is desirable for the design of CTL induction protocols towards the development of ex vivo CTL therapies. The present application describes that an endogenous MHC-binding peptide is APC
A method is disclosed for peeling from the surface of, followed by loading the desired peptide.

A stable MHC class I molecule is a trimeric complex formed from the following elements: 1) a peptide, usually 8-10 residues, 2) a peptide binding site in the α1 and α2 domains. Β ₂ microblobulin, which is a transmembrane heavy chain polymorphic protein chain carrying 3) and 3) a non-covalently linked non-polymorphic light chain. Removing bound peptides and / or dissociating β ₂ microglobulin from the complex renders MHC class I molecules non-functional and unstable, resulting in rapid degradation. All MHC class I molecules isolated from PBMC have an endogenous peptide bound to them. Therefore, the first stage is MHC on APC
The removal of all endogenous peptides bound to class I molecules without causing their degradation before exogenous peptides can be added to it.

Two possible ways to release MHC class I molecules of bound peptides are to destabilize β ₂ microglobulin by lowering the culture temperature from 37 ° C. to 26 ° C. overnight. And stripping the endogenous peptide from the cells using a mild acid treatment. This method liberates previously bound peptides into the extracellular environment, allowing new exogenous peptides to bind to empty class I molecules. The low temperature incubation method allows exogenous peptides to bind efficiently to the MHC complex but requires overnight culture at 26 ° C., which can slow the cell's metabolic rate. . Cells that do not actively synthesize MHC molecules (eg,
It is also likely that resting PBMCs do not yield large amounts of empty surface MHC molecules by low temperature procedures.

Stringent acid stripping involves extraction of the peptide with trifluoroacetic acid (pH 2) or acid denaturation of immunoaffinity purified Class I peptide complexes.
These methods are not capable of CTL induction. This is because it is important to remove the endogenous peptide while maintaining the viability and optimal metabolic state of APC, which is important for antigen presentation. Mildly acidic solutions at pH 3 (eg glycine or citrate-phosphate buffer) have been used to identify endogenous peptides and to identify tumor associated T cell epitopes. This treatment is particularly advantageous in that only MHC class I molecules are destabilized (and bound peptides are released), while other surface antigens (including MHC class II molecules) remain intact. It is valid. Most importantly, treatment of cells with mildly acidic solutions does not affect cell viability or metabolic status. Mild acid treatment is rapid. Because endogenous peptide stripping occurs at 4 ° C. for 2 minutes, and APC is ready to perform its function after being loaded with the appropriate peptide. This technique is utilized herein to generate peptide-specific APCs to generate primary antigen-specific CTL. Obtained A
PC is efficient in inducing peptide-specific CD8 + CTL.

Activated CD8 + cells can be effectively separated from stimulator cells using one of a variety of known methods. For example, monoclonal antibodies specific for stimulator cells, peptides loaded on stimulator cells, or CD8 + cells (or segments thereof) can be utilized to bind their appropriate complementary ligands. The antibody-tagged molecule can then be extracted from the stimulator cell / effector cell mixture by suitable means, such as well known immunoprecipitation or immunoassay methods.

Effective, cytotoxic amounts of activated CD8 + cells can vary between in vitro and in vivo applications, and the amount of cells that are the ultimate target of those killer cells and It can vary depending on the type. This amount may also vary depending on the condition of the patient and should be determined by the practitioner in light of all appropriate factors. However, compared to about 5 × 10 ^{6 to} 5 × 10 ⁷ cells used in mice, preferably about 1 × 10 ⁶ to about 1 × 10 ¹² and more preferably about 1 × 10 ⁸ to
About 1 × 10 ¹¹ , and even more preferably about 1 × 10 ⁹ to about 1 × 10 ¹⁰ activated CD8 + cells are utilized for adult humans.

Preferably, as described above, activated CD8 + cells are harvested from cell culture prior to administration of CD8 + cells to the individual to be treated. However, unlike other current and proposed treatment modalities, it is important to note that the methods of the present invention employ cell culture systems that are not tumorigenic. Therefore, if complete separation of stimulator cells from activated CD8 + cells is not achieved, there is no inherent risk known to be associated with the administration of small numbers of stimulator cells, whereas mammalian tumor-promoting cells Administration of can be extremely dangerous.

Methods of reintroducing cellular components are known in the art, and include, for example, US Pat. No. 4,844,893 to Honsik et al. And Rose.
Procedures such as those illustrated in US Pat. No. 4,690,915 to Nberg. For example, administration of activated CD8 + cells by intravenous infusion is suitable.

The immunogenic peptides of the invention can also be used to make monoclonal antibodies. Such antibodies may be useful as potential diagnostic or therapeutic agents.

These peptides may also find use as diagnostic reagents. For example, the peptides of the invention can be used to determine the susceptibility of a particular individual to a treatment regimen with a peptide or related peptides, and thus in modifying existing treatment protocols or in prognosing for affected individuals. It can be useful in making decisions. Furthermore, these peptides can also be used to predict which individuals are at significant risk of developing a chronic infection.

To identify the peptides of the invention, class I antigen isolation, as well as isolation and sequencing of naturally processed peptides, was performed as described in the related application. 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 above. These motifs, set forth in Tables 6-9 below, are defined from a sequencing data pool of naturally processed peptides, as described in the related application.

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[Table 8]

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[Table 9]

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[Table 10]

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[Table 11] Example 2 Identification of Immunoatom Peptides Using the motifs identified above for various MHC class I alleles, amino acid sequences from various pathogens and tumor-associated proteins were analyzed for the presence of these motifs. did. The screening described in the related application was performed. Table 12 provides the search results for these antigens.

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[Table 12] Example 3 Identification of Immunogenic Peptides Using the B7-like supermotif identified in the related application above, sequences from various pathogen and tumor associated proteins were analyzed for the presence of these motifs. . Screening was performed as described in the related application. Table 13 provides the search results for these antigens.

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[Table 13] The above description is provided to illustrate the invention, but not to limit its scope. Other modifications of the invention will be readily apparent to those skilled in the art and are covered by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 105 A61P 43/00 105 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, E, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Cast, W. Martin Netherlands Nuel-2331 Neck Sleiden, Maria Rutgersvek 106 (72) Inventor Southwood, Scott United States California 92071, Santee, Strathmore Drive 10679 F term (reference) 4C085 AA03 BB11 CC32 GG02 GG03 GG04 GG05 GG08 4H045 AA11 AA30 BA15 BA16 DA86 EA20 EA50 FA74

Claims (6)

[Claims] 1. A composition comprising an immunogenic peptide having an HLA-A2.1 binding motif, wherein the immunogenic peptide is selected from the group consisting of: ] 2. Use of an immunogenic peptide for the manufacture of a medicament for inducing a cytotoxic T cell response against a given antigen in a patient expressing a HLA-A2.1 MHC product, said immunization. The antigenic peptide is selected from the group consisting of the use: 3. A composition containing an immunogenic peptide selected from the group consisting of: 4. Use of an immunogenic peptide for the manufacture of a medicament for inducing a cytotoxic T cell response against a predetermined antigen in a patient, said immunogenic peptide being from the group consisting of: Selected, use: 5. A composition comprising an immunogenic peptide selected from the group consisting of the peptides listed in Table 13. 6. Use of an immunogenic peptide for the manufacture of a medicament for inducing a cytotoxic T cell response against a given antigen in a patient, the immunogenic peptide being listed in Table 13. Use, selected from the group consisting of peptides.